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Cancer is one of the leading global causes of death in both men and women. Colorectal cancer (CRC) alone accounts for ∼10 % of total new global cases and poses an over 4% lifetime risk of developing cancer. Recent advancements in the field of biotechnology and microbiology concocted novel microbe-based therapies to treat various cancers, including CRC. Microbes have been explored for human use since centuries, especially for the treatment of various ailments. The utility of microbes in cancer therapeutics is widely explored, and various bacteria, fungi, and viruses are currently in use for the development of cancer therapeutics. The human gut hosts about 100 trillion microbes that release their metabolites in active, inactive, or dead conditions. Microbial secondary metabolites, proteins, immunotoxins, and enzymes are used to target cancer cells to induce cell cycle arrest, apoptosis, and death. Various approaches, such as dietary interventions, the use of prebiotics and probiotics, and fecal microbiota transplantation have been used to modulate the gut microbiota in order to prevent or treat CRC pathogenesis. The present review highlights the role of the gut microbiota in CRC precipitation, the potential mechanisms and use of microorganisms as CRC biomarkers, and strategies to modulate microbiota for the prevention and treatment of CRC.

Objective: To compare the conventional ways of teaching to new teaching methodologies thus finding their efficacy. It also highlights the significance of incorporating technology into teaching thereby creating a rich learning experience for students. Study Design: Cross-sectional Survey. Setting: Different Medical and Nursing Colleges and Universities of Punjab, Pakistan; Faisalabad Medical University Faisalabad, University Medical and Dental College Faisalabad, Ameer-ud-din Medical College Lahore, Nishtar Medical and Dental College Multan, Shareef Medical and Dental College, Continental Medical College Lahore. Period: June 2020 to August 2020. Material & Methods: Was conducted amongst 1600 medical, dental and nursing students from various medical and dental colleges across Pakistan. A questionnaire was circulated among students after briefing them about the objective of the study. Result: Out of 1600, 816 were MBBS students, 715 were BDS and 69 were nursing students. About various teaching methods, 53% in MBBS, 56.78% in BDS and 30.43% of nursing students were aware about new teaching methodologies. Tutorials were least known by MBBS students, online classes for BDS students whereas both methods of teaching were least popular amongst nursing students. A significant percentage of students were more in favor of group discussion as an effective method of teaching. Conclusion: This study concluded that student-centered learning should be focused on and new technologies should be incorporated in order to make medical education more interactive and interesting.

Herein, the one-step, multi-component reaction (MCR) of a series of spirooxindole-pyranopyrazole derivatives (5a-g), via a Knoevenagel condensation and Michael addition cascade, under mild and green reaction conditions, is reported. The newly synthesized derivatives were screened for in vitro anti-cancer activity against 60 human cancer cell lines at the National Cancer Institute (NCI), USA. We found that compounds 5c, 5d, and 5g showed good activity against the HOP-92 (lung cancer), UO-31 (renal cancer), KM-12, SW-620 (colon cancer), and HS578T (breast cancer) cell lines. Compound 5c showed 43.19% and 21.18% growth inhibition at 10 µM for HOP-92 and UO-31 cell lines, respectively, while compound 5g showed 82.02% growth inhibition for the KM12 cell line at the same concentration. Therefore, the compound 5g could be further derivatized as a futuristic lead molecule for colorectal cancer.

Exosomes or small extracellular vesicles (EVs) are natural nanoparticles and known to play essential roles in intercellular communications, carrying a cargo of a broad variety of lipids, proteins, metabolites, RNAs (mRNA, miRNA, tRNA, long non-coding RNA), and DNAs (mtDNA, ssDNA, dsDNA) [...]

