Effect Of Phenformin Research Articles

Progress in antitumor mechanisms and applications of phenformin (Review).

Phenformin, a biguanide compound, has attracted increased attention due to its prominent antitumor activity. As a multi‑target agent, the antitumor effects of phenformin involve a wide range of factors, including inhibition of mitochondrial complex I, activation of AMP‑activated protein kinase, impact on the tumor microenvironment, suppression of cancer stem cells and others. In addition, phenformin has been shown to markedly augment the effectiveness of various clinical treatment methods, including radiotherapy, chemotherapy, targeted therapy and immunotherapy. It is noteworthy that breakthrough progress has been made in the treatment of cancer with phenformin with application in clinical trials for the treatment of melanoma. Phenformin not only reduces the lesion area of patients, but also enhances the efficacy of dalafinib/trimetinib. In the present review, the novel breakthroughs in the antitumor effects and mechanisms of phenformin were discussed. In addition, the current review focuses on the clinical development value of phenformin, striving to provide new insights into the future research direction of phenformin in the field of tumor treatment.

Phenformin attenuates the oxidative-nitrosative stress in the liver of rats under long-term ethanol administration

Modulation of the AMP-activated protein kinase (AMPK) pathway activity is considered to be a promi­sing option in the development of approaches to chronic alcoholic hepatitis treatment. Phenformin, which is a biguanide, has been reported to increase AMPK activity. The aim of this work was to estimate the effect of phenformin as AMPK activator on the development of oxidative-nitrosative stress in the liver of rats under conditions of long-term ethanol administration. The experiments were performed on 24 male Wistar rats, divided into 4 groups: control; animals, which received phenformin hydrochloride orally at a dose of 10 mg/kg daily for 63 days; animals with a forced intermittent alcoholization for 5 days by intraperitoneal administration of 16.5% ethanol solution in 5% glucose at the rate of 4 ml/kg b.w. and subsequent transfer to 10% ethanol as the only source of drinking; animals with chronic alcohol hepatitis simulation and phenformin administration. Superoxide dismutase, catalase, NO synthase isoforms activity, superoxide anion radical production, concentration of malonic dialdehyde, peroxynitrite, nitrites, nitrosothiols concentration and oxidative modification of proteins (OMP) were estimated in liver homogenates. The increased production of oxygen and nitrogen active forms and OMP intensification in the liver of rats under long-term administration of ethanol was detected. Phenformin introduction under long-term ethanol administration was shown to limit the excess peroxynitrite formation and to prevent oxidative damage to rat liver proteins. Keywords: AMP-activated protein kinase., chronic alcoholic hepatitis, liver, oxidative and nitrosative stress, phenformin

Cytotoxic effects of Phenformin on ovarian cancer cells: expression of HIF-1α and PDK1 in the hypoxic microenvironment.

Today, many anticancer drugs are used clinically for ovarian cancer, one of the leading causes of cancer-related deaths in women. Phenformin is an antidiabetic drug of the biguanide class. It improves the antiproliferative activity in cancer cells. Hypoxia is an important component associated with ovarian cancer and its tumor microenvironment. The aim of this study was to investigate the anticancer effects of Phenformin in SKOV-3 human ovarian cancer cells under hypoxic conditions. SKOV-3 human ovarian cancer cells treated with different doses of Phenformin (0.5 mM, 1 mM, 2 mM, 5 mM) for 24 hours were subjected to WST-1 cell viability assay and Annexin V apoptosis assay. A dose-dependent decrease in cell viability with Phenformin treatment was observed. In addition, Phenformin activated percentage of apoptotic SKOV-3 cancer cells in a dose-dependent manner. In this study, Cobalt(II) chloride (CoCl2) treatment leads to increased hypoxia-inducible factor-1alpha (HIF-1α) expression and Phenformin can recover hypoxic condition. HIF-1α protein expression was significantly correlated with cell viability assay and apoptosis assay. We also found that Phenformin inhibits expression of phosphoinositide-dependent kinase 1 (PDK1) in SKOV-3 ovarian cancer cells. The ability to migrate to cancer cells was significantly reduced in a dose-dependent manner with Phenformin. This data demonstrates that Phenformin treatment can induce apoptosis and inhibit proliferation in ovarian cancer cells under hypoxic conditions. The findings reveal that HIF-1α is a new target for the treatment of ovarian cancer.

