Caffeic Acid Targets AMPK Signaling and Regulates Tricarboxylic Acid Cycle Anaplerosis while Metformin Downregulates HIF-1α-Induced Glycolytic Enzymes in Human Cervical Squamous Cell Carcinoma Lines

Abstract

The small molecules, natural antioxidant Caffeic Acid (trans-3,4-Dihydroxycinnamic acid CA) and anti-diabetic drug Metformin (Met), activate 5′-adenosine monophosphate-activated protein kinase (AMPK) and interfere with metabolic reprogramming in human cervical squamous carcinoma cells. Here, to gain more insight into the ability of CA, Met and the combination of both compounds to impair aerobic glycolysis (the “Warburg effect”) and disrupt bioenergetics of cancer cells, we employed the cervical tumor cell lines C-4I and HTB-35/SiHa. In epithelial C-4I cells derived from solid tumors, CA alleviated glutamine anaplerosis by downregulation of Glutaminase (GLS) and Malic Enzyme 1 (ME1), which resulted in the reduction of NADPH levels. CA treatment of the cells altered tricarboxylic acid (TCA) cycle supplementation with pyruvate via Pyruvate Dehydrogenase Complex (PDH), increased ROS formation and enhanced cell death. Additionally, CA and CA/Met evoked intracellular energetic stress, which was followed by activation of AMPK and the impairment of unsaturated FA de novo synthesis. In invasive HTB-35 cells, Met inhibited Hypoxia-inducible Factor 1 (HIF-1α) and suppressed the expression of the proteins involved in the “Warburg effect”, such as glucose transporters (GLUT1, GLUT3) and regulatory enzymes of glycolytic pathway Hexokinase 2 (HK2), 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4), Pyruvate Kinase (PKM) and Lactate Dehydrogenase A (LDH). Met suppressed the expression of c-Myc, BAX and cyclin-D1 (CCND1) and evoked apoptosis in HTB-35 cells. In conclusion, both small molecules CA and Met are capable of disrupting energy homeostasis, regulating oxidative metabolism/glycolysis in cervical tumor cells in regard to specific metabolic phenotype of the cells. CA and Met may provide a promising approach in the prevention of cervical cancer progression.

Metformin in Cervical Cancer: Metabolic Reprogramming

“…The glycolytic phenotype of tumor cells is triggered mainly by a master regulator HIF-1α and its downstream proteins. Our study showed that Met alleviated the hypoxia-induced activation of HIF-1α, which was followed by decreased expression of HIF-1α downstream protein effectors in HTB-35 cells, as demonstrated in [22]. In particular, Met downregulated GLUT transporters (solute carrier family 2 member receptors, SLC2A), specifically GLUT1 and GLUT3.…”

“…As measured using qPCR analysis, Met decreased c-MYC transcript level in HTB-35 cells [22], which was in compliance with inhibition of GLS protein expression [11]. The treatment of cervical tumor cells with Met decreased mRNA level for another c-Myc downstream protein, CCND1 (cyclin D1), which regulates cell cycle progression [22]. Zhang et al [24] reported that Met caused a substantial decrease of cyclin D1 expression in bladder cancer cells.…”

“…The overexpression of oncogene cyclin D1 is positively correlated with chemotherapeutic resistance and apoptosis avoidance in squamous cell cancers [23]. The inhibition of CCND1 expression in aggressive cervical tumor cells resulted in enhanced apoptosis [22].…”

“…In particular, the upregulated c-Myc enhances glutamine catabolism in tumor cells, since the oncogene controls glutaminase (GLS) expression [23]. As measured using qPCR analysis, Met decreased c-MYC transcript level in HTB-35 cells [22], which was in compliance with inhibition of GLS protein expression [11]. The treatment of cervical tumor cells with Met decreased mRNA level for another c-Myc downstream protein, CCND1 (cyclin D1), which regulates cell cycle progression [22].…”

“…Met triggered another pro-apoptotic mechanism in cervical carcinoma cells via regulation of Bcl-2 (B-cell lymphoma 2) protein family members' expression [22]. Bcl-2 proteins are key players in the regulation of mitochondrial-dependent programmed cell death.…”