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Physiological signals obtained from electroencephalography (EEG), electromyography (EMG), and electrocardiography (ECG) provide valuable clinical information but pose challenges for analysis due to their high-dimensional nature. Traditional machine learning techniques, relying on hand-crafted features from fixed analysis windows, can lead to the loss of discriminative information. Recent studies have demonstrated the effectiveness of deep convolutional neural networks (CNNs) for robust automated feature learning from raw physiological signals. However, standard CNN architectures require two-dimensional image data as input. This has motivated research into innovative signal-to-image (STI) transformation techniques to convert one-dimensional time series into images preserving spectral, spatial, and temporal characteristics. This paper reviews recent advances in strategies for physiological signal-to-image conversion and their applications using CNNs for automated processing tasks. A systematic analysis of EEG, EMG, and ECG signal transformation and CNN-based analysis techniques spanning diverse applications, including brain-computer interfaces, seizure detection, motor control, sleep stage classification, arrhythmia detection, and more, are presented. Key insights are synthesized regarding the relative merits of different transformation approaches, CNN model architectures, training procedures, and benchmark performance. Current challenges and promising research directions at the intersection of deep learning and physiological signal processing are discussed. This review aims to catalyze continued innovations in effective end-to-end systems for clinically relevant information extraction from multidimensional physiological data using convolutional neural networks by providing a comprehensive overview of state-of-the-art techniques.

Background: Central airway obstruction (CAO) can lead to acute respiratory failure (RF) necessitating positive pressure ventilation (PPV). The efficacy of airway stenting to aid liberation from PPV in patients with severe acute RF has been scarcely published. We present a systematic review and our recent experience., Methods: A systematic review of PubMed was performed, and a retrospective review of cases performed at our two institutions from 2018 to 2022 in adult patients who needed stent insertion for extrinsic or mixed CAO complicated by RF necessitating PPV., Results: Fifteen studies were identified with a total of 156 patients. The weighted mean of successful liberation from PPV post-stenting was 84.5% and the median survival was 127.9 days. Our retrospective series included a total of 24 patients. The most common etiology was malignant CAO (83%). The types of PPV used included high-flow nasal cannula (HFNC) (21%), non-invasive ventilation (NIV) (17%) and Invasive Mechanical Ventilation (62%). The overall rate of successful liberation from PPV was 79%, with 55% of HFNC and NIV cases being liberated immediately post-procedure. The median survival of the patients with MCAO that were successfully liberated from PPV was 74 days (n = 16, range 3-893 days), and for those with that failed to be liberated from PPV, it was 22 days (n = 4, range 9-26 days)., Conclusion: In patients presenting with acute RF from extrinsic or mixed morphology CAO requiring PPV, airway stenting can successfully liberate most from the PPV. This may allow patients to receive pathology-directed treatment and better end-of-life care., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose of Review: Treatment of obstructive sleep apnea (OSA) has historically been centered on outpatients given sleep testing is performed on an outpatient basis. Much of this practice originates from insurers only covering sleep testing on an outpatient basis. Over the last decade, there have been innovations made in the portability of sleep monitors which have allowed sleep testing on inpatients to be facilitated. There is also emerging data that inpatient sleep testing may reduce readmissions and healthcare costs in certain cardiovascular conditions. Accordingly, this review aims to provide comprehensive coverage of recent advances in the practice of inpatient sleep medicine and its effect on reducing the burden of cardiovascular disease., Recent Findings: Chief cardiovascular diseases that intersect with OSA in inpatients are stroke, atrial fibrillation, and heart failure. There is data from the National Inpatient Sample comparing arrhythmia burdens in patients with OSA and HFpEF showing that OSA patients have higher mortality rates, hospital durations, and medical costs. Also, OSA is associated with higher burdens of arrhythmia. It is currently unknown whether treatment of inpatients with PAP therapy lowers the occurrence of arrhythmias. Recent data suggests that costs for heart failure patients with OSA that are readmitted are higher than those for heart failure patients without OSA. A recent analysis of patients with HFpEF (heart failure with preserved ejection fraction) and OSA showed that the PAP adherent patients had fewer healthcare related costs, lower readmission rates, and fewer emergency room visits than those that were nonadherent. In broader terms, rapid initiation of PAP therapy in a large administration database query of 23 million Medicare patients appears to reduce annual healthcare costs and reduce readmissions although further study is required., Summary: OSA is globally underdiagnosed, with an estimated one billion individuals affected. OSA's pathogenesis involves a combination of risk factors, such as obesity, age, and increased neck circumference that contribute to fragmented sleep patterns and in turn, numerous cardiovascular comorbidities, such as stroke, atrial fibrillation, and coronary artery disease. Recently, inpatient sleep medicine programs have emerged as a promising avenue for improving diagnosis, patient safety, and potentially reducing readmissions. Integrating inpatient sleep medicine into healthcare systems to address the significant health and economic burden associated with undiagnosed OSA. Improved coverage of inpatient sleep testing and services will be a key driver of addressing inpatient gaps in sleep medicine care. The current research findings provide a bedrock from which further investigations may proceed in a prospective and randomized, controlled fashion to further clarify the effects of treatment of OSA on cardiovascular outcomes of inpatients., Competing Interests: Conflict of Interest: The authors declare that they have no conflict of interest.

