Ridhwi Mukerji

Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma.

As part of a program to discover drug leads from plant biodiversity, the present investigation was undertaken to explore the anticancer potential of compounds derived from selected Latin American plants. Bioassay-guided fractionation of a crude extract of the aerial parts of Vassobia breviflora led to the isolation of the withanolide-type steroidal lactone withaferin A (1). This compound was tested for antiproliferative activity against the head and neck squamous cell carcinoma (HNSCC) cell lines, MDA1986, JMAR, UM-SCC-2, and JHU011. The inhibitory concentrations to reduce cell viability to 50% (IC(50)) were determined by the MTS cytotoxicity assay, and 1 reduced cell viability with IC(50) values in the range 0.5-2.2 μM. A mechanistic study showed that 1 induces apoptosis and cell death in HNSCC cells as well as a cell-cycle shift from G(0)/G(1) to G(2)/M. Cells treated with 1 exhibited inactivation of Akt and a reduction in total Akt concentration. This investigation constitutes the first report of the antiproliferative activity of withaferin A (1) against head and neck squamous carcinoma.

Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells.

BackgroundWithaferin A (WA), a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα) and its associated molecules in vitro as novel mechanisms of WA action.MethodsThe effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function.ResultsWA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation.ConclusionsTaken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells in vitro. Down-regulation of ERα protein levels by WA is caused by proteasome-dependent ERα degradation.

A novel C-terminal HSP90 inhibitor KU135 induces apoptosis and cell cycle arrest in melanoma cells

Heat shock protein 90 (Hsp90) is differentially expressed in tumor cells including melanoma and involved in proper folding, stabilization and regulation of cellular proteins. We investigated a novobiocin-derived Hsp90 C-terminal inhibitor, KU135, for anti-proliferative effects in melanoma cells. The results indicate that KU135 reduced cell viability and cell proliferation in melanoma cells and IC(50) values for A735(DRO), M14(NPA), B16F10 and SKMEL28 cells were 0.82, 0.92, 1.33 and 1.30μM respectively. KU135 induced a more potent anti-proliferative effect in most melanoma cells versus N-terminal Hsp90 inhibitor 17AAG. KU135 induced apoptosis in melanoma cells, as indicated by annexin V/PI staining, reduction in the mitochondrial membrane potential, mitochondrial cytochrome C release and caspase 3 activation. KU135 reduced levels of Hsp90 client proteins Akt, BRAF, RAF-1, cyclin B and cdc25. Additionally, levels of Hsp90 and Hsp70 did not increase, while the levels of phosphorylated HSF1 levels decreased. KU135 induced strong G2/M cell cycle arrest, associated with decreased expression of cdc25c, cyclin B and increased phosphorylation of cdc25c. These finding show that KU135 reduced cell survival, proliferation, and induces apoptosis in melanoma cells. We suggest that KU135 may be a potential candidate for cancer therapy against melanoma.

A novel RET inhibitor with potent efficacy against medullary thyroid cancer in vivo

BACKGROUND Most medullary thyroid carcinomas (MTC) recur or progress despite curative resection. Current targeted therapies show promise but lack durable efficacy and tolerability. The purpose of this study was to build on previous in vitro work and evaluate withaferin A (WA), a novel RET inhibitor, in a metastatic murine model of MTC. METHODS A total of 5 million DRO-81-1 human MTC cells injected in the left posterior neck of nu/nu mice generated metastases uniformly to the liver, spleen, and/or lungs. Treatment with WA (8 mg/kg/day, intraperitoneally, for 21 days) was started for neoplasms > 100 mm(3). Endpoints were survival, neoplasm > 15,00 mm(3), decreased body weight, or body score (all measured three times/wk). RESULTS All controls (saline; n = 5) died or deteriorated from metastatic disease by 7 weeks postinjection. All treated animals were alive (WA; n = 5), having tumor regression and growth delay without toxicity or weight loss at 6 weeks posttreatment (P < .01). Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose-dependent manner (almost complete inhibition with treatment of 5 μM WA) as well as potent inhibition of phospho-ERK and phospho-Akt levels. CONCLUSION WA is a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. Further long-term efficacy/toxicity studies are warranted to evaluate this compound for clinical translation.

