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- Materials and Methods
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- Supporting Information
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 α-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.
Withaferin 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, colon cancer, pancreatic cancer, glioma, renal cancer and leukemia. Moreover, a clinical trial of WA has been launched for treatment of metastatic melanoma. 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”. 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 α-ketoglutarate, consists of three isoforms. Mutations in IDH1 and IDH2 have been found in glioma and leukemia, and IDH2 mutation is much less common than IDH1 mutation. Mutations in IDH1 impair its enzymatic activity, leading to decreased production of α-ketoglutarate; the latter serves as an inhibitor of hypoxia-inducing factor (HIF-1) via α-ketoglutarate-dependent dioxygenases. Recent studies also demonstrate a gain-of-function role of the IDH1 mutation. Instead of producing α-ketoglutarate, mutant IDH generates 2-hydroxyglutarate, one of the so-called “onco-metabolites”.
Although it is still controversial whether the shift from OXPHOS to aerobic glycolysis in tumor cells is because of malfunctions in mitochondria, we hypothesized that maintaining mitochondrial function and inhibiting aerobic glycolysis can be effective targets for chemoprevention.
Based on our previous study, IDH1, but not IDH2, was inactivated by tumor promoter TPA treatment in skin epidermal JB6 cells and skin tissues, and IDH1 inactivation promoted skin cell transformation. Whether IDH1 and mitochondrial function might be a potent target of WA, which leads to suppression of skin cell transformation, will be investigated using JB6 cells and skin epidermal tissues. These models are well established for screening cancer prevention agents and for mechanistic studies, and therefore suitable for our purpose.
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- Materials and Methods
- Disclosure Statement
- Supporting Information
Withaferin A, a major withanolide found in the traditional Indian medicine Ashwagandha, has been widely studied for treating human cancers and currently is in clinical trial for melanoma therapy. In summary, WA suppresses tumor growth by inhibiting cell proliferation, the cell cycle, inflammation and angiogenesis, or by promoting apoptosis and oxidative stress. In particular, WA inhibits Notch-1 signaling and downregulates prosurvival pathways in colon cancer cell lines (HCT-116, SW-480 and SW-620). Withaferin A downregulates the expression of mammalian target of rapamycin signaling components, pS6K and p4EBP1, and activates JNK-mediated apoptosis in colon cancer cells. Withaferin A also causes G2 and M phase cell cycle arrest in human breast cancer cells, and induces apoptosis in human leukemia U937 cells and prostate cancer cells. The antiangiogenic effect of WA relies on targeting the intermediate filament protein vimentin. However, the chemopreventive activity of withaferin A is likely to be mediated by different mechanisms.
Neoplastic transformation of a normal cell requires many gene changes, mainly in two categories: activation of oncogenes and/or inactivation of tumor suppressor genes. Therefore, as neoplastic transformation occurs during the early stage of carcinogenesis, transformation inhibition could be an effective approach for chemoprevention. In our present study, we confirmed that WA significantly suppressed tumor promoters TPA and UVC-induced neoplastic transformation in JB6 P+ cells. In contrast, the balance between proliferation and cell death is pivotal for carcinogenesis, the uncontrolled and rapid proliferation of cells could be a characteristic of benign tumors. As a tumor promoter, TPA induces neoplastic transformation; in addition, accumulating evidence has shown that TPA could stimulate cell proliferation.[28, 29] We detected the expression levels of proliferating cell nuclear antigen, which is a widely used proliferation marker, and found that WA suppressed TPA-induced cell proliferation (Fig. S3a). Next we tested the possibility of WA inducing apoptosis as the mechanism action. As shown in Figure S3b,c, WA did not induce apoptosis in skin epidermal tissues. Based on the present study, the mechanism of the chemopreventive activity of WA might be mediated by inhibiting neoplastic transformation and cell proliferation but not by inducing apoptosis.
