Cumin Prevents 17ß-Estradiol-Associated Breast Cancer in ACI Rats.

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 mm3) 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.

Development of a goat model for evaluation of withaferin A: Cervical implants for the treatment of cervical intraepithelial neoplasia.

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.

Milk-derived exosomes for oral delivery of paclitaxel.

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.

Chemoprevention of Rat Mammary Carcinogenesis by Apiaceae Spices.

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 17ß-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.

Exosomal miRNAs as biomarkers of recurrent lung cancer.

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.

Exosomal formulation enhances therapeutic response of celastrol against lung cancer.

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.

Controlled-release systemic delivery - a new concept in cancer chemoprevention.

Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.

Antioxidant and antiproliferative activities of anthocyanin/ellagitannin-enriched extracts from Syzygium cumini L. (Jamun, the Indian Blackberry).

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.

Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk.

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

Exosomes as Emerging Drug Delivery and Diagnostic Modality for Breast Cancer: Recent Advances in Isolation and Application.

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.

Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells.

Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 µM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.

Berry anthocyanidins inhibit intestinal polyps and colon tumors by modulation of Src, EGFR and the colon inflammatory environment.

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.

Anthocyanidins Inhibit Growth and Chemosensitize Triple-Negative Breast Cancer via the NF-κB Signaling Pathway.

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.

Exosome-mediated delivery of RNA and DNA for gene therapy.

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.

Synergistic combinations of paclitaxel and withaferin A against human non-small cell lung cancer cells.

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.

Chemoprevention of Colorectal Cancer by Anthocyanidins and Mitigation of Metabolic Shifts Induced by Dysbiosis of the Gut Microbiome.

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

Significance of multiple HPV infection in cervical cancer patients and its impact on treatment response.

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.

Milk exosomes - Natural nanoparticles for siRNA delivery.

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'-32P-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 KRASG12S, in A549 cells. We observed a dose-dependent anti-proliferative activity against A549 cells treated with exosomes carrying siKRASG12S. 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.

Withaferin A inhibits Epithelial to Mesenchymal Transition in Non-Small Cell Lung Cancer Cells.

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 TGFß1 (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 TGFß1 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.

Exosomes for the Enhanced Tissue Bioavailability and Efficacy of Curcumin.

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.