Abstract: Central sleep apnea (CSA) is common in patients with heart failure. Recent studies link ticagrelor use with CSA. We aimed to evaluate CSA prevalence in patients with coronary heart disease (CHD) and whether ticagrelor use is associated with CSA. We reviewed consecutive patients with CHD who underwent a polysomnography (PSG) test over a 5-year period from 3 sleep centers. We sampled patients who were on ticagrelor or clopidogrel during a PSG test at a 1:4 ticagrelor:clopidogrel ratio. Patients with an active opioid prescription during PSG test were excluded. Age, left ventricle (LV) dysfunction, and P2Y12 inhibitor use were included in a multivariate logistic regression. A total of 135 patients were included with 26 on ticagrelor and 109 on clopidogrel (age 64.1 ± 11.4, 32% male). High CSA burden (12%) and strict CSA (4.4%) were more common in patients on ticagrelor than in those on clopidogrel (27% vs. 8.3% and 10.0% vs. 1.8%). Ticagrelor use (vs. clopidogrel) was associated with high CSA burden (OR 3.53, 95% CI 1.04-12.9, P = 0.039) and trended toward significance for strict CSA (OR 6.32, 95% CI 1.03-51.4, P = 0.052) when adjusting for age and LV dysfunction. In an additional analysis also adjusting for history of atrial fibrillation, ticagrelor use and strict CSA became significantly associated (OR 10.0, 95% CI 1.32-117, P = 0.035). CSA was uncommon in patients with CHD undergoing sleep studies. Ticagrelor use (vs. clopidogrel) was associated with high CSA burden and trended toward significance for strict CSA., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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.

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 IC 50 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.

Colorectal cancer is the third most common form of cancer diagnosed and the third leading class for cancer-related deaths. Given the prevalence of colon cancer worldwide, further insight into developing novel and 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 and therapeutic effects. A limitation to current clinical applications of anthocyanins is the high doses that are required. In order to overcome this limitation, we tested the active moiety, anthocyanidins for chemopreventive and therapeutic effects against colorectal cancer in vivo and in vitro . Treatment with native anthocyanidin mixture (Anthos) from bilberry yielded significant antiproliferative activity against colon cancer cells. Anthos treatment led to significant reductions in polyp and tumor counts in vivo . Reduced Src and EGFR phosphorylation was observed with Anthos treatment, which correlated with downstream targets such as PD-L1 and modulation of the colon inflammatory environment. These results provide a promising outlook on the impact of berry Anthos for the treatment and prevention of familial adenomatous polyposis and colorectal cancer. Results from this study also provide novel mechanistic insight into the chemopreventive and therapeutic activities of Anthos., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w / w ) or dried ethanolic cumin extract (1%, w / w ), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm 3 ) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Gene therapy promises to revolutionize biomedicine and personalized medicine by modulating or compensating the expression of abnormal genes. The biggest obstacle for clinical application is the lack of an effective, non-immunogenic delivery system. We show that bovine colostrum exosomes and polyethyleneimine matrix (EPM) delivers short interfering RNA (siRNA) or plasmid DNA (pDNA) for effective gene therapy. KRAS, a therapeutic focus for many cancers, was targeted by EPM-delivered KRAS siRNA (siKRAS) and inhibited lung tumor growth (>70%) and reduced KRAS expression (50%-80%). Aberrant p53 is another therapeutic focus for many cancers. EPM-mediated introduction of wild-type (WT) p53 pDNA (pcDNA-p53) resulted in p53 expression in p53-null H1299 cells in culture, subcutaneous lung tumor, and tissues of p53-knockout mice. Additionally, chemo-sensitizing effects of paclitaxel were restored by exogenous WT p53 in lung cancer cells. Together, this novel EPM technology represents an effective 'platform' for delivery of therapeutic nucleic acids to treat human disease., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interests., (Copyright: © 2020 Kyakulaga et al.)

