We investigated the effect of microRNA-375 (miR-375) on tumour necrosis factor-alpha (TNF-α)-induced cell death in head and neck squamous cell carcinoma, and further explored the potential molecular mechanism underlying this phenomenon. Cal27 cells were transfected with miR-375 mimic and subsequently treated with or without TNF-α (10 ng/ml). An additional group of cells were treated with TNF-α alone. The resulting morphological changes were observed, and the percentage of sub-G1 cells was measured. The protein expression and cleavage of caspase 3, caspase 8, and poly(ADP ribose) polymerase (PARP) were determined through Western blotting. The results showed a significant increase in cell death in the combination group, but not in the groups treated with miR-375 mimic, TNF-α alone, or control. The data obtained from sub-G1 cells supported the notion that miR-375 increases the accumulation of sub-G1. In the combination group, the degradation of caspase 3, caspase 8, and PARP was observed and the cleavage of these enzymes was detected. The pan-caspase inhibitor, Z-VAD, inhibited the apoptosis of Cal27 cells treated with a combination of miR-375 mimic and TNF-α. In addition, the apoptosis inhibitory proteins, cFLIP-L and cIAP1, were down-regulated in a time-dependent manner. Taken together, these data suggest that miR-375 sensitizes TNF-α-induced apoptosis, and the reduction in the expression of the apoptosis inhibitory proteins cFLIP-L and cIAP2 plays an important role in this sensitization.
Head and neck squamous cell carcinoma (HNSCC) in the oral cavity, oropharynx, larynx, or hypopharynx remains one of the most common malignancies worldwide, despite the steady decrease in the incidence of this cancer type. The clinical outcome of HNSCC has gradually improved, resulting in a decline in deaths from this disease; however, the prognosis of patients with advanced stage HNSCC remains dismal, and the overall 5-year survival rate for patients with HNSCC is low.
Multimodal therapy, including surgery, radiotherapy, chemotherapy, and biotherapy, has been applied to this disease. Because of the nature of squamous cell carcinoma, surgery has become the primary treatment, with other therapies considered as secondary. However, the appropriate selection and application of other therapies undoubtedly improves the effects of surgical treatment and increases the survival rate of patients.
Apoptosis is an inducible form of controlled cell death essential for normal human embryogenesis and homeostatic and protective processes. The failure of apoptosis has been implicated in tumour development and resistance to cancer therapy; thus, the apoptosis of cancer cells would be a beneficial and feasible method for cancer prevention and cancer therapy.
Tumour necrosis factor-alpha (TNF-α), which was first identified as an anti-tumour therapy, is a crucial inflammatory cytokine. TNF-α induces apoptosis via endogenous mitochondrial cytochrome C and exogenous death receptors. TNF-α also activates nuclear transcription factor-kappaB (NF-κB), which controls apoptosis through the regulation of the expression of apoptosis inhibitor genes, such as Bcl-2, Bcl-xL, cIAPs, xIAP, and cFLIP. As an inflammatory cytokine, TNF-α enhances cell viability. The long-term low-dose administration of TNF-α activates NF-κB, which promotes cell survival. TNF-α induces tumour cell death at concentrations greater than those that a normal cell or the body can tolerate, and this severe toxicity limits the application of TNF-α in cancer therapy. Thus, the use of TNF-α in combination with a sensitizer is important to increase the susceptibility of tumour cells to TNF-α and reduce the toxicity of this cytokine.
The potential application of microRNAs (miRNAs) for cancer therapy has been demonstrated in many studies, although the functions of these molecules as sensitizers has rarely been observed. miRNAs are non-protein-coding small RNAs of 19–25 nucleotides cleaved from 70- to 100-nucleotide hairpin pre-miRNA precursors. miRNAs bind to complementary sequences in target mRNAs and induce mRNA degradation or translational repression, which greatly affects various biological processes, such as cellular differentiation, proliferation, and death. In cancer, miRNAs are profoundly involved in oncogenesis, tumour suppression, the epithelial–mesenchymal transition, and resistance or sensitivity to medical drugs.