A novel combination of withaferin A and sorafenib shows synergistic efficacy against both papillary and anaplastic thyroid cancers.

BACKGROUND Sorafenib (SO), a multikinase-targeted inhibitor in clinical trials for papillary and anaplastic cancers, shows limited efficacy with moderate toxicity. Withaferin A (WA), a natural withanolide, shows potent preclinical anticancer activity in thyroid cancers through multiple cytotoxic mechanisms including heat-shock protein inhibition. We hypothesized that combination therapy (WA + SO) would have a synergistic effect against anaplastic and papillary carcinoma cells at lower sorafenib doses. METHODS Human papillary (BCPAP) and anaplastic (SW1736) thyroid cancer cell lines were evaluated after treatment with SO, WA, or their combination at different doses. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and trypan blue exclusion; apoptosis and cell-cycle arrest was measured by flow cytometry. Western analysis confirmed apoptosis (Poly ADP ribose polymerase [PARP] and caspase-3 cleavage) and Raf inhibition. Experiments were repeated in triplicate and were evaluated statistically with significance set at a P value of less than .05. RESULTS The concentration of drug at which 50% of the cells are inhibited (IC(50)) in BCPAP were 6.3 μmol/L (SO), .155 μmol/L (WA), and .055 μmol/L (IC(50)WA + 50% IC(50)SO), whereas in SW1736 cells the concentration was 7.6 μmol/L (SO), 2.5 μmol/L (WA), and 1.4 μmol/L (IC(50)WA + 50% IC(50)SO). Combination (WA + SO) at IC(50) decreased cell viability to 19% (from 50% individually). Apoptosis levels on flow cytometry in anaplastic cells increased significantly from 0% to 2% (SO or WA alone) to 89% (combo at IC(50), P < .001). Combination therapy apoptosis (PARP cleavage and caspase-3 inactivation) and BRAF/Raf-1 down-regulation were dose-dependent starting at 50% IC(50) levels. Cell-cycle modulation was significant with combination treatment (35% increase in G2 arrest at 50% IC(50)SO + WA and 70% increase at 75% IC(50)SO + WA; P < .01). CONCLUSIONS Combination therapy with sorafenib + withaferin showed synergistic efficacy in papillary and anaplastic cancers in vitro with significant induction of apoptosis. This combination achieved potent anticancer activity with lower overall doses of sorafenib, indicating a potential strategy to decrease sorafenib toxicity in future translational studies.

Subcutaneous delivery of nanoconjugated doxorubicin and cisplatin for locally advanced breast cancer demonstrates improved efficacy and decreased toxicity at lower doses than standard systemic combination therapy in vivo

BACKGROUND Combination cytotoxic agents in breast cancer carry dose-limiting toxicities. The aim of this study was to test the hypothesis that nanocarrier-conjugated doxorubicin and cisplatin would have improved tumor efficacy with decreased systemic toxicity over standard drugs, even at lower doses. METHODS Female Nu/Nu mice were injected in the breast with human MDA-MB-468LN cells and treated with either standard or nanocarrier-conjugated combination therapy (doxorubicin plus cisplatin) at 50% or 75% maximum tolerated dose (MTD) and monitored for efficacy and toxicity over 12 weeks. RESULTS Efficacy results for mice treated with hyaluronan-conjugated doxorubicin and cisplatin at 50% MTD were as follows: 5 complete responses, 2 partial responses, and 1 case of stable disease. For hyaluronan-conjugated doxorubicin and cisplatin at 75% MTD, efficacy results were as follows: 7 complete responses, 1 partial response. All complete responses were confirmed histologically. In comparison, mice given standard doxorubicin and cisplatin at 50% MTD demonstrated progressive disease in 6, stable disease in 1, and partial response in 1. For standard doxorubicin and cisplatin at 75% MTD, there were 5 cases of progressive disease and 3 of stable disease (P < .0001 on multivariate analysis of variance). At 75% MTD, standard drug-treated mice had significant weight loss compared to nanocarrier drug-treated mice (P < .001). CONCLUSIONS Subcutaneous nanocarrier delivery of doxorubicin and cisplatin demonstrated significantly improved efficacy with decreased toxicity compared with standard agent combination therapy at all doses tested, achieving complete pathologic tumor response.

