Biguanide MC001, a Dual Inhibitor of OXPHOS and Glycolysis,Shows Enhanced Antitumor Activity Without Increasing Lactate Production

Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising antitumor effects. However, stronger inhibition of mitochondrial complex I is generally associated with upregulation of glycolysis and higher risk of lactic acidosis. Herein we report a novel biguanide derivative, N-cystaminylbiguanide (MC001), which was found to inhibit mitochondrial complex I with higher potency while inducing lactate production to a similar degree as metformin.Furthermore, MC001 was found to efficiently inhibit a panel of colorectal cancer (CRC) cells in vitro and to suppress tumor growth in a HCT116 xenograft nude mouse model, while not enhancing lactate production relative to metformin, exhibiting a superior safety profile to other potent biguanides such as phenformin. Mechanistically, MC001 efficiently inhibits mitochondrial complex I, activates AMPK, and represses mTOR, leading to cell-cycle arrest and apoptosis. Notably, MC001 inhibits both oxidative phosphorylation (OXPHOS) and glycolysis. We therefore propose that MC001 warrants further investigation in cancer treatment.     

BRAF Controls the Effects of Metformin on Neuroblast Cell Divisions in C. elegans

Metformin has demonstrated substantial potential for use in cancer treatments. Liver kinase B (LKB)-AMP-activated protein kinase (AMPK) and mTOR are reported to be the main targets of metformin in relation to its ability to prevent cancer cell proliferation. However, the role of metformin in the control of neoplastic cancer cell growth is possibly independent of LKB-AMPK and mTOR. Using C. elegans as a model, we found that the neuronal Q-cell divisions in L1-arrested worms were suppressed following metformin treatment in AMPK-deficient mutants, suggesting that the mechanism by which metformin suppresses these cell divisions is independent of AMPK. Our results showed that the mTOR pathway indeed played a role in controlling germ cell proliferation, but it was not involved in the neuronal Q-cell divisions occurring in L1-arrested worms. We found that the neuronal Q-cells divisions were held at G1/S cell stage by metformin in vivo. Additionally, we demonstrated that metformin could reduce the phosphorylation activity of BRAF and block the BRAF-MAPK oncogenesis pathway to regulate neuronal Q-cell divisions during L1 arrest. This work discloses a new mechanism by which metformin treatment acts to promote neuronal cancer prevention, and these results will help promote the study of the anticancer mechanisms underlying metformin treatments.     

Metformin as a Treatment Strategy for Sjögren’s Syndrome

Sjögren’s syndrome (SS), a chronic inflammatory disease involving the salivary and lacrimal glands, presents symptoms of sicca as well as systemic manifestations such as fatigue and musculoskeletal pain. Only a few treatments have been successful in management of SS; thus treatment of the disease is challenging. Metformin is the first-line agent for type 2 diabetes and has anti-inflammatory potential. Its immunomodulatory capacity is exerted via activation of 5’ adenosine monophosphate-activated protein kinase (AMPK). Metformin inhibits mitochondrial respiratory chain complex I which leads to change in adenosine mono-phosphate (AMP) to adenosine tri-phosphate (ATP) ratio. This results in AMPK activation and causes inhibition of mammalian target of rapamycin (mTOR). mTOR plays an important role in T cell differentiation and mTOR deficient T cells differentiate into regulatory T cells. In this manner, metformin enhances immunoregulatory response in an individual. mTOR is responsible for B cell proliferation and germinal center (GC) differentiation. Thus, reduction of B cell differentiation into antibody-producing plasma cells occurs via downregulation of mTOR. Due to the lack of suggested treatment for SS, metformin has been considered as a treatment strategy and is expected to ameliorate salivary gland function.     

The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21^waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.     

Multifaceted Mechanisms of Action of Metformin Which Have Been Unraveled One after Another in the Long History

While there are various kinds of drugs for type 2 diabetes mellitus at present, in this review article, we focus on metformin which is an insulin sensitizer and is often used as a first-choice drug worldwide. Metformin mainly activates adenosine monophosphate-activated protein kinase (AMPK) in the liver which leads to suppression of fatty acid synthesis and gluconeogenesis. Metformin activates AMPK in skeletal muscle as well, which increases translocation of glucose transporter 4 to the cell membrane and thereby increases glucose uptake. Further, metformin suppresses glucagon signaling in the liver by suppressing adenylate cyclase which leads to suppression of gluconeogenesis. In addition, metformin reduces autophagy failure observed in pancreatic β-cells under diabetic conditions. Furthermore, it is known that metformin alters the gut microbiome and facilitates the transport of glucose from the circulation into excrement. It is also known that metformin reduces food intake and lowers body weight by increasing circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15). Furthermore, much attention has been drawn to the fact that the frequency of various cancers is lower in subjects taking metformin. Metformin suppresses the mechanistic target of rapamycin (mTOR) by activating AMPK in pre-neoplastic cells, which leads to suppression of cell growth and an increase in apoptosis in pre-neoplastic cells. It has been shown recently that metformin consumption potentially influences the mortality in patients with type 2 diabetes mellitus and coronavirus infectious disease (COVID-19). Taken together, metformin is an old drug, but multifaceted mechanisms of action of metformin have been unraveled one after another in its long history.     