DIPG-02. A NOVEL STRATEGY TO RADIOSENSITISE DIFFUSE MIDLINE GLIOMAS VIA DUAL-TARGETING OF GLUCOSE METABOLISM

Abstract Diffuse Midline Gliomas (DMG) is the leading cause of brain tumor-related death in children. Currently, radiation is the only treatment that offers transient benefit but almost all DMG relapse due to radioresistance. Compared to normal brain tissue, DMG are hypo-perfused with tumor cells being exposed to hypoxia, a low oxygen microenvironment serves as a potent barrier to effective radiotherapy. Biguanides are hypoglycemic agents that can reduce the oxygen consumption rate (OCR) in mitochondria, thereby sparing more oxygen and alleviating hypoxia. Our previous study has shown that metformin significantly improves the radiosensitivity of DMG and extends survival in a patient-derived xenograft (PDX) model. In comparison with metformin, phenformin demonstrated greater efficacies in anti-proliferation, OCR and hypoxia inhibition, and radiosensitization in a panel of DMG cultures. These effects were possibly induced by phenformin through inhibition of mitochondrial complex I in DMG cells. The anti-tumor effect of phenformin was further enhanced by combining a second drug dichloroacetate that simultaneously attenuated phenformin-induced acidification. Specifically, the combination treatment induced higher levels of reactive oxygen species and DNA damage, which subsequently resulted in a much stronger effect in cell-cycle and proliferation arrest, apoptosis, and hypoxia inhibition. Metabolically, the co-treatment simultaneously targeted multiple pathways, which significantly reduced ATP production and further triggered metabolic catastrophe. RNA sequencing demonstrated significant alterations induced by the combination in cell-cycle, DNA repair, unfolded protein response and alternative energetic pathways. Furthermore, the combination treatment significantly improved the radiosensitivity of DMG cells, evidenced by higher levels of DNA damage, apoptosis, and clonogenic inhibition. Our preliminary in vivo data demonstrated a possible reduction in tumor burden following 4 weeks of combination treatment, with no elevation of serum L-lactate level in phenformin-treated groups. This therapeutic efficacy is currently being validated in multiple orthotopic DMG models with optimized treatment schedules.

Phenformin Inhibits the Proliferation of MCF-7 and MDA-MB-231 Human Breast Cancer Cell Lines

Aim: Breast cancer is the most common malignancy in women. This disease is a critical public health problem and further research at the molecular level would define its prognosis and specific treatment. Phenformin is an antidiabetic agent. Recent studies showed that it should be considered as a potential agent for the prevention and treatment of cancer cell lines. This study aimed to investigate the antiproliferative effect of phenformin on MDA-MB-231 and MCF-7 breast cancer cell lines. Materials and Methods: MDA-MB-231 and MCF-7 breast cancer cell lines were used in the study. Experimental groups; control group, 1 µM, 2.5 µM, and 5 µM phenformin administered groups were planned. WST-1 analysis was performed to evaluate the viability of cells 24 h after the treatments. Statistical analysis was carried out using the SPSS 17.0 statistical program. p<0.05 was considered to indicate a statistically significant difference.Results: In the MDA-MB-231 breast cancer cell line, there were statistically significant differences among all groups (p<0.05, for all), except between the 1 µM phenformin-treated and control groups (p>0.05). In the MCF-7 breast cancer cell line; there were statistically significant differences between the control group and 5 µM phenformin-treated, and between 1 µM phenformin-treated and 5 µM phenformin-treated (p<0.05, for all) groups. Conclusion: Phenformin seems to exert antiproliferative effects on MDA-MB-231 and MCF-7 breast cancer cell lines. It was observed that the antiproliferative effect was stronger in the MDA-MB-231 cell line. It showed a stronger antiproliferative effect in the MDA-MB-231 cell line of antiproliferative effect.

Phenformin Down-Regulates c-Myc Expression to Suppress the Expression of Pro-Inflammatory Cytokines in Keratinocytes.

