Jillian N. Eskra

Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo

SCOPE Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue-specific activity have not been analyzed. METHODS AND RESULTS An orally administered hop extract and subcutaneously injected XH were administered to Sprague-Dawley rats over 4 days. LC-MS-MS analysis of plasma, liver, and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH-induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, (i) XH modified Kelch-like ECH-associated protein leading to nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation and antioxidant response element (ARE) activation; (ii) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; (iii) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to estrogen receptorα, recruiting Nrf2, and downregulating ARE-regulated genes. CONCLUSION XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.

Interactive effects of 9-cis-retinoic acid and androgen on proliferation, differentiation, and apoptosis of LNCaP prostate cancer cells.

9-cis-Retinoic acid (9cRA), which binds to both retinoic acid receptors and retinoic X receptors, inhibits prostate cancer induction in rats and reduces growth of prostate cancer cells. However, the nature of this growth inhibition and the interactive influence of androgens are not well defined and are the subject of this report. LNCaP and PC-3 cells were cultured and treated with a range of 9cRA concentrations for 3–6 days in the absence or presence of 5&agr;-dehydrotestosterone. 9cRA inhibited cell proliferation in a dose-dependent manner, plateauing at 10−7 mol/l. Treatment of cells with 10−6 mol/l 9cRA inhibited 5&agr;-dihydroxytestosterone (DHT)-stimulated proliferation, the effect of which was maximal at 10−9 mol/l DHT. Treatment of DHT (10−9 mol/l)-exposed cells with 9cRA caused a dose-dependent increase in prostate-specific antigen in the medium after 6 days, but not 3 days. 9cRA caused a dose-dependent increase in apoptotic cells stained with H33258 after 3 days, but not 6 days; however, on using flow cytometry, apoptosis was apparent at both 3 and 6 days. Flow cytometry also revealed interference of G0/G1 to S phase transition by 9cRA. Inhibition by 9cRA of anchorage-independent growth of PC-3 cells was also found; LNCaP cells did not grow colonies in soft agar. 9cRA inhibited growth and induced differentiation of human LNCaP prostate cancer cells in vitro and inhibited anchorage-independent growth of PC-3 cells. Because 9cRA and 13-cis-retinoic acid, which is retinoic acid receptor-selective, prevent prostate carcinogenesis in rats, and 13-cis-retinoic acid also inhibits growth of human prostate cancer cells, the RAR is a potential molecular target for prostate cancer prevention and therapy.

Abstract 2131: Effects of black raspberries and their constituents on rat prostate carcinogenesis and human prostate cancer cell growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Black raspberries (BRBs) and their constituents may inhibit human GI tract cancers and prevent breast cancer in a rat model. Anthocyanins and polyphenols in BRBs are responsible for their anti-cancer effects. Berries and other fruits are used as dietary supplements by cancer patients. As prostate cancer remains a major health problem, it is thus important to determine whether BRBs or BRB constituents may prevent or treat this malignancy. We previously reported (AACR Ann Mtg 2013) that the BRB anthocyanin metabolite protocatechuic acid (PCA) was detectable in the prostate of rats fed BRB, indicating that it reaches this tissue, but BRB consumption by Wistar rats did not prevent prostate cancer induced by MNU and chronic testosterone treatment. We now studied the effect of feeding AIN-93M diet containing 5% or 10% lyophilized BRB powder (exchanged for starch) on induction of prostate cancer in NBL rats by chronic testosterone & 17β-estradiol treatment via SC Silastic implants. Incidence of adenocarcinomas arising from periurethral prostatic ducts was similar in rats fed control diet and those fed berry-containing diets (73-90%), as was tumor multiplicity (1.7-1.8 tumors per prostate). In contrast, we previously found that cyanidin-3-rutinoside, the most abundant anthocyanin in BRBs, inhibits growth of LAPC-4 human prostate cancer cells. We now used PC-3 and LNCaP prostate cancer cells to study effects of ethanolic extract of lyophilized BRB powder and major BRB constituents: the polyphenol ellagic acid (EA), cyanidin-3-rutinoside, and PCA. Growth rates were measured by hemocytometer counting with trypan blue exclusion. BRB extract (1 µg/ml to 1 mg/ml) inhibited viability of LNCaP cells dose-dependently, but linear trend was not significant (p=0.09). Similar, but also not quite significant, decreases in LNCaP cell numbers were caused by PCA and EA, while EA increased the number of dead cells (ANOVA p=0.0471, linear trend p=0.0385). No significant changes occurred in PC-3 cell viability with any of the treatments. Effects on anchorage independent growth of PC-3 cells were assessed by soft agar colony forming assays. A dose-dependent reduction in the number of colonies occurred after EA treatment (ANOVA p=0.0065, linear trend p=0.0007) and BRB extract (ANOVA p<0.0001, linear trend p<0.0001), but no change in colony formation of cells treated with cyanidin-3-rutinoside or PCA. Compared to vehicle, BRB at 1 mg/ml maximally reduced colonies by 50%, and 3.0 µg/ml EA reduced colonies by 39%. No effects on cell migration of PC-3 cells, assessed by wound healing assays, were found for any of the treatments. Thus, BRBs may be beneficial for treatment of prostate cancer and EA is at least partially responsible for the anti-cancer activity of BRBs, but, surprisingly, cyanidin-3-rutinoside and protocatechuic acid did not have significant effects. (Supported by NIH Grant R21 CA152879). Citation Format: Jillian N. Eskra, Michael J. Schlicht, Maarten C. Bosland. Effects of black raspberries and their constituents on rat prostate carcinogenesis and human prostate cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2014-2131

