Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Basal Autophagy and Feedback Activation of Akt Are Associated with Resistance to Metformin-Induced Inhibition of Hepatic Tumor Cell Growth

While accumulating evidence has shown that the use of the diabetic drug metformin may be beneficial against various tumors in some epidemiological studies, a few studies failed to show the same beneficial effects. The molecular and cellular mechanisms for these conflicting observations are not clear. In this study, we compared the inhibitory effects of cell growth by metformin on several hepatic tumor cell lines: SMMC-7721, HCC-97L, HCC-LM3 and HepG2. While metformin inhibited cell growth in all these cells, we found that SMMC-7721, HCC-97L and HCC-LM3 cells were more resistant than HepG2 cells. Mechanistically, we found that metformin inhibited mTOR in all these hepatic tumor cells. However, SMMC-7721 cells had higher levels of basal autophagy and mTORC2-mediated feedback activation of Akt than HepG2 cells, which may render SMMC-7721 cells to be more resistant to metformin-induced inhibition of cell growth. Similarly, HCC-97L and HCC-LM3 cells also had higher feedback activation of AKT than HepG2 cells, which may also account for their resistance to metformin-induced inhibition of cell growth. Therefore, the various basal autophagy and mTOR activity in different cancer cells may contribute to the controversial findings on the use of metformin in inhibition of cancers in humans.     

Inhibiting ROS-TFE3-dependent autophagy enhances the therapeutic response to metformin in breast cancer

Autophagy modulation is a potential therapeutic strategy for breast cancer, and a previous study indicated that metformin exhibits significant anti-carcinogenic activity. However, the ability of metformin to induce autophagy and its role in breast cancer cell death remains unclear. In this study, we exposed MCF-7 cells to different concentrations of metformin (2.5, 5, and 10?mM) for 48?h, and metformin-induced significant apoptosis in the MCF-7 cells. The expression levels of CL-PARP (poly(ADP-ribose) polymerase 1) and the ratio of BAX to BCL-2 were significantly increased. In addition to apoptosis, we showed that metformin increased autophagic flux in MCF-7 cells, as evidenced by the upregulation of LC3-II and downregulation of P62/SQSTM1. Moreover, pharmacological or genetic blocking of autophagy increased metformin-induced apoptosis, indicating a cytoprotective role of autophagy in metformin-treated MCF-7 cells. Mechanistically, metformin-induced TFE3^(Ser321) dephosphorylation activated TFE3 nuclear translocation and increased of TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes and, subsequently, initiated autophagy in MCF-7 cells. Importantly, we found that metformin triggered the generation of reactive oxygen species (ROS) in MCF-7 cells. Furthermore, N-acetyl-l-cysteine (NAC), a ROS scavenger, abrogated the effects of metformin on TFE3-dependent autophagy. Notably, TFE3 expression positively correlated with breast cancer development and poor prognosis in patients. Taken together, these data demonstrate that blocking ROS-TFE3-dependent autophagy to enhance the activity of metformin warrants further attention as a treatment strategy for breast cancer.     

Metformin targets Stat3 to inhibit cell growth and induce apoptosis in triple-negative breast cancers

A distinct group of breast cancers, called “basal” or “triple-negative” (TN) cancers express both basal cytokeratins and the epidermal growth factor receptor, but fail to express estrogen receptors, progesterone receptors or HER2 and have stem-like or mesenchymal features. They are particularly aggressive, are frequently chemo-resistant, with p53 mutation, up-regulation of IL-6 and Stat3. Because TN cells are particularly sensitive to the anti-diabetic agent metformin, we hypothesized that it may target JAK2/Stat3 signaling. The effects of metformin upon Stat3 expression and activation were examined in four human TN cell lines. Metformin’s effects were also studied in sublines with forced over-expression of constitutively active (CA) Stat3, as well as lines with stable knockdown of Stat3. Metformin inhibited Stat3 activation (P-Stat3) at Tyr705 and Ser727 and downstream signaling in each of the four parental cell lines. CA-Stat3 transfection attenuated, whereas Stat3 knockdown enhanced, the effects of metformin upon growth inhibition and apoptosis induction. A Stat3 specific inhibitor acted synergistically with metformin in reducing cell growth and inducing apoptosis. An mTOR inhibitor showed no significant interaction with metformin. In summary, Stat3 is a critical regulator of metformin action in TN cancer cells, providing the potential for enhancing metformin’s efficacy in the clinical setting.     

Discovery of indoline derivatives that inhibit esophageal squamous cell carcinoma growth by Noxa mediated apoptosis

A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent inhibition activity against esophageal cancer cells (Kyse30, Kyse450, Kyse510 and EC109). Cellular mechanism studies in esophageal squamous cell carcinoma (ESCC) cells elucidated compound 20 inhibited cell growths in vitro and in vivo, reduced colony formation, arrested cell cycle at M phase, and induced Noxa-dependent apoptosis in ESCC. Importantly, compound 20 was identified as a novel Noxa mediated apoptosis inducer. These results suggested that compound 20 might be a promising anticancer agent with potential for development of further clinical applications.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo. Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo, metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2–Beclin1 interaction at endoplasmic reticulum

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (−)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2–Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (−)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (−)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (−)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.     