Efficacy and Toxicity of Peritumoral Delivery of Nanoconjugated Cisplatin in an In Vivo Murine Model of Head and Neck Squamous Cell Carcinoma

IMPORTANCE Treatment of locally advanced head and neck squamous cell carcinoma (HNSCC) uses a multidisciplinary approach often limited by the toxicity and drug resistance of platinum agents. OBJECTIVES To test whether a nanocarrier-conjugated cisplatin boosting locoregional drug delivery improves tumor efficacy while decreasing systemic toxicity over systemic cisplatin in a murine model of locally advanced HNSCC. DESIGN A randomized, controlled, in vivo study compared standard cisplatin with nanocarrier (hyaluronan [HA])-conjugated cisplatin (HA-cisplatin) each at 50% of the maximum tolerated doses in a murine model of locally advanced HNSCC (10 mice/arm, each injected with 1 × 106 MDA-1986 HNSCC cells, with phosphate-buffered saline and HA-only control arms). Mice were treated for 3 weeks and observed for 3 additional weeks. SETTING Academic medical center. PARTICIPANTS Forty female Nu/Nu mice. Randomization and treatment arms were initiated once tumor volumes reached 30 mm3. INTERVENTION Injection with MDA-1986 HNSCC cells followed by 3 weeks of treatment with cisplatin, HA-cisplatin, phosphate-buffered saline, or HA only. MAIN OUTCOMES AND MEASURES Animal weights and tumor volumes were measured 3 times each week (modified RECIST [Response Evaluation Criteria in Solid Tumors]). At necropsy, animal kidneys were examined for nephrotoxic effects and cochleae were examined for ototoxic effects. RESULTS The mice treated with HA-cisplatin showed superior tumor efficacy (1 with complete clinical response, 3 with partial response, 1 with stable disease, and 5 with progressive disease) compared with standard cisplatin (no animals with complete clinical response, 1 with partial response, 1 with stable disease, and 8 with progressive disease), which was statistically significant (P = .003). All control animals developed progressive disease. Weight loss and body score were surrogate measures of treatment toxicity. The HA-cisplatin group had the least weight loss (mean [SD], 10.8% [4.7%]) compared with the cisplatin group (13.6% [5.6%]; P = .25). Body score dropped to 2 or less in all cisplatin-treated mice but not in any HA-cisplatin-treated mice, which also lacked any histologic signs of nephrotoxic or ototoxic effects. CONCLUSIONS AND RELEVANCE Nanoconjugated HA-cisplatin significantly improves tumor efficacy with lower toxicity compared with standard cisplatin in locally advanced HNSCC in vivo, justifying additional translational studies.

Abstract B84: Withaferin‐A is a novel HSF‐1 inhibitor with potent antitumor effects in melanoma in vivo