Lack of combination effects of soy isoflavones and taxane chemotherapy of castration-resistant prostate cancer

Patients with cancer, including prostate cancer, often use dietary supplements, such as soy or isoflavones, before, during, or after therapy. There is little information about possible interactions between supplements and cancer chemotherapy. There are some reports suggesting enhancement by genistein of taxane chemotherapy for castrate‐resistant prostate cancer (CRPC).

Abstract 317: Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy

Ellagic acid (EA) is a polyphenolic compound found in berries, pomegranates and other foods, which has cancer preventive and therapeutic properties. EA inhibits proliferation, induces apoptosis, blocks cell cycle progression and inhibits ABC drug transporters. Based on these anti-cancer activities of EA, we hypothesized that EA could enhance the efficacy of taxane chemotherapy on prostate cancer cells and inhibit drug resistance. We used castration-resistant 22Rv1, VCaP, and C4-2 prostate cancer cells and androgen-independent PC-3 cells to study effects of EA on efficacy of the taxane drug cabazitaxel (CBZ). Cells were treated with EA (0.1 μM - 30 μM) and after 72 hr proliferation was measured using the SRB assay. EA inhibited proliferation at levels greater than 10 μM. At 30 μM the greatest inhibition was observed in the most proliferative cell lines (22Rv1: 66.7%, PC-3: 82.7%), with a more modest effect on the slower growing cells (VCaP: 25.2%, C4-2: 31.7%). We next tested if growth inhibitory concentrations of EA could enhance the microtubule stabilizing effects of CBZ. In a cell-free assay, CBZ (1 nM - 1 μM) dose dependently increased tubulin polymerization, and EA (10 μM) increased polymerization by 61.0%. However, we observed an antagonistic response with the combination of CBZ and EA, reducing polymerized tubulin by 13.9% compared to CBZ alone. To confirm these results in cell culture, we treated 22Rv1 cells with EA (10 μM) and CBZ (10 nM) and determined levels of polymerized tubulin by western blotting. We observed a 1.6 fold increase in polymerized tubulin by EA, a 5.5 fold increase by CBZ, which was reduced to a 4.6 fold increase by combination of CBZ with EA. Contrary to our hypothesis, these results suggest that EA may interfere with taxanes, leading to a reduction in drug effectiveness. Additionally, the ability of EA alone to induce polymerization suggests the possibility that the growth inhibitory activity of EA is a consequence of its effects on microtubule dynamics, potentially through an interaction with taxane binding site of tubulin. This is consistent with our observation that EA has the greatest growth inhibitory effect on very proliferative cell lines, since microtubule targeting agents are most effective at killing rapidly dividing cells. To our knowledge, this is the first evidence of microtubule targeting as an anticancer mechanism of EA. We also studied the effects of EA on drug efflux to determine if EA could inhibit development of taxane drug resistance. We treated C4-2 cells with EA (0.01 μM - 30 μM) and measured calcein retention as marker of the activity of the P-glycoprotein (P-gp) pump. There was a dose dependent increase in calcein retention by EA, with a 1.9 fold increase at the highest concentration (30 μM), suggesting an inhibition of P-gp pump activity with increasing amounts of EA. Thus, EA may increase intracellular taxane levels, but at the same time reduce their microtubule-polymerization effects. Citation Format: Jillian N. Eskra, Alaina Dodge, Maarten C. Bosland. Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 317.