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 Apc Min/+ 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., (©2019 American Association for Cancer Research.)

Gene-silencing with targeted siRNAs has great potential as a therapeutic approach for various diseases including cancer. However, intracellular delivery of siRNA is challenging. We used bovine milk exosomes as a novel system for siRNA delivery. First, we demonstrated that exosomes can deliver endogenous RNA payloads into recipient cells. Next, we loaded siRNA against specific genes including VEGF, EGFR, AKT, MAPK, and KRAS. We utilized 5' -32 P-labeled siKRAS as a tracer and found exosome loading with siRNA could be variable. We demonstrated that the siRNA of loaded exosomes is stable and resist degradation. Our results indicated that siRNAs against target genes ranged from 2 to 10-fold knockdown in expression levels in various cancers. Since mutated KRAS has been implicated in the development of various cancers including lung cancer, we tested a mutant-allele specific siRNA against KRAS G12S , in A549 cells. We observed a dose-dependent anti-proliferative activity against A549 cells treated with exosomes carrying siKRAS G12S . We observed significant inhibition of A549 tumor xenografts in animals treated with folic acid-functionalized exosomes carrying siKRAS. In summary, milk-derived exosomes represent a viable natural nano-carrier for the delivery of siRNA for therapeutic application against cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 (CIN 2+ ) 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, was chosen due to similarities in cervical dimensions to the human. Estrus was induced with progesterone CIDR® vaginal implants for 14days followed by the administration of chorionic gonadotropins 48h 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 12-16ng 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., (Copyright © 2017. Published by Elsevier Inc.)

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 IC 50 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.

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 < 0.01) of the cervical tumor xenograft. No gross or systemic toxicity was observed in the rats administered with the exosomes or ExoCUR. These results suggest that exosomes can be developed as potential nano carriers for delivering curcumin which otherwise has encountered significant tissue bioavailability issues in the past.

