Metformin-induced Apoptosis Research Articles

Estrogen Receptor Is Required for Metformin-Induced Apoptosis in Breast Cancer Cells Under Hyperglycemic Conditions.

About 25% to 30% of estrogen receptor (ER)-positive breast cancer patients develop resistance to endocrine therapy. Human epidermal growth factor receptor 2 (HER2) has been shown to cooperate with several growth factors that regulate cellular energy metabolism, including the insulin-like growth factor 1 receptor (IGF-1R). As the first-line therapy for type 2 diabetes mellitus (T2DM) patients, metformin is widely known to inhibit the metabolic reprogramming of cancer cells. This study aims to investigate metformin's efficacy in inhibiting endocrine resistance related to genes regulating energy metabolism in both ER-positive and ER-negative breast cancer cell lines under hyperglycemic conditions. MDA-MB-361 (ER-positive, HER2-positive) and SKBR3 (ER-negative, HER2-positive) cancer cell lines were used to represent ER status. Cell viability and cell survival rate were measured using the colorimetric assay of Cell Counting Kit-8. All mRNA levels were quantified using real-time quantitative polymerase chain reaction preceded by reverse transcription. A P value of <.05 was considered statistically significant. Unlike MDA-MB-361, SKBR3 were found to acquire resistance upon metformin treatment in hyperglycemic conditions. Moreover, the mRNA expression of IGF-1R and its downstream signaling, such as the mammalian target of rapamycin (mTOR), was not affected by metformin. Meanwhile, the mRNA expression level of ribosomal S6 kinase 1 (S6K1) was upregulated, whereas forkhead box O1 (FOXO1) was downregulated after metformin treatment in hyperglycemic conditions. This preliminary study suggests that an alternative pathway of metformin resistance may exist in the absence of ERα. Therefore, relying solely on metformin may be inadequate to inhibit the aggressiveness of breast cancer cells.

Inhibition of Dipeptidyl Peptidase-4 Activates Autophagy to Promote Survival of Breast Cancer Cells via the mTOR/HIF-1α Pathway.

Autophagy plays a complex role in breast cancer cell survival, metastasis, and chemotherapeutic resistance. Dipeptidyl peptidase (DPP)-4, a therapeutic target for type 2 diabetes mellitus, is also involved in autophagic flux. The potential influence of DPP-4 suppression on cancer biology remains unknown. Here, we report that DPP-4 deficiency promotes breast cancer cell survival via the induction of autophagy by the C-X-C motif chemokine 12 (CXCL12)/C-X-C receptor 4 (CXCR4)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor (HIF)-1α axis. DPP-4 knockdown and DPP-4 inhibitor KR62436 (KR) treatment both increased the levels of LC3II and HIF-1α in cultured human breast and mouse mammary cancer cells. The KR-induced autophagic phenotype in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 and rapamycin. HIF-1α knockdown also suppressed breast cancer autophagy induced by KR. The autophagy inhibitor 3-methyladenine significantly blocked the KR-mediated suppression of cleaved caspase-3 levels and apoptosis in breast cancer cell lines. Finally, we found that the metformin-induced apoptosis of DPP-4-deficient 4T1 mammary cancer cells was associated with the suppression of autophagy. Our findings identify a novel role for DPP-4 inhibition in the promotion of breast cancer survival by inducing CXCL12/CXCR4/mTOR/HIF-1α axis-dependent autophagy. Metformin is a potential drug that counteracts the breast cancer cell survival system.

Metformin sensitizes AML cells to venetoclax through endoplasmic reticulum stress-CHOP pathway.

Venetoclax inhibits acute myeloid leukaemia by inhibiting BCL-2 targeting, and a combination regimen with venetoclax has been explored. Although these regimens produce better clinical results, the vast majority of patients still suffer from disease recurrence or primary drug resistance. Metformin has been demonstrated to induce apoptosis in cancer cells. However, whether it can synergize with venetoclax and the underlying mechanisms of metformin-induced apoptosis are not fully understood. In this study, we investigated the effect of metformin and venetoclax on the growth of AML cells in vitro and in vivo. In both Molm13 and THP-1 cell lines, metformin and venetoclax synergistically inhibited the proliferation and induced apoptosis of leukaemia cells. Most importantly, the combination of metformin and venetoclax treatment significantly increased the expression levels of the endoplasmic reticulum (ER) stress-related marker CHOP, for example, in AML cell lines. Knockdown of CHOP markedly attenuated the metformin- and venetoclax-induced cell apoptosis. Moreover, the combination of metformin and venetoclax demonstrated prominent anti-leukaemia effects in xenograft models and bone marrow samples from AML patients. In summary, the combination of metformin and venetoclax showed enhanced anti-leukaemia activity with acceptable safety in AML patients, representing a new combinatorial strategy worth further clinical investigation to treat AML.

