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Diets rich in fat, smoking, as well as exposure to environmental pollutants and dysbiosis of gut microbiota, increase the risk of developing colorectal cancer. Much progress has been made in combating colorectal cancer. However, options for chemoprevention from environmental insult and dysbiosis of gut microbiota remain elusive. We investigated the influence of berry-derived anthocyanidins (Anthos), with and without encapsulating them in bovine milk–derived exosomes (ExoAnthos), on the chemoprevention of bacteria-driven colon tumor development. Anthos and ExoAnthos treatment of colon cancer cells showed dose-dependent decreases in cell viability. Calculated selectivity index (SI) values for Anthos and ExoAnthos suggest that both treatments selectively targeted cancer over normal colon cells. In addition, ExoAnthos treatment yielded higher SI values than Anthos. Anthos and ExoAnthos treatment of ApcMin/+ mice inoculated with enterotoxigenic Bacteriodes fragilis (ETBF) bacteria led to significant decreases in colon tumor numbers over mice receiving vehicle treatments. Western blot analysis of normal colon, colon tumor, and liver tissue lysates showed that mice inoculated with ETBF featured increased expression of phase I enzymes in normal colon tissue and decreased expression of phase II enzymes in liver tissue. Treatment with the Anthos and ExoAnthos reverted the modulation of phase I and phase II enzymes, respectively; no significant changes in phase II enzyme expression occurred in colon tumor tissue. Treatment of HCT-116 cells with the ubiquitous carcinogen, benzo[a]pyrene (B[a]P) led to similar modulation of phase I and II enzymes, which was partially mitigated by treatment with Anthos. These results provide a promising outlook on the impact of berry Anthos for prevention and treatment of bacteria- and B[a]P-driven colorectal cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) is the most common type accounting for 84% of all lung cancers. Paclitaxel (PAC) is a widely used drug in the treatment of a broad spectrum of human cancers, including lung. While efficacious, PAC generally is not well tolerated and its limitations include low aqueous solubility, and significant toxicity. To overcome the dose-related toxicity of solvent-based PAC, we utilized bovine colostrum-derived exosomes as a delivery vehicle for PAC for the treatment of lung cancer. Colostrum provided higher yield of exosomes and could be loaded with higher amount of PAC compared to mature milk. Exosomal formulation of PAC (ExoPAC) showed higher antiproliferative activity and inhibition of colony formation against A549 cells compared with PAC alone, and also showed antiproliferative activity against a drug-resistant variant of A549. To further enhance its efficacy, exosomes were attached with a tumor-targeting ligand, folic acid (FA). FA-ExoPAC given orally showed significant inhibition (&gt, 50%) of subcutaneous tumor xenograft while similar doses of PAC showed insignificant inhibition. In the orthotopic lung cancer model, oral dosing of FA-ExoPAC achieved greater efficacy (55% growth inhibition) than traditional i.v. PAC (24–32% growth inhibition) and similar efficacy as i.v. Abraxane (59% growth inhibition). The FA-ExoPAC given i.v. exceeded the therapeutic efficacy of Abraxane (76% growth inhibition). Finally, wild-type animals treated with p.o. ExoPAC did not show gross, systemic or immunotoxicity. Solvent-based PAC caused immunotoxicity which was either reduced or completely mitigated by its exosomal formulations. These studies show that a tumor-targeted oral formulation of PAC (FA-ExoPAC) significantly improved the overall efficacy and safety profile while providing a user-friendly, cost-effective alternative to bolus i.v. PAC and i.v. Abraxane.

Simple Summary Breast cancer is the most common female cancer diagnosed in the U.S. and the second most common cause of cancer death in women. Chemotherapeutics used to treat breast cancer often have side effects, which are sometimes life-threatening. Moreover, the tumors can develop resistance over time, making breast cancer treatment challenging. In this paper, we show that the oral administration of colored pigments isolated from bilberry/blueberry, called anthocyanidins (Anthos), significantly decrease MDA-MB-231 orthoxenograft tumor volume, inhibit the growth and metastasis of breast cancer, sensitize drug-resistant tumor cells, and exhibit a lower rate of lymph node and lung metastasis, compared to control. Our results also suggest regulation of cell-cycle progression and inhibition of NF-κB activation as mechanisms underpinning the anti-proliferative activity of Anthos in breast cancer. These mechanistic insights are expected to be valuable for clinical translation of berry Anthos, either alone or as adjuvant to chemotherapy, for the treatment of breast cancer patients. Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is the only systemic treatment option. Although chemotherapeutic drugs respond initially in TNBC, many patients relapse and have a poor prognosis. Poor survival after metastatic relapse is largely attributed to the development of resistance to chemotherapeutic drugs. In this study, we show that bilberry-derived anthocyanidins (Anthos) can inhibit the growth and metastasis of TNBC and chemosensitize paclitaxel (PAC)-resistant TNBC cells by modulating the NF-κB signaling pathway, as well as metastatic and angiogenic mediators. Anthos administered orally significantly decreased MDA-MB-231 orthoxenograft tumor volume and led to lower rates of lymph node and lung metastasis, compared to control. Treatment of PAC-resistant MDA-MB-231Tx cells with Anthos and PAC in combination lowered the IC50 of PAC by nearly 20-fold. The combination treatment also significantly (p < 0.01) decreased the tumor volume in MDA-MB-231Tx orthoxenografts, compared to control. In contrast, Anthos and PAC alone were ineffective against MDA-MB-231Tx tumors. Our approach of using Anthos to inhibit the growth and metastasis of breast cancers, as well as to chemosensitize PAC-resistant TNBC, provides a highly promising and effective strategy for the management of TNBC.

Over the last two decades, berries and berry bioactives, particularly anthocyanins and their aglycones anthocyanidins (Anthos) have demonstrated excellent anti-oxidant, anti-proliferative, apoptotic and anti-inflammatory properties. However, their physicochemical and pharmacokinetic limitations such as, low permeability, and poor oral bioavailability are considered as unfavorable properties for development as drugs. Therefore there is a need to develop systems for efficient systemic delivery and robust bioavailability. In this study we prepared nano-formulation of bilberry-derived Anthos using exosomes harvested from raw bovine milk. Exosomal formulation of Anthos enhanced antiproliferative and anti-inflammatory effects compared with the free Anthos against various cancer cells in vitro. Our data also showed significantly enhanced therapeutic response of exosomal-Anthos formulation compared with the free Anthos against lung cancer tumor xenograft in nude mice. The Anthos showed no signs of gross or systemic toxicity in wild-type mice. Thus, exosomes provide an effective alternative for oral delivery of Anthos that is efficacious, cost-effective, and safe, and this regimen can be developed as a non-toxic, widely applicable therapeutic agent.

Despite optimal diagnosis and early therapeutic interventions, the prognosis for ovarian cancer patients remains dismal because the efficacy of chemotherapy is limited by the development of resistance and off-site toxicity. Berry bioactives indicate preventive and therapeutic activities against various cancer types. Here, we examined the antiproliferative activity of berry anthocyanidins (Anthos) against drug-sensitive (A2780) and drug-resistant (A2780/CP70, OVCA432 and OVCA433) ovarian cancer cells. These drug-resistant ovarian cancer cell lines overexpress p-glycoproteins (PgP) and show >100-fold resistance to the chemotherapeutic drug cisplatin compared to A2780. We observed a dose-dependent growth inhibition of ovarian cancer cells with the Anthos. Furthermore, the treatment of drug-resistant ovarian cancer (OVCA433) cells with cisplatin in combination with the Anthos (75 μM) resulted in significantly higher cell killing. The cisplatin dose required to achieve this effect was 10 to 15-fold lower than the IC50 of cisplatin alone. However, many plant bioactives including Anthos face the challenge of poor oral bioavailability and stability. Recently, we have developed strategies to overcome these limitations by delivering Anthos via milk-derived exosomes. The exosomal Anthos (ExoAnthos) significantly enhanced the antiproliferative activity against the growth of ovarian cancer cells and inhibited tumor growth more efficiently compared to Anthos alone and a vehicle control. Often patients with cisplatin-resistant tumors retain sensitivity to paclitaxel (PAC). We prepared exosomal formulations of PAC (ExoPAC) for oral delivery as the systemic administration of PAC has severe side effects. ExoPAC delivered orally showed the same therapeutic efficacy as the free PAC delivered intraperitoneally. Finally, we report that the combination of the Anthos and PAC decreased the PgP level in a dose-dependent manner in OVCA432 cells. A significantly enhanced antitumor activity was observed with the combination of ExoPAC and ExoAnthos against A2780 tumor xenografts. Together, our data indicate that the berry Anthos are highly effective against ovarian cancer and that the milk exosomes serve as an excellent nano-carrier to enhance the drug's oral bioavailability for the management of ovarian cancer.

