Abstract
Background/Aim: The present study aimed to investigate the apoptotic effects of phenformin, a therapeutic agent for diabetes, on head and neck squamous cell carcinoma (HNSCC). Materials and Methods: Cytotoxicity was measured by the MTT and live/dead cell assay. Phenformin-induced apoptotic FaDu cell death and its associated cellular signaling pathways were investigated by hematoxylin and eosin staining, 4’,6-diamidino-2-phenylindole staining, caspase-3 activity assay, fluorescence-activated cell sorting analysis, and western blotting. Results: Phenformin promoted death of and apoptotic processes in FaDu cells, including morphological alterations and nuclear condensation. Furthermore, treatment with phenformin increased caspase-3 activity and apoptotic populations via the caspase cascade through cleavage of capspase-8, -9, and -3 and poly(ADP-ribose) polymerase in FaDu cells. Moreover, phosphorylation levels of mitogen-activated protein kinases, nuclear factor-κB, and AKT were down-regulated in FaDu cells by phenformin. Conclusion: Phenformin induced death of FaDu cells via caspase-dependent extrinsic and intrinsic apoptosis pathways and is a promising novel therapeutic agent for HNSCC.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer (1, 2). Although HNSCC is unexpectedly complex in terms of pathophysiological etiology, morphological characteristics, and clinical features, it generally arises from tissues of the oral cavity, including the oropharynx, larynx, and hypopharynx (2, 3). Not only are tobacco smoking and alcohol consumption traditionally implicated as representative risk factors for HNSCC (4), but infection with human papillomavirus has also recently been proven as one of its pathophysiological etiologies (5). Lymphatic metastatic spread of HNSCC significantly reduces the 5-year overall survival rate by approximately 40 to 50% (6, 7). Various clinical interventions, including surgery, radiotherapy, and chemotherapy, are performed for patients with HNSCC (8). Although there are still grave concerns in regard to the balance between drug response and adverse drug reactions, new clinical treatment strategies for HNSCC are warranted (8). However, despite advances in clinical interventions for HNSCC, the frequency of distant metastasis and the overall survival rate of patients with HNSCC, has not significantly improved over the past few decades (9). Hence, there is an urgent need to develop effective chemotherapeutic agents with minimal side-effects for patients with HNSCC.
Phenformin (CAS number 114-86-3, C10H15N5), shown in Figure 1, is a biguanide with anti-diabetic activity (10). Previous preclinical and clinical studies suggested that phenformin not only has a high safety profile (11), but also shares similar mechanisms of action with metformin in inhibiting cell proliferation, and inducing cell-cycle arrest, and apoptosis of various cancer cells (12). Recently, phenformin was found to exert antitumor effects, including the suppression of cell proliferation and the induction of apoptosis in various cancer types, including breast (11), lung (13), melanoma (14), glioblastoma (15) and colon (16). Thus, the present study aimed to evaluate potential anti-tumorigenic effects and cellular signaling pathways that mediate the effects of phenformin in HNSCC cells.
Materials and Methods
Cell culture. FaDu cells, a human HNSCC cell line, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). FaDu cells were grown in minimum essential medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and were cultured in a humidified incubator at 37°C with 5% CO2 according to the ATCC guidelines.
Cell viability assay. In order to determine the half-maximal inhibitory concentration (IC50) of phenformin, FaDu cells (1×105 cells/ml) were cultured in 96-well plates and treated with 0, 0.25, 0.5, 1, 2, and 4 mM phenformin for 24 h at 37°C. Thereafter, the cells were incubated for a further 4 h with 20 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Life Technologies). The supernatant was removed, and 200 μl/well dimethyl sulfoxide were added to the samples with to dissolve the MTT crystals. The optical density (OD) of each well was measured at 570 nm using a spectrophotometer (Epoch microplate spectrophotometer; BioTek instruments, Winooski, VT, USA).