Metformin Restores Parkin-Mediated Mitophagy,Suppressed by Cytosolic p53

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.     

Impact of Sodium Dichloroacetate Alone and in Combination Therapies on Lung Tumor Growth and Metastasis

Metabolic reprogramming has been recognized as an essential emerging cancer hallmark. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been reported to have anti-cancer effects by reversing tumor-associated glycolysis. This study was performed to explore the anti-cancer potential of DCA in lung cancer alone and in combination with chemo- and targeted therapies using two non-small cell lung cancer (NSCLC) cell lines, namely, A549 and LNM35. DCA markedly caused a concentration- and time-dependent decrease in the viability and colony growth of A549 and LNM35 cells in vitro. DCA also reduced the growth of tumor xenografts in both a chick embryo chorioallantoic membrane and nude mice models in vivo. Furthermore, DCA decreased the angiogenic capacity of human umbilical vein endothelial cells in vitro. On the other hand, DCA did not inhibit the in vitro cellular migration and invasion and the in vivo incidence and growth of axillary lymph nodes metastases in nude mice. Treatment with DCA did not show any toxicity in chick embryos and nude mice. Finally, we demonstrated that DCA significantly enhanced the anti-cancer effect of cisplatin in LNM35. In addition, the combination of DCA with gefitinib or erlotinib leads to additive effects on the inhibition of LNM35 colony growth after seven days of treatment and to synergistic effects on the inhibition of A549 colony growth after 14 days of treatment. Collectively, this study demonstrates that DCA is a safe and promising therapeutic agent for lung cancer.     

Metformin Actions on the Liver: Protection Mechanisms Emerging in Hepatocytes and Immune Cells against NASH-Related HCC

Nonalcoholic fatty liver disease (NAFLD) is strongly linked to the global epidemic of obesity and type 2 diabetes mellitus (T2DM). Notably, NAFLD can progress from the mildest form of simple steatosis to nonalcoholic steatohepatitis (NASH) that increases the risk for hepatocellular carcinoma (HCC), which is a malignancy with a dismal prognosis and rising incidence in the United States and other developed counties, possibly due to the epidemic of NAFLD. Metformin, the first-line drug for T2DM, has been suggested to reduce risks for several types of cancers including HCC and protect against NASH-related HCC, as revealed by epidemical studies on humans and preclinical studies on animal models. This review focuses on the pathogenesis of NASH-related HCC and the mechanisms by which metformin inhibits the initiation and progression of NASH-related HCC. Since the functional role of immune cells in liver homeostasis and pathogenesis is increasingly appreciated in developing anti-cancer therapies on liver malignancies, we discuss both the traditional targets of metformin in hepatocytes and the recently defined effects of metformin on immune cells.     

New Insight into the Effects of Metformin on Diabetic Retinopathy,Aging and Cancer: Nonapoptotic Cell Death,Immunosuppression, and Effects beyond the AMPK Pathway

Under metabolic stress conditions such as hypoxia and glucose deprivation, an increase in the AMP:ATP ratio activates the AMP-activated protein kinase (AMPK) pathway, resulting in the modulation of cellular metabolism. Metformin, which is widely prescribed for type 2 diabetes mellitus (T2DM) patients, regulates blood sugar by inhibiting hepatic gluconeogenesis and promoting insulin sensitivity to facilitate glucose uptake by cells. At the molecular level, the most well-known mechanism of metformin-mediated cytoprotection is AMPK pathway activation, which modulates metabolism and protects cells from degradation or pathogenic changes, such as those related to aging and diabetic retinopathy (DR). Recently, it has been revealed that metformin acts via AMPK- and non-AMPK-mediated pathways to exert effects beyond those related to diabetes treatment that might prevent aging and ameliorate DR. This review focuses on new insights into the anticancer effects of metformin and its potential modulation of several novel types of nonapoptotic cell death, including ferroptosis, pyroptosis, and necroptosis. In addition, the antimetastatic and immunosuppressive effects of metformin and its hypothesized mechanism are also discussed, highlighting promising cancer prevention strategies for the future.     

Alisertib Induces Cell Cycle Arrest,Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G₂/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5′ AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells.     

Current Status and Application of Metformin for Prostate Cancer: A Comprehensive Review

Metformin, an oral biguanide used for first-line treatment of type 2 diabetes mellitus, has attracted attention for its anti-proliferative and anti-cancer effects in several solid tumors, including prostate cancer (PCa). Liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) activation, inhibition of the mammalian target of rapamycin (mTOR) activity and protein synthesis, induction of apoptosis and autophagy by p53 and p21, and decreased blood insulin level have been suggested as direct anti-cancer mechanisms of metformin. Research has shown that PCa development and progression are associated with metabolic syndrome and its components. Therefore, reduction in the risk of PCa and improvement in survival in metformin users may be the results of the direct anti-cancer mechanisms of the drug or the secondary effects from improvement of metabolic syndrome. In contrast, some research has suggested that there is no association between metformin use and PCa incidence or survival. In this comprehensive review, we summarize updated evidence on the relationship between metformin use and oncological effects in patients with PCa. We also highlight ongoing clinical trials evaluating metformin as an adjuvant therapy in novel drug combinations in various disease settings.     

Dichloroacetate Radiosensitizes Hypoxic Breast Cancer Cells

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.     

Metformin Improves Ovarian Cancer Sensitivity to Paclitaxel and Platinum-Based Drugs: A Review of In Vitro Findings

Ovarian cancer is one of the most dangerous gynecologic cancers worldwide, showing a high fatality rate and recurrence due to diagnosis at an advanced stage of the disease and the occurrence of chemoresistance, which weakens the therapeutic effects of the chemotherapeutic treatments. In fact, although paclitaxel and platinum-based drugs (carboplatin or cisplatin) are widely used alone or in combination to treat ovarian cancer, the occurrence of chemoresistance significantly reduces the effects of these drugs. Metformin is a hypoglycemic agent that is commonly used for the treatment of type 2 diabetes mellitus and non-alcoholic fatty liver disease. However, this drug also shows anti-tumor activity, reducing cancer risk and chemoresistance. This review analyzes the current literature regarding the role of metformin in ovarian cancer and investigates what is currently known about its effects in reducing paclitaxel and platinum resistance to restore sensitivity to these drugs.     

Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by Wes^TM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.     

HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.     

Targeting Lactate Dehydrogenase A with Catechin Resensitizes SNU620/5FU Gastric Cancer Cells to 5-Fluorouracil

Resistance to anticancer therapeutics occurs in virtually every type of cancer and becomes a major difficulty in cancer treatment. Although 5-fluorouracil (5FU) is the first-line choice of anticancer therapy for gastric cancer, its effectiveness is limited owing to drug resistance. Recently, altered cancer metabolism, including the Warburg effect, a preference for glycolysis rather than oxidative phosphorylation for energy production, has been accepted as a pivotal mechanism regulating resistance to chemotherapy. Thus, we investigated the detailed mechanism and possible usefulness of antiglycolytic agents in ameliorating 5FU resistance using established gastric cancer cell lines, SNU620 and SNU620/5FU. SNU620/5FU, a gastric cancer cell harboring resistance to 5FU, showed much higher lactate production and expression of glycolysis-related enzymes, such as lactate dehydrogenase A (LDHA), than those of the parent SNU620 cells. To limit glycolysis, we examined catechin and its derivatives, which are known anti-inflammatory and anticancer natural products because epigallocatechin gallate has been previously reported as a suppressor of LDHA expression. Catechin, the simplest compound among them, had the highest inhibitory effect on lactate production and LDHA activity. In addition, the combination of 5FU and catechin showed additional cytotoxicity and induced reactive oxygen species (ROS)-mediated apoptosis in SNU620/5FU cells. Thus, based on these results, we suggest catechin as a candidate for the development of a novel adjuvant drug that reduces chemoresistance to 5FU by restricting LDHA.     

Maternal Metformin Treatment Reprograms Maternal High-Fat Diet-Induced Hepatic Steatosis in Offspring Associated with Placental Glucose Transporter Modifications

Maternal high-fat (HF) diet exposure in utero may affect fetal development and cause metabolic problems throughout life due to lipid dysmetabolism and oxidative damage. Metformin has been suggested as a potential treatment for body weight reduction and nonalcoholic fatty liver disease, but its reprogramming effect on offspring is undetermined. This study assesses the effects of maternal metformin treatment on hepatic steatosis in offspring caused by maternal HF diet. Female rats were fed either a control or an HF diet before conception, with or without metformin treatment during gestation, and placenta and fetal liver tissues were collected. In another experiment, the offspring were fed a control diet until 120 d (adult stage). Metformin treatment during pregnancy ameliorates placental oxidative stress and enhances placental glucose transporter 1 (GLUT1), GLUT3, and GLUT4 expression levels through 5’ adenosine monophosphate-activated protein kinase (AMPK) activation. Maternal metformin treatment was shown to reprogram maternal HF diet-induced changes in offspring fatty liver with the effects observed in adulthood as well. Further validation is required to develop maternal metformin therapy for clinical applications.