Abstract 910: Genistein does not alter the effectiveness of taxane chemotherapeutics on human prostate cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Prostate cancer (PCa) continues to be a major health problem in the United States. Despite the general effectiveness of androgen deprivation therapy, nearly all cases will progress to become resistant to this first-line treatment. The standard of care for castrate-resistant prostate cancer (CRPC) includes a regimen of chemotherapy with taxane-based agents. Patients with CRPC commonly make dietary modifications and/or use nutritional supplements in addition to their standard of care therapy. Soy products are commonly used by men with PCa, but there are only few publications addressing either the safety or efficacy of soy products in combination with chemotherapy. Previously published studies (Prostate 2013;73:1681-9) suggest that the soy isoflavone genistein has synergistic activity when combined the taxane drug cabazitaxel. We studied the effects of the combination of taxane chemotherapy with genistein. PC-3, 22Rv1, and C4-2 prostate cancer cells were treated with genistein (1, 10, and 100 μM) in combination with docetaxel or cabazitaxel (0.1, 1.0, and 10.0 nM); after 72 hours proliferation was measured using the SRB assay and combination index (CI) analysis was performed to identify synergistic or antagonistic activity. Similar dose response curves were observed for docetaxel and cabazitaxel as expected since both drugs have the same mechanism of action. Compared to cells treated only with taxanes (either docetaxel or cabazitaxel), treatment with physiologically relevant concentrations genistein (1 μM and 10 μM) did not yield additional inhibition or enhancement of proliferation in any of the cell lines studied. There was slight synergistic activity at 100 μM in combination with 1 nM docetaxel in C4-2 (CI = 0.03), PC-3 (CI = 0.60), and 22Rv1 (CI = 0.20) cells. Similarly, slight synergism with 1 nM cabazitaxel was observed in all cell lines: C4-2 (CI = 0.29), PC-3 (CI = 0.40), and 22Rv1 (CI = 0.53). We did not observe any antagonism at clinically relevant concentration combinations of genistein and taxanes. These findings suggest that genistein supplementation is not harmful for PCa patients undergoing chemotherapy, but do not fully support findings of others that demonstrate synergistic activity. Although we did observe an enhancement of growth inhibition, it only occurred at the highest concentration of genistein (100 μM) tested, which is beyond what is achievable through dietary consumption of soy products. Quantities of genistein consumed in the diet or by supplementation are likely not sufficient to enhance the cytotoxicity of chemotherapy, but can be used without reducing the effectiveness of taxane drugs. Citation Format: Jillian N. Eskra, Alaina Dodge, Maarten C. Bosland. Genistein does not alter the effectiveness of taxane chemotherapeutics on human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 910. doi:10.1158/1538-7445.AM2015-910

Abstract 2585: Effects of 9-cis-retinoic acid on proliferation, differentiation, and apoptosis of LNCaP prostate cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer is one of the most commonly diagnosed cancer types in US men. Consequently, there is an urgent need to identify preventive agents which can inhibit the prostate carcinogenesis process. 9-cis-retinoic acid (9cRA) is a naturally occurring retinol metabolite and pan retinoic acid receptor agonist which is of interest as a potential chemopreventive compound. Previous studies have demonstrated preventive activity against breast and prostate cancer in animal models and inhibitory effects on growth of cancer cell lines of this retinoid, but the mechanisms by which 9cRA can influence growth of prostate cancer cells remains unclear. In this study we used LNCaP cells to investigate the effects of 9cRA on cell proliferation, differentiation, and apoptosis. Cells were cultured with and without 10−1 or 10−9M dihydrotestosterone and treated with 9cRA at non-cytotoxic concentrations ranging from 10−8M to 10−5M. After 3 and 6 days of incubation, we determined cell growth, 3H-thymidine incorporation, and secretion of prostate specific antigen (PSA), and performed cell cycle analysis and apoptosis assays. Cell growth assessed by cell counts was inhibited on 3 and 6 days in a dose-dependent manner by treatment with 9cRA, and 3H-thymidine incorporation was reduced, reaching a maximum at day 3. PSA secretion was increased in a dose-related fashion after 6 days of treatment, suggesting a differentiation effect. Treatment with 9cRA increased the number of apoptotic cells observed microscopically by staining with H33258 by 3 to 7-fold peaking on day 3. When apoptosis was measured using flow cytometry and staining with PI and FITC-Annexin V, a maximal increase of apoptotic cells was observed on day 6. A cell cycle analysis by flow cytometry revealed up to a 70% increase in cells in S-phase and a decrease in cells in G0/G1 after 3 and 6 days of 9cRA treatment. The results suggest that 9cRA acts on LNCaP cells by interfering with the completion of S-phase or S to G2-phase transition and inducing cellular differentiation and apoptosis. These observations together indicate that the growth inhibitory mechanism of 9cRA on LNCaP prostate human cancer cells is a combination of the ability of 9cRA to inhibit cell proliferation, promote differentiation, and induce apoptosis. Although these findings suggest that 9cRA may be an attractive treatment for option for the prevention of prostate cancer development, this is limited by its human toxicity. A search for 9cRA analogues or other selective retinoid acid receptor modulators with similar anti-prostate cancer activity is thus warranted. Citation Format: Jillian N. Eskra, Maarten C. Bosland, Nur Ozten. Effects of 9- cis -retinoic acid on proliferation, differentiation, and apoptosis of LNCaP prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2585. doi:10.1158/1538-7445.AM2013-2585