Cell survival assay. To verify the survival of FaDu cells treated with phenformin, cell survival assay was performed using a live/dead cell viability assay kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Briefly, FaDu cells (1×105 cells/ml) were cultured in an eight-well chamber slide (Sigma-Aldrich, St. Louis, MO, USA) and treated with 0, 0.5, and 1 mM phenformin for 24 h at 37°C. Thereafter, FaDu cells were stained with green calcein-AM to stain the live cells (green fluorescence) and with ethidium homodimer-1 to stain the dead cells (red fluorescence). The cells were imaged using a fluorescence microscope (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).
Hematoxylin and eosin (H & E) staining. H & E staining was performed to allow observation of the morphological alterations in phenformin-treated FaDu cells. Briefly, FaDu cells (1×105 cells/ml) were cultured in an eight-well chamber slide (Sigma-Aldrich) and then treated with 0, 0.5, and 1 mM phenformin for 24 h at 37°C. Thereafter, FaDu cells were fixed with 4% paraformaldehyde for 30 min at 4°C and H&E staining was performed. The cells were observed and imaged using a microscope (Leica DM750, Leica Microsystems, Heerbrugg, Switzerland) equipped with a 3MP Color Diagnostic Monitor (CX30p, Wide, Yongin, Republic of Korea).
Nuclear staining. To determine whether phenformin induced nuclear condensation in FaDu cells, 4’,6-diamidino-2-phenylindole (DAPI) staining was performed. Briefly, FaDu cells (1×105 cells/ml) were cultured in an eight-well chamber slide (Sigma-Aldrich) and treated with 0, 0.5, and 1 mM phenformin for 24 h at 37°C. Thereafter, cells were stained with 1 mg/ml DAPI (Sigma-Aldrich) for 20 min. Nuclear condensation was observed and imaged using a fluorescence microscope (Eclipse TE200; Nikon Instruments).
Caspase-3/-7 activity assay. To determine whether phenformin increases the activity of caspase-3/-7 in FaDu cells, caspase-3/-7 activity was assayed using the cell-permeable fluorogenic substrate PhiPhiLux-G1D2 kit (OncoImmunin Inc.; Gaithersburg, MD, USA) according to the manufacturer's instructions. Cells were imaged using fluorescence microscopy (Eclipse TE200; Nikon Instruments).
Fluorescence-activated cell sorting analysis (FACS). In order to verify whether phenformin increases the proportion of apoptotic FaDu cells, FACS was performed using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Cell Signaling Technology, Danvers, MA, USA). Briefly, FaDu cells (5×105 cells/ml) were cultured on a six-well plate for 24 h, after which cells were treated with 0, 0.5, and 1 mM phenformin for 24 h. Thereafter, the collected FaDu cells were stained with annexin V-FITC and PI and incubated for 15 min at 37°C. Afterwards, the apoptotic populations were analyzed using BD Cell Quest® version 3.3 (Becton Dickinson, San José, CA, USA).
Western blot analysis. FaDu cells (5×105 cells/ml) cultured on a six-well plate were treated with 0, 0.5, and 1 mM phenformin for 24 h. Thereafter, cell lysates were prepared using a cell lysis buffer (Cell Signaling Technology) according to the manufacturer's instructions. Protein concentrations were measured by performing the bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of the cell lysates were then electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then analyzed by western blotting using antibodies to Fas ligand (FASL, 40 kDa; Cell Signaling Technology), caspase-8 (18, 43, and 57 kDa; Cell Signaling Technology), BH3-interacting domain death agonist (BID, 20 kDa; Santa Cruz Biotechnology Inc., Dallas, TX, USA), B-cell lymphoma-2 (BCL2, 26 kDa, Santa Cruz Biotechnology Inc.), BCL-extra large (BCL-xL, 30 kDa; Cell Signaling Technology), BCL2-associated death promoter (BAD, 23 kDa; Cell Signaling Technology), BCL2-associated X protein (BAX, 20 kDa; Cell Signaling Technology), p53 (53 kDa; Cell Signaling Technology), cleaved caspase-3 (17 and 19 kDa; Cell Signaling Technology), poly(ADP-ribose) polymerase (PARP, 89 and 116 kDa; Cell Signaling Technology), β-actin (45 kDa; Santa Cruz Biotechnology Inc.), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2, 42 and 44 kDa; Santa Cruz Biotechnology Inc.), total ERK (42 and 44 kDa; Santa Cruz Biotechnology Inc.), phospho-p38 (38 kDa; Santa Cruz Biotechnology Inc.), total p-38 (38 kDa; Santa Cruz Biotechnology Inc.), phospho-protein kinase B (AKT, 60 kDa; Cell Signaling Technology), total-AKT (60 kDa; Cell Signaling Technology), phospho-nuclear factor kappa B (NFκB, 65 kDa; Cell Signaling Technology), and total NFκB (65 kDa; Cell Signaling Technology). The immunoreactive bands were visualized using the ECL System (Amersham Biosciences, Piscataway, NJ, USA), exposed on radiographic film or MicroChemi 4.2 (Dong-Il SHIMADZE Crop., Seoul, Korea).