Potential Mechanisms of Metformin-Induced Apoptosis in HeLa Cells

Metformin is a traditional antidiabetic drug that also shows potential antitumor effects in cervical cancer. However, some of its apoptosis-related mechanisms are still unclear. In this study, flow cytometry, western blotting, and RNA sequencing (RNA-seq) were used to evaluate the molecular mechanisms of metformin in HeLa cells. The results showed that metformin inhibited cell viability and promoted apoptosis, the protein expression level of Caspase-3 (CASP3) was increased and that of BCL-2 was decreased in HeLa cells treated with metformin. The RNA-seq results indicated a total of 239 differentially expressed genes between the metformin and control check (CK) groups, with 136 genes upregulated and 103 genes downregulated, and 14 of them were found to be associated with apoptosis signaling pathways. The DDIT3 and HRK genes were robustly upregulated in HeLa cells by the endoplasmic reticulum (ER) stress and the mitochondrial pathway of apoptosis. Metformin also affects the expression of PPP2R5C, PPP2R5A, and RRAGA, which participate in biological processes such as PI3K-AKT, mTOR, and AMPK signaling pathways. Metformin mediates the expression of related genes to induce apoptosis.

Inhibition of Autophagy Promotes the Anti-Tumor Effect of Metformin in Oral Squamous Cell Carcinoma.

Simple SummaryIn this study, we found that metformin could inhibit the cell proliferation of Oral Squamous Cell Carcinoma (OSCC) by promoting apoptosis and blocking the cell cycle. Furthermore, inhibiting autophagy with hydroxychloroquine (HCQ) could enhance the anti-tumor effect of metformin in OSCC.Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor in the head and neck. Due to its high malignancy and easy recurrence, the five-year survival rate is only 50–60%. Currently, commonly used chemotherapy drugs for OSCC include cisplatin, paclitaxel, and fluorouracil, which are highly cytotoxic and cause drug resistance in patients. Therefore, a safe and effective treatment strategy for OSCC is urgent. To address this issue, our study investigated the anti-tumor activity of metformin (the first-line diabetes drug) in OSCC. We found that metformin could inhibit OSCC cell proliferation by promoting apoptosis and blocking the cell cycle in G1 phase. Additionally, we also found that metformin could induce protective autophagy of OSCC cells. After inhibiting autophagy with hydroxychloroquine (HCQ), the metformin-induced apoptosis was enhanced. In vitro, metformin inhibited the growth of subcutaneous xenograft tumor in nude mice and HCQ enhanced this effect of metformin. Therefore, metformin combined with HCQ may become a safe and effective treatment strategy for OSCC.

AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma.

AXL receptor tyrosine kinase promotes an invasive phenotype and chemotherapy resistance in esophageal adenocarcinoma (EAC). AXL has been implicated in the regulation of autophagy, but the underlying molecular mechanism remains poorly understood. Herein, we investigate the mechanistic role of AXL in autophagy as well as metformin-induced effects on the growth and survival of EAC. We demonstrate that AXL mediates autophagic flux through activation of AMPK-ULK1 signaling in a reactive oxygen species (ROS)-dependent mechanism by glucose starvation. AXL positively regulates basal cellular ROS levels without significantly affecting mitochondrial ROS production in EAC cells. Pharmacological inhibition of cellular ROS using Trolox abrogates glucose starvation-induced AMPK signaling and autophagy. We demonstrate that AXL expression is required for metformin-induced apoptosis in EAC cells in vitro. The apoptosis induction by metformin is markedly attenuated by inhibition of autophagy through genetic silencing of Beclin1 or ATG7 autophagy mediators, thereby confirming the requirement of intact autophagy for enhancing metformin-induced apoptosis in EAC cells. Our data indicate that metformin-induced autophagy displays a pro-apoptotic function in EAC cells. We show that the metformin-induced suppression of tumor growth in vivo is highly dependent on AXL expression in a tumor xenograft mouse model of EAC. We demonstrate that AXL promotes metformin-induced apoptosis through activation of autophagy in EAC. AXL may be a valuable biomarker to identify tumors that are sensitive to metformin. Therefore, AXL expression could inform the selection of patients for future clinical trials to evaluate the therapeutic efficacy of metformin in EAC.

Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line.

The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

Plantamajoside promotes metformin-induced apoptosis, autophagy and proliferation arrest of liver cancer cells via suppressing Akt/GSK3β signaling.

Metformin, a well-known antidiabetic drug, exhibits anticancer effect in a variety of cancers, including liver cancer. Plantamajoside (PMS), a phenylethanoid glycoside compound isolated from Plantago asiatica, is proved to possess anticancer effects, too. In our study, we hypothesized that PMS might promote metformin mediated anticancer effects on liver cancer. The half maximal inhibitory concentration (IC50) of metformin was evaluated by cell viability assay. The influence of PMS on proliferation, migration, invasion and apoptosis of metformin-treated cells was evaluated by BrdU incorporation assay, flow cytometry, western blot, wound scratch healing assay, transwell cell migration assay and immunofluorescence. A fasting/feeding mouse model was built to evaluate the influence of PMS on metformin sensitivity in vivo. PMS (2.5, 10 or 40μg/mL) treatment reduced the IC50 of metformin under different glucose concentrations. PMS (10μg/mL) promoted metformin (5mm) induced apoptosis and autophagy, and inhibition on proliferation, migration and invasion of HepG2 and HuH-7 cells. In the fasting/feeding mouse model, PMS (50mg/kg) promoted metformin (200mg/kg) induced proliferation arrest and apoptosis in vivo. Meanwhile, PMS reduced the level of pAkt(ser473) and GSK3β(ser9) in HepG2 and HuH-7 cells. Restoration of Akt/GSK3β signaling by a constitutively activated myr-Akt1 abrogated the effects of PMS on metformin-treated liver cancer cells. Our results demonstrated that PMS promoted the anticancer effects of metformin on liver cancer in vitro and in vivo.

Bak instead of Bax plays a key role in metformin-induced apoptosis s in HCT116 cells

Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.

Abstract PS19-18: Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis

Abstract Introduction: Triple-negative breast cancer (TNBC) does not respond to conventional targeted therapy, necessitating novel treatment options. Metformin possesses unique anti-proliferative and pro-apoptotic properties in TNBC cells. However, the molecular mechanism through which metformin may induce apoptosis is incompletely understood. In the current study, we aim to determine whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptotic signaling is involved in metformin-induced cytotoxicity on TNBC cells. Methods: TNBC cell lines HCC70, MDA-MB-468, BT549 and MDA-MB-231 were used in the study. A cell death detection ELISA kit was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). Western blot analyses were carried out to assess the expression of TRAIL, TRAIL receptors DR5 and DR4, and apoptosis-related proteins (PARP, Caspase-8 and Caspase-3). Flow cytometric analyses were performed to define the expression of cell surface TRAIL receptors DR5 and DR4. Secreted TRAIL protein level in the conditioned medium (CM) of cell culture was measured by using a highly specific ELISA and western blot. MDA-MB-231 cells were treated with concentrated CM from metformin-treated cells to test the activity of secreted TRAIL in inducing cell apoptosis. A soluble recombinant TRAIL decoy receptor, which contains a normal extracellular domain of death receptor 5 (DR5) but a truncated intracellular domain and thus is unable to transduce death signals, was used to block TRAIL function. Infection with lentivirus containing specific shRNAs was performed to knockdown TRAIL expression. Results: Metformin induced apoptosis in a panel of TNBC cell lines in a dose-dependent manner, as evidenced by the increase of cytoplasmic histone-associated DNA fragments and the cleavage of PARP, caspase-8 and caspase-3. Interestingly, during the process of TNBC cells undergoing apoptosis, metformin elicited a dose-dependent upregulation of TRAIL protein levels (both cellular TRAIL and secreted TRAIL) without altering the expression of TRAIL receptors DR5 and DR4. The secreted TRAIL from concentrated CM of metformin-treated cells showed activity in inducing apoptosis of MDA-MB-231 cells. To determine if the induction of TRAIL was essential for metformin-induced cytotoxicity effects on TNBC cells, we examined whether inhibition of TRAIL function by a decoy receptor or specific knockdown of TRAIL expression with shRNAs would affect metformin-induced apoptosis. In all three cell lines (HCC70, MDA-MB-468, BT549) we tested, both the TRAIL decoy receptor and TRAIL-specific shRNAs significantly attenuated metformin-induced DNA fragmentation and cleavage of PARP, caspase-8, and caspase-3. Conclusion: We demonstrate that metformin promotes TNBC cells undergoing apoptosis via induction of TRAIL expression. Our data suggest that TRAIL-mediated apoptosis critically contributes to metformin's antitumor activity against TNBC. Keywords: Metformin, TRAIL, Apoptosis, triple-negative breast cancer Citation Format: Shuang Liu, Erik V Polsdofer, Lukun Zhou, Susan Edgerton, Ann Thor, Bolin Liu. Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-18.