BACKGROUND: Blueberry (BB) and black raspberry (BRB) have been shown to be chemopreventive against estrogenmediated breast cancer in pre-clinical studies. However, therapeutic efficacy of these berries against lung cancer is not known. METHODS: In this study we investigated i) relative efficacy of individual anthocyanidins vs. respective anthocyanins, ii) relative antiproliferative activity of mixture of anthocyanidins compared to individual anthocyanidins, iii) antitumor activity of dietary BB, iv) Tumor inhibitory activity of diet supplemented with BB, alone and in combination with BRB, against lung tumor xenograft using nude mice, and finally, v) the efficacy of select polyphenolics present in BB and BRB against lung tumor xenograft. RESULTS: Our findings indicated that individual anthocyanidins (aglycones) were significantly more potent (2-3 fold lower IC50) in inhibiting the non-small-cell lung cancer (NSCLC) cell growth vs. respective anthocyanins (glycones). Further, anthocyanidins mixture at equimolar concentrations exhibited synergistic antiproliferative activity vs. individual anthocyanidins. When tested against NSCLC (A549 and H1299) cells in nude mice, dietary BB (7.5%, w/w) showed >40% reduction in tumor volume against H1299 xenografts. The maximal growth inhibition occurred with 5% BB dose, with no additional protection occurring at a higher dose (7.5%). However, somewhat lower protection was found when the BB diet initiated prior to tumor cell inoculation. The mixture of BB (5%, w/w) and BRB (2.5%) resulted in higher inhibition of tumor growth vs. BB alone (71% vs 42%). Likewise, a combination of delphinidin (bioactive of BB) and punicalagins (a bioactive of BRB, which gets converted to ellagic acid in vivo) showed higher tumor growth inhibition compared to delphinidin. CONCLUSIONS: The therapeutic effects of the berries and berry polyphenolics observed against lung cancer in this study are highly encouraging. Further investigation into the mechanism of action of the combinations of the berry bioactives will be valuable for clinical use of this potent natural product against lung cancer.

Spices are used worldwide, particularly, in the Asian and Middle-Eastern countries and considered protective against degenerative diseases, including cancer. Here, we report the efficacy of aqueous and non-aqueous extracts of eleven Apiaceae spices for free radical-scavenging activity and to inhibit cytochrome P450s in two separate reactions involving: i) 4-hydroxy-17β-estradiol (4E2), DNA and CuCl2 and ii) 17β-estradiol, rat liver microsomes, co-factors, DNA and CuCl2. Oxidative DNA adducts resulting from redox cycling of 4E2 were analyzed by 32P-postlabeling. Aqueous (5 mg/ml) and non-aqueous extracts (6 mg/ml) substantially inhibited (83% – 98%) formation of DNA adducts in the microsomal reaction. However, in non-microsomal reaction, only aqueous extracts showed the inhibitory activity (83% – 96%). Adduct inhibition was also observed at 5-fold lower concentrations of aqueous extracts of cumin (60%) and caraway (90%), and 10-fold lower concentrations of carrot seeds (76%) and ajowan (90%). These results suggests the presence of two groups of phytochemicals - polar compounds that have free radical-scavenging activity, and lipophilic compounds that selectively inhibit P450 activity associated with estrogen metabolism. Because most of these Apiaceae spices are used widely with no known toxicity, the phytochemicals from the Apiaceae spices used in foods may be potentially protective against estrogen-mediated breast cancer.

Human papilloma virus (HPV) is the well-established etiological factor of cervical cancer. E6 and E7 oncoproteins expressed by HPV are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in the traditional Chinese medicine Danshen (Salvia sp.). Tan IIA has been shown to possess anti-tumor activity against several cancer types. In this study we show that Tan IIA potently inhibited proliferation of the human cervical cancer CaSki, SiHa, HeLa and C33a cells. Mechanistically in HPV positive CaSki cells, Tan IIA was found to (i) downregulate expression of HPV E6 and E7 genes and modulate associated proteins E6AP and E2F1, (ii) cause S phase cell cycle arrest, (iii) induce accumulation of p53 and alter expression of p53-dependent targets, (iv) modulate pRb and related proteins, and (v) cause p53-mediated apoptosis by moderating Bcl2, Bax, caspase-3, and PARP cleavage expressions. In vivo, Tan IIA resulted in over 66% reduction in tumor volume of cervical cancer xenograft in athymic nude mice. Tan IIA treated tumor tissues had lower expression of proliferation marker PCNA and changes in apoptosis targets were in agreement with in vitro studies, further confirming reduced proliferation and involvement of multiple targets behind anti-cancer effects. This is the first demonstration of Tan IIA to possess significant anti-viral activity by repressing HPV oncogenes leading to inhibition of cervical cancer. Together, our data suggest that Tan IIA can be exploited as a potent therapeutic agent for the prevention and treatment of cervical and other HPV-related cancers.

Cervical cancer is caused by human papillomavirus (HPV). The disease develops over many years through a series of precancerous lesions. Cervical cancer can be prevented by HPV-vaccination, screening and treatment of precancer before development of cervical cancer. The treatment of high-grade cervical dysplasia (CIN2+) has traditionally been by cervical conization. Surgical procedures are associated with increased risk of undesirable side effects including bleeding, infection, scarring (stenosis), infertility and complications in later pregnancies. An inexpensive, non-invasive method of delivering therapeutics locally will be favorable to treat precancerous cervical lesions without damaging healthy tissue. The feasibility and safety of a sustained, continuous drug-releasing cervical polymeric implant for use in clinical trials was studied using a large animal model. The goat (Capra hircus), non-pregnant adult female Boer goats (n=7), was chosen due to similarities in cervical dimensions to the human. Estrus was induced with progesterone CIDR® vaginal implants for 14 days followed by the administration of chorionic gonadotropins 48 hours prior to removal of the progesterone implants to relax the cervix to allow for the placement of the cervical implant. Cervical implants, containing 2% and 4% withaferin A (WFA), with 8 coats of blank polymer, provided sustained release for a long duration and were used for the animal study. The ‘mushroom’-shaped cervical polymeric implant, originally designed for women required redesigning to be accommodated within the goat cervix. The cervical implants were well tolerated by the animals with no obvious evidence of discomfort, systemic or local inflammation or toxicity. In addition, we developed a new method to analyze tissue WFA levels by solvent extractions and LS/MS-MS. WFA was found to be localized to the target and adjacent tissues with 12 – 16 ng WFA/g tissue, with essentially no detectable WFA in distant tissues. This study suggests that the goat is a good large animal model for the future development and evaluation of therapeutic efficacy of continuous local drug delivery by cervical polymeric implants to treat precancerous cervical lesions.

Exosomes are extracellular microvesicles with a particle size of 30–100 nm and carry a cargo of proteins, lipids, RNA, and DNA. Their properties of shuttling in-and-out of the cells suggest that these particles can be exploited as a nano drug carrier. In this manuscript, we show that curcumin can be delivered effectively using milk-derived exosomes. Curcumin when mixed with exosomes in the presence of 10% ethanol:acetonitrile (1:1) provided a drug load of 18–24%, and the formulation stored at − 80°C was stable for 6 months as determined by particle size analysis, drug load, and antiproliferative activity. The uptake of exosomes by cancer cells involved caveolae/clathrin-mediated endocytosis. Oral administration of exosomal curcumin (ExoCUR) in Sprague-Dawley rats demonstrated 3–5 times higher levels in various organs versus free agent. ExoCUR showed enhanced antiproliferative activity against multiple cancer cell lines including, breast, lung, and cervical cancer compared with the free curcumin. ExoCUR showed significantly higher anti-inflammatory activity measured as NF-κB activation in human lung and breast cancer cells. To determine in vivo antitumor activity, nude mice bearing the cervical CaSki tumor xenograft were treated with ExoCUR by oral gavage, curcumin diet, exosomes alone, and PBS as controls. While curcumin via dietary route failed to elicit any effect, exosomes had a modest (25–30%) tumor growth inhibition. However, ExoCUR showed significant inhibition (61%; p

Highlights • Polymeric implants successfully achieved continuous exposure to PCBs in rats. • PCB126 resulted in significant oxidative DNA damage (8-oxodG) in rat liver and lung. • PCB126 or in combination with PCB153 induced PON1 and its activity in the liver. • The induction was even greater for PON3 and AhR gene transcription. • Co-treatment reduced mammary PCB153 and increased liver PCB126 and PCB153 levels., A new delivery method via polymeric implants was used for continuous exposure to PCBs. Female Sprague-Dawley rats received subcutaneous polymeric implants containing PCB126 (0.15% load), PCB153 (5% load), or both, for up to 45 d and release kinetics and tissue distribution were measured. PCB153 tissue levels on day 15 were readily detected in lung, liver, mammary and serum, with highest levels in the mammary tissue. PCB126 was detected only in liver and mammary tissues. However, a completely different pharmacokinetics was observed on co-exposure of PCB153 and PCB126, with a 1.8-fold higher levels of PCB153 in the liver whereas a 1.7-fold lower levels in the mammary tissue. PCB126 and PCB153 caused an increase in expression of key PCB-inducible enzymes, CYP 1A1/2 and 2B1/2, respectively. Serum and liver activities of the antioxidant enzymes, PON1 and PON3, and AhR transcription were also significantly increased by PCB126. 32P-postlabeling for polar and lipophilic DNA-adducts showed significant quantitative differences: PCB126 increased 8-oxodG, an oxidative DNA lesion, in liver and lung tissues. Adduct levels in the liver remained upregulated up to 45 d, while some lung DNA adducts declined. This is the first demonstration that continuous low-dose exposure to PCBs via implants can produce sustained tissue levels leading to the accumulation of DNA-adducts in target tissue and induction of indicator enzymes. Collectively, these data demonstrate that this exposure model is a promising tool for long-term exposure studies.

