Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

Abstract

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancers worldwide, continues to maintain high levels of resistance to standard therapy. As clinical data revealed poor response rates, the need for developing new methods has increased to improve the overall wellbeing of patients with HCC. Furthermore, a growing body of evidence shows that cancer metabolic changes are a key feature of many types of human malignancies. Metabolic reprogramming refers to cancer cells’ ability to change their metabolism in order to meet the increased energy demand caused by continuous growth, rapid proliferation, and other neoplastic cell characteristics. For these reasons, metabolic pathways may become new therapeutic and chemopreventive targets. The aim of this study was to investigate the metabolic alterations associated with metformin (MET), an anti-diabetic agent when combined with two antifolate drugs: trimethoprim (TMP) or methotrexate (MTX), and how metabolic changes within the cancer cell may be used to increase cellular death. In this study, single drugs and combinations were investigated using in vitro assays including cytotoxicity assay (MTT), RT-qPCR, annexin V/PI apoptosis assay, scratch wound assay and Seahorse XF analysis, on a human HCC cell line, HepG2. The cytotoxicity assay showed that the IC50 of MET as single therapy was 44.08 mM that was reduced to 22.73 mM and 29.29 mM when combined with TMP and MTX, respectively. The co-treatment of both drugs increased p53 and Bax apoptotic markers, while decreased the anti-apoptotic marker; Bcl-2. Both combinations increased the percentage of apoptotic cells and halted cancer cell migration when compared to MET alone. Furthermore, both combinations decreased the MET-induced increase in glycolysis, while also inducing mitochondrial damage, altering cancer cell bioenergetics. These findings provide an exciting insight into the anti-proliferative and apoptotic effects of MET and anti-folates on HepG2 cells, and how in combination, may potentially combat the aggressiveness of HCC.

Methotrexate-Induced Liver Injury Is Associated with Oxidative Stress, Impaired Mitochondrial Respiration, and Endoplasmic Reticulum Stress In Vitro

“…The decrease in cell viability plateauing at around 20% toxicity across a wide range of concentrations confirmed the broad therapeutic window of MTX. Similar responses were seen in 2D HepG2, HeLa, and HEK293 cells [ 40 , 41 , 42 ]. In 2D HepaRG, 100–150,000 nM MTX induced the accumulation of neutral lipids and reduced the viability to 70% [ 43 ].…”

“…Our findings further support the key role of mitochondrial stress in hepatocellular toxicity by MTX. Previous publications using HepG2 cells also reported a decrease in SRC by MTX but at much higher concentrations (3 mM) [42]. Having shown the antioxidant effects of NAC in the MitoSOX TM assay, we tested whether pre-incubation with NAC could also reverse the effects of MTX on OCR and SRC.…”

A potential Role of Metformin in Hepatocellular Carcinoma.

“…studies, but with higher concentrations of metformin (in millimoles) applied to HCC, also showed harmonious results (Cai et al, 2013), (Miyoshi et al, 2014), (Ferretti et al, 2019), (Tawfik et al, 2022). Other studies using metformin on other types of cancer cell types confirm the same concept, including surgically resected glioblastoma tissue samples (Guarnaccia et al, 2022) and human gastric cancer cells (Lu et al, 2019).…”

The Emerging Role of Metformin in the Treatment of Hepatocellular Carcinoma: Is There Any Value in Repurposing Metformin for HCC Immunotherapy?

“…It is worth mentioning that mounting evidence suggests that metformin could synergize with other molecules or modalities to enhance their cytotoxic capacity. Several studies have shown that combining metformin with other drugs can increase its antitumor effects in HCC, such as sodium glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin [ 88 ], aloin [ 89 ], antifolates [ 90 ], dichloroacetate (DCA) [ 91 ], celastrol [ 92 ] and systemic hypoxia [ 93 , 94 ]. Zhang et al developed a new pH-responding magnetic nanocomposite based on reduced graphene oxide that combines cisplatin and metformin to kill HepG2 and Caco-2 cells.…”