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. 2017 Feb 21;18(2):462.
doi: 10.3390/ijms18020462.

Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis

Malgorzata Tyszka-Czochara  1 , Pawel Konieczny  2 , Marcin Majka  3
Affiliations

Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis

Malgorzata Tyszka-Czochara et al. Int J Mol Sci. .

Abstract

The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug _targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL-1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment.

Keywords: 5′-adenosine monophosphate-activated protein kinase (AMPK); Metformin; caffeic acid; cervical cancer; metabolic reprogramming.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Metformin (Met) and caffeic acid (CA) exert an anti-proliferative effect on HTB-34 human cervical cancer cells. Sensitivity of HTB-34 to Met ((A) 100 μM to 100 mM) and CA ((B) 1 μM to 10 mM) after 24 h treatment as measured with MTT assay. Effect of Met and CA treatment on (C) cell proliferation and (D) LDH release; (E) Cell culture morphology under phase contrast light microscope after CA (100 µM), Met (10 mM), and Met/CA treatment. The detrimental effect caused by Met and CA alone is mainly due to necrosis, while combination of Met and CA significantly increase apoptosis in cancer cells (F) followed by a shift towards S and G2/M phases of cell cycle in population of treated cells (G). Experiments were repeated three times with similar results.
Figure 2
Figure 2
CA activates AMPK in HTB-34 cells along with increasing pyruvate decarboxylation via PDH complex and decreasing glucose consumption and lactate production. Immunoblot analysis (the details described in Materials and Methods) reveals enhanced phosphorylation of AMPK on T172 residue by CA alone and Met/CA co-treatment along with activation of AMPK downstream ACC-1 and decrease of ATP content (A); Met and CA have slight, opposite effect on glucose uptake via GLUT-1. CA and Met/CA cause loss of PFK-2 activity. CA increases PDH-E1α phosphorylation on S293 and inhibits PDH kinase (PDK) activity facilitating pyruvate flux via PDH complex. Note the opposing effect of Met on PDH phosphorylation (caused by PDK activation) compared with CA and recovery of metformin-inhibited PDH complex by co-treatment with CA. CA, Met, and Met/CA treatment has no effect on Glutaminase (GLS) expression (B); For Western blot analyses β-actin was used as the protein loading control, band intensities were quantified by densitometry analysis and expressed relative to the control (* p < 0.05 vs. untreated control). CA decreases glucose consumption and lactate release into culture medium (C) after 24 h of incubation and attenuates the effect of Met. CA was used at 100 µM and Met at 10 mM for 24 h. Experiments were repeated three times with similar results.
Figure 2
Figure 2
CA activates AMPK in HTB-34 cells along with increasing pyruvate decarboxylation via PDH complex and decreasing glucose consumption and lactate production. Immunoblot analysis (the details described in Materials and Methods) reveals enhanced phosphorylation of AMPK on T172 residue by CA alone and Met/CA co-treatment along with activation of AMPK downstream ACC-1 and decrease of ATP content (A); Met and CA have slight, opposite effect on glucose uptake via GLUT-1. CA and Met/CA cause loss of PFK-2 activity. CA increases PDH-E1α phosphorylation on S293 and inhibits PDH kinase (PDK) activity facilitating pyruvate flux via PDH complex. Note the opposing effect of Met on PDH phosphorylation (caused by PDK activation) compared with CA and recovery of metformin-inhibited PDH complex by co-treatment with CA. CA, Met, and Met/CA treatment has no effect on Glutaminase (GLS) expression (B); For Western blot analyses β-actin was used as the protein loading control, band intensities were quantified by densitometry analysis and expressed relative to the control (* p < 0.05 vs. untreated control). CA decreases glucose consumption and lactate release into culture medium (C) after 24 h of incubation and attenuates the effect of Met. CA was used at 100 µM and Met at 10 mM for 24 h. Experiments were repeated three times with similar results.
Figure 3
Figure 3
CA and Met/CA causes significantly enhanced generation of mitochondrial ROS measured with MitoSox Red probe by flow cytometry (A) and visualized by fluorescence microscopy after incubation of HTB-34 cells with Met at 10 mM and CA at 100 µM for 24 h; (B) note the induction of mitochondrial oxidative stress after incubation of cells with CA and Met/CA (red fluorescence indicate mitochondrial superoxide). Experiments were repeated three times with similar results.
Figure 4
Figure 4
Met decreases expression of lipogenic genes (A) and unsaturated FA content (B) in human cervical carcinoma HTB-34 cells. Total cell lysates were subjected to SDS-PAGE followed by Western blot analysis and chemiluminescent detection (band intensities were quantified by densitometry analysis; the details described in Material and Methods, * p < 0.05 vs. untreated control). Note that co-treatment of cells with Met and CA for 24 h restrains the expression of ACLY, FASN, ELOVL6, and SCD1 (A) and significantly decreases unsaturated FA content in cancer cells, as measured by spectrophotometric assay. Data shown here are from a representative experiment repeated three times with similar results and presented as mean values ± SD (B).
Figure 5
Figure 5
The effects of Met (green path), CA (yellow path), and Met/CA co-treatment (purple path) on oxidative metabolism and fatty acids biosynthesis in HTB-34 cells. Met promotes glycolysis to lactate and regulates FA de novo synthesis. CA activates AMPK, enhances oxidative metabolism of glucose via PDH and ROS formation in mitochondria and apoptosis. Met/CA activates AMPK, inhibits FA biosynthesis in the short-term via ACC1 and in the long-term by downregulation of ACLY, FAS, ELOVL6, and SCD1 enzymes expression.
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