Role of autophagy in metformin-induced apoptosis of H4IIE hepatocellular carcinoma cells

Role of autophagy in metformin-induced apoptosis of H4IIE hepatocellular carcinoma cells

Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells.

Bladder cancer (BC) is the sixth most common cancer in men. Moreover, chemotherapy for BC leads to various side effects. Metformin is known to induce apoptosis in vitro in many types of cancer. Furthermore, it has feasibility as a drug repositioning used for the treatment of cancer. The molecular mechanism of metformin mediating apoptosis in BC is still unclear. In this study, we showed that metformin stimulated the caspase-dependent apoptotic signaling pathway in T24 cells, a human BC cell line. Moreover, the induced apoptosis was partially inhibited by a general caspase inhibitor, z-VAD-fmk, which suggested that metformin-induced apoptosis in T24 cells is partially caspase-independent. Notably, we observed the nuclear translocation of apoptosis-inducing factors (AIFs) in metformin-promoted apoptosis, which is a typical characteristic of the caspase-independent apoptotic pathway. In addition, we found that metformin-mediated apoptosis occurred via degradation of the cellular FADD-like interleukin-1β-converting enzyme inhibitory protein (c-FLIP) by facilitating ubiquitin/proteasome-mediated c-FLIPL degradation. Furthermore, treatment with the reactive oxygen species scavenger N-acetylcysteine, failed to suppress metformin-induced apoptosis and c-FLIPL protein degradation in metformin-treated T24 cells. In conclusion, these results indicate that metformin-induced apoptosis was mediated through AIF-promoted caspase-independent pathways as well as caspase-dependent pathways in T24 cells. As such, metformin could be used as a possible apoptotic agent for the treatment of BC.

Full title: High glucose protects mesenchymal stem cells from metformin-induced apoptosis through the AMPK-mediated mTOR pathway

Micro- and macro-vascular events are directly associated with hyperglycemia in patients with type 2 diabetes mellitus (T2DM), but whether intensive glucose control decreases the risk of diabetic cardiovascular complications remains uncertain. Many studies have confirmed that impaired quality and quantity of mesenchymal stem cells (MSCs) plays a pathogenic role in diabetes. Our previous study found that the abundance of circulating MSCs was significantly decreased in patients with T2DM, which was correlated with the progression of diabetic complications. In addition, metformin-induced MSC apoptosis is one of the reasons for the decreased quantity of endogenous or exogenous MSCs during intensive glucose control. However, the role of glucose in metformin-induced MSC apoptosis during intensive glucose control in T2DM remains unknown. In this study, we found that metformin induces MSC apoptosis during intensive glucose control, while high glucose (standard glucose control) could significantly reverse its adverse effect in an AMPK-mTOR pathway dependent manner. Thus, our results indicate that the poorer clinical benefit of the intensive glucose control strategy may be related to an adverse effect due to metformin-induced MSC apoptosis during intensive glucose control therapy in patients with T2DM.

The mechanism of aspirin combined with metformin induced apoptosis of thyroid cancer TPC-1 cells