Alzheimer's disease (AD) has been the most extensively studied neurological disorders that affects millions of individuals globally and is associated with misfolding of proteins in the brain. Amyloid-β and tau are predominantly involved in the pathogenesis of AD. Therapeutic interventions and nanotechnological advancements are useful only in managing the AD symptoms and the cure for this disease remains elusive. Exosomes, originating from most cell and tissue types are regarded as a double-edged sword, considering their roles in the progression and treatment of AD. Exosomes can be manipulated as drug delivery vehicles for a wide range of therapeutic cargos-both small molecules and macromolecules. Herein, we review the roles of exosomes in the pathology, diagnosis, and treatment of AD and highlight their application as a drug carrier to the brain for AD treatment.

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.

The rise of antimicrobial resistance (AMR) impacts public health due to the diminished potency of existing antibiotics. The microbiome plays an important role in the host's immune system activity and shows the history of exposure to antimicrobials and its manipulation in combating antimicrobial resistance. Advancements in gene technologies, DNA sequencing, and computational biology have emerged as powerful platforms to better understand the relationship between animals and microorganisms (MOs). The past few years have witnessed an increase in the use of nanotechnology, both in industry and in academia, as tools to tackle antimicrobial resistance. New strategies of microbiome manipulation have been developed, such as the use of prebiotics, probiotics, peptides, antibodies, an appropriate diet, phage therapy, and the use of various nanotechnological techniques. Owing to the research outcomes, targeted delivery of antimicrobials with some modifications with nanoparticles can lead to the destruction of resistant microbial cells. In addition, nanoparticles have been studied for their potential antimicrobial effects both in vitro and in vivo. In this review, we highlight key opportunistic areas for applying nanotechnologies with the aim of manipulating the microbiome for the treatment of antimicrobial resistance. Besides providing a detailed review on various nanomaterials, technologies, opportunities, technical needs, and potential approaches for the manipulation of the microbiome to address these challenges, we discuss future challenges and our perspective.

Lung cancer is the second most common cancer in both men and women. Despite several treatment options, chemotherapy remains a major standard-of-care treatment modality. Over time cancer cells acquire drug resistance and evade its effects for survival. The combination of chemotherapeutic drugs with the non-overlapping mechanism of actions are one alternative approach to treat drug-resistant cancers. Paclitaxel (PAC) and cisplatin (CisPt) are widely used to treat non-small cell-lung cancer (NSCLC). However, dose-limiting toxicity, metastasis and chemoresistance have restricted its use. Our recent findings suggested that PAC, when loaded onto folic acid (FA)-functionalized colostrum exosomes (FA-ExoPAC) and administered orally, exceeded the efficacy of i.v. PAC but matched efficacy of i.v. albumin-bound PAC nanoformulation (Abraxane [Abx]) against A549 orthotopic lung tumors. Furthermore, unlike i.v. PAC and i.v. Abx, oral FA-ExoPAC lacked immune toxicity. In order to advance the FA-ExoPAC to clinical studies, we determined if the exosomal formulation will interfere in CisPt therapy. MTT assay showed similar antiproliferative activity of ExoPAC and PAC (IC50 6.25 nM) against drug-sensitive A549 lung cancer cells. However, the antiproliferative activity of ExoPAC (IC50 38 nM) was much greater than for PAC (IC50 >>100 nM) against drug-resistant A549TR lung cancer cells. The activity was further enhanced with FA-functionalized ExoPAC. The enhanced efficacy of the drug presumably resulted from higher cell uptake of ExoPAC and FA-ExoPAC by both drug-sensitive and drug-resistant cells. To determine the effect of the combination of ExoPAC and CisPt, A549 and A549TR cells were treated with increasing concentrations of both the drugs, alone and in combination, for 72 h. MTT assay showed that CisPt (20 µM) and ExoPAC (80 nM) inhibited the growth of A549 cells by 47% and 75%, respectively, the effect was significantly increased with the combination (>95% inhibition). The combination effect was likewise enhanced against the A549TR, except the respective concentration of CisPt (40 µM) and ExoPAC (200 nM) was increased to achieve >95% growth inhibition; the inhibition was lower (50–60%) when treated individually. Together, these data suggest that targeted oral therapy with FA-ExoPAC can potentially eliminate drug resistance and inhibit metastasis with minimal or no side effects and warrants testing in the clinical settings (Supported from the NIH grant R44-CA-221487 and Agnes Brown Duggan Endowment). Citation Format: Raghuram Kandimalla, Disha N. Moholkar, Jayaprakash Jeyabalan, Wendy Spencer, Ramesh C. Gupta, Farrukh Aqil. Exosomal paclitaxel formulation, alone and in combination with cisplatin, enhances drug’s efficacy against lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 371.

