Evaluating synergistic effects of metformin and simvastatin on ovarian cancer cells.

BackgroundOvarian Cancer (OC) stands as the most lethal gynecological malignancy, presenting an urgent clinical challenge in the quest to improve response rates. One approach to address this challenge is through drug repurposing, exemplified by the investigation of metabolic-modulating drugs such as Metformin (MTF) and Simvastatin (SIM). This study aims to explore the molecular mechanisms contributing to the potential synergistic anti-cancer effects between MTF and SIM on ovarian cancer cells.MethodsWe assessed the effects of the combination on the proliferation and viability of two cell lines OVCAR-3 and SKOV-3. IC50 concentrations of MTF and SIM were determined using a proliferation assay, followed by subtoxic concentrations to explore the potential synergistic effects on the viability of both cell lines. Transcriptomic analysis was conducted on OVCAR-3 treated cells, and the findings were validated by assessing the expression levels of differentially expressed genes (DEGs) through real-time PCR in both cell lines SK-OV-3 and OVCAR-3.ResultsCytotoxicity analysis guided the selection of treatment concentrations as such MTF 10 mM and SIM 5 μM. The combined treatment of MTF and SIM demonstrated a synergistic inhibition of proliferation and viability in both cell lines. In OVCAR-3, exclusive identification of 507 DEGs was seen in the combination arm. Upregulation of FOXO3, RhoA, and TNFα, along with downregulation of PIK3R1, SKP2, and ATP6V1D levels, was observed in OVCAR-3 treated cells. Real-time PCR validation confirmed the consistency of expression levels for the mentioned DEGs.ConclusionOur data strongly supports the presence of synergy between MTF and SIM in OC cells. The combination's effect is associated with the dysregulation of genes in the key regulators AMPK and mTOR alongside other interconnected pathways

S1 Fig -

A–AMPK signaling pathway: AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK system acts as a sensor of cellular energy status. It is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (eg, deprivation for glucose or oxygen) or that accelerate ATP consumption (eg, muscle contraction). Several upstream kinases, including liver kinase B1 (LKB1), calcium/calmodulin kinase kinase-beta (CaMKK beta), and TGF-beta-activated kinase-1 (TAK-1), can activate AMPK by phosphorylating a threonine residue on its catalytic alpha-subunit. Once activated, AMPK leads to a concomitant inhibition of energy-consuming biosynthetic pathways, such as protein, fatty acid and glycogen synthesis, and activation of ATP-producing catabolic pathways. B–mTOR signaling pathway: The mammalian (mechanistic) target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase, which exists in two complexes termed mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 contains mTOR, Raptor, PRAS40, Deptor, mLST8, Tel2 and Tti1. mTORC1 is activated by the presence of growth factors, amino acids, energy status, stress and oxygen levels to regulate several biological processes, including lipid metabolism, autophagy, protein synthesis and ribosome biogenesis. On the other hand, mTORC2, which consists of mTOR, mSin1, Rictor, Protor, Deptor, mLST8, Tel2 and Tti1, responds to growth factors and controls cytoskeletal organization, metabolism and survival. (ZIP)</p

Primer sequences.

Table showing the forward and reverse sequences of the primers for the chosen differentially expressed genes used for real-time PCR. Primers were designed using Primer-BLAST designing tool. (PDF)</p

Proliferation effects.

Dose-response curves of (a) Metformin [MTF] (b) Simvastatin [SIM] in OVCAR-3 cells. And (c) MTF and (d) SIM in SKOV-3 cells. Both showed a decrease in proliferation in dose and time dependent manners. (*p<0.05 **p<0.01 ****p<0.0001).</p

Validation of selected DEGs by real-time PCR in OVCAR-3.