Caspase-dependent cell survival assay. FaDu cells (1×105 cells/ml) cultured in a 96-well plate for 24 h. After incubation, FaDu cells were treated with 1 mM phenformin in the presence or absence of the cell-permeant pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-VAD-fmk; Santa Cruz Biotechnology Inc.) and were incubated for 24 h. Thereafter, western blotting of caspase-3 and MTT assay were conducted to verify whether phenformin-induced FaDu cell death is mediated by the caspase cascade.
Statistical analysis. Experimental data are presented as the mean±standard deviation of at least three independent experiments and were analyzed by analysis of variance, followed by Student's t-tests. Values of p<0.05 were considered statistically significant.
Results
The MTT assay was performed to verify whether phenformin promotes cell cytotoxicity. As shown in Figure 2A, FaDu cells treated with 0.25, 0.5, 1, 2, and 4 mM phenformin for 24 h increased cell cytotoxicity in a dose-dependent manner by 8.8±2.5% (p<0.05), 13.7±4.7% (p<0.05), 27.8±1.3% (p<0.01), 67.2±0.9% (p<0.01), and 79.3±1.6% (p<0.01) relative to the untreated control. Furthermore, the IC50 value of phenformin in FaDu cells value was estimated to be approximately 1.5 mM phenformin. However, to confirm that phenformin induced FaDu cell death by increasing cytotoxicity, live and dead cells were stained using a live/dead cell assay kit, in which cell-permeable green calcein AM stains live cells to produce green fluorescence, while dead cells are stained with ethidium homodimer-1 and produce red fluorescence. As shown in Figure 3A, the number of FaDu cells was reduced by treatment with 0.5 and 1 mM phenformin compared to those of the control. Furthermore, phenformin increased the number of dead FaDu cells in a dose-dependent manner compared to the controls. Taken together, our results indicate that phenformin induces cell death in a dose-dependent manner in FaDu cells.
To determine whether phenformin-mediated FaDu cell death is involved in apoptosis, the morphological alterations of FaDu cells treated with phenformin were observed with H & E staining. As shown in Figure 3B, phenformin not only reduced the number of FaDu cells, which is consistent with the results of live/dead cell assay, but also increased the number of FaDu cells with altered morphology, including cell shrinkage, which is closely associated with apoptotic cell death. Next, results of DAPI staining showed that phenformin promoted nuclear condensation in FaDu cells, as shown in Figure 3C. Furthermore, phenformin treatment increased the activity of capspase-3/-7 in FaDu cells in a dose-dependent manner, as shown as Figure 3D. Next, in order to verify whether phenformin induced apoptosis of FaDu cells, FACS analysis was performed using annexin-V-FITC and PI. Results of FACS analysis, as shown as Figure 3E, showed that the total proportion of dead cells was approximately 15.95% and 39.2% in FaDu cells treated with 0.5 and 1 mM phenformin, respectively. Among these, cells in the early and late stages of apoptosis were verified to comprise 12.1% and 21.52%, respectively, in FaDu cells treated with 0.5 and 1 mM phenformin. Taken together, the above findings consistently indicated that apoptosis is involved in phenformin-induced FaDu cell death.