Lung cancer remains the leading cause of cancer-related deaths in the United States and worldwide. Non-small cell-lung cancer (NSCLC) accounts for 85% of all lung cancer cases. Plant-derived compounds have historically led to some of our most useful cancer drugs (e.g., paclitaxel, vincristine, etc). Celastrol (CEL), a triterpenoid isolated from Tripterygium wilfordii, has attracted significant attention for its strong anticancer properties. CEL demonstrate time- and dose-dependent anti-proliferative activity against H1299 and A549 cells with IC50 0.63 and 1.8 µM, respectively and shows dose dependent inhibition of A549 colony formation. CEL has minimal effect against the normal bronchial epithelium Beas2b. We have also demonstrated that CEL loaded onto bovine milk-derived exosomes (ExoCEL) enhanced therapeutic response vs. the free CEL in tumor xenograft model and lacks systemic and immunotoxicity. Our data indicate that the wound healing and cell migration rate was significantly reduced in the CEL treatment. In fact, the effect was time and dose dependent as analyzed by Wimasis software. CEL is an inhibitor of TAK1 and has shown to suppress basal TGF-β1 and TNF-α induced NF-κB activity, however, the precise mechanisms remain to be identified. Here, we systemically explored the mechanism of CEL by which it inhibits the growth and metastasis of NSCLC using RNAseq analysis. RNAseq followed by differential expression of genes demonstrate a total of over 600 genes modulated by the CEL treatment of which 287 genes were upregulated while 314 genes were downregulated. Gene Ontology analysis, genome analysis along with reactome pathway enrichment analyses indicated that CEL-treated genes are involved in cell cycle, EMT, PI3K-AKT and p53 signaling and pathways related to lung cancer and its metastasis. The molecules of major pathways involved in the RNAseq analysis were analyzed by western blot. Our findings indicated significant decrease in TGFβ-induced migratory behavior of cancer cells and reversal of TGFβ-mediated changes in EMT proteins such as ZO1, Vimentin, β-catenin and claudin by CEL in H1299 lung cancer cells. Cancer cells exhibited up-regulated N-cadherin with loss of E-cadherin called the "cadherin switch" and downregulation of E-cadherin is one of the hallmarks of EMT. We also confirmed that CEL inhibit TGFβ-induced cMyc expression by decreasing PI3K and AKT phosphorylation via TGF-β/non-Smad pathway and show dose- dependent reduction of pAKT, PI3K, cMyc and Smad4 in the absence and presence of TGF-β. In summary, the analysis of biological functions of the most differentially expressed genes revealed possible mechanisms of CEL for its strong activity against lung cancer. Pathways identification provide information about the potential use of CEL to inhibit tumorigenesis, progression, and metastasis of lung cancer. (Supported by Research and Development Office, KSA Grant# RDO-2003). Citation Format: Farrukh Aqil, Raghuram Kandimalla, Neha Tyagi, Mohd Saeed, Ramesh Gupta. Celastrol targets multiple pathways and suppress cell invasion in lung cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 8.