In this report milk-derived exosomes have been investigated for oral delivery of the chemotherapeutic drug paclitaxel (PAC) as an alternative to conventional i.v. therapy for improved efficacy and reduced toxicity. PAC-loaded exosomes (ExoPAC) were found to have a particle size of ~108 nm, a narrow particle size distribution (PDI ~0.190), zeta potential (~ -7 mV) and a practical loading efficiency of ~8%. Exosomes and ExoPAC exhibited excellent stability in the presence of simulated-gastrointestinal fluids, and during the storage at -80 °C. A sustained release of PAC was also observed up to 48 h in vitro using PBS (pH 6.8). Importantly, ExoPAC delivered orally showed significant tumor growth inhibition (60%; P<0.001) against human lung tumor xenografts in nude mice. Treatment with i.p. PAC at the same dose as ExoPAC, however, showed modest but statistically insignificant inhibition (31%). Moreover, ExoPAC demonstrated remarkably lower systemic and immunologic toxicities as compared to i.v. PAC., (Published by Elsevier Inc.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Prognosis of lung cancer still remains grim largely due to recurrence and aggressive metastasis of the disease. In this study, we examined the potential of exosomal miRNAs as biomarkers of recurrent lung cancer. Initially, in vitro miRNA profiles of normal lung (Beas-2b) and lung cancer (H1299) cells and of exosomes isolated from conditioned media were determined. In vivo study involved establishing subcutaneous primary and recurrent lung cancer xenografts in nude mouse model and examining tumor and serum exosomal miRNA alteration in secondary/recurrent lung tumors. A total of 77 miRNAs were observed to be significantly modulated in the H1299 cells (47 miRNA upregulated and 30 downregulated) compared to the Beas-2b cells. The exosomes isolated from conditioned media indicated several miRNAs which were in agreement with cells of origin. A similarity was also observed between miRNAs from serum exosomes and tumors, indicating their origin from the lung tumors. Two miRNAs, miR-21 and miR-155, were found to be significantly upregulated in recurrent tumors compared to primary tumors. These miRNAs were also upregulated in serum exosomes of recurrent tumor-bearing animals versus non-tumor- or primary tumor-bearing animals. Increased expression of the recurrent disease markers were also observed in recurrent tumors compared with primary tumors. Serum exosomes from recurrent tumor mice mirrored its tumor profile in expressing higher levels of these proteins compared with exosomes from primary tumor mice. Our data suggest that exosomal miRNA signatures may be a true representation of a pathological profile of lung cancer; thus, miRNAs could serve as promising biomarkers for non-invasive diagnosis of the disease.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Scope: 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 mm(3) ) 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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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.

Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E2) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E2 in human breast cancer, as well as developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E2 exposure and identified a 'signature' of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E2 treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E2-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR-127 and miR-183 detected with E2 treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E2 treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E2-mediated mammary tumorigenesis is also established. These observations provide mechanistic insights into the new molecular events behind the chemopreventive action of ellagic acid and treatment of breast cancer., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cucurbitacin B (CuB), has recently emerged as a potent anticancer agent; however, its efficacy in non-small-cell lung cancer (NSCLC) and the mechanism(s) initiating its biological effects remain largely unclear. In this study, CuB potently suppressed the growth of four NSCLC cells (H1299, A549, HCC-827 and H661) in vitro and the highly aggressive H1299 xenograft in vivo. CuB significantly altered the actin cytoskeletal assembly, induced G2/M cell-cycle arrest and mitochondrial apoptosis through the modulation of several key molecular targets mediating the aforementioned processes. Interestingly, all cellular effects of CuB were completely attenuated only by the thiol antioxidant N-acetylcysteine (NAC). Furthermore, pretreatment with glutathione synthesis inhibitor butithione-sulfoxime (BSO), significantly exacerbated CuB's cytotoxic effects. To this end, cells treated with CuB revealed a rapid and significant decrease in the levels of protein thiols and GSH/GSSG ratio, suggesting disruption of cellular redox balance as the primary event in CuB's cytotoxic arsenal. Using UV and FTICR mass spectrometry we also demonstrate for the first time a physical interaction of CuB with NAC and GSH in a cell-free system suggesting that CuB interacts with and modulates cellular thiols to mediate its anti-cancer effects. Collectively, our data sheds new light on the working mechanisms of CuB and demonstrate its therapeutic potential against NSCLC., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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 Cu 2+ -catalyzed redox cycling of 4-hydroxy-17β-estradiol as analyzed by 32 P-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 (H 2 O 2 ) scavenging and ferrous ion-chelating activities (IC 50 =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, antiproliferative, and antiinflammatory activities. We report the 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% total 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 5 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. Their high antioxidant potential was also reflected by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)- and 2,2-diphenyl-1-picrylhydrazyl-scavenging and ferrous ion-chelating activities. We also analyzed antiproliferative 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 5 anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.