Dietary polyphenols may contribute to the prevention of several degenerative diseases, including cancer. Anthocyanins have been shown to possess potential anticancer activity. The aim of this study was to determine anthocyanin bioavailability in lung tissue of mice fed a blueberry diet (5% w/w) for 10 days or a bolus dose (10 mg/mouse; po) of a native mixture of bilberry anthocyanidins. All five anthocyanidins present in the blueberry were detected in the lung tissue using improved methods. The effect of various solvents on the stability of anthocyanins and their recovery from the biomatrix was analyzed. Detection of anthocyanins and their metabolites was performed by UPLC and LC-MS. Although anthocyanins were not detected, cyanidin was detected by UPLC-PDA and other anthocyanidins were detected by LC-MS, following conversion to anthocyanidins and selective extraction in isoamyl alcohol. The results show that anthocyanins can be detected in lung tissue of blueberry-fed mice and thus are bioavailable beyond the gastrointestinal tract.

Polymeric implants (millirods) have been tested for local delivery of chemotherapeutic agents in cancer treatment. Modeling of drug release profiles is critical as it may provide theoretical insights on rational implant design. In this study, a biodegradable poly (ε-caprolactone) (PCL) polymeric implant delivery system was tested to deliver green tea polyphenols (GTPs), both in vitro and in vivo. Factors including polymer compositions, supplements, drug loads and surface area of implants were investigated. Our data showed that GTPs were released from PCL implants continuously for long durations, and drug load was the main determining factor of GTPs release. Furthermore, the rates of in vitro release and in vivo release in the rat model followed similar kinetics for up to 16 months. A mathematical model was deduced and discussed. GTPs implants have the potential to be used locally as an alternative strategy. GTP implants have the potential to be used systemically and locally at the tumor site as an alternative strategy.

Scientific evidence suggests that many herbs and spices have medicinal properties that alleviate symptoms or prevent disease. In this study, we examined the chemopreventive effects of the Apiaceae spices, anise, caraway, and celery seeds against 17β-estrogen (E2)-mediated mammary tumorigenesis in an ACI (August-Copenhagen Irish) rat model. Female ACI rats were given either control diet (AIN 93M) or diet supplemented with 7.5% (w/w) of anise, caraway, or celery seed powder. Two weeks later, one half of the animals in each group received subcutaneous silastic implants of E2. Diet intake and body weight were recorded weekly, and animals were euthanized after 3 and 12 weeks. E2-treatment showed significantly (2.1- and 3.4-fold) enhanced growth of pituitary gland at 3 and 12 weeks, respectively. All test spices significantly offset the pituitary growth by 12 weeks, except celery which was effective as early as three weeks. Immunohistochemical analysis for proliferative cell nuclear antigen (PCNA) in mammary tissues showed significant reduction in E2-mediated mammary cell proliferation. Test spices reduced the circulating levels of both E2 and prolactin at three weeks. This protection was more pronounced at 12 weeks, with celery eliciting the highest effect. RT-PCR and western blot analysis were performed to determine the potential molecular targets of the spices. Anise and caraway diets significantly offset estrogen-mediated overexpression of both cyclin D1 and estrogen receptor α (ERα). The effect of anise was modest. Likewise, expression of CYP1B1 and CYP1A1 was inhibited by all test spices. Based on short-term molecular markers, caraway was selected over other spices based on its enhanced effect on estrogen-associated pathway. Therefore, a tumor-end point study in ACI rats was conducted with dietary caraway. Tumor palpation from 12 weeks onwards revealed tumor latency of 29 days in caraway-treated animals compared with first tumor appearance at 92 days in control group. At the end of the study (25 weeks), the tumor incidence was 96% in the control group compared with only 70% in the caraway group. A significant reduction in tumor volume (661 ± 123 vs. 313 ± 81 mm³) and tumor multiplicity (4.2 ± 0.4 vs. 2.5 ± 0.5 tumors/animal) was also observed in the caraway group compared with the control group. Together, our data show dietary caraway can significantly delay and prevent the hormonal mammary tumorigenesis by modulating different cellular and molecular targets.

The study of anthocyanins and carotenoids is an active area of research due to their various medicinal uses and growing interest in alternatives to synthetic colorants [1]. Anthocyanins (of the Greek anthos = flower and kians = blue) are important pigments of the vascular plants as they have shown various promising biological activities [2]. Anthocyanins are abundant in berries such as blackberry, raspberry, huckleberry, and blueberry [3]. The genus Eugenia is one of 75 genera (approximately 3000 species) belonging to the family Myrtaceae. Eugenia jambolana commonly known as jamun/jambu/jambula/jamboola originated in Asia, specifically India and other tropical regions [4]. Jamun fruit is a rich source of anthocyanins. Major anthocyanins of jamun include delphinidin-3,5-diglucoside, petunidin-3,5-diglucoside, and malvidin-3,5-diglucoside, while cyanidin-3,5-diglucoside and peonidin-3,5-diglucoside are minor constituents. The fruits are used as stomachic, astringent, antiscorbutic, diuretic, antidiabetic, chronic diarrhea, and enlargement of spleen [5]. Medicinal uses and pharmacological activities of plant extracts necessitate the development of analytical methods for their standardization and quality control. E. jambolana fruits possess numerous pharmacological activities, such as protection from DNA cleavage, estrogenic activity, enzyme inhibition, boosting production of cytokines, anti-inflammatory activity, lipid peroxidation, decreasing capillary permeability, fragility and membrane strengthening [6–10]. Petunidin and its glycosides induce apoptosis, whereas cyanidin and its glycosides show growth inhibitory and apoptosis-inducing effects. Delphinidin and its glycosides show antiproliferativity, photo-chemopreventive activity, inhibition of UVB (ultraviolet type B rays)-mediated oxidative stress, etc. [11–15]. Recently, we have reported antiproliferative activity of anthocyanins and anthocyanidin-enriched extract prepared from jamun pulp, with the extract eliciting higher activity than individual anthocyanins [16]. This may be due to synergism between anthocyanins and other phenolics present in the extract [17]. Therefore, a robust method to quantify individual anthocyanins and to standardize the crude jamun extract is necessary for quality control. In the present study, we report a simple high-performance thinlayer chromatography (HPTLC) method for the quantification of major anthocyanins of E. jambolana fruit extract. Earlier, HPLC and pH differential methods have been routinely used for quantification of anthocyanins in various plants. HPLC methods use solvents containing formic acid, phosphoric acid, trifluoroacetic acid, etc. in high concentration. Moreover, sample preparations are tedious and also require the addition of acid followed by solid phase extraction. The pH differential method determines only the total anthocyanin content, whereas HPTLC method can be used to determine individual as well as total anthocyanin content in the crude extract. In addition, different samples prepared in any solvent can be directly applied to the HPTLC plate and easily developed in developing chamber using commercial grade solvents.

Issues of poor oral bioavailability of cancer chemopreventives have hindered progress in cancer prevention. Novel delivery systems that modulate the pharmacokinetics of existing drugs, such as nanoparticles, cyclodextrins, niosomes, liposomes and implants, could be used to enhance the delivery of chemopreventive agents to target sites. The development of new approaches in prevention and treatment of cancer could encompass new delivery systems for approved and newly investigated compounds. In this review, we discuss some of the delivery approaches that have already made an impact by either delivering a drug to target tissue or increasing its bioavailability by many fold.