The treatment of many skin inflammation diseases, such as psoriasis and atopic dermatitis, is still a challenge and inflammation plays important roles in multiple stages of skin tumor development, including initiation, promotion and metastasis. Phenformin, a biguanide drug, has been shown to play a more efficient anti-tumor function than another well-known biguanide drug, metformin, which has been reported to control the expression of pro-inflammatory cytokines; however, little is known about the effects of phenformin on skin inflammation. This study used a mouse acute inflammation model, ex vivo skin organ cultures and in vitro human primary keratinocyte cultures to demonstrate that phenformin can suppress acute skin inflammatory responses induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and significantly suppresses the pro-inflammatory cytokines IL-1β, IL-6 and IL-8 in human primary keratinocytes in vitro. The suppression of pro-inflammatory cytokine expression by phenformin was not directly through regulation of the MAPK or NF-κB pathways, but by controlling the expression of c-Myc in human keratinocytes. We demonstrated that the overexpression of c-Myc can induce pro-inflammatory cytokine expression and counteract the suppressive effect of phenformin on cytokine expression in keratinocytes. In contrast, the down-regulation of c-Myc produces effects similar to phenformin, both in cytokine expression by keratinocytes in vitro and in skin inflammation in vivo. Finally, we showed that phenformin, as an AMPK activator, down-regulates the expression of c-Myc through regulation of the AMPK/mTOR pathways. In summary, phenformin inhibits the expression of pro-inflammatory cytokines in keratinocytes through the down-regulation of c-Myc expression to play an anti-inflammation function in the skin.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

EXTH-41. IMPROVING THE RADIOSENSITIVITY OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG) BY TARGETING HYPOXIA AND MITOCHONDRIAL METABOLISM

Abstract DIPG is the leading cause of brain tumor-related death in children. Radiotherapy is the only treatment that offers transient benefit and provides palliative care. DIPGs are hypo-perfused with tumor cells being exposed to hypoxia, a potent barrier to effective radiotherapy. Reducing the oxygen consumption rate (OCR) is therefore a potential strategy to reduce tumor hypoxia. We therefore performed a high-throughput screening with 1963 FDA-approved drugs that could reduce tumor hypoxia and OCR, thereby improving radiosensitivity of DIPGs. A subset of anti-diabetic drugs was identified, with phenformin amongst the most potent. It significantly reduced OCR in a panel of DIPG cultures and subsequently suppressed hypoxia in 3-dimensional DIPG neurosphere models that mimic the hypoxic microenvironment. In addition, phenformin demonstrated greater anti-tumor activity and radiosensitizing effect with much lower doses compared with metformin, a less potent biguanide that improved radiosensitivity of DIPG in a patient-derived xenograft (PDX) model in our previous study. The effect of phenformin was further enhanced by combining a second drug dichloroacetate that simultaneously attenuated phenformin-induced acidification rate. Specifically, the combination of phenformin and dichloroacetate induced higher levels of hypoxia inhibition, reactive oxygen species, DNA damage and apoptosis. RNA sequencing demonstrated significant changes induced by the combination treatment in cell-cycle, DNA repair, unfolded protein response and alternative energetic pathways. These changes were further validated by PCR array and western blotting at mRNA and protein levels. In addition, two master regulators that enhance the metabolic capacity of tumor cells through increased glycolysis thus contributing to radioresistance, HIF-1α and c-Myc, were also significantly suppressed by combination treatment. Ultimately, the triple combination of phenformin, DCA and irradiation demonstrated the most potent efficacy in inducing DNA damage, apoptosis and clonogenic inhibition in DIPG cultures. This promising triple combination therapy is currently being tested in our PDX cohort in vivo.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using β-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

054 Phenformin promotes keratinocyte differentiation via the calcineurin/NFAT pathway

Phenformin is a drug in the biguanide classify that was previously used to treat type 2 diabetes. We have reported the anti-tumor activities of phenformin to enhance the efficacy of BRAF-MEK-ERK pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the DMBA/TPA two stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacological or genetic inhibition of calcineurin/NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an anti-tumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.

