Srivani Ravoori

Inhibition of Estrogen-Mediated Mammary Tumorigenesis by Blueberry and Black Raspberry

We previously demonstrated the protective effects of blueberry (BB) and black raspberry (BRB) supplemented at 2.5% dose in an ACI rat mammary tumor model. Here, we assessed a dose-related alteration in tumor indices with diet supplemented with 5% BB or BRB powder. The diet was well tolerated. Tumor palpation from 12 weeks revealed first tumor appearance by 84 days in the control group, that was delayed by 24 and 39 days with the BB and BRB diets, respectively (p = 0.04). Ellagic acid detected in the plasma of rats fed the BRB diet was in the range of 96.6-294.2 ng/mL. While the BB diet showed better efficacy in reducing mammary tissue proliferation and tumor burden, tumor latency was delayed efficiently by BRB. Furthermore, BB was effective in downregulating CYP1A1 expression, while BRB downregulated ERα expression effectively. Distinct anticarcinogenic effects of the two berries correspond to their distinct phytochemical signatures.

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.

Cigarette smoke condensate-induced oxidative DNA damage and its removal in human cervical cancer cells

Exposure to cigarette smoke is well documented to increase oxidative stress and could account for higher risk of cervical cancer in smokers. Cervical pre-cancerous lesions that are initiated by human papillomavirus (HPV) infection generally regress in the absence of known risk factors such as smoking. 8-oxodeoxyguanosine (8-oxodG) is a highly mutagenic oxidative DNA lesion that is formed by the oxidation of deoxyguanosine. In the present study, we examined: a) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in and its removal from HPV-transfected (ECT1/E6 E7), HPV-positive (CaSki) and HPV-negative (C33A) human cervical cancer cells, and b) the cell cycle progression and apoptosis in CSC-treated ECT1/E6 E7 cells. CSC induced 8-oxodG in a dose- (p=0.03) and time (p=0.002)-dependent fashion in ECT1/E6 E7 cells as determined by flow cytometry. A 2.4-fold higher level of 8-oxodG was observed in HPV-positive compared with HPV-negative cells. However, 8-oxodG lesions were almost completely removed 72 h post-exposure in all cell lines as determined by ImageStream analysis. This observation correlates with the 2- and 5-fold increase in the p53 levels in ECT1/E6 E7 and CaSki cells with no significant change in C33A cells. We conclude that: a) cigarette smoke constituents induce oxidative stress with higher burden in HPV-positive cervical cancer cells and b) the significant increase observed in p53 levels in wild-type cervical cells (ECT1/E6 E7 and CaSki) may be attributed to the p53-dependent DNA repair pathway while a p53-independent pathway in C33A cells cannot be ruled out.

Controlled systemic delivery by polymeric implants enhances tissue and plasma curcumin levels compared with oral administration.

Curcumin possesses potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ∼2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ∼3.3 ng/ml on day 1, which dropped to ∼0.2 ng/ml after 3 months, whereas only 0.2-0.3 ng/ml concentration was observed from 4-12 days with diet and was undetected subsequently. Almost 10-fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1 ± 7.3 vs 2.7 ± 0.8 ng/g) and were still significant even after 90 days (7.7 ± 3.8 vs 2.2 ± 0.8 ng/g). Although curcumin levels were similar in liver from both the routes (∼25-30 ng/g from day 1-4 and ∼10-15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ∼28-fold lower doses at 90 days. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.

Protective effects of selenium against DNA adduct formation in inuit environmentally exposed to PCBs.

Dietary habits that expose populations to potential toxicants as well as protective agents simultaneously are a realistic scenario where a meaningful assessment of the interactions and net benefit or damage can be made. A group of Inuit from Salluit, Northern Canada are exposed to high levels of PCBs and selenium, both present in the Inuit traditional foods such as blubber from sea mammals and fatty fish. Blood samples were collected from 83 Inuit, 22-70 years old. Blood selenium and PCB levels were determined previously and ranged from 227 to 2069µg/L and 1.7 to 143µg/L, respectively. DNA isolated from white blood cells were analyzed by modified (32)P-postlabeling adductomics technology that detects a multitude of highly polar to lipophilic adducts. The levels of 8-oxodG adducts ranged from 470 to 7400 adducts/10(9) nucleotides. Other as yet unidentified polar adducts showed a 30 to 800-fold inter-individual variability. Adduct levels were negatively associated with PCB and selenium levels. The subjects were classified into high and low ratio groups, with respect to selenium/PCB. In the high ratio group, the coefficient of selenium is significantly negatively correlated with 8-oxodG (r = -0.38, p = 0.014) and total adducts (r = -0.41, p = 0.009) while there was no correlation within the low selenium/PCB group. This study suggests that increasing selenium has mitigating effect in reducing DNA adducts and therefore, possible negative effects of PCB were not seen. A protective effect of selenium is highlighted.

Chemoprevention of mammary carcinogenesis by sustained systemic delivery of ellagic acid.

Many chemopreventives that show efficacy in vitro show little/no response or require bolus doses in animal models and clinical trials because of limited bioavailability. Ellagic acid has been tested in various animal models with mixed results. We report the efficacy of ellagic acid delivered by a subcutaneous implant compared with a dietary route against estrogen-induced mammary tumors. Ellagic acid delayed the first tumor appearance by 2 and 3 weeks by implant and diet routes, respectively. The tumor incidence was 75% and 69% by the implant and dietary routes when the control group had 100% palpable tumors by 26 weeks. Ellagic acid also significantly reduced the tumor burden by both implant (855±242 mm3; P=0.0375) and dietary (599±169 mm3; P=0.0133) routes compared with control (1522±299 mm3). Similar reductions were observed in tumor multiplicity (4.8±0.5; P=0.0042 and 4.5±0.4; P=0.0031 tumors/rat with implant and diet, respectively, vs. 8.9±1.2 in control). The total amount of ellagic acid administered by implant was 5.92±3.48 whereas it was 800±40 mg/rat through diet. Thus, over 130-fold dose reduction produced similar biological responses when delivered by implant. The anticarcinogenicity effects corroborated the observed reduction in levels of pituitary prolactin. This novel approach opens new avenues to test agents individually or as mixtures for their chemopreventive potential that are discontinued, either due to lack of bioavailability or toxicity potentially associated with high doses or due to lack of availability of sufficient quantities.

DNA damage associated with PCBs in the whole blood cells of Inuit

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

Curcumin Implants, Not Curcumin Diet, Inhibit Estrogen-Induced Mammary Carcinogenesis in ACI Rats

Curcumin is widely known for its antioxidant, anti-inflammatory, and antiproliferative activities in cell-culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvent oral bioavailability issues, and tested their potential against 17β-estradiol (E2)–mediated mammary tumorigenesis. Female Augustus Copenhagen Irish (ACI) rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2 cm; 200 mg each; 20% drug load) 4 days before grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Curcumin implants were changed after 4.5 months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months, and after the tumor incidence reached >80% (∼6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2 ± 1 vs. 5 ± 3; P = 0.001) and tumor volume (184 ± 198 mm3 vs. 280 ± 141 mm3; P = 0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity, whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in the presence of E2. Because CYP1A and CYP3A4 metabolize most of the E2 to its noncarcinogenic 2-OH metabolite, and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by high-performance liquid chromatography showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered. Cancer Prev Res; 7(4); 456–65. ©2014 AACR.

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 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.

Polymeric Implants for the Delivery of Green Tea Polyphenols

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, 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. GTP implants have the potential to be used systemically and locally at the tumor site as an alternative strategy.