(a-g): Relative expression of the differentially expressed genes (DEGs) found to be enriched in AMPK and mTOR pathway. (h,i): Relative expression of the most up and downregulated genes respectively. Determined with Real time PCR Measurements with GAPDH as internal control. Treatment conditions were compared to untreated samples. Experiments were performed in triplicate. Graphs were plotted as mean SEM using graphpad Prism. (a) FOXO3 (ANOVA, Tukey’s HSD p = 0.0482), (b) PCK2 (ANOVA, Tukey’s HSD p = 0.0483), (c) TNFα (ANOVA, Tukey’s HSD p = 0.0229) and (d) RhoA (ANOVA, Tukey’s HSD p = 0.0302), (e) PIK3R1 (ANOVA, Tukey’s HSD p = 0.0016), (f) SKP2 (ANOVA, Tukey’s HSD p = 0.0423), and (g) ATP6V1D (ANOVA, Tukey’s HSD p = 0.0485), (h) SEMA7A (ANOVA, Tukey’s HSD p = 0.0367), (i) PHACTR2 (ANOVA, Tukey’s HSD p = 0.0380). Abbreviations: MTF: metformin; SIM: simvastatin; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, FOXO3: fockhead box O3; PCK2: Phosphoenolpyruvate Carboxykinase 2; TNFα: tumor necrosis factor alpha; RhoA: Ras homolog family member A; PIK3R1: Phosphoinositide-3-Kinase Regulatory Subunit 1; SKP2: S-phase kinase-associated protein 2; ATP6V1D: ATPase H+ Transporting V1 Subunit D; SEMA7A: Semaphorin 7A; PHACTR2: Phosphatase and Actin Regulator 2.</p

Cytotoxicity analysis and IC50 values.

Dose-response curves of (a) Metformin [MTF] (b) Simvastatin [SIM] in OVCAR-3 at 24, 48, and 72 hours. MTF showed logIC50 values of 1.37mM, 1.16 mM, and 0.9 mM after 24, 48 and 72 hours respectively. SIM showed logIC50 values of 0.98μM, 0.88μM, and 0.69μM after 24, 48, and 72 hours respectively. (c) Metformin [MTF] (d) Simvastatin [SIM] in SK-OV-3 at 24, 48, and 72 hours. MTF showed logIC50 values of 1.45mM, 1.2 mM, 0.98 mM after 24, 48 and 72 hours respectively. SIM showed logIC50 values of 1.395μM, 0.96μM, and 0.77μM after 24, 48 and 72 h respectively. Dose-response curves are p2lotted as normalized mean SEM using GraphPad Prism (n = 3).</p

Crosstalk between mTOR and AMPK.

mTOR (mammalian target of rapamycin) and AMPK (AMP-activated protein kinase) are found to be enriched in KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. Colored genes are found to be enriched in those pathways and are found to be regulated in the combination arm exclusively. Green highlighted genes are found to be upregulated by the combination. Red highlighted genes are found to be downregulated by the combination. Yellow highlighted gene (RhoA) was shown to be upregulated in OVCAR-3 but downregulated in SK-OV-3. DEGs enriched in AMPK include PI3K and FOXO3. According to KEGG, FOXO3 is a direct target of AMPK forming a positive feedback loop. DEGs enriched in mTOR signaling pathway include ATP6V1D, PI3K, SKP2, TNF that control mTOR signaling pathway through different trails. KEGG analysis shows that RhoA is a direct target of mTORC2. PI3K is activated by insulin receptor substrate 1 (IRS1), a signaling protein that is phosphorylated and activated upon the interaction between insulin growth factor and its receptor or even by SCF (c-kit ligand) that is also shown to be targeted by the combination of MTF and SIM. Activated PI3K activates mTORC2 by direct phosphorylation or by indirect activation via IKKα. Second it activates AKT which in turn activates mTORC1 directly or indirectly via inhibition of TSC2 or IKKα. SKP2 inhibits FOXO3, p21, p27, RagA/B, C/D and activates cyclin E, CDK2 and AKT. ATP6V1D activates RagA/B and Rag C/D; dotted arrow: indirect activation (Original figure).</p

Synergy analysis.

(a) Fa-CI plot illustration of one biological replicate of OVCAR-3 cell line with its (b) corresponding values. (c) Fa-CI plot illustration of one biological replicate of SK-OV cell line with its (d) corresponding values. The graphs were generated by the computerized software CompuSyn. The circles in the graph indicate the CI values. Tables b and d summarize the compuSyn results of the specific concentrations for each drug when combined with their relative Fa and CI values. CI10 Very strong antagonism. Fa: fraction affected, CI combination index.</p

DEGs combination vs control group.

Table showing the full list of the DEGs found in the combination vs control group in an ascending order of p-values. Columns show the following: Probe ID (array ID), gene ID, gene symbol, fold change for each group (with insignificant values for single therapies), p-values with adjusted p-values. (XLSX)</p