Enhanced Sensitivity of Nonsmall Cell Lung Cancer with Acquired Resistance to Epidermal Growth Factor Receptor‐Tyrosine Kinase Inhibitors to Phenformin: The Roles of a Metabolic Shift to Oxidative Phosphorylation and Redox Balance

Background Although the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR- TKI) therapy has been proven in non-small cell lung cancer (NSCLC) patients, acquired resistance to EGFR-TKIs presents a serious clinical problem. Hence, the identification of new therapeutic strategy is needed to treat EGFR-TKI-resistant NSCLC. Methods Acquired EGFR-TKI-resistant lung cancer cell lines (HCC827, H1993, and H292 cells with acquired resistance to gefitinib or erlotinib) were used for cell-based studies. IncuCyte live cell analysis system and XFp analyzer were used for the determination of cell proliferation and energy metabolism, respectively. In vivo anticancer effect of phenformin was assessed in xenografts implanting HCC827 and gefitinib-resistant HCC827 (HCC827 GR) cells. Results HCC827 GR and erlotinib-resistant H1993 (H1993 ER) cells exhibited different metabolic properties compared with their respective parental cells, HCC827, and H1993. In EGFR-TKI-resistant NSCLC cells, glycolysis markers including the glucose consumption rate, intracellular lactate level, and extracellular acidification rate were decreased; however, mitochondrial oxidative phosphorylation (OXPHOS) markers including mitochondria-driven ATP production, mitochondrial membrane potential, and maximal OXPHOS capacity were increased. Cell proliferation and tumor growth were strongly inhibited by biguanide phenformin via targeting of mitochondrial OXPHOS complex 1 in EGFR-TKI-resistant NSCLC cells. Inhibition of OXPHOS resulted in a reduced NAD+/NADH ratio and intracellular aspartate levels. Recovery of glycolysis by hexokinase 2 overexpression in erlotinib-resistant H292 (H292 ER) cells significantly reduced the anticancer effects of phenformin. Conclusion Long-term treatment with EGFR-TKIs causes reactivation of mitochondrial metabolism, resulting in vulnerability to OXPHOS inhibitor such as phenformin. We propose a new therapeutic option for NSCLC with acquired EGFR-TKI resistance that focuses on cancer metabolism.

Abstract IA10: Targeting melanoma metabolism to overcome resistance to treatment

Abstract Despite adequate oxygen levels, cancer cells undergo anaerobic glycolysis converting glucose to lactate (the Warburg effect). It is now thought that the advantage of this for the cancer cell is that it provides the carbon and nitrogen precursors critical for macromolecule synthesis (amino acids, nucleotides, fatty acids) while still providing an adequate supply of ATP needed for growth. Understanding this metabolic landscape may provide strategies to overcome mechanisms of resistance to therapy in melanoma. AMP-activated protein kinase (AMPK) has been shown to inactivate BRAF by phosphorylating Ser 729, and in preclinical models, this can be used to enhance inhibitors of BRAFV600E. In the high ATP environment of a melanoma cell, AMPK is typically low but inducers of AMPK, such as biguanides, increase AMPK. The biguanide metformin does not enter melanoma cells efficiently due to the lack of organic cation transporter (OCT) expression on melanoma cells. However, phenformin does not require OCT for uptake. We are conducting a phase I trial in BRAF V600E-mutated melanoma patients using dabrafenib/trametinib and escalating doses of phenformin. One of the pharmacodynamic effects of phenformin is weight loss, which we are measuring. Interestingly, obesity has been associated with improved outcome in male melanoma patients treated with anti-PD1 antibodies. In contrast, our data in melanoma patients treated with anti-PD1 antibody immunotherapy do not show an association between outcome and obesity. Another consequence of anaerobic glycolysis is generation and excretion of large amounts of lactate. There is evidence that lactate can inhibit T-cell function, and we will discuss strategies to block lactate transport to enhance immunotherapy. Citation Format: Allison Betoff, Jonathan Zippin, Taha Merghoub, Paul B. Chapman, Jedd Wolchok. Targeting melanoma metabolism to overcome resistance to treatment [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA10.