Biodistribution studies are relevant models for understanding the fundamental preclinical information of the distribution of drugs to the potential target organs, which provide insight on which on-target or off-target effects might be expected. One of our research focuses is the study of uptake and distribution of bovine milk- and colostrum-derived exosomes, functionalized exosomes and exosomes in polyethyleneimine (PEI) matrix (EPM) using near-infrared fluorescent dye in rodents. Previously, when DiR dye was loaded onto milk exosomes, biodistribution studies showed that route of administration had a significant influence on the tissue distribution with somewhat uniform biodistribution with oral gavage while predominated liver accumulation with the i.v. route. In this study, we show biodistribution of colostrum exosomes, EPM and tumor targeting by attaching tumor targeting ligand, folic acid (FA). We studied the biodistribution of these formulations using exosomes labeled with Alexa Fluor 750 (AF750) in wild-type mice and subcutaneous lung tumor-bearing mice. In various studies we tested: i) biodistribution of exosomes vs EPM, ii) effect of different administration routes such as intravenous (i.v.), oral (p.o.), subcutaneous (s.c.), intranasal (i.n.) and intramuscular (i.m.) on biodistribution, and iii) tumor targeting using FA-functionalized exosomes and EPM. Uniform tissue distribution was observed upon oral administration of exosomes while predominant hepatic accumulation was observed with i.v. administration. The i.n. route resulted in pre-dominant accumulation in lung, whereas i.m. and s.c. delivery had almost similar distribution as observed with i.v. route. The distribution of exosomes and EPM matrix was largely similar. We observed that the fluorescent signals from AF750-labeled FA-Exo and FA-EPM treatment revealed higher tumor accumulation of exosomes as compared to non-functionalized exosomes and EPM, respectively due to overexpression of folate receptors. Time-dependent distribution showed accumulation of EPM in tumors at later time point. The EPM formulations could be detected at the sites otherwise difficult to target such as brain and lymph nodes after systemic administration, thus indicating suitability of these formulations to cross physiological barriers. To validate the therapeutic potential, FA-EPM was loaded with 15 μg siKRAS and injected intravenously to orthotopic A549 lung tumor-bearing mice. Significant reduction in tumor volume (67%; p Citation Format: Disha Nagesh Moholkar, Raghuram Kandimalla, Farrukh Aqil, Ramesh Gupta. Biodistribution and tumor targeting of exosomes using mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 372.

Lung cancer continues to be a major cause of cancer mortality. Emerging evidence suggests that cancer likely involves multiple mutations. Therefore, compounds targeting multiple signal transduction pathways would be ideal for the prevention and treatment of cancer. Mahanine, a carbazole alkaloid isolated from an Indian folklore medicinal plant Murraya koenigii (known as Curry Patta), widely consumed in traditional dishes, have been shown to possess anticancer activities and confound many oncogenic signaling events in breast, colon, and prostate cancers. In the current study, we determined the anticancer potential of mahanine against drug-sensitive and drug-resistant lung cancer cells and postulate the possible mechanism of action. Mahanine was isolated from the dried leaves of Murraya koenigii by hydro alcoholic extraction and fractionation with ethyl acetate, followed by preparative UPLC to isolate pure (>95%) compound and its identity was confirmed by LC-MS/MS and NMR spectroscopy. Antiproliferative activity of mahanine was determined against drug- sensitive (A549 and H1299) and taxol-resistant (A549-TR) human lung cancer cells. Mahanine showed a dose- and time-dependent inhibition of both drug-sensitive and drug-resistant lung cancer cell growth. Interestingly, the IC50 values of mahanine were essentially the same for drug sensitive (A549) (12.5 µM) and drug- resistant (12.5 µM) lung cancer cells; the IC50 value was somewhat lower (10 µM) against highly metastatic H1299 lung cancer cells. Mahanine attenuated the colony formation and reduced the wound healing of lung cancer cells. Mechanistically, mahanine dose dependently impeded the oncogenic markers such as KRAS, MET, AKT, mTOR and cMYC. Together, mahanine demonstrate ability to inhibit both drug- sensitive and drug-resistant lung cancer cells and modulate distinct oncogenic molecular targets providing potential to be developed for the treatment of drug-resistant and metastatic NSCLC (Supported from Duggan Endowment funds). Keywords: Mahanine, Lung cancer, Oncogenes, Plant therapeutics. Citation Format: Raghuram Kandimalla, Farrukh Aqil, Disha N. Moholkar, Suman K. Samanta, Ramesh C. Gupta. Mahanine, a carbazole alkaloid attenuates lung cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 707.