Berry anthocyanidins (cyanidin, malvidin, peonidin, petunidin and delphinidin) have increasingly been explored for their anticancer effects; however, their combinatorial effects as a mixture, as present in blueberry, bilberry and Indian blackberry ('Jamun') remain untested. In this study, we demonstrate for the first time that the combination of suboptimal concentrations of equimolar anthocyanidins synergistically inhibited growth of two aggressive non-small-cell lung cancer cell lines, with minimal effects on non-tumorigenic cell viability. The induction of cell-cycle arrest, apoptosis and suppression of NSCLC cell invasion and migration were also significantly greater with the mixture than individual anthocyanidins. The superior effects of the combinatorial treatment presumably resulted from its effects on the oncogenic Notch and WNT pathways and their downstream targets (β-catenin, c-myc, cyclin D1, cyclin B1, pERK, MMP9 and VEGF proteins), enhanced cleavage of the apoptotic mediators Bcl2 and PARP and enhanced inhibition of TNFα-induced NF-kappa B activation. In vivo, both the native mixture of anthocyanidins from bilberry (0.5mg/mouse) and the most potent anthocyanidin, delphinidin (1.5mg/mouse) significantly inhibited the growth of H1299 xenografts in nude miceby ≈60%. Notably, the effective dose of delphinidin in the anthocyanidin mixture was 8-fold lower than delphinidin alone, further emphasizing synergism. Our results thus demonstrate therapeutic potential of berries rich in this mixture of diverse anthocyanidins for non-small-cell lung cancer treatment and to prevent its future recurrence and metastasis.

Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant formulation (“coated” implants) using curcumin, individual curcuminoids, withaferin A and oltipraz. This method involves the coating of blank polycaprolactone implants with 20–30 layers of 10–20% polycaprolactone solution in dichloromethane containing 0.5–2% of the test agent. The in vitro release showed that while oltipraz was released with almost zero-order kinetics over 8 weeks, curcumin, individual curcuminoids and withaferin A were released with some initial burst. The in vivo release was determined by grafting implants subcutaneously in A/J mice. When delivered by coated implants, oltipraz significantly diminished lung DNA adducts in mice treated with dibenzo[ a,l ]pyrene compared with sham treatment (28 ± 7 versus 54 ± 17 adducts/10 9 nucleotides). Withaferin A also diminished DNA adducts, but it was insignificant. Curcumin and individual curcuminoids were ineffective. Analysis of lung, liver and brain by UPLC-fluorescence showed the presence of the three test curcuminoids indicating effectiveness of the implant delivery system. Further, based on its known antitumor activity in vivo , withaferin A given via the implants significantly inhibited human lung cancer A549 xenograft in athymic nude mice, while it was ineffective when the same total dose was administered i.p. and required over 2-fold higher dose to elicit effectiveness. Together, our data suggest that coated polymeric implants can accommodate heat-labile compounds, can furnish sustained release for long duration, and elicit DNA damage-inhibiting and anti-tumor activities.

Celastrol (CEL), a plant-derived triterpenoid, is a known inhibitor of Hsp90 and NF-κB activation pathways and has recently been suggested to be of therapeutic importance in various cancers. However, the molecular mechanisms of celastrol-mediated effects in lung cancer are not systematically studied. Moreover, it suffers from poor bioavailability and off-site toxicity issues. This study aims to study the effect of celastrol loaded into exosomes against two non-small cell-lung carcinoma (NSCLC) cell lines and explore the molecular mechanisms to determine the proteins governing the cellular responses. We observed that celastrol inhibited the proliferation of A549 and H1299 NSCLC cells in a time- and concentration-dependent manner as indexed by MTT assay. Mechanistically, CEL pre-treatment of H1299 cells completely abrogated TNFα-induced NF-κB activation and upregulated the expression of ER-stress chaperones Grp 94, Grp78, and pPERK. These changes in ER-stress mediators were paralleled by an increase in apoptotic response as evidenced by higher annexin-V/PI positive cells evaluated by FACS and immunoblotting which showed upregulation of the ER stress specific pro-apoptotic transcription factor, GADD153/CHOP and alteration of Bax/Bcl2 levels. Exosomes loaded with CEL exhibited enhanced anti-tumor efficacy as compared to free CEL against lung cancer cell xenograft. CEL did not exhibit any gross or systemic toxicity in wild-type C57BL6 mice as determined by hematological and liver and kidney function test. Together, our data demonstrate the chemotherapeutic potential of CEL in lung cancer and that exosomal formulation enhances its efficacy and reduces dose related toxicity.

cope Syzygium cumini (jamun) is perhaps the only berry that has the diversity of anthocyanidins of blueberry and bilberry and the abundance of ellagitannins/ellagic acid of black raspberry. Here, we report the potential of jamun against 17β-estrogen-mediated breast cancer and the role of miRNAs and other targets in disease inhibition. Methods and results Female August-Copenhagen Irish rats were given AIN-93M diet or diet supplemented with jamun. Two weeks later, animals received 17β-estradiol and were palpated weekly for the mammary tumors. At the end of 26 weeks, the jamun-diet significantly delayed the first tumor appearance by 21 days, and reduced the tumor incidence (65% versus 96%), tumor burden (313 ± 95 versus 661 ± 123 mm3) and tumor multiplicity (1.8 ± 0.3 versus 4.2 ± 0.4 tumors/rat) compared to control. The experimental diet significantly reduced the estrogen-associated growth of pituitary prolactinomas, circulating prolactin and estradiol levels and offset estrogen-associated increases in mammary cell-proliferation, estrogen receptor-alpha (ER-α), and cyclinD1. miRNAs that were either overexpressed (miR-182 and miR-375) or underexpressed (miR-127 and miR-206) following estrogen-treatment were significantly protected by jamun diet. Conclusions Together, our data show that jamun significantly offset estrogen-mediated alterations in mammary cell-proliferation, ER-α, cyclinD1, and candidate miRNAs, and that the modulation of these biomarkers correlated with a reduction in mammary carcinogenicity.

Curcumin, an anti-oxidant, anti-inflammatory, and anti-neoplastic agent, exhibited limited oral efficacy due to its poor bioavailability. To overcome this limitation, polymeric implants for continuous systemic delivery of curcumin were developed and tested for their safety and biocompatibility. Two 2-cm polycaprolactone implants containing polyethylene glycol and 20% (w/w) curcumin were grafted subcutaneously at the back of the Augustus Copenhagen Irish rats. Rats were euthanized and blood was analyzed for various hematological parameters; biochemical markers of liver/kidney function and local tissues were analyzed for local inflammatory reactions. Curcumin implants exhibited biphasic release kinetics with ∼3.6 + 0.8, 5.8 ± 1.1, 13.1 ± 2.1, 21.8 ± 0.3, 38.1 ± 0.6, and 47.2 ± 1.6 mg cumulative curcumin being released from both the implants after 1, 4, 12, 25, and 90 days. No significant differences in various hematological parameters (like white blood cells, red blood cells, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin), liver enzymes (like aspartate transaminase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, amylase, or lipase), or biochemical parameters of kidney function (like blood urea nitrogen, creatinine, Ca(2+), Na(+), and Cl(-) levels) were observed at any of these time points. However, a significant increase in serum phosphorus levels was observed at all the time points in sham implants as well as in curcumin diet and implant groups. Local implantation site showed foreign body granulomatous reaction with influx of histiocytes and occasional multi-nucleated giant cells with sham implants and was minimal around the curcumin implants. These polymeric implants were found to have little or no systemic toxicity with an acute reaction at local site which was reduced significantly by curcumin implants.

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383±46 adducts/10(6) nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2'-deoxyguanosine (122±19 adducts/10(6) nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H(2)O(2), Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible.