Next, to investigate phenformin-induced apoptosis pathways in FaDu cells, we performed western blotting using specific antibodies to proteins associated with the extrinsic and intrinsic apoptosis pathways. As shown in Figure 4A, phenformin up-regulated FASL (40 kDa) expression in FaDu cells in a dose-dependent manner. Phenformin treatment led to a dose-dependent increase in the protein level of cleaved caspase-8 (18 and 43 kDa), a downstream substrate of FASL, in FaDu cells. Furthermore, the up-regulation of cleaved caspase-8 the activation of caspase-3 (17 and 19 kDa) and PARP (89 kDa) in FaDu cells treated with phenformin, as shown in Figure 4C. Hence, the above findings indicate that phenformin-induced cell death is mediated by the extrinsic apoptosis pathway, which involves the cascade activation of caspase-8, caspase-3, and PARP via FASL up-regulation in FaDu cells. Furthermore, phenformin down-regulated the expression of BID (20 kDa) in a dose-dependent manner. The expression levels of anti-apoptotic factors BCL2 (26 kDa) and BCL-xL (30 kDa), were down-regulated by phenformin in a dose-dependent manner (Figure 4B). On the other hand, phenformin treatment induced a dose-dependent increase in the expression levels of pro-apoptotic factors BAD (23 kDa) and BAX (20 kDa), and the tumor-suppressor p53 (53 kDa) in FaDu cells. Phenformin also increased the protein level of cleaved caspase-9 (17 and 38 kDa) in a dose-dependent manner. Hence, our findings indicate that phenformin-induced FaDu cell death also involves the mitochondria-dependent intrinsic apoptosis pathway.
Next, to verify whether phenformin-induced FaDu cell death is mediated by the caspase cascade, we conducted western blotting of capsase-3 and MTT assay on FaDu cells treated with phenformin in the presence or absence of the cell-permeant pan-caspase inhibitor Z-VAD-fmk. As shown in Figure 5A, although the cleaved caspase-3 level was up-regulated in FaDu cells treated with 1 mM phenformin, it cleaved caspase-3 levels were lower in the presence of 20 μM Z-VAD-fmk. Furthermore, the viability of FaDu cells significantly decreased (p<0.01) relative to those of control cells as shown in Figure 5B. On the other hand, Z-VAD-fmk increased the viability of FaDu cells by approximately 23% (p<0.01) in the presence of 1 mM phenformin. Taken together, the above findings indicate that phenformin-induced FaDu cell death is dependent on the activation of the caspase cascade.
Next, to investigate the cellular signaling pathways related to phenformin-induced apoptosis, we performed western blotting using specific antibodies to mitogen-activated protein kinases (MAPKs), NFκB, and AKT, as shown in Figure 6. Interestingly, phenformin reduced the phosphorylation levels of ERK1/2, p38, NFκB, and AKT in FaDu cells in a dose-dependent manner.
Discussion
In present study, the results showed that phenformin-induced FaDu cell death is mediated by caspases via the FASL-triggered extrinsic and intrinsic apoptosis pathways. Furthermore, phenformin-induced FaDu cell death is closely involved in alterations in MAPK, including the ERK1/2 and p38, NFκB, and AKT signaling pathways.
Phenformin is a biguanide with anti-diabetic activity that is almost 50-fold more potent than metformin, and is the most widely prescribed treatment for type II diabetes (17, 18). Metformin is a biguanide drug that has a similar chemical structure and similar pharmacological effects to those of phenformin (19, 20). Although the anticancer efficacy of metformin in diabetic patients remains to be investigated, epidemiological studies have suggested that metformin reduces incidence and risk of types of cancer (21), including gastric, colorectal (22), pancreatic (23), and lung cancer (24). Furthermore, recent studies reported that metformin suppressed cell proliferation (25, 26), migration (27), and angiogenesis (11, 28) and induces cell arrest (29) and apoptosis (30) in various types of cancer cell. Currently, clinical trials that investigate metformin-induced anticancer efficacy in various types of cancer were conducted (31).