Breast cancer (BC) is the most common type of malignancy which covers almost one-fourth of all the cancers diagnosed in women. Conventionally, chemo-, hormonal-, immune-, surgery, and radiotherapy are the clinically available therapies for BC. However, toxicity and other related adverse effects are still the major challenges. A variety of nano platforms have been reported to overcome these limitations, among them, exosomes provide a versatile platform not only for the diagnosis but also as a delivery vehicle for drugs. Exosomes are biological nanovesicles made up of a lipidic bilayer and known for cell-to-cell communication. Exosomes have been reported to be present in almost all bodily fluids, viz., blood, milk, urine, saliva, pancreatic juice, bile, peritoneal, and cerebrospinal fluid. Such characteristics of exosomes have attracted immense interest in cancer diagnosis and therapy. They can deliver bioactive moieties such as protein, lipids, hydrophilic as well as hydrophobic drugs, various RNAs to both distant and nearby recipient cells as well as have specific biological markers. By considering the growing interest of the scientific community in this field, we comprehensively compiled the information about the biogenesis of exosomes, various isolation methods, the drug loading techniques, and their diverse applications in breast cancer diagnosis and therapy along with ongoing clinical trials which will assist future scientific endeavors in a more organized direction.

Exosomes are unique biogenic nanocarriers of endocytic origin that are generated from most of the cells and found in biofluids like milk, plasma, saliva, and urine. Bovine milk represents the largest and an economic source for the production of exosomes. In recent past, the utility of the milk exosomes as drug carriers is intensified. Exosomes are emerging for delivery of both small and large therapeutics due to their biocompatibility. In this article, we highlighted the various exosomal isolation techniques, physicochemical properties, their biodistribution, and utility of milk exosomes in delivering the small drug molecules and siRNA to combat cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide and in the United States. Despite recent advancements in treatment approaches, metastasis remains a major therapeutic challenge in lung cancer and explains the extremely poor prognosis. Epithelial to mesenchymal transition (EMT), a complex process of cellular reprogramming has become an attractive drug target because it plays a crucial role in the metastasis of non-small cell lung cancer (NSCLC). In the present study, we examined the effects of withaferin A (WFA), a plant-derived steroidal lactone on EMT in human NSCLC cell lines. First, we demonstrated that WFA displayed time- and concentration-dependent cytotoxicity on A549 and H1299 NSCLC cells. Then, cells were exposed to ≤ 0.5 µM WFA for ≤ 4 h to minimize cytotoxicity and determined its effects on EMT, cell adhesion, motility, migration, and invasion. EMT induction was performed by culturing cells in serum-free media containing TGFβ1 (5 ng/mL) and TNFα (25 ng/mL) for 48 h. We observed that pretreatment of cells with WFA inhibited cell adhesion, migration, and invasion of A549 and H1299 cells. Using western blot, immunofluorescence, and qRT-PCR analysis, we demonstrated that WFA suppressed TGFβ1 and TNFα-induced EMT in both cell lines. Mechanistically, WFA suppressed the phosphorylation and nuclear translocation of Smad2/3 and NF-κB in A549 and H1299 cells. Together, our study provides additional evidence demonstrating the inhibitory effects of WFA on EMT induction in NSCLC cells and further demonstrates the therapeutic potential of WFA against the metastasis in NSCLC.

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.