Familial adenomatous polyposis (FAP) is an inherited disorder caused by mutations in the adenomatous polyposis coli (APC) gene. If left untreated, nearly 100% of patients with FAP will eventually develop colorectal cancer, which is the third most common form of cancer diagnosed in men and women and the third leading class for cancer-related deaths within the United States. Given the prevalence of colon cancer both in the United States and worldwide, further insight into developing novel and more effective prevention and treatment strategies are warranted. The family of plant pigments known as the anthocyanins has been identified with a variety of health benefits including chemopreventive as well as therapeutic effects. However, a limitation to current clinical applications of anthocyanins is the high doses that are required. In order to elucidate the chemo-preventive/therapeutic potential and mechanism of action for the active moiety, anthocyanidins (Anthos), a series of in vitro and in vivo experiments were undertaken. The antiproliferative effects of Anthos native mixture isolated from bilberry, as well as individual Anthos and anthocyanins against APC mutant (HT-29), APC wild-type (HCT116) colon cancer cells and CCD-18Co normal colon cells were assessed using an MTT assay. To assess chemopreventive effects, the impact of the Anthos on polyp number was investigated in the APCMin/+ mouse model for FAP. Therapeutic efficacy of Anthos treatment on colorectal tumor number was assessed using an APCMin/+ ETBF mouse tumor model. Mechanistic studies were undertaken to assess the impact of Anthos on Src and EGFR phosphorylation and modulation of downstream targets such as STAT1, STAT3, STAT5, COX-2, p21, Cyclin D1, Cyclin D2 by western blot analysis using cell lysates of colon adjacent normal and tumor tissue from control and Anthos treated animals. Antiproliferation studies showed that the anthocyanidin, delphinidin, yielded greater potency compared to the anthocyanin, delphinidin-3,5-diglucoside against HCT-116 colon cancer cells. Treatment with native Anthos mixture yielded selective antiproliferative activity against colon cancer over normal colon cells. Furthermore, Anthos treatment led to significant reductions in polyp and tumor counts in vivo. Reduced Src and EGFR phosphorylation was observed in Anthos-treated tissue samples, with correlating favorable modulation of downstream targets including STAT1, STAT3, STAT5, COX-2, p21, Cyclin D1, and Cyclin D2. Overall, these results provide a promising outlook on the future of berry Anthos for treatment and prevention of both FAP and CRC. Additionally, results from this study provide highly novel mechanistic insight into the chemopreventive and therapeutic activities of Anthos. Translation of these findings to the clinical setting is one of the main goals moving forward with this research. Citation Format: Ashley M. Mudd, Tao Gu, Jeyaprakash Jeyabalan, Radha Munagala, Nejat K. Egilmez, Ramesh C. Gupta. Novel mechanistic insight into the prevention and treatment of colorectal cancer and familial adenomatous polyposis by anthocyanidins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-172.

Lower chlorinated PCBs can damage DNA directly or via free radical mechanisms. In order to assess the DNA-damaging potential of PCBs in humans, blood samples were collected from Inuit population from Salluit, Northern Canada. Their diet comprises blubber from sea mammals and fatty fish, which accumulate non-biodegradable PCBs at varying levels. The 103 samples thus collected were categorized into low-, medium- and high-PCB exposure groups. A comprehensive (32)P-postlabeling adductomics technology, which allows measure differences in DNA adduct profiles of polar and lipophilic adducts between control and exposure groups, was applied to these samples to assess the effect of PCB on DNA damage. The adduct patterns obtained were qualitatively similar to other human tissues studied previously. A range of highly polar to lipophilic subgroups of adducts were detected. The known oxidative lesion, 8-oxodG was predominant. While some individual adducts appear to accumulate with increasing PCB levels, a definitive association could not be made. A possible confounder effect of selenium is discussed.

Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated (32)P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N(2)-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.

Exosomes are biological nanovesicles that are involved in cell-cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs.

Despite significant advances in treatment and understanding of the molecular basis of the disease, cancer is still the second leading cause of death in the United States. Environmental, dietary, or lifestyle factors have been linked with increased cancer risks. The incidence of cancer is expected to increase more than 75% by the year 2030 in developed countries, and over 90% in developing nations. Therefore, search for alternatives that are effective, nontoxic, and cost-effective in prevention and treatment of cancer are of utmost importance. Traditional medicinal plants provide such a source. Increased consumption of dark-colored fruits and vegetables generally has been found to be protective against a variety of cancers. The anticancer potential of berries is gaining considerable attention lately due to the presence of a multitude of bioactive phytochemicals, including anthocyanins and polyphenols. In this chapter we discuss phytochemical composition of jamun, the Indian blackberry (Syzygium cumini), which contains appreciable amounts of polyphenols, ellagic acid, and ellagitannins, in addition to an abundance and diversity of anthocyanins. We also discuss the antioxidant properties and cancer chemopreventive potential of its bioactive constituents.

Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers. In earlier studies, a blueberry-supplemented diet has shown protection against 17β-estradiol (E2)-mediated mammary tumorigenesis. This study tested both preventive and therapeutic activities of diet supplemented with whole blueberry powder (50:50 blend of Tifblue and Rubel). Animals received 5% blueberry diet, either 2 weeks prior to or 12 weeks after E2 treatment in preventive and therapeutic groups, respectively. Both interventions delayed the tumor latency for palpable mammary tumors by 28 and 37 days, respectively. Tumor volume and multiplicity were also reduced significantly in both modes. The effect on mammary tumorigenesis was largely due to down-regulation of CYP 1A1 and ER-α gene expression and also favorable modulation of microRNA (miR-18a and miR-34c) levels. These data suggest that the blueberry blend tested is effective in inhibiting E2-mediated mammary tumorigenesis in both preventive and therapeutic modes.

Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity > 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10-40 µM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 µM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17β-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 µmol of TE/g), as well as by 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 µg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.

Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. We report cancer chemoprotective potential of Syzygium cumini L., commonly known as ‘jamun’ or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol, and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins and 1.15% polyphenolics. Jamun seed contained no detectable anthocyanins, but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded five anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445±64 μmol of trolox equivalent (TE)/g) and seeds (3,379±151 μM of TE/g) showed high oxygen radical absorbance capacity (ORAC). Their high antioxidant potential was also reflected by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, and ferrous ion-chelating activities. We also analyzed anti-proliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to five anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 μg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17β-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.

Spices are highly chemoprotective because of their antioxidant, antiproliferative, and anti-inflammatory activities. We have previously demonstrated significant chemoprotective effects of cumin seed powder (7.5%, w/w) against estrogen-induced mammary tumorigenesis in ACI rats by inhibiting cell proliferation as well as cellular and molecular markers. Here we report the inhibition of E2-mediated mammary tumorigenesis by cumin extract (1%, w/w) that is equivalent to the dietary dose in our previous study. Three extractions of cumin seeds with 80% aqueous ethanol followed by evaporation provided 13% of total dried extract (w/w). Groups of female ACI rats were given either AIN-93M diet or diet supplemented with cumin extract and then treated with subcutaneous 17ß-estradiol silastic implants (1.2 cm; 9 mg). Two additional groups were maintained on control or experimental diet. Rats received diet and water ad libitum. Five-six animals from each group were euthanized three weeks after the E2 treatment to determine the effect of interventions on early biomarkers. The remaining animals (n = 20-22) were euthanized when estrogen-treated group on control diet reached over 90% tumor incidence. Diet consumption and body weight gain was found to be largely unaffected by the cumin extract, suggesting that it was well tolerated. The first tumor incidence in control group was after 92 days, this was significantly delayed by 35 days in cumin extract treated group. Experimental diet significantly offset estrogen-related growth of pituitary prolactinomas as early as after three weeks (p = 0.019), the effect was more pronounced at tumor time point (p < 0.0001). At the end of the study (25 wks), the tumor incidence was found to be 96% in control group whereas only 45% animals had tumors in the intervention group. Significant reduction in average tumor volume (474 vs. 75 mm3) and tumor multiplicity (4.1 vs. 0.8 tumors/animal) was also observed by cumin extract compared to control. The mechanistic studies including effect on the plasma prolactin levels, and the cell proliferation marker, PCNA, estrogen-associated expression of the ERα and cyclin D1 in the mammary tissues are being evaluated. Together, our data show that cumin extract can significantly delay and prevent estrogen-mediated mammary tumorigenesis. This data further support our ultimate goal to develop these spice bioactives as clinically translatable chemopreventives and therapeutics against breast cancer. (Supported by CA-125152, Helmsley Fund, and Agnes Brown Duggan Endowment). Citation Format: Farrukh Aqil, Jeyaprakash Jeyabalan, Radha Munagala, Ramesh C. Gupta. Cumin extract prevents estrogen-associated breast cancer in ACI rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4646. doi:10.1158/1538-7445.AM2015-4646

