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. 2020 Aug;31(7):655-662.
doi: 10.1097/CAD.0000000000000966.

Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells

Ji Hoon Jang  1 , Eon-Gi Sung, In-Hwan Song, Tae-Jin Lee, Joo-Young Kim
Affiliations

Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells

Ji Hoon Jang et al. Anticancer Drugs. 2020 Aug.

Abstract

Bladder cancer (BC) is the sixth most common cancer in men. Moreover, chemotherapy for BC leads to various side effects. Metformin is known to induce apoptosis in vitro in many types of cancer. Furthermore, it has feasibility as a drug repositioning used for the treatment of cancer. The molecular mechanism of metformin mediating apoptosis in BC is still unclear. In this study, we showed that metformin stimulated the caspase-dependent apoptotic signaling pathway in T24 cells, a human BC cell line. Moreover, the induced apoptosis was partially inhibited by a general caspase inhibitor, z-VAD-fmk, which suggested that metformin-induced apoptosis in T24 cells is partially caspase-independent. Notably, we observed the nuclear translocation of apoptosis-inducing factors (AIFs) in metformin-promoted apoptosis, which is a typical characteristic of the caspase-independent apoptotic pathway. In addition, we found that metformin-mediated apoptosis occurred via degradation of the cellular FADD-like interleukin-1β-converting enzyme inhibitory protein (c-FLIP) by facilitating ubiquitin/proteasome-mediated c-FLIPL degradation. Furthermore, treatment with the reactive oxygen species scavenger N-acetylcysteine, failed to suppress metformin-induced apoptosis and c-FLIPL protein degradation in metformin-treated T24 cells. In conclusion, these results indicate that metformin-induced apoptosis was mediated through AIF-promoted caspase-independent pathways as well as caspase-dependent pathways in T24 cells. As such, metformin could be used as a possible apoptotic agent for the treatment of BC.

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Conflict of interest statement

There are no conflictss of interest.

Figures

Fig. 1
Fig. 1
Metformin induces apoptosis in T24 cells dose-dependently. (a) T24 cells were treated with the indicated concentration of metformin. After 24 h, cell viability was assessed using the XTT assay. (b) The morphological changes were examined by inverted microscope (magnification, ×200). (c) T24 cells were treated with metformin (0, 3, 6, 9, 12 and 15 mM) for 24 h. FACS analysis is shown in the upper panel. Apoptosis confirmed by flow cytometry is shown in the middle panel. Cell cycle phase is shown in the lower panel. (d) T24 cells were treated with metformin. PARP, cleaved-caspase-3 and β-actin expression levels were detected by western blot. β-actin was used as a control. Arrows indicate PARP and caspase-3 cleavage form. Data are representative from three independent experiments. The data are expressed as mean ± SD (n = 3). *P < 0.05 compared to non-treated cells. FACS, fluorescence-activated cell sorting.
Fig. 2
Fig. 2
Metformin suppresses the expression of c-FLIPL proteins in T24 cells. (a) T24 cells were treated with various concentrations of metformin for 24 h. c-FLIPL, Bcl-2, Mcl-1, XIAP and β-actin expression levels were detected by western blot. β-actin was used as a control. (b) T24 cells were cultured with the concentrations of metformin. After 24 h, the c-FLIPL mRNA level was analyzed by RT-PCR. Data are representative from three independent experiments.
Fig. 3
Fig. 3
Metformin-mediated apoptosis in T24 cells is partially blocked by inhibition of caspase-dependent signaling pathway. (a) T24 cells were pretreated with 50 μM z-VAD-fmk or a solvent for 30 minutes and incubated with 12 and 15 mM metformin for 24 h. Cell viability was assessed using the XTT assay. (b) T24 cells were incubated with 50 μM z-VAD-fmk or a vehicle for 30 min before treatment with metformin (12 and 15 mM). After 24 h, the morphological changes were examined using an inverted microscope (magnification, ×200). (c) Cells were pretreated with 50 μM z-VAD-fmk or a solvent for 30 minutes and incubated with 12 and 15 mM metformin for 24 h. The sub-G1 cell fraction was confirmed by flow cytometry. (d) T24 cells were treated with 50 μM z-VAD-fmk or a vehicle for 30 minutes before treatment with metformin for 24 h. PARP, cleaved-caspase-3, c-FLIPL and β-actin expression levels were detected by western blot. β-actin was used as a control. Arrows indicate PARP and caspase-3 cleavage form. Data are representative from three independent experiments. The data are expressed as mean ± SD (n = 3). *P < 0.05 compared to non-treated cells, #P < 0.01 compared with metformin-treated cells.
Fig. 4
Fig. 4
ROS are not involved in metformin-induced apoptosis. (a) Cells were cultured with 5 mM NAC or a vehicle for 30 minutes before treatment with metformin (12 and 15 mM). After 24 h, cell viability was determined by XTT assay. (b) T24 cells were pretreated with 5 mM NAC or a solvent for 30 minutes and then incubated with metformin for 24 h. Morphological features were examined by inverted microscope (magnification, ×200). (c) Cells were cultured with 5 mM NAC or a vehicle for 30 minutes before treatment with metformin (12 and 15 mM). After 24 h, the sub-G1 fraction was confirmed by flow cytometry. (d) T24 cells were incubated with different concentrations of metformin for 24 h in the presence or absence of 5 mM NAC. PARP, cleaved-caspase-3 and β-actin expression levels were detected by western blot. β-actin was used as a control. Arrows indicate PARP and caspase-3 cleavage form. Data are representative from three independent experiments. The data are expressed as mean ± SD (n = 3). *P < 0.05 compared to non-treated cells. NAC, N-acetylcysteine; ROS, reactive oxygen species.
Fig. 5
Fig. 5
AIF translocation is required for metformin-induced apoptosis in T24 cells. Cells were treated with metformin for 24 h. Next, cytoplasmic and nucleus fractions were prepared and detected by western blot using AIF, α-tubulin (cytosolic loading control) and lamin B (nucleus loading control) antibodies. Data are representative from three independent experiments. The data are expressed as mean ± SD (n = 3). *P < 0.05 compared to non-treated cells. AIF density was performed by the ImageJ software.
Fig. 6
Fig. 6
Metformin-mediated apoptosis occurs via proteasomal signaling pathways in T24 cells. (a) Cells were treated with or without metformin 15 mM in the presence or absence of 20 μg/ml cycloheximide (CHX) for the indicated times. c-FLIPL and β-actin expression levels were detected by western blot. β-actin was used as a control for western blot. (b) T24 cells were incubated with 0.5 μM MG132 or a vehicle for 1 h before treatment with metformin for 24 h. Data are representative from three independent experiments. The data are expressed as mean ± SD (n = 3). *P < 0.05 compared to non-treated cells, #P < 0.01 compared to metformin-treated cells. The c-FLIPL density was analyzed by the ImageJ software.
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