Oncogenic Features of PHF8 Histone Demethylase in Esophageal Squamous Cell Carcinoma

Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. It has been reported that histone demethylases are involved in the carcinogenesis of certain types of tumors. Here, we studied the role of one of the histone lysine demethylases, plant homeodomain finger protein 8 (PHF8), in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Using short hairpin RNA via lentiviral infection, we established stable ESCC cell lines with constitutive downregulation of PHF8 expression. Knockdown of PHF8 in ESCC cells resulted in inhibition of cell proliferation and an increase of apoptosis. Moreover, there were reductions of both anchorage-dependent and -independent colony formation. In vitro migration and invasion assays showed that knockdown of PHF8 led to a reduction in the number of migratory and invasive cells. Furthermore, downregulation of PHF8 attenuated the tumorigenicity of ESCC cells in vivo. Taken together, our study revealed the oncogenic features of PHF8 in ESCC, suggesting that PHF8 may be a potential diagnostic marker and therapeutic target for ESCC.     

Overexpression of S-adenosylhomocysteine hydrolase (SAHH) in esophageal squamous cell carcinoma (ESCC) cell lines: effects on apoptosis,migration and adhesion of cells

S-adenosylhomocysteine hydrolase (SAHH) is the sole enzyme that catalyses the hydrolysis of S-adenosylhomocysteine (SAH) in methylation reaction. Previous studies have shown that its inhibition or deficiency leads to several human disorders such as severe coagulopathy, hepatopathy and myopathy. However, the effects of SAHH on esophageal squamous cell carcinoma (ESCC) cells have not been explored so far. To determine whether SAHH is involved in carcinogenesis of the esophagus, we investigated the expression of SAHH in ESCC and normal esophageal epithelial cells and found that SAHH was downregulated in ESCC cells compared with normal esophageal epithelial cells (P < 0.05). The overexpressed SAHH in ESCC cells promoted cell apoptosis, inhibited cell migration and adhesion, but did not affect the cell proliferation and cell cycle. Furthermore, an interaction of SAHH with receptor of activated C kinase 1 (RACK1) protein was detected by coimmunoprecipitation and an increased RACK1, which is caused by overexpression of SAHH, was verified by Western blotting. The findings mentioned above demonstrate that SAHH can promote apoptosis, inhibit migration and adhesion of ESCC cells suggesting that it may be involved in carcinogenesis of the esophagus.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Apoptosis inhibition by Bcl-2 gives way to autophagy in glucocorticoid-treated lymphocytes

Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.     

Apoptosis inhibition by Bcl-2 gives way to autophagy in glucocorticoid-treated lymphocytes

Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.     

Chloride intracellular channel 1 promotes esophageal squamous cell carcinoma proliferation via mTOR signalling

ObjectivesTo investigate the clinical significance of Chloride Intracellular Channel 1 (CLIC1) expression in esophageal squamous cell carcinoma (ESCC) and its functional contribution and molecular mechanisms to the progression of ESCC.MethodsCLIC1 expression was analyzed by immunohistochemistry (IHC) in a cohort of 86 ESCC tissue specimens and paired normal adjacent esophageal tissues. Associations between clinicopathological features of ESCC and CLIC1 expression were determined. In vitro analyses examined CLIC1 expression in the ESCC cell lines KYSE150 and TE1 using RT-PCR and Western blotting. The downstream pathways of CLIC1 were detected by lentiviral shRNA knockdown and subsequent proteomic analyses. CLIC1 siRNA knockdown was performed in ESCC cell lines KYSE150 and TE1 and the functional effects of CLIC1 on the growth and proliferation of ESCC cells were evaluated combined with cell viability and colony formation assays; the mTOR signaling pathway-related proteins were detected by Western blotting based on the previous proteomic data.ResultsCLIC1 expression was significantly increased in ex vivo ESCC tissues compared with corresponding normal tissues, and the up-regulation was associated with clinical tumor node metastasis (TNM) classifications. Knockdown of CLIC1 inhibited in vitro cell proliferation of ESCC cell lines KYSE150 and TE1. CLIC1 knockdown down-regulated the protein expression of p-mTOR and the downstream targets Rictor and p-4EBP1 in both KYSE150 and TE1 cell lines. And the CLIC1 knockdown induced inhibition of cell proliferation on ESCC cells could be rescued by mTOR overexpression.ConclusionsCLIC1 expression increases during esophageal carcinogenesis and it may functionally contribute to the progression of ESCC through growth promotion effects by promoting the mTOR and downstream signaling pathway. CLIC1 therefore constitutes a candidate molecular biomarker of ESCC.     

4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Promotes Esophageal Squamous Cell Carcinoma Growth via Beta-Adrenoceptors In Vitro and In Vivo

Cigarette smoke is a risk factor for esophageal squamous cell carcinoma (ESCC). It contains several carcinogens known to initiate and promote tumorigenesis as well as metastasis. The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the strongest carcinogens in tobacco and our previous studies have shown its proliferation-promoting role in the progression of ESCC. Recently, NNK was identified as an agonist for both beta1- and beta2-adrenoceptors. Thus, we hypothesized that the cancer-promoting effect of NNK was likely mediated through beta-adrenoceptors in ESCC. Therefore, we investigated the comprehensive role of NNK in ESCC in vitro and in vivo, and found that NNK promoted many oncogenic features including ESCC cell proliferation and xenograft tumor growth as well as ESCC cell migration and invasion. Western blotting showed that NNK induced significant up-regulation of phosphorylated ERK1/2, cyclin D1, Bcl-2, and vascular endothelial growth factor as well as down-regulation of Bax. Importantly, the oncogenic effects of NNK in ESCC and the altered protein expression were reversed to some extent by down-regulation of beta1- and beta2-adrenoceptors with the beta2-adrenoceptor showing a greater rescue effect. Taken together, our in vitro and in vivo results demonstrate that NNK plays an oncogenic role in ESCC through beta-adrenoceptors. Furthermore, beta2-adrenoceptor might play a more important role in this process. Our findings might provide a chemoprevention and therapy strategy for cigarette smoke-related ESCC carcinogenesis.