Exosomes are biological nanoparticles and found in almost all the body fluids. They shuttle in and out of cells, are comprised of lipid bilayers, and carry a cargo of macromolecules. Because of the nano size (30-100 nm) these natural vesicles are being explored as nanodevices for the development of new therapeutic applications. Here we show bovine milk as a scalable source of exosomes and as a drug delivery vehicle for small molecules - both lipophilic and hydrophilic. The exosomes were isolated by differential centrifugation with the size generally ranging from 30-100 nm as analyzed by NanoSight and confirmed by electron microscopy and atomic force microscopy. The identity of exosomes was confirmed by surface protein markers (CD63, CD81), buoyant floatation density (1.12 g/ml) determined by sucrose density gradient, and high content of immune-related miRNAs and mRNA as determined by PCR. Exosomes, suspended in PBS, and stored frozen (-80°C) were found to be stable in terms of their size and biological activities. Milk exosomes loaded with fluorescent dye were readily taken up by macrophages and human cancer cells in vitro and distributed in the various tissues of nude mice following treatment with gavage and i.v. routes. Imaging of various tissues collected after euthanasia showed a broad tissue distribution of the exosomes delivered via the oral route, whereas exosome concentration predominated in the liver when administered by the i.v. route. Both lipophilic and hydrophilic chemopreventive (curcumin, withaferin A, anthocyanidins, and punicalagins, etc.) and chemotherapeutic (paclitaxel and docetaxel) agents could be loaded onto exosomes with 8%-60% drug load with respect to exosomal protein content. Drug-exosomal formulations showed significantly higher antiproliferative activity against lung, breast and ovarian cancer cells versus the free drugs. The drug formulation also showed significantly higher tissue drug levels, and enhanced anti-inflammatory and antitumor activity versus the free drug. Intriguingly, in the absence of any drug, the milk exosomes showed anti-proliferative and anti-inflammatory activities in cancer cells, suggesting the presence of protective factors in the milk exosomes (e.g., miRNAs); this notion corroborates the epidemiological and experimental findings of the presence of built-in immune factors in milk. (Supported from USPHS grants CA-118114 and CA-125152, Kentucky Lung Cancer Research Program, Agnes Brown Duggan Endowment, and Helmsley Funds). Citation Format: Farrukh Aqil, Radha Munagala, Jeyaprakash Jeyabalan, Ramesh C. Gupta. Milk derived exosomes: Scalable source of biologically active drug delivery nanoparticles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2014-5407

Lung cancer is the leading cause of all cancer deaths in the U.S., Europe and the World. With only an expected 16% relative 5-year survival rate in the U.S., treatment of this aggressive and progressive disease continues to remain a fundamental problem. Moreover, resistance to chemotherapy is a major problem in cancer treatment. In an attempt to overcome drug resistance and improve treatment efficacy, new molecular targeted drugs are being developed and tested in clinical studies. Over the recent years various epidemiological studies have provided compelling evidence for the chemopreventive/therapeutic efficacies of various functional foods and natural products. We hypothesize that non-toxic natural compounds such as a native mixture of blueberry anthocyanidins (BB anthos) and withaferin A (WFA), alone and in combination with a suboptimal dose of the chemo drug paclitaxel (PAC) will have therapeutic response and also chemosensitize the lung cancer cells. We evaluated whether BB anthos could enhance the sensitivity of H1299 cells to PAC and reduce its effective dose to inhibit cell proliferation. Data show significantly enhanced cell death by PAC at a suboptimal concentration (1 nM) (IC50 ≈ 40 nM) when combined with 5 μM (25% vs. 5% inhibition) and 10 μM (49% vs. 5% inhibition) BB anthos (1:1 mixture of Dp, Cy, Mv, Pe and Pt). The significant inhibition of H1299 lung cancer xenografts in nude mice was also evident from the same combination, while no significant growth inhibition was observed with the individual entities. Since BB is a known antioxidant, we determined if BB anthos would attenuate oxidative stress-induced by PAC in H1299 lung cancer cells by measuring intracellular ROS production. Our data showed that addition of BB anthos (10 μM each) did not affect the ROS levels produced by PAC (10 nM), as determined by cell permeable fluorescence probe DCFH-DA. Similarly, we also determined the antiproliferative potential of WFA alone and in combination with chemo drugs, PAC and docetaxel (DOC) against non-small-carcinoma lung cancer cells. WFA demonstrated high antiproliferative activity against the growth of both H1299 and A549 cells as determined by MTT assay. When suboptimal concentrations of WFA were tested in combination with suboptimal concentration of DOC and PAC, significantly enhanced antiproliferative activities were observed against H1299 and A549 compared with individual entities. The combination effect was found to be synergistic as analyzed using the CalcuSyn software. Together, our results suggest that both BB anthos and WFA enhanced therapeutic response of chemo drugs against lung cancer and reduce their required dose thus minimizing their toxicity. (Supported from Duggan endowment fund.) Citation Format: Farrukh Aqil, Radha Munagala, Hina Kausar, Jeyaprakash Jeyabalan, Ramesh Gupta. Enhanced activity of chemotherapeutic drugs by blueberry anthocyanidins and withaferin A against human lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3678. doi:10.1158/1538-7445.AM2013-3678

Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers in pre-clinical studies. In our earlier studies, blueberry-supplemented diet has shown chemopreventive protection against 17β-estradiol-mediated mammary tumorigenesis. The 5 anthocyanidins in blueberry are presumable bioactives. In this study, we tested both preventive and therapeutic activities of high anthocyanin-content blueberry diet in ACI rat model against 17β-estradiol-mediated mammary tumorigenesis. The blueberry used was a blend of Tifblue and Rubel blueberry (50:50) (U.S. Highbush Blueberry Council, CA) which has substantially higher amount of anthocyanins than Berkley/Blue Jay/Bluecrop blueberries used in our earlier studies. To study the chemopreventive effect, female ACI rats (5-6-week old) were administered AIN-93M diet or diets supplemented with blueberry (2.5 and 5%, w/w) 2 weeks prior to 17β-estradiol implantation. To test for the therapeutic activity, when 5% of the animals in the control group developed palpable tumors, one half of the animals were provided diet supplemented with 5% blueberry while the other half were continued on control diet. Animals received diet and water ad libitum and palpated weekly to determine the effect of berry intervention on tumor latency and tumor growth. When the tumor incidence reached >90% in estrogen-treated group on control diet, all the animals were euthanized. Our data revealed i) the first tumor in control group appeared after 85 days, while this was delayed by 28 and 37 days in groups receiving 5% blueberry in chemopreventive and therapeutic mode, respectively. The effect was less pronounced (delay by 7 days) with 2.5% berry; ii) the tumor incidence was also decreased dose dependently (96% in control versus 85% and 61% with 2.5% and 5% blueberry in chemopreventive mode, 55% with 5% blueberry in therapeutic mode); iii) a 25% and 51% reduction in tumor multiplicity with 2.5% and 5% blueberry, respectively (57% reduction with 5% blueberry in therapeutic mode); and iv) 37% and 45% reduction in tumor volume with 5% blueberry in chemopreventive and therapeutic modes, respectively; the effect with 2.5% blueberry was only modest. These data suggest that the test blueberry is effective in inhibiting estrogen-mediated mammary tumorigenesis in both chemopreventive and therapeutic modes. In view of our recent findings of miRNA ‘signature’ in estrogen-induced mammary tumors and its favorable modulation by ellagic acid, we are analyzing modulation of miRNAs in the current study and its correlation with the tumor inhibition. (Supported by Highbush Blueberry Council, CA, Agnes Brown Duggan Fund and Helmsley Foundation). Citation Format: Jeyaprakash Jeyabalan, Farrukh Aqil, Radha Munagala, Ramesh Gupta. Chemopreventive and therapeutic activity of high anthocyanin-content blueberry against estrogen-mediated breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3705. doi:10.1158/1538-7445.AM2013-3705

Dark-colored berries are gaining increasing attention lately for their chemopreventive and chemotherapeutic activities. Jamun (Syzygium cumini L.), the Indian blackberry is perhaps the only berry that has the diversity of anthocyanins of blueberry and abundance of ellagic acid present in black raspberry. We previously reported chemoprotective activities in jamun pulp extract and powder in vitro and short-term in vivo studies (Aqil et al. AACR 2010, Vol. 51, Abst. # 5688). Here we report anti-cancer potential of jamun pulp powder against estrogen mediated breast cancer and the role of miRNAs and other targets in the disease inhibition. Groups of female ACI rats were given AIN-93M diet or diet supplemented with jamun pulp powder (5%, w/w). Two weeks later, animals from each group were treated with subcutaneous silastic implants of 17β-estradiol (1.2 cm; 9 mg). Additional groups of animals received control or experimental diet in the absence of estrogen. Diet and water were provided ad libitum. Twelve weeks later animals were palpated weekly for the mammary tumors, and all groups were euthanized when tumors incidence reached over 90% in control group. Compared to control, jamun-supplemented diet delayed the first tumor appearance by 21 days, and significantly reduced the tumor incidence (65% vs 96%), tumor burden (313±95 vs 661±123 mm3) and tumor multiplicity (1.8±0.3 vs 4.2±0.4 tumors/rat). The experimental diet also significantly reduced the estrogen-associated growth of pituitary prolactinomas, circulating prolactin and estradiol levels and the mammary cell proliferation (PCNA) markers. Mechanistically, the jamun diet significantly offset estrogen-associated increases in mammary cell proliferation, ER-α and cyclin D1 as determined by western blot analysis. We have recently identified ‘miRNA signature’ associated with estrogen treatment (Munagala et al. AACR 2011, Vol. 52, Abst. # 155). All miRNAs that were either significantly overexpressed (miR-182 and miR-375) or underexpressed (miR-127 and miR-206) as a result of estrogen treatment were significantly protected by intervention with the jamun diet. Targets for these miRNAs remain to be identified. Together, our data show that jamun significantly offset estrogen-mediated alterations in i) mammary cell proliferation; ii) ER-α; iii) cyclin D1, and iv) candidate miRNAs, and that the modulation of these biomarkers correlated with the reduction in mammary carcinogenicity. (Supported by CA-125152, CA-118114 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5447. doi:1538-7445.AM2012-5447