Phenformin Promotes Keratinocyte Differentiation via the Calcineurin/NFAT Pathway

Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the antitumor activities of phenformin to enhance the efficacy of BRAF-MAPK kinase-extracellular signal-regulated kinase pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacologic or genetic inhibition of calcineurin and NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an antitumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.

Phenformin Attenuates Renal Injury in Unilateral Ureteral Obstructed Mice without Affecting Immune Cell Infiltration.

Phenformin and metformin are antihyperglycemic drugs that belong to the class of biguanides. Previously, we demonstrated that metformin elicits renoprotective effects in unilateral ureteral obstructed mice by reducing the infiltration of immune cells into the kidney. Since phenformin is a more potent drug as compared to metformin, we investigated the renoprotective properties of phenformin. We studied the efficacy of both drugs using mice that underwent unilateral ureteral obstruction. Renal damage was evaluated on RNA and protein level by qPCR, Western blotting, and immunohistochemistry. Moreover, we studied immune cell infiltration using flow cytometry. Both biguanides significantly reduced UUO-induced kidney injury, as illustrated by a reduction in KIM-1 protein expression. In addition, both metformin and phenformin impacted the gene expression of several inflammatory markers but to a different extent. Moreover, in contrast to metformin, phenformin did not impact immune cell infiltration into UUO kidneys. In conclusion, we demonstrated that phenformin has similar renoprotective effects as metformin, but the mechanism of action differs, and phenformin is more potent. The beneficial effects of phenformin are probably due to inhibition of the STAT3 pathway and mitochondrial complex I. Further research is needed to unveil the therapeutic potential of phenformin for the treatment of renal injury, either at low, non-toxic concentrations or as part of a combination therapy.

The Surprising Effect of Phenformin on Cutaneous Darkening and Characterization of Its Underlying Mechanism by a Forward Chemical Genetics Approach.

Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human skin cultures to search for effective materials to regulate skin pigmentation. Since a previous study reported the potent inhibitory effect of metformin on melanogenesis, we evaluated several biguanide compounds. The unexpected effect of phenformin, once used as an oral anti-diabetic drug, on cutaneous darkening motivated us to investigate its underlying mechanism utilizing a chemical genetics approach, and especially to identify alternatives to phenformin because of its risk of severe lactic acidosis. Chemical pull-down assays with phenformin-immobilized beads were performed on lysates of human epidermal keratinocytes, and subsequent mass spectrometry identified 7-dehydrocholesterol reductase (DHCR7). Consistent with this, AY9944, an inhibitor of DHCR7, was found to decrease autophagic melanosome degradation in keratinocytes and to intensely darken skin in ex vivo cultures, suggesting the involvement of cholesterol biosynthesis in the metabolism of melanosomes. Thus, our results validated the combined utilization of the phenotypic screening system and chemical genetics as a new approach to develop promising materials for brightening/lightening and/or tanning technologies.

Phenformin Inhibits Hedgehog-Dependent Tumor Growth through a Complex I-Independent Redox/Corepressor Module

The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.

Protective effects of phenformin on zebrafish embryonic neurodevelopmental toxicity induced by X-ray radiation

Radiotherapy (RT) is a common treatment for head and neck cancers, but central nervous system function can be impaired by clinical radiation doses. This experimental study evaluated the protective efficacy of the anti-hyperglycaemic/anti-neoplastic agent phenformin against radiation-induced developmental toxicity in zebrafish embryos. Zebrafish embryos pre-treated with 25 μM phenformin 1 h before x-ray irradiation were compared to irradiation-only embryos for mortality, hatching rate, morphology, spontaneous movement, heart beat, larval swimming, activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), malondialdehyde content (MDA, a by-product of membrane lipid oxidation), and acetylcholinesterase (AChE) activity. In addition, expression levels of multiple genes related to neural development and apoptosis (sod2, bdnf, ache, p53, bax, and bcl-2) were compared by RT-PCR and associated protein expression levels by western blotting. Pre-treatment with phenformin increased hatching rate, spontaneous movement, heart beat, and larval motor activity, decreased mortality and malformation rate, increased SOD, CAT, and AChE activities, and reduced MDA compared to irradiation-only embryos. The mRNA expression levels of anti-apoptotic sod2, bdnf, ache, and bcl-2 were enhanced while mRNA expression of p53 and pro-apoptotic bax were reduced in the phenformin pre-treatment group. Further, p53, Bax, and γ-H2AX (a biomarker of DNA damage) were downregulated while Bcl-2 and BDNF were upregulated by phenformin pre-treatment. Taken together, this study supports the protective efficacy of phenformin against radiation toxicity in zebrafish embryos by suppressing oxidative stress and ensuing apoptosis.