Platinum-taxane combination chemotherapy still represents the standard of care for advanced non-small cell lung cancer (NSCLC) with no targetable driver mutations. However, the efficacy of these drugs has plateaued at 10-14 months primarily due to dose-limiting toxicity, chemoresistance, and metastasis. Here, we explored the effects of withaferin A (WFA) alone and in combination with paclitaxel (PAC) on the growth, proliferation, migration, and invasion of human NSCLC cells. We show that the sensitivity of H1299 and A549 cells to concomitant treatment with PAC and WFA was greater than that of either PAC or WFA alone. Using the combination index and dose-reduction index, we demonstrated that various combinations (1:40, 1:20, 1:10) of PAC to WFA, respectively, were highly synergistic. In addition, PAC+WFA co-treatment synergistically inhibited colony formation, migration, invasion and increased the induction of apoptosis in H1299 and A549 cells. Interestingly, the synergism of PAC and WFA was not schedule-dependent but was enhanced when cells were pretreated with WFA indicating a chemo-sensitizing effect. Importantly, WFA was active against both PAC-sensitive (TS-A549) and PAC-resistant (TR-A549) cells both in vitro and in vivo. Mechanistically, WFA inhibits the proliferation of NSCLC cells via thiol oxidation. The effects of WFA were inhibited in the presence of N-acetyl cysteine and other thiol donors. Taken together, our results demonstrate the efficacy of WFA alone or alongside PAC on NSCLC cells and provide a strong rationale for further detailed testing in clinically relevant models for the development of PAC+WFA combination as an alternative therapeutic strategy for advanced NSCLC.

In this paper, we report the development of the nanotube-CTC-chip for isolation of tumor-derived epithelial cells (circulating tumor cells, CTCs) from peripheral blood, with high purity, by exploiting the physical mechanisms of preferential adherence of CTCs on a nanotube surface. The nanotube-CTC-chip is a new 76-element microarray technology that combines carbon nanotube surfaces with microarray batch manufacturing techniques for the capture and isolation of tumor-derived epithelial cells. Using a combination of red blood cell (RBC) lysis and preferential adherence, we demonstrate the capture and enrichment of CTCs with a 5-log reduction of contaminating WBCs. EpCAM negative MDA-MB-231/luciferase-2A-green fluorescent protein (GFP) cells were spiked in the blood of wild mice and enriched using an RBC lysis protocol. The enriched samples were then processed using the nanotube-CTC-chip for preferential CTC adherence on the nanosurface and counting the GFP cells yielded anywhere from 89% to 100% capture from the droplets. Electron microscopy (EM) studies showed focal adhesion with filaments from the cell body to the nanotube surface. We compared the nanotube preferential adherence to collagen adhesion matrix (CAM) scaffolding, reported as a viable strategy for CTC capture in patients. The CAM scaffolding on the device surface yielded 50% adherence with 100% tracking of cancer cells (adhered vs. non-adhered) versus carbon nanotubes with >90% adherence and 100% tracking for the same protocol. The nanotube-CTC-chip successfully captured CTCs in the peripheral blood of breast cancer patients (stage 1-4) with a range of 4-238 CTCs per 8.5 ml blood or 0.5-28 CTCs per ml. CTCs (based on CK8/18, Her2, EGFR) were successfully identified in 7/7 breast cancer patients, and no CTCs were captured in healthy controls (n = 2). CTC enumeration based on multiple markers using the nanotube-CTC-chip enables dynamic views of metastatic progression and could potentially have predictive capabilities for diagnosis and treatment response.

Cancer chemoprevention involves the chronic administration of a natural, synthetic, or biological agent to delay or prevent the occurrence of malignancy. Since ancient times, plant-derived compounds, the so-called phytocompounds, have been used to prevent or treat several diseases including cancer. The potential use of phytocompounds against various cancers like lung, breast, colon, etc. has been demonstrated in preclinical and clinical studies. In the last decade, strategies of chemoprevention have changed significantly, and now have rigorous selection criteria involving more extensive preclinical mechanistic evaluation of agents before clinical trials are instituted and a focus on defining biomarkers of activity that can be used as early predictors of efficacy. In this review, we discuss the preclinical and clinical studies of selected promising chemopreventive agents and also summarize newly emerging compounds and highlight potential new developments in the field of chemoprevention.