We have previously demonstrated the chemoprotective effects of dietary Apiaceae spices against estrogen induced mammary tumorigenesis in ACI rats by inhibiting cell proliferation and few cellular and molecular markers as early as three weeks of estrogen administration. Here we report inhibition of 17ß-estradiol-mediated mammary carcinogenicity by fennel, cumin and caraway and potential mechanisms. Groups of female ACI rats were given either AIN-93M diet or diet supplemented with fennel, cumin and caraway (7.5% each, w/w) and then treated with subcutaneous 17ß-estradiol silastic implants (1.2 cm; 9 mg). Four additional groups were maintained on control or experimental diets. Rats received diet and water ad libitum. Animals were euthanized when estrogen-treated group on control diet reached over 90% tumor incidence. Tumor palpation from 12 wks revealed first tumor incidence by 92 days in control group that was significantly delayed by 22, 37 and 29 days by fennel, cumin and caraway, respectively. At the end of the study (26 wks), the tumor incidence was found to be 96% in control group whereas only 83%, 55% and 90% animals had tumors in fennel, cumin and caraway groups, respectively. Significant reduction in tumor volume (661±123 vs 196±64, 138±50 and 313±81 mm3) and tumor multiplicity (4.2±0.4 vs 2.2±0.4, 1.1±0.3 and 2.5±0.5 tumors/animal) was also observed by fennel, cumin and caraway, respectively compared to control. All experimental diets significantly offset estrogen-related growth of pituitary prolactinomas, the plasma prolactin levels, and the cell proliferation marker, PCNA in the mammary tissues. The spice intervention had no significant effects on any of these markers in the absence of estrogen treatment. Mechanistically, all the experimental diets resulted in significant downregulation of estrogen-associated increase in the expression of the ERα and cyclin D1. We have recently reported ‘miRNA signature’ associated with estrogen treatment in the ACI rat model. Of the four candidate miRNAs, miR-182 and miR-375 which were overexpressed by 4- and 7-fold and miR-127 and miR-206 were downregulated by 17- and 35-fold, respectively with estrogen treatment. These miRNAs were significantly modulated favorably by dietary cumin analyzed thus far; molecules downstream of these miRNAs remain to be explored. Together, our data show that apiaceae spices can significantly delay and prevent estrogen-mediated mammary tumorigenesis, with maximal protective effects observed with cumin, by modulating different cellular and molecular targets, including key dysregulated miRNAs. (Supported by CA-125152 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5443. doi:1538-7445.AM2012-5443

Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. We previously reported a novel concept in which polycaprolactone (PCL) implants embedded with test agents provide sustained delivery for long duration (months to >1year), reduce effective dose substantially and increase bioavailability. While tested successfully for various agents, this formulation results in an initial burst release and does not apply to heat-labile compounds. Furthermore, it takes 2 or more years for the polymeric implant to biodegrade. To overcome these limitations, we hypothesized that formulation of low mol wt polymeric implants will provide more sustained and higher release compared with higher mol. wt polymers; the low mol. wt polymers are also expected to biodegrade in weeks to months. To test part of this hypothesis, we used three different mol wt PCL polymers and two co-polymers, poly(D, L-lactide-co-glycolide) (PLGA). The method involves i) preparation of blank PCL implants (1.4 mm dia), and ii) coating of 30-40 layers by dipping blank implants, with intermittent drying, in 10-20% of different mol wt PCL solutions in dichloromethane containing 10% curcumin in tetrahydrofuran. The curcumin implants when tested for in vitro release showed that i) the burst release effect and the amount released varied with mol wt of the polymer. Implants prepared with PCL of 3,600 mol wt (PCL-3.6K) did not show any burst release phenomenon. Instead, these implants took nearly 7 days to render maximum daily release and thereafter the rate of release declined gradually over a period of two weeks. Implants prepared from higher mol wt polymers (PCL-31K and PCL-112K) were accompanied with some initial burst release and the rate of release declined gradually. Cumulative release measured over two weeks was found to be inversely proportional to the mol wt of PCL: PCL-3.6K (30%) > PCL-31K (24%) > PCL-112K (21%). On the other hand, the co-polymers, PLGA (50:50) and PLGA (75:25) showed only 1% and 0.5% total release, respectively. To determine if the implant delivery reduced the effective dose in a tumor model, nude mice were inoculated with human lung cancer (A549) cells and then treated with coated implants of withaferin A, a potent triterpenoid isolated from the traditional Ayurvedic herb “Ashwagandha”. Withaferin A given via the coated implants significantly inhibited (>50%) human lung cancer (A549) xenograft, while it was ineffective when the same dose was administered i.p. Together, our data suggest that coated implants of low mol wt PCL can accommodate heat-labile compounds, enhance bioavailability, furnish more sustained and higher cumulative release, and elicits anti-tumor activity. Our ultimate goal is to use low mol polymeric implants that will biodegrade in few months and provide continuous release of the drug. (Supported from CA-118114, KLCRP and Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2883. doi:1538-7445.AM2012-2883

Curcumin, a naturally occurring compound, is widely known for its anti-oxidant, anti-inflammatory, anti-proliferative and various other activities in cell culture studies. However, it has elicited only limited efficacy in animal and in clinical studies despite high doses, due to its poor oral bioavailability. Recently, we showed that continuous systemic delivery of curcumin by subcutaneous polymeric implants can circumvent issues associated with bioavailability. In this study we investigated potential of curcumin delivered via the implants and diet against 17β-estradiol (E2)-mediated mammary carcinogenesis. Implants were prepared using polycaprolactone and polyethylene glycol (65:35) alone or with curcumin (20%, w/w). The implants exhibited biphasic release kinetics in vitro with a burst release (7%) in the first week, followed by a more controlled release. In vivo drug release followed the same kinetics as the in vitro release, except that the total amount released was 1.8-fold higher presumably due to sink conditions. To determine the anticarcinogenicity potential, female ACI rats were either administered curcumin via the diet or were subcutaneously grafted with two 2-cm implants (20% drug load) 4 days prior to grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Implants were changed after nearly 4 ½ months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months and after the tumor incidence reached >80% (6 months) in control animals. Curcumin administered via implants resulted in significant reduction in both the tumor volume (184±198 mm3 vs 280±141 mm3; p=0.0283) and tumor multiplicity (5±3 versus 2±1; p=0.001); the dietary curcumin, however, was ineffective. Further, serum E2 concentration and E2-mediated increase in plasma prolactin were also found to be significantly reduced by curcumin implants but not with dietary curcumin. Analysis of plasma by HPLC showed higher curcumin levels (2.4 nM) via implant route as compared to diet at ∼10-20 fold lower doses both after 3 weeks and 3 months of treatment. Similarly, higher levels of curcumin were detected in liver tissue also by the implant route than dietary administration after both 3 weeks (60±28 versus 40±20 ng/g) and 6 months (32±15 versus 21±9 ng/g). Dietary curcumin increased hepatic CYP1A and CYP1B1 activity without any effect on CYP3A4 activity whereas curcumin implants increased both CYP1A and CYP3A4 activity but decreased CYP1B1 activity in presence of E2. Since, CYP1A and 3A4 metabolize most of the E2 to its non-carcinogenic 2-OH metabolite and CYP1B1 produces potentially carcinogenic 4-OH metabolite, it appears that favorable modulation of these CYPs via systemic curcumin delivery could be one of the mechanisms that resulted in the reduced E2 mediated mammary carcinogenesis (Supported from CA-118114 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1863. doi:10.1158/1538-7445.AM2011-1863