Isocitrate dehydrogenases convert isocitrate to α-KG to generate NADPH, which supplies reducing energy in enzymatic reactions as a cofactor. They consist of three isoforms, IDH1, IDH2 and IDH3; the former two act in the cytoplasm and mitochondria, respectively, and the latter acts in the TCA cycle. Our previous study showed that tumor promoter decreased the expression and activity levels of IDH1, but not IDH2, in the tumor promotable JB6 P+ cell model. In the present study, our data show that WA suppresses the decreases in IDH1 protein expression and activity during the early stage of skin carcinogenesis. Importantly, we found that WA directly increased IDH1 activity, suggesting IDH1 might be a potential target of WA. However, how WA and IDH1 might interact is not known, which will be examined in our future studies.
Can WA affect IDH1 at the transcription level? Next we treated JB6 P+ cells with WA and/or TPA and found that the mRNA levels of IDH1 remained the same (Fig. S4), and similar results were found for IDH2. These data suggest that WA may act on IDH1 at the post-translational level instead of the transcriptional level, or WA might directly interact with IDH1 and increase its activity (as suggested by Fig. 3). We will work on this interesting hypothesis in our future studies.
In the JB6 cell model, TPA treatment has been shown to activate the ERK pathway, which contributes to neoplastic transformation. In addition, the activated AKT signaling pathway by TPA also contributes to skin tumor promotion. We detected the phosphorylation status of ERK, JNK (both are the members of the MAPK family) and AKT. As shown in Figure S5, WA did not affect TPA-induced phosphorylation of ERK, JNK or AKT. These data suggest that the MAPK and AKT signaling pathways might not contribute to the mechanism of action of WA during the early stage of skin carcinogenesis. As a pivotal sensor of energy status, AMP-activated protein kinase (AMPK) plays an important role in cancer cell metabolism. In our future studies, the link between IDH1 and the AMPK pathway will be addressed.
Cell growth requires energy. How to obtain the energy makes a difference between cancer cells and normal cells. Even with ample oxygen, cancer cells prefer glycolysis over oxidative phosphorylation to produce ATP. On one hand, accumulating evidence has shown that there is mitochondrial malfunction in cancer cells, so do mutations in mtDNA and metabolic enzymes. In particular, Warburg first hypothesized that there is mitochondrial dysfunction in cancer cell development. mtDNA mutations have been found in various kinds of cancer, such as breast and colorectal cancer. Mutations of metabolic enzymes have also been identified, including succinate dehydrogenase, fumarate hydratase and IDH.[11, 37] On the other hand, some studies have shown that there is natural mitochondrial function in several cancer cells.[38, 39] Although the role of mitochondrial metabolism in cancer development remains a disputed question, our in vitro studies show that tumor promoters induced mitochondrial dysfunction and WA could reverse the effects in the early stage of skin carcinogenesis.
Wild-type IDH1 converts isocitrate to α-KG, which activates dioxygenases. Mutant IDH1 gains a new function, which produces 2-hydroxyglutatarate. Based on our previous studies, both UV irradiation (in vitro) and tumor promoter TPA (in vitro and in vivo) cause downregulation of IDH1 expression and activity in skin epidermal cells. In addition, knockdown of IDH1 enhances whereas overexpression of IDH1 suppresses skin cell transformation, suggesting a tumor suppressive role for IDH1 in the early stage of skin carcinogenesis. In addition, the expression and activity levels of IDH2 are not altered by tumor promoter treatment. In the present study, we found that WA promotes the conversion of isocitrate to α-KG. However, as an important metabolic enzyme, glutamate dehydrogenase (GDH) converts glutamate to α-KG as well. We detected levels of GDH activity using JB6 P+ cells. As shown in Figure S6, GDH activity was not significantly altered by either the TPA or WA treatment. Furthermore, our result (Fig. S7) demonstrated that WA did not alter the protein expression of IDH2. These results suggest that WA exerts its chemopreventive activity by maintaining IDH1 activation during early tumorigenesis.
In summary, our studies indicate that IDH1 and mitochondrial dysfunction will be induced during early skin tumorigenesis. Withaferin A can preserve IDH1 activity and mitochondrial function, which might provide a novel mechanism for WA as a chemopreventive agent.