Objective: To explore the effect of aspirin combined with metformin on the apoptosis of thyroid cancer TPC-1 cells and its mechanism. Methods: The proliferation and apoptosis of TPC-1 cells treated with different concentrations of aspirin and metformin were detected using cell count kit-8 (CCK-8) assay and flow cytometry, respectively. Western blot was used to detect the expressions of microtubule-associated protein light chain 3 (LC3), p62 and cysteinyl aspartate specific proteinase 3 (caspase-3) after treatment with aspirin, metformin and 3-Methyladenine (3-MA). Results: The relative cell viability of TPC-1 cells treated with 0.5, 1.0, 2.0, 4.0 mmol/L aspirin for 24 and 48 hours were (85.6±9.1)%, (79.9±8.6)%, (57.0±5.3)%, (55.7±5.4)%; (76.7±2.8)%, (75.4±6.1)%, (46.1±4.1)%, (36.3±3.2)%, respectively. The value of half maximal inhibitory concentration (IC(50)) for 24 and 48 hours were 4.297 mmol/L, 2.133 mmol/L, respectively. The apoptotic rate in the 1 mmol/L aspirin treatment group and negative control group were (29.2±8.5)%, (4.2±2.9)%, respectively (P<0.05). Moreover, treatment with metformin increased the protein expression of LC3Ⅱ/Ⅰ ratio, and decreased the expression of p62, while treatment with aspirin decreased the expression of LC3Ⅱ/Ⅰ ratio and increased the expression of p62. The relative cell viability of TPC-1 cells treated with metformin, 3-MA, an autophagy inhibitor, and 3-MA combined with metformin were (73.2±9.2)%, (95.8±3.3)%, (59.9±9.2)%, respectively. The apoptotic rates in these groups were (35.5±1.5)%, (12.3±1.4)%, (49.9±5.4)%, respectively. Compared with the metformin group, the relative cell viability in metformin combined with 3-MA group was significantly lower while the apoptotic rate was higher (P<0.05), which indicated that treatment with 3-MA enhanced the metformin-induced apoptosis of TPC-1 cells. The relative cell viability of TPC-1 cells in metformin group, aspirin group, metformin combined with aspirin group were (87.3±11.8)%, (85.7±9.6)%, (72.4±8.8)%, respectively. The apoptotic rates in these groups were (29.7±4.0)%, (30.5±6.5)%, (52.5±4.6)%, respectively. Compared with the metformin or aspirin group, the relative cell viability in metformin combined with aspirin group was significantly lower, while the apoptotic rate was higher (P<0.05), which indicated that aspirin enhanced the metformin-induced apoptosis of TPC-1 cells. Conclusions: Our findings indicate that metformin-mediated autophagy plays a protective role in metformin-induced apoptosis and proliferation inhibition. Aspirin enhances the metformin-induced apoptosis of thyroid cancer TPC-1 cells through inhibition of autophagy.

Fibroblasts rescue oral squamous cancer cell from metformin-induced apoptosis via alleviating metabolic disbalance and inhibiting AMPK pathway

ABSTRACT Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Growing evidence suggests that it may exert antitumor effects in vivo and in vitro. However, even with the promising potency on defeating cancer cells, the pre-clinical and epidemiological studies of metformin on various kinds of cancers are not satisfactory, and the reasons and underlying mechanisms remain unknown. Since cancer is a complex system, dependent on a promoting microenvironment, we hypothesize that the interactions between cancer cells and their neighborhood fibroblasts are essential for metformin resistance. To test this, we used a cell co-culture model closely mimicking the in vivo interactions and metabolic exchanges between normal stromal cells (NOFs) and oral squamous cancer cells (OSCC). Here we show that while metformin can significantly inhibit cell growth and induce apoptosis of OSCC cultured alone in a dose-dependent manner through activating p-AMPKT172 and modulating Bcl-2, Bax, and cleaved PARP. However, when OSCC are co-cultured with NOFs the metformin effects on OSCC cells are annihilated. NOFs are rescuing OSCC from metformin – induced apoptosis, at least partially, through inhibiting the activity of AMPK and PARP, maintaining mitochondrial membrane potential and increasing the oxidative stress. Our results indicate that metformin effects on oral cancer cells are modulated by the microenvironment and that this has to be taken into consideration in the context of developing a new combination of drugs for oral cancer treatment.

Low glucose and metformin-induced apoptosis of human ovarian cancer cells is connected to ASK1 via mitochondrial and endoplasmic reticulum stress-associated pathways

BackgroundMetformin, a first-line drug for type 2 diabetes, could induce apoptosis in cancer cells. However, the concentration of glucose affects the effect of metformin, especially low glucose in the culture medium can enhance the cytotoxicity of metformin on cancer cells. Since mitochondria and endoplasmic reticulum is vital for maintaining cell homeostasis, we speculate that low glucose and metformin-induced cell apoptosis may be associated with mitochondria and endoplasmic reticulum. ASK1, as apoptosis signaling regulating kinase 1, is associated with cell apoptosis and mitochondrial damage. This study was designed to investigate the functional significance of ASK1, mitochondria and endoplasmic reticulum and underlying mechanism in low glucose and metformin-induced cell apoptosis.MethodsAn MTT assay was used to evaluate cell viability in SKOV3, OVCAR3 and HO8910 human ovarian cancer cells. Cell apoptosis was analyzed by flow cytometry. The expression of ASK1 was inhibited using a specific pharmacological inhibitor or ASK1-siRNA. Immunofluorescence was used to detect mitochondrial damage and ER stress. Nude mouse xenograft models were given metformin or/and NQDI-1, and ASK1 expression was detected using immunoblotting. In addition, subcellular fractionation of mitochondria was performed to assay the internal connection between ASK1 and mitochondria.ResultsThe present study found that low glucose in culture medium enhanced the anticancer effect of metformin in human ovarian cancer cells. Utilization of a specific pharmacological inhibitor or ASK1-siRNA identified a potential role for ASK1 as an apoptotic protein in the regulation of low glucose and metformin-induced cell apoptosis via ASK1-mediated mitochondrial damage through the ASK1/Noxa pathway and via ER stress through the ROS/ASK1/JNK pathway. Moreover, ASK1 inhibition weakened the antitumor activity of metformin in vivo. Thus, mitochondrial damage and ER stress play a crucial role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells.ConclusionsThese data suggested that low glucose and metformin induce cell apoptosis via ASK1-mediated mitochondrial damage and ER stress. These findings indicated that the effect of metformin in anticancer treatment may be related to cell culture conditions.

Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1

BackgroundMetformin is the most commonly used first-line medicine for type II diabetes mellitus. Acting via AMP-activated protein kinase, it has been used for more than 60 years and has an outstanding safety record. Metformin also offers protection against cancer, but its precise mechanisms remain unclear.MethodsWe first examined the cytotoxic effects of metformin in the HeLa human cervical carcinoma and ZR-75-1 breast cancer cell lines using assays of cell viability, cleaved poly-ADP-ribose polymerase, and Annexin V-fluorescein isothiocyanate apoptosis, as well as flow cytometric analyses of the cell cycle profile and reactive oxygen species (ROS). We later clarified the effect of metformin on p53 protein stability using transient transfection and cycloheximide chase analyses.ResultsWe observed that metformin represses cell cycle progression, thereby inducing subG1 populations, and had induced apoptosis through downregulation of p53 protein and a target gene, differentiated embryo chondrocyte 1 (DEC1). In addition, metformin increased intracellular ROS levels, but N-acetyl cysteine, a ROS scavenger, failed to suppress metformin-induced apoptosis. Further results showed that metformin disrupted the electron transport chain and collapsed the mitochondrial membrane potential, which may be the cause of the elevated ROS levels. Examination of the mechanisms underlying metformin-induced HeLa cell death revealed that reduced stability of p53 in metformin-treated cells leads to decreases in DEC1 and induction of apoptosis.ConclusionThe involvement of DEC1 provides new insight into the positive or negative functional roles of p53 in the metformin-induced cytotoxicity in tumor cells.

Metformin induces apoptosis in mesenchymal stromal cells and dampens their therapeutic efficacy in infarcted myocardium

BackgroundCardiovascular complications, especially myocardial infarctions (MIs), are the leading mortality cause in diabetic patients. The transplantation of stem cells into damaged hearts has had considerable success as a treatment for MI, although whether antidiabetic drugs affect the therapeutic efficacy of stem cell transplantation is still unknown. This study aims to understand whether and how metformin, one of the first-line drugs used to treat type 2 diabetes mellitus (T2DM), induces mesenchymal stromal cell (MSC) apoptosis and dampens their cardioprotective effect after transplantation into infarcted hearts.MethodsA mouse MI model was generated via permanent ligation of the left anterior descending (LAD) coronary artery. MSCs with or without metformin treatment were transplanted after MI in diabetic mice. Echocardiography was used to assess cardiac function and determine cardiac remodeling, and TTC staining was performed to evaluate infarction size. A mouse gavage model was performed to evaluate bone marrow MSCs for flow cytometry assay.ResultsMetformin dampened MSC therapeutic efficacy, which increased infarct size and restricted functional cardiac recovery. Specifically, metformin induced the activation of AMP-activated protein kinase (AMPK)-mediated apoptosis through the inhibition of S6K1-Bad-Bcl-xL cell survival signaling, resulting in the upregulated expression of apoptosis-associated proteins and increased MSC apoptosis. Accordingly, counteracting AMPK attenuated metformin-induced apoptosis in MSCs and partially restored their cardioprotective effects in diabetic mice with MI. Furthermore, a decrease in peripheral blood MSCs was found in patients with T2DM who had a metformin medication history.ConclusionsOur results highlight an unexpected adverse effect of metformin-induced MSC apoptosis through AMPK-mediated mTOR suppression, which is attenuated by an AMPK inhibitor. Moreover, AMPK inhibition may be a novel strategy for enhancing the effectiveness of stem cell therapy after MI in diabetes.

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.