In recent years, there has been a significant improvement in the understanding of molecular events and critical pathways involved in breast cancer. This has led to the identification of novel targets and development of anticancer therapies referred to as targeted therapy. Targeted therapy has high specificity for the molecules involved in key molecular events that are responsible for cancer phenotype such as cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression, and angiogenesis. Targeted agents that have been approved for breast cancer include trastuzumab and lapatinib, directed against human epidermal growth factor receptor 2 (HER2) and bevacizumab, directed against vascular endothelial growth factor (VEGF). Several other targeted agents currently under evaluation in preclinical and clinical trials include inhibitors of epidermal growth factor receptor (EGFR), dual EGFR and HER2 inhibitors, VEGF/VEGFR inhibitors, and agents that interfere with crucial signaling pathways such as PI3K/AKT/mTOR and RAS/MEK/ERK; agents against other tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that promote apoptosis such as Poly ADP ribose polymerase inhibitors; agents that target invasion and metastasis such as matrix metalloproteinases inhibitors and others. In this review, we highlight the most promising targeted agents and their combination with mainstream chemotherapeutic drugs in clinical trials.

The purpose of this study was to investigate the prognostic factors, such as clinical, histological and socio-demographic features affecting the event-free and overall survival of the patients with stage I-III carcinoma of the cervix. Eighty-nine patients with International FIGO stage I-III cervical cancer were treated radiation therapy and follow-up of 5-7 years were analyzed for various clinical, histopathological and socio-demographic factors influencing prognosis. Survival estimations were performed using the Kaplan-Meier method, and were compared using the un-weighted log-rank test and multivariable analysis using the Cox proportional hazards model. The median age was 46 years (range, 28-65 years). The 5-year event-free survival (EFS) and overall survival (OAS), along with standard error (SE), were 65.2% (7.0%) and 81.4% (6.1%), respectively. Significant prognostic factors for EFS include, stage (P=0.019), pelvic lymph node metastasis (P=0.013), parametrial (PMT) involvement (P=0.025), number of parametria involved (P=0.000) and tumor size (P=0.034). However, number of parametrial invasion was only significant prognostic factors for overall survival (P=0.015); 5-year survival rate was significantly lower in patients with both PMT involved (58%) than with one PMT involved (>85%). Using a multivariable analysis, we found that number of PMT involved being the only independent significant factor for the development of recurrent disease. None of the socio-demographic factors analyzed were of prognostic importance on event-free and overall survival in cervical cancer patients. Several clinicopathological factors were of prognostic significance but none of the socio-demographic factors analyzed had any role in determining patient outcome. Hence, in cervical cancer, prognosis is more likely dependent on clinical than socio-demographic factors unlike several other cancers where their significant role is well documented. Study of clinical and demographic characteristics for their influence on patient survival could help design better patient management strategies.

Human papilloma virus (HPV) is the major cause of invasive cervical cancer (ICC). The study aim was to determine the prevalence of HPV genotypes and to correlate HPV types with response to radiotherapy. A total of 43 cervical biopsies collected from sequentially enrolled patients were analyzed by DNA amplification with MY09/MY11 primers and sequenced to determine the HPV genotype. Samples with multiple infections were resolved by multiplex PCR, combined with array primer extension (APEX). HPV DNA was detected in 40 of 43 (93%) samples. Nine different HPVs, including the most common types -16 (53%) and -18 (13%) were detected. Other types were HPV 31, 33, 45, 52, 58, 66 and 68. Single HPV types were found in 33 of 40 samples (82%) and multiple types in 7 of 40 samples (18%). The following significant predictors were identified: a) HPV 58 was most significant (p=0.02), followed by HPV 18 (p=0.04) associated with lack of treatment response; b) tumor size (p=0.042) and treatment response (p=0.025) elicited association with HPV infection type; c) treatment failure were found to be nearly 5-fold higher in case of multiple infections than of single infection (57% versus 12%) (odds ratio = 9.66; 95% CI 1.6-6.00). d) Multiple HPV infections correlated most prominently with lack of treatment compared with single type infection (p=0.005). Hence, patients with multiple infections, large tumor size, and HPV 58 and/or 18, are at risk of treatment failure and need to be followed for response and suitable inter-ventions done for a favorable outcome.