Apoptosis, also known as programmed cell death, is generally characterized by distinct morphological characteristics, including cellular shrinkage, condensation and margination of the chromatin, ruffling plasma membrane, and the formation of apoptotic bodies (32, 33). Apoptosis is a crucial cellular mechanism that maintains tissue homeostasis via the elimination of harmful or unnecessary cells (34). Hence, there is an urgent need to develop novel chemotherapeutic agents that can trigger cancer-specific cell death such as via apoptosis (34). Interestingly, recent studies reported that phenformin, which has similar chemical structure and physiological effects to those of metformin, not only inhibits cell proliferation and angiogenesis, but also induces cell arrest and apoptosis in various cancer types, including cholangiocarcinoma, breast, and non-small cell lung cancer (11, 13, 35-37). Consistent with the findings of previous studies associated with phenformin-induced cancer cell apoptosis, phenformin-induced FaDu cell death was found to be mediated by apoptotic processes, such as cellular shrinkage and nuclear condensation (Figure 2, and 3A-C). Furthermore, our results based on the caspase-3/-7 activity assay using cell-permeable fluorogenic substrate PhiPhiLux-G1D2 kit (OncoImmunin Inc., Gaithersburg, MD, USA) and FACS analysis using PI and annexin-V showed that phenformin increased the activity of caspase-3 (Figure 3D) and the proportion of apoptotic FaDu cells (Figure 3E). In particular, caspase-3 is a key pro-apoptotic factor that induces apoptotic cell death by catalyzing the cleavage of specific cellular proteins (38). Therefore, these findings indicated that phenformin-induced FaDu cell death involves apoptosis. Generally, cellular mechanisms underlying apoptosis can be classified into the extrinsic death receptor-mediated and the intrinsic mitochondria-dependent pathways (33). These are closely regulated by apoptotic initiators caspase-8 and -9 of the extrinsic and intrinsic apoptosis pathways, respectively. Furthermore, the activation of both caspase-8 and -9 can induce the ‘executive’ caspases include caspase-3, -6, and -7 (39). Hence, the activation of caspases has being considered as an important chemotherapeutic strategy. As shown in Figure 4, phenformin-induced FaDu cell death was mediated by the activation of caspase-3 through the activation of apoptotic initiators caspase-8 and -9 located on extrinsic death receptor-mediated and intrinsic mitochondria-dependent pathways, respectively. Furthermore, Figure 5 showed that treatment with the pan-caspase inhibitor Z-VAD-fmk not only counteracted phenformin-induced FaDu cell death, but also suppressed the expression of cleaved caspase-3 in phenformin-treated FaDu cells. These findings demonstrated that phenformin induced a caspase-dependent apoptotic cell death of FaDu cells through the extrinsic death receptor-mediated and intrinsic mitochondria-dependent pathways.
Recent studies that investigated the anticancer effects of phenformin reported the suppression of ERK phosphorylation in tumor suppressor neurofibromin-mutant melanoma (40) and breast cancer cells (36). Furthermore, Wang et al. reported that phenformin inhibited cell growth and angiogenesis through the suppression of ERK and AKT phosphorylation in non-small cell lung cancer cells (13). Similarly to previous studies, phenformin suppressed the phosphorylation of MAPKs ERK, p38, NFκB, and AKT in FaDu cells. Hence, these cellular signaling pathways appear to be closely associated with phenformin-induced apoptosis of FaDu cells.
In conclusion, our findings demonstrated that phenformin induces caspase-dependent FaDu cell death via FASL-induced extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis. Hence, our results highlighted the potential use of phenformin and other biguanides as HNSCC therapeutics.
Acknowledgements
This study was supported by research fund from Chosun University Dental Hospital, 2018.
Footnotes
Authors' Contributions
Y.S.S., T.H.K., H.L., and J.S.K. contributed to the experimental work; All authors including J.S.O., J.S.Y., G.J.L., S.K.Y., D.K.K., H.J.K., C.S.K., S.Y.L., S.G.W., and J.S.K. participated in data analysis and interpretations; Y.S.S. and J.S.K drafted the article. All Authors gave final approval for publication.
Conflicts of Interest
The Authors have no conflicts of interest.
- Received April 29, 2019.
- Revision received June 2, 2019.
- Accepted June 4, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved