Renal cell carcinoma (RCC) is one of the most common types of cancer in adults. Previous studies have reported that the survival rate was significantly lower for renal cancer patients with diabetes than for those without diabetes. Metformin is a well-known anti-diabetic agent used for the treatment of type 2 diabetes mellitus (T2DM). It also inhibits cell proliferation and angiogenesis and is known to possess antitumor effects. However, the molecular mechanism for metformin-induced apoptosis in renal cell carcinoma is not understood. In the present study, treatment with metformin induced apoptosis in A498 cells in a dose-dependent manner. It was revealed that degradation of cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) and activation of procaspase-8 were associated with metformin-mediated apoptosis. By contrast, treatment with metformin did not affect the mRNA level of c-FLIPL in A498 cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk, a pan-caspase inhibitor) almost completely blocked metformin-induced apoptosis and degradation of c-FLIPL protein. However, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, did not inhibit metformin-mediated apoptosis in A498 cells. Taken together, the results of the present study demonstrated that metformin-induced apoptosis involved degradation of the c-FLIPL protein and activation of caspase-8 in human renal cell carcinoma A498 cells and suggested that metformin could be potentially used for the treatment of renal cancer.
Renal cell carcinoma (RCC), a neoplastic lesion of the kidney in humans, accounts for ~90% of kidney tumors (
Metformin is the most widely used biguanide drug for treating type 2 diabetes mellitus patients (
The cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) gene makes three isoforms, namely c-FLIPL, c-FLIPS and c-FLIPR, via alternative splicing in humans. These proteins are well known as anti-apoptotic proteins; each exert this effect via different mechanisms (
In the present study, the mechanism of metformin-mediated apoptosis in human renal cell carcinoma A498 cells was investigated. It was revealed that degradation of c-FLIPL protein and activation of caspase-8 were associated with metformin-induced apoptosis.
A498 human renal carcinoma cells were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM; catalog no. LM 001-05; Welgene, Inc., Kyungsan, Korea) containing 10% fetal bovine serum (FBS; catalog no. S001-07; Welgene, Inc.), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; catalog no. H0887; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) buffer and 100 µg/ml gentamicin (catalog no. 15710-072; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as the culture medium. The cells were cultured in an incubator at 37°C with humidified 5% CO2.
A498 human renal carcinoma cells were treated with an inhibitor in either the absence or presence of metformin (10 mM). Following 24 h incubation, morphological changes were visualized with light microscopy (catalog no. DFC495; Leica Microsystems GmbH, Wetzlar, Germany) at ×200 magnification. The images were analyzed using the i-Solution program (IMT i-Solution, Burnaby, BC, Canada).
Cell counting was performed using a hemocytometer. Metformin was immediately added to cell cultures at the indicated concentrations. Approximately 0.4×106 cells were resuspended in 100 µl PBS (catalog no. 17-517Q; Lonza, Walkersville, MD, USA), and 200 µl of 95% ethanol (catalog no. 1.00983.1011; Merck KGaA) was added during vortexing. The cells were incubated at 4°C for 1 h, washed in PBS, and resuspended in 250 µl 1.12% sodium citrate buffer (pH 8.4) along with 12.5 µl RNase. Incubation was continued for 30 min at 37°C. Cellular DNA was stained with 250 µl (1:1 dilution) propidium iodide (50 µg/ml; catalog no. p4170; Sigma-Aldrich; Merck KGaA) for 30 min at 37°C, and the relative DNA contents of the stained cells were analyzed using fluorescence-activated cell sorting (FACS) on the BD FACS Cato II flow cytometer (BD Biosciences, San Jose, CA, USA).
A498 whole-cell lysates were prepared by resuspending 0.4×106 cells in 50 µl lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, 100 µM phenylmethylsulfonyl fluoride and 20 µM leupeptin, adjusted to pH 7.2). The cells were disrupted by sonication and protein extracted at 4°C for 30 min. Protein concentrations were quantified using the BCA assay kit (catalog no. 23225; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The proteins (50 µg) were separated using 10% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes (catalog no. 23225; GE Healthcare, Chicago, IL, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) for 30 min at room temperature. The anti c-FLIPL (dilution, 1:700; catalog no. ALX-804-961) antibody was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). The anti-PARP (dilution, 1:1,000; catalog no. 9542) antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-B-cell lymphoma-2 (Bcl-2); (dilution, 1:700; catalog no. sc-783), anti-B-cell lymphoma-extra-large (Bcl-xL); (dilution, 1:1,000; catalog no. sc-634), anti-myeloid cell leukemia-1 (Mcl-1); (dilution, 1:1,000; catalog no. sc-819), anti-cellular inhibitor of apoptosis 2 (cIAP-2); (dilution, 1:1,000; catalog no. sc-7944) and anti-actin (dilution, 1:2,000; catalog no. sc-1616) antibodies were procured from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The anti-XIAP (dilution, 1:5,000; catalog no. 610762) antibody was supplied by BD Biosciences (San Jose, CA, USA). Membranes were incubated with the primary antibodies overnight at 4°C. Following six washes with TBS (each for 5 min), the membranes were incubated with the indicated secondary antibody for 1 h at room temperature and washed six times with TBS. The secondary goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugated (dilution 1:1,000; catalog no. sc-2004) and goat anti-mouse IgG-HRP (dilution 1:1,000; catalog no. sc-2005) antibodies were procured from Santa Cruz Biotechnology, Inc. Specific proteins were detected using an ECL western blotting kit (catalog no. WBKLS0500; Merck KGaA). Proteins were detected using by ImageQuant LAS 4000 Mini Imaging System (GE Healthcare).
A498 cells were seeded onto 6-well plates at a concentration of 0.2×106 cells/well and incubated overnight at 37°C. The pcDNA 3.1 vector and pcDNA 3.1 c-FLIPL plasmid were provided by Professor Tae-Jin Lee (Yeungnam University, South Korea). They were then transfected with control plasmid pcDNA 3.1 vector or pcDNA 3.1-c-FLIPL plasmid for 5 h using lipofectamine 2000 (catalog no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) in Opti-MEM medium (catalog no. 31985-070; Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection, the cells were cultured in DMEM supplemented with 10% FBS for 12 h. Next, the cells were treated with metformin for 24 h. Finally, the cells were analyzed for c-FLIPL expression using western blotting.
c-FLIPL mRNA expression was determined by RT-PCR. Total RNA was extracted from A498 cells using the EasyBlue reagent (catalog no. 17061; Thermo Fisher Scientific, Inc.). cDNA was prepared using M-MLV reverse transcriptase (catalog no. 18057018; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. In addition, the total cellular RNA was reverse-transcribed using a random primer and subsequently amplified using PCR. PCR primers were purchased from GenoTech (Daejeon, Korea). GAPDH was used as the internal control. The PCR cycling conditions used were as follows: For c-FLIPL: 95°C for 45 sec, 54°C for 45 sec, 72°C for 45 sec (33 cycles) and for GAPDH: 94°C for 30 sec, 58°C for 45 sec, 72°C for 42 sec (25 cycles). The following primer sequences were used to amplify c-FLIPL and GAPDH: For c-FLIPL: 5′-CGGACTATAGAGTGCTGATGG-3′ (forward) and 5′-GATTATCAGGCAGATTCCTAG-3′ (reverse); and for GAPDH: 5′-AGGTCGGAGTCAACGGATTTG-3′ (forward) and 5′-GTGATGGCATGGACTGTGGT-3′ (reverse). PCR products were analyzed by electrophoresis using 1.5% agarose gels and visualized by ethidium bromide using UV light gel (catalog no. WGD30; DAIHAN Scientific, Seoul, Korea).
A498 cells were plated in 6 well plates at a density of 0.4×106 cells/well and incubated for 24 h. The cells were incubated with metformin for 1 h and loaded with 10 µM 2′,7′-dichlorofluorescin diacetate (H2DCFDA; Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. Next, they were washed three times with PBS. Fluorescence was measured using flow cytometry. ROS generation was assessed by the dichlorofluorescence in fluorescence intensity (FL-1, 530 nm) of 10,000 cells using the BD FACS Cato II flow cytometer (BD Biosciences).
Data were analyzed using one-way ANOVA followed by post hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences 8.0 (SPSS, Inc., Chicago, IL, USA). At least three independent experiments were performed. The data were expressed as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.
Previous studies reported that metformin induces apoptosis in renal cancer and breast cancer cell lines (
Caspases are important regulators of apoptotic cell death associated with apoptotic signaling pathways in various cancer cells (
The role of the caspase signaling pathway in metformin-mediated apoptosis was investigated. As presented in
The association between caspase-8 activation and metformin-mediated apoptosis was investigated. Treatment with metformin led to c-FLIPL degradation, which was recovered by z-VAD-fmk (
Metformin has been used as a therapeutic agent for diabetic patients (
Apoptosis is the process of programmed cell death that is closely associated with caspase activation. Caspases can be divided into two main groups, namely initiator caspases (caspase-2, −8, −9 and −10) and executioner caspases (caspase-3, −6 and −7) (
Reactive oxygen species (ROS) are important mediators of apoptosis in several cancer cell lines (
Taken together, these results demonstrated that metformin-induced apoptosis was mediated by the degradation of c-FLIPL protein via activation of caspase-8 in A498 human renal cell carcinoma cells. This suggested that metformin can serve the role of a chemotherapeutic agent for diabetes, as well as an anti-cancer agent.
Not applicable.
The present study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant no. 2017R1D1A1B3030961).
All data generated or analyzed during present study are included within the article.
JJ and JK designed the study, collected and analyzed the data. TL, IS and ES advised on the morphological images and performed the data analysis. JJ and JK drafted and wrote the manuscript. TL and JK revised the manuscript critically for intellectual content. All authors gave intellectual input to the study and approved the final version of the manuscript.
This research was approved by the Ethics Committee of Yeungnam University (Daegu, South Korea). All procedures were performed according to the ethical standards of Ethics Committee of Yeungnam University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Not applicable.
The authors declare that they have no competing interests.
Metformin induces apoptosis in a dose-dependent manner in A498 cells. (A) A498 human renal carcinoma cells were treated with the indicated concentration of metformin. After 24 h, morphological changes were observed using a microscope at ×200 magnification. (B) A498 cells were treated with metformin (0, 5, 7.5 and 10 mM) for 24 h. Apoptosis was determined by flow cytometry. Representative FACS histograms are presented in the upper panel, cumulative data are presented in the lower panel. (C) A498 cells were treated with metformin for 24 h, and PARP, procaspase-3 and β-actin expression was analyzed using western blotting. β-actin was used as the loading control. All data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 compared with untreated cells. FACS, Fluorescence-activated cell sorting; PARP, poly (ADP-ribose) polymerase.
Metformin-induced apoptosis is associated with activation of procaspase-8 and degradation of c-FLIPL. (A) A498 cells were treated with metformin for 24 h, and procaspase-2 and procaspase-8 expression was analyzed using western blotting. β-actin was used as the loading control. (B) A498 cells were treated with various concentrations of metformin for 24 h. c-FLIPL, Bcl-2, Bcl-xL, Mcl-1, XIAP and c-IAP2 expression was determined using western blot analysis. β-actin served as the loading control. c-FLIP, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra-large; Mcl-1, myeloid cell leukemia-1, XIAP, X-linked inhibitor of apoptosis protein; c-IAP, cellular inhibitor of apoptosis protein, baculoviral IAP repeat containing 3.
Metformin induces apoptosis by a caspase-dependent signaling pathway in A498 cells. (A) A498 cells were pretreated with 50 µM z-VAD-fmk or a solvent for 30 min and incubated with 10 mM metformin for 24 h. Morphological features were analyzed using light microscopy at ×200 magnification. (B) A498 cells were cultured with 50 µM z-VAD-fmk or the vehicle for 30 min prior to treatment with metformin (10 mM) for 24 h. The sub-G1 fraction was determined using flow cytometry. Representative FACS histograms are presented in the upper panel, cumulative data are presented in the lower panel. (C) A498 cells were incubated with 10 mM metformin for 24 h in the presence or absence of 50 µM z-VAD-fmk. PARP, procaspase-3 and β-actin expression was analyzed using western blotting. β-actin was used as the loading control. Band density of procaspase-3 was analyzed using ImageJ software. All data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 compared with untreated cells, #P<0.05 compared with metformin-treated cells. z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluorometyhlketone; PARP, poly(ADP-ribose) polymerase.
Degradation of c-FLIPL protein is regulated via a caspase-dependent signaling pathway. (A) A498 cells were treated with 10 mM metformin for 24 h in the presence or absence of 50 mM z-VAD-fmk. c-FLIPL and β-actin expression was analyzed using western blotting. β-actin was used as the loading control. Band density of the c-FLIPL protein was determined using the ImageJ program. (B) A498 cells were incubated with the indicated concentrations of metformin for 24 h. c-FLIPL mRNA level was determined using reverse transcription-polymerase chain reaction. (C) A498 cells transfected with pcDNA3.1 or c-FLIPL plasmid were treated for 24 h with different concentrations of metformin. The sub-G1 cell population was analyzed using flow cytometry. (D) The pcDNA3.1 and c-FLIPL transfected cells were treated with varying concentrations of metformin. PARP, procaspase-3 and c-FLIPL expression levels were determined using western blotting. β-actin was used as the loading control. All data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 compared with untreated cells, &P<0.05 compared with metformin-treated pcDNA3.1 cells. z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl ketone; c-FLIP, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; PARP, poly(ADP-ribose) polymerase.
Metformin-mediated apoptosis in A498 cells was not affected by reactive oxygen species generation. (A) Cells were pretreated with 5 mM NAC for 30 min and incubated with 10 mM metformin for 1 h. H2DCFDA fluorescence was quantified with flow cytometry. (B) A498 cells were incubated with 5 mM NAC or the vehicle for 30 min prior to treatment with 10 mM metformin for 24 h. Morphological changes were analyzed using a light microscope at ×200 magnification. (C) A498 cells were pretreated with 5 mM NAC or the solvent for 30 min and incubated in the presence or absence of 10 mM metformin for 24 h. The sub-G1 cell fraction was analyzed using flow cytometry. (D) A498 cells were pretreated with 5 mM NAC or the solvent for 30 min and incubated with 10 mM metformin for 24 h. PARP, procaspase-3, c-FLIPL and β-actin expression levels were analyzed using western blotting. β-actin was used as the loading control. All data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 compared with untreated cells, #P<0.05 compared with metformin-treated cells. NAC, N-acetyl-L-cysteine; PARP, poly(ADP-ribose) polymerase; c-FLIP, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; ROS, reactive oxygen species.