The anti-cancer effects of phenformin in thyroid cancer cell lines and in normal thyrocytes.

Phenformin is a biguanide drug which, besides the original anti-diabetic effect, also exerts anti-cancer effects. The aim of this study was to further characterize these latter in terms of both cell-viability and modulation of the secretion of the pro-tumorigenic chemokine CXCL8. Normal human thyrocytes in primary cultures (NHT) and thyroid cancer cell lines, TPC-1 and 8505C (RET/PTC and BRAFV600E mutated, respectively) were treated with increasing concentrations of phenformin at different times. Cell-viability was assessed by WST-1 and further characterized by AnnexinV/PI staining and cell proliferation colony-assay. CXCL8 levels were measured in cell supernatants. Phenformin reduced cell-viability in TPC-1 and 8505C and their ability to form colonies. In NHT cells, phenformin affected cell-viability only at the maximal dose but interestingly it inhibited CXCL8 secretion at all the concentrations not affecting cell-viability. Phenformin had no effect on CXCL8 secretion in thyroid cancer cell lines. Thus, phenformin exerts anti-cancer effects on both cancer cells (cell death induction) and surrounding normal cells (inhibition of CXCL8 secretion). These results highlight that the anti-cancer effects of phenformin are multifaceted and effective on both solid and soluble components of the tumor-microenvironment.

Phenformin Induces Caspase-dependent Apoptosis of FaDu Head and Neck Squamous Cell Carcinoma Cells.

The present study aimed to investigate the apoptotic effects of phenformin, a therapeutic agent for diabetes, on head and neck squamous cell carcinoma (HNSCC). Cytotoxicity was measured by the MTT and live/dead cell assay. Phenformin-induced apoptotic FaDu cell death and its associated cellular signaling pathways were investigated by hematoxylin and eosin staining, 4',6-diamidino-2-phenylindole staining, caspase-3 activity assay, fluorescence-activated cell sorting analysis, and western blotting. Phenformin promoted death of and apoptotic processes in FaDu cells, including morphological alterations and nuclear condensation. Furthermore, treatment with phenformin increased caspase-3 activity and apoptotic populations via the caspase cascade through cleavage of capspase-8, -9, and -3 and poly(ADP-ribose) polymerase in FaDu cells. Moreover, phosphorylation levels of mitogen-activated protein kinases, nuclear factor-κB, and AKT were down-regulated in FaDu cells by phenformin. Phenformin induced death of FaDu cells via caspase-dependent extrinsic and intrinsic apoptosis pathways and is a promising novel therapeutic agent for HNSCC.

Abstract A68: Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade

Abstract Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that differentiate from bone marrow precursors and expand in cancer-bearing hosts. MDSCs significantly contribute to tumor-induced immune suppression and represent a promising target for cancer immune therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin, a mitochondrial complex I inhibitor, selectively inhibits granulocytic myeloid-derived suppressor cells (G-MDSCs) in spleens of tumor bearing mice and ex vivo. Metabolomics analysis reveal that, compared to CD11b+Ly6G- cells, CD11b+Ly6G+ MDSCs have lower levels of glutathione and NADPH/NADP+ ratio, suggesting these cells have higher level of endogenous ROS. Interestingly, phenformin further induces production of ROS in G-MDSC, whereas co-treatment of the antioxidant N-acetylcysteine dampens ROS levels in these cells and rescues the effect of phenformin on reducing the numbers of G-MDSCs. Importantly, co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8+ T cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of MDSCs. Our findings demonstrate a selective, inhibitory effect of phenformin on G-MDSCs-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma. Citation Format: Sun Hye Kim, Man Li, Sebastian Trousil, bin zheng. Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A68.