Withaferin A suppresses tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate‐induced decreases in isocitrate dehydrogenase 1 activity and mitochondrial function in skin epidermal JB6 cells

Abstract

Withaferin A (WA) is a bioactive compound derived from Withania somnifera. The antitumor activity of WA has been well studied in human cancer models; however, its chemopreventive potential is unclear. In the present study, we used the skin epidermal JB6 P+ cells, a well-established model for tumor promotion, and demonstrated that WA suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation and cell proliferation. Interestingly, TPA inactivated isocitrate dehydrogenase 1 (IDH1), which was reversed by WA. Similar results were also observed in mouse skin tissue. Therefore, we focused on metabolism as the potential mechanism of action. We found that mitochondrial functions were downregulated by TPA treatment, as indicated by reduced mitochondrial membrane potential, complex I activity and mitochondrial respiration. However, all of these downregulations were inhibited by WA. In addition, we examined the levels of a-ketoglutarate, a product of IDH1, and WA blocked its reduction upon TPA treatment. Finally, we detected the lactate level as a glycolysis marker, and WA suppressed its elevation caused by tumor promoter treatment. Altogether, these results suggest that WA might exert its chemopreventive activity via inhibiting not only oncogenic activation, but also IDH1 inactivation and mitochondrial dysfunction in early tumorigenesis. (Cancer Sci 2013; 104: 143-148) W ithaferin A (WA; Supporting Information Fig. S1) belongs to a group of biologically active constituents known as withanolides. The antitumor activity of WA has been reported, which shows that WA suppresses human breast cancer, (1,2) prostate cancer, (3) colon cancer, (4) pancreatic cancer,glioma, (6) renal cancer (7) and leukemia. (8) Moreover, a clinical trial of WA has been launched for treatment of metastatic melanoma.(9) Not surprising, Withania somnifera is within the high-priority topics from the National Center for Complementary and Alternative Medicine. However, the chemopreventive potential of WA has not been well studied. Therefore, testing the mechanism of action of WA in chemoprevention using well-established skin cell transformation and skin carcinogenesis models is important.Carcinogenesis is often associated with a metabolic shift. Cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of oxygen, known as the "Warburg effect".(10) Although glycolysis is inefficient at producing ATP compared with oxidative phosphorylation (OXPHOS), the metabolic intermediates produced during glycolysis might provide a growth advantage for cancer cells. Associated with this metabolic switch is dysregulation of important metabolic enzymes. Isocitrate dehydrogenase (IDH), as a metabolic enzyme converting isocitrate to a-ketoglutarate, consists of three isoforms. Mutations in IDH1 and IDH2 have been found in glioma (11) and leukemia, (12) and IDH2 mutation is much less common than IDH1 mutation. Mutations in IDH1 impair its enzymatic activity, l...

Withaferin A suppresses the up‐regulation of acetyl‐coA carboxylase 1 and skin tumor formation in a skin carcinogenesis mouse model

“…As shown in Figure B, a time‐course study revealed that ACC1 expression was up‐regulated 30 min after TPA treatment and reached a peak at the 6 and 24 h time‐point. Next, JB6 cells were treated with WA (0.625 μM, has been used in our previous study and skin cell transformation can be inhibited at this concentration . A lower concentration of WA [0.125 μM] has also been tested and cell transformation was not inhibited (data not shown) 30 min before TPA treatment (24 h).…”

“…Tumor promoter TPA induced colony formation in soft agar, whereas the number of colonies was increased by 75% in ACC1 stably transfected cells ( Figure 2B). TPA treatment is known to induce the activation of activator protein one (AP-1) during skin carcinogenesis and Fra-1, as a subunit of AP-1, contributes to AP-1 DNA binding activity in these models [12,24]. The results ( Figure 2C) show that ACC1 overexpression increased the protein expression levels of Fra-1, and TPA treatment further enhanced this increase.…”

“…However, the chemopreventive potential of WA has not been well studied, and the mechanisms of cancer prevention could be different from WA's anti‐tumor activities. Our preliminary studies have shown that WA could inhibit skin cell transformation via blocking tumor promoter‐induced metabolic alterations [, providing a rationale and a potential novel mechanism for WA being a chemopreventive agent. Herein, we aim to study the mechanism of action of WA in chemoprevention using the well‐developed chemically induced skin carcinogenesis models.…”

Mitochondrial dysfunction in cancer chemoprevention by phytochemicals from dietary and medicinal plants

“…It is also important to establish if inhibition of OXPHOS and complex III by WA treatment is not specific for breast cancer cells. In this regard, a recent study using skin epidermal JB6 P+ cells showed reversal of 12- O -tetradecanoylphorbol 13-acetate (TPA)-induced changes by WA (Li and Zhao 2013). TPA treatment inhibited mitochondrial membrane potential, complex I activity and mitochondrial respiration, which were reversed by WA (Li and Zhao 2013).…”

Anticancer activities ofWithania somnifera: Current research, formulations, and future perspectives

“…In prostate cancer, dysfunctioning and irregularity in cell cycle generally occur, which can be regulated by WA (Roy et al, 2013), in which, WA acts as a regulator of cell cycle functioning at the G2/M phase, to inhibit prostate and some other kind of cancers. Li and Zhao (2013) studied the potential of WA for chemo-preventive activity and reported the activity of oncogenic cells; mitochondrial dysfunctioning and inactivation of IDH1 are inhibited during early tumorogenesis. Hahm and Singh (2013) studied the mechanism of apoptosis induction by WA in breast cancer cell line and divulged that mitogenactivated protein kinases act as target specific with WA.…”