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.

Fruits, vegetables and medicinal herbs rich in phenolics antioxidants contribute toward reduced risk of age-related diseases and cancer. In this study, Psidium guajava leaf extract was fractionated in various organic solvents viz. petroleum ether, benzene, ethyl acetate, ethanl and methanol and tested for their antioxidant and antimutagenic properties. Methanolic fraction showed maximum antioxidant activity comparable to ascorbic acid and butylated hydroxyl toluene (BHT) as tested by DPPH free radical scavenging, phosphomolybdenum, FRAP (Fe3 + reducing power) and CUPRAC (cupric ions (Cu2+) reducing ability) assays. The fraction was analyzed for antimutagenic activities against sodium azide (NaN3), methylmethane sulfonate (MMS), 2-aminofluorene (2AF) and benzo(a)pyrene (BP) in Ames Salmonella tester strains. The methanol extracted fraction at 80 μg/ml concentration inhibited above 70% mutagenicity. Further, phytochemical analysis of methanol fraction that was found to be most active revealed the pr...

Antioxidant properties of methanol extracts of 20 plants were studied by four different assays over a range of concentrations (12.5–400 µg mL−1), and compared with standard antioxidants’ ascorbic acid and butylated hydroxytoluene. Ascorbic acid–equivalent antioxidant activity of extracts by the phosphomolybdenum method ranged from 392.8 to 2942.7 µmoles g−1 at 400 µg mL−1, whereas DPPH radical scavenging was between 30.9% and 95.2%; extracts also showed strong activity by FRAP and CUPRAC assays. The extracts demonstrated dose-dependent activity and strong correlation between antioxidant activities and total phenolics. Total phenolic content was the highest in Syzygium aromaticum (279 mg GAE g−1) and the lowest in Sesame indicum (28.16 mg GAE g−1).

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

Quorum sensing (QS) is a global gene regulatory mechanism in bacteria for various traits including virulence factors. Disabling QS system with anti-infective agent is considered as a potential strategy to prevent bacterial infection. Mangifera indica L. (mango) has been shown to possess various biological activities including anti-QS. This study investigates the efficacy of leaf extracts on QS-regulated virulence factors and biofilm formation in Gram negative pathogens. Mango leaf (ML) extract was tested for QS inhibition and QS-regulated virulence factors using various indicator strains. It was further correlated with the biofilm inhibition and confirmed by electron microscopy. Phytochemical analysis was carried out using ultra performance liquid chromatography (UPLC) and gas chromatography–mass spectrometry (GC-MS) analysis. In vitro evaluation of anti-QS activity of ML extracts against Chromobacterium violaceum revealed promising dose-dependent interference in violacein production, by methanol extract. QS inhibitory activity is also demonstrated by reduction in elastase (76%), total protease (56%), pyocyanin (89%), chitinase (55%), exopolysaccharide production (58%) and swarming motility (74%) in Pseudomonas aeruginosa PAO1 at 800 μg/ml concentration. Biofilm formation by P. aeruginosa PAO1 and Aeromonas hydrophila WAF38 was reduced considerably (36–82%) over control. The inhibition of biofilm was also observed by scanning electron microscopy. Moreover, ML extracts significantly reduced mortality of Caenorhabditis elegans pre-infected with PAO1 at the tested concentration. Phytochemical analysis of active extracts revealed very high content of phenolics in methanol extract and a total of 14 compounds were detected by GC-MS and UPLC. These findings suggest that phytochemicals from the ML could provide bioactive anti-infective and needs further investigation to isolate and uncover their therapeutic efficacy.

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.