Introduction: Withaferin A (WA) is a natural product withanolide compound with historical anti‐inflammatory activity and recent anticancer activity in vitro. The purpose of this study was to evaluate the mechanism of action of withaferin A in melanoma cancer cells and test its efficacy in a murine orthotopic model of metastatic melanoma. Methods: Human melanoma cells lines SKMEL28, NPA, DRO and the murine B16F10 cell line were used for in vitro analysis. MTS cell viability assay and cell proliferation analysis was performed to determine the concentration‐dependent effect of withaferin A on melanoma cells. Effects on cell cycle arrest and apoptosis were evaluated by PI/Annexin V staining with flow cytometry and confirmed through Western analysis. MAPkinase and heat shock protein expression after WA treatment was evaluated by Western analysis. Inhibitors of MEK1/2, JNK and p38 MAP kinase were used to examine the relationship between WA‐mediated MAPkinase protein expression and modulation of heat shock proteins. In vivo analysis was performed on BalbC mice injected s.c. with 5 million B16F10 melanoma cells, allowing tumors to grow and metastasize. Treatment was with 5mg/kg/d WA i.p. for 3 weeks. Tumor growth and survival was followed prospectively. In vitro and in vivo variables were statistically evaluated using SPSS 17.0 software and significance was defined for p Results: Withaferin A induces reduction of cell viability and cell proliferation in all melanoma cell lines tested [Range of IC50(B16F10 =209.7±16nM); (SKMEL‐28=650.1 ±25nM)]. Mechanistic studies demonstrate that WA induced a cell cycle shift from G0/G1 arrest to G2/M arrest in all melanoma cells lines tested. Annexin V /PI staining on FC as well as caspase activation and PARP cleavage on Western studies confirm that WA induces apoptosis in melanoma cells. WA reduced p‐Akt levels and total Akt levels in all cell lines tested in a concentration and time‐dependent manner. Withaferin A modulates MAP kinase proteins, inducing activation of p‐ERK1/2 , p38 MAPkinase and SAPK/JNK in all melanoma cell lines. HSP expression analysis yielded strong induction of HSP72 expression in all cell lines, reduction of HSP90 in SKMEL28 cells, and strong reduction of HSF1 cellular levels in a dose dependant manner with almost complete reduction at IC50 concentrations. Confocal microscopy analysis showed that withaferin A‐induced expression of HSP72 was increased in the cytoplasm and that HSP72 was transported to the nucleus upon withaferin A stimulation. In vivo studies demonstrate almost complete tumor regression with CR in 80% of animals treated with 5 mg/kg/day WA compared to controls (p Conclusion: Our present results show that withaferin A is a potent therapy against melanoma cells in vitro and in vivo through induction of cell cycle arrest, apoptosis, and modulation of MAPkinase and HSP expression. This is the first report of inhibition of HSF1 expression in cancer cells induced by withaferin A treatment. These results support future proof of concept studies for translation to potential therapies for melanoma patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B84.

Abstract A214: A novobiocin analogue HSP90 inhibitor induces apoptosis and has potent antitumor effects in head and neck squamous cell cancer in vivo

Introduction: Small molecule n‐terminal inhibitors of heat shock protein 90 (HSP90i) have been evaluated as anticancer agents in various stages of human clinical trials. This study evaluates a novel C‐terminal HSP90 inhibitor derived as an analogue of novobiocin for its anti‐tumor activity against head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Methods: Head and neck squamous cell carcinoma cells MDA1986 and JMAR were used to examine the efficacy of KU174 as an anti‐cancer agent. Cell viability assay MTS and cell proliferation analysis were used to examine the concentration‐dependent effect of KU174 on HNSCC cells. Cells were stained with propidium iodide and evaluated on flow cytometry (FC) to study the effects on cell cycle progression. Analysis of KU174 ‐ induced apoptosis of HNSCC was evaluated first by annexin V/PI staining on FC after treatment with drug and confirmed by Western blot analysis for caspase activation and PARP cleavage. KU174 ‐ induced modulation of kinases Akt and ERK1/2 was studied using Western blot analysis for levels of protein expression following treatment. Finally in vivo efficacy of KU174 was evaluated in an orthotopic mouse model of HNSCC following 3 weeks of daily i.p. injection with 5 mg/kg/d of drug. Results: KU174 inhibited cell viability in both MDA1986 and JMAR cells (IC50 = 7.5 and 4.3 M respectively; novobiocin IC50>700 M for each; p Conclusion: These results show that the c‐terminal HSP90 inhibitor KU174 is significantly more potent in antiproliferative activity in HNSCC cells than its parent compound novobiocin. Mechanistic studies suggest that the HNSCC cells treated with KU174 are likely dying by a combination of apoptosis, cell cycle arrest and modulation of proteins such as prosurvival kinases chaperoned by HSP90. Additionally this compound has potent antitumor efficacy in an orthotopic mouse model of HNSCC in vivo lending support for future preclinical proof‐of‐concept studies to translate its application as a potential human therapy in HNSCC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A214.