Colored fruits, particularly berries, are emerging as highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. In this study, we investigated the chemoprotective potential of Eugenia jambolana Lam., commonly known as ‘jamun’ or Indian blackberry. Jamun, a widely available fruit in India and other Asian countries, has also been used in the treatment of various ailments, including diabetes mellitus. To determine its antioxidant potential, jamun pulp was collected from freshly harvested fruits, dried, and the powder was extracted with acidic ethanol, and enriched by amberlite XAD7/HP20 (1:1) column chromatography. The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins and 1.15% total phenolics using standard assays. HPLC analysis of acid-hydrolyzed extract showed the presence of five anthocyanidins – malvidin, petunidin, delphinidin, cyanidin and peonidin. The unhydrolyzed extract was found to have significant antioxidant activity as reflected by high ORAC value (1,445±64 μmol of TE/g) as well as high ABTS- and DPPH-scavenging activities, and ferrous ions-chelating activity; the acid-hydrolyzed extract showed higher activity than unhydrolyzed extract. Since some of the jamun bioactives (ellagitannins and anthocyanidins/anthocyanins) have significant antiproliferative activity, we also analyzed these extracts for their antiproliferative potential using MTT assay against human lung cancer A549 cells in cell culture. The hydrolyzed jamun extract showed significant antiproliferative activity (IC50=79 µg/mL). However, unhydrolyzed extract showed almost no activity. These data suggest that the antiproliferative activity may result from anthocyanidins (present in the hydrolyzed extract) rather than anthocyanins (present in unhydrolyzed extract). Together, our data show high antioxidant, and antiproliferative activities in jamun pulp and may be a viable candidate for chemoprevention of lung cancer (Supported from Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2011-4619

MicroRNAs (miRNAs) play important role in a wide range of normal biological processes. Dysregulated miRNA expression has been reported with the development and progression of cancers, including breast cancer. The role of estrogens in regulation of cell proliferation and breast carcinogenesis is well known. Recent reports have associated several miRNAs with estrogen receptors in breast tumors. However, identification of miRNA ‘signature’ during estrogen-mediated carcinogenesis may be critical for understanding the effect of estrogen in human breast cancer, as well as for developing strategies for cancer chemoprevention. In this study we analyzed miRNAs at 3 weeks, 12 weeks close to the appearance of first palpable tumor and 28 weeks when tumor incidence reaches 85-90% following treatment of female ACI rats with 17ß-estradiol (E2) silastic implants. Total RNA from mammary tissues and tumors were isolated using mirVana microRNA kit and analyzed using Affymetrix GeneChip miRNAarray. This array had 351 rat specific miRNAs probes. E2 treatment resulted in significant up regulation or repression (2 fold change) of 33 miRNAs at two or more time points. A total of 9 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2011-155

Ellagitannins are the most abundant polyphenols present in pomegranate (Punica granatum) peel and black raspberry and contribute greatly towards reported biological properties. In this study, we report on i) the bulk isolation of punicalagins from pomegranate peel, and ii) various chemopreventive/chemotherapeutic activities. The pre-enriched pomegranate peel powder was extracted repeatedly with water. The pooled extract was concentrated using rotovapor and then further purified by an amberlite XAD-16 column. Punicalagins were eluted using a gradient of methanol. Fractions eluted with 20-25% methanol yielded 1.2 g light brown powder from a total of 40 g extract. This fraction was identified as punicalagins by HPLC using reference compounds and confirmed by LC/MS analysis. The punicalagins (40 µM) were found to inhibit oxidative DNA products (70% inhibition; p=0.0017) from Cu2+-catalyzed redox cycling of 4-hydroxy-17ß-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of punicalagins was also obtained based on ORAC assay (1556 ± 79 μmol of TE/mg), as well as by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, and ferrous ions-chelating activities (IC50 1.2 µg/ml, 19 µg/ml, and 46.5 µg/ml respectively). We further investigated the effect of punicalagins on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo: Incubation of BP (1 µM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagins (1-40 µM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40 µM. To determine its efficacy in vivo, female S/D rats were treated with punicalagins via diet (1,500 ppm) or by subcutaneous polymeric implants (two 2-cm implants; 20% drug load) and then challenged with BP by subcutaneous polymeric implants (2-cm; 10% load) and euthanized after 1 week. Analysis of the lung DNA by 32P-postlabeling showed significant (60%; p = 0.029) inhibition of DNA adducts by punicalagins administered by the implants; the dietary administration of punicalagins also showed modest but statistically insignificant inhibition (34%). Measurement of the residual compound in the implants recovered from the animals showed that the total dose administered by the implants was 40-fold lower than the oral route. Together, our data show that punicalagins isolated from pomegranate peel had significant antioxidant activity and was highly effective against BP-induced DNA adducts in vivo only when administered by sustained-release polymeric implants (Supported from the USPHS grants CA-118114 and CA-125152 and the Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2011-3711

Berries, particularly blueberry (BB), black raspberry (BRB), are gaining increased attention for their chemopreventive and chemotherapeutic activities and have been tested in limited clinical studies with mixed results. Previous studies with strawberry and BRB failed to elicit protection against NNK/PAH-induced lung carcinogenicity in the A/J mice (the Stoner studies). However, a low dose of a mixture of different berries (BB, BRB, blackberry, and strawberry) elicited modest inhibition of cigarette smoke-mediated lung carcinogenicity in our study. In this study we investigated chemopreventive and chemotherapeutic activities of BB and BRB using two different animal models: To determine the DNA adduct-inhibiting potential female A/J mice were provided AIN-93M diet or diets supplemented with lyophilized powder of BB or BRB (5% each, w/w) and then challenged with continuous low doses of dibenzo-[a,l]pyrene (DBP) by subcutaneous polymeric implants (two, 20 mm long, 1.4 mm dia; DBP load 5%). Three weeks later animals were euthanized and the target tissue (lung) DNA was analyzed by 32P-postlabeling assay. The DNA adduct burden (as adducts/109 nucleotides) in animals receiving the BB (67 ± 16), BRB (68 ± 9) diets was found to be unaffected compared with control diet (54 ± 17), indicating that the berry bioactives were ineffective against the DNA adduct-forming ability of DBP under the conditions used. To determine the chemotherapeutic potential, female nude mice received AIN-93M (control) diet or diet supplemented with a mixture of BB and BRB (2:1) and 4 days later inoculated with human lung cancer A549 cells (3×106 cells). Four weeks into the ongoing study, the berry diet has shown significant (nearly 60%) inhibition of the lung tumor xenograft (186 ± 117 versus 79 ± 47). The anti-tumor activity elicited by the berry diet in xenograft model is consistent with the anti-tumor activity of anthocyanidins reported by us in cell culture and nude mice (Kausar et al. AACR 51: 1884, 2010). In conclusion, our data suggest that the anti-lung cancer potential of the berries may be associated with mechanisms other than inhibition of carcinogen-induced DNA adducts. (Supported from CA-118114, KLCRP and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2011-4597

The past and current trends in determining the DNA-damaging and carcinogenicity potential of environmental toxicants rely largely on treatment of rodents with single or multiple bolus doses. To simulate the human scenario where the exposure is generally continuous to low doses, we have developed polymeric implants embedded with carcinogens. When grafted subcutaneously, polymeric implants provide continuous (“24/7”) low dose exposure. In this study we report the effects of sustained low doses of a highly potent environmental carcinogen, dibenzo[a,l]pyrene (DBP) on DNA adduct accumulation. Different size DBP implants (10 – 20 mm long, 1.4 – 2.4 mm dia; 5% DBP load) were tested first in a pilot study using female A/J mice to optimize the implant surface area. Two 20-mm implants (1.4 mm dia), which produced maximal lung DNA adducts, were used subsequently. Groups of female A/J mice (n = 4 – 6) were grafted with sham or DBP implants. For comparison, animals were also treated in parallel with a bolus carcinogenic dose of DBP intraperitoneally (6 mg/kg) or vehicle alone. Animals from both the treatment groups were euthanized at different time intervals and selected tissues were collected. Analysis of the target (lung) DNA adducts by 32P-postlabeling showed qualitative identical adduct patterns by the two routes of administration; the sham treatments showed no detectable adducts. As expected, the bolus dose treatment resulted in higher DNA adducts (in adducts /109 nucleotides) (72 ± 18) compared with the implant group (33 ± 17) one week post-treatment. However, 3 weeks later, adduct levels in the implant group increased (54 ± 17) but a significant declined occurred in the i.p treatment group (19 ± 13). The adduct levels after 4 weeks in the implant group (66 ± 19) were somewhat higher than the 3 week group, suggesting that the adduct levels plateaued. The steady accumulation of DNA adducts was consistent with continuous release of DBP from the implant (about 10 µg/day based on in vitro release). The study is ongoing to determine if the low-dose continuous exposure which provides steady accumulation of DNA adducts in the target tissue will be tumorigenic. In summary, the steady-state adduct level reflect human exposure scenario and this approach will be ideal to determine both carcinogenicity and biological/pathological changes due to low-dose exposure as well as for screening potential chemopreventive agents. This exposure system has been used to determine efficacy of chemopreventive agents (accompanying abstracts by Aqil et al.; Gupta et al.) (Supported from CA-118114, KLCRP and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5557. doi:10.1158/1538-7445.AM2011-5557