According to comprehensive miRNA profiling for HNSCC, miRNA-375 (miR-375) is down-regulated and acts as a tumour suppressor in HNSCC progression. This has also been confirmed in other squamous cell carcinomas, such as gastric carcinoma.
In this study, we examined whether miR-375 augments TNF-α-induced cell death and explored some underlying molecular mechanisms.
Materials and methods
Reagents and antibodies
miR-375 mimic was purchased from RiboBio Company (Guangdong, China) and propidium iodine (PI) was from Sigma (St. Louis, MO, USA). Human recombinant TNF-α was obtained from R&D Systems (Minneapolis, MN, USA) and dissolved in 1% bovine serum albumin as a stock solution (TNF-α 100 μg/ml). The Lipo Max Transfection Reagent used for the transfection of miR-375 mimic was purchased from Life Technologies. The pan-caspase inhibitor, Z-VAD, was obtained from Calbiochem (San Diego, CA, USA). Anti-caspase 3 and anti-Bcl-xL antibodies were obtained from Cell Signalling Technology (Beverly, CA, USA). Anti-xIAP, anti-poly(ADP-ribose) polymerase (PARP) antibodies, anti-A20, and anti-cIAP2 were purchased from BD Transduction Laboratories (San Diego, CA, USA). Anti-cIAP1 and anti-tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FLIP antibody was obtained from Alexis, and anti-caspase 8 antibody was purchased from Pharmingen.
Cell culture and treatment
The HNSCC cell line, Cal27, was obtained from Sun Yat-sen University (Guangzhou, China). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) F12 (Invitrogen) supplemented with 10% foetal bovine serum (PAA Laboratories) and 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were cultured at 37 °C with 5% CO 2 and saturated humidity.
Four groups of cells were used in this study. The first group was used as a control. The second group was transfected with 50 nM miR-375 mimic, in accordance with the manufacturer’s instructions. The third group was treated with TNF-α alone, and the fourth group was treated with a combination of both effectors. The cells were transfected with mimic at 24 h after seeding and cultured for another 48 h. Subsequently, the cells were treated with or without TNF-α for an additional 24 h.
Measurement of cell death and apoptosis
Changes in the cell morphology were observed using an inverted phase contrast microscope. At the end of treatment, the cells were stained with a fluorescent dye, Hoechst 33258, and the type of cell death was analyzed.
Cells were harvested and centrifuged (1000 rpm, 10 min), and washed twice with phosphate buffered saline (PBS; pH 7.0). The precipitate was subsequently fixed in cold 70% ethanol solution for at least 2 h at −20 °C. The cell pellets were suspended in PBS (500 μl) and incubated with 2.4 μl RNase A (10 μg/ml) in the same volume of PI (40 μg/ml) in the dark for 30 min at room temperature. The stained cells were analyzed using a Becton–Dickinson FACSCalibur flow cytometer.
Apoptosis suppression and examination
We used the pan-caspase inhibitor Z-VAD to block the caspase cascade apoptosis. Cal27 cells were pretreated for 30 min with Z-VAD before individual treatments with either miR-375 mimic or TNF-α, or the combination of these effectors. The cells were collected for the detection of caspase 8, caspase 3, and PARP using Western blotting.
Western blotting analysis
After treatment, the cells were collected by scraping and washed twice with ice-cold PBS. Total protein was extracted from the cells using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, China) containing 1% of the protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Beyotime). The supernatant was collected after centrifugation at 16,000 × g for 15 min for Western blotting; equal amounts of proteins were fractionated on a sodium dodecyl sulfate (SDS) polyacrylamide gel in the Mini-PROTEAN II system (Bio-Rad, Hercules, CA, USA) and blotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in TBST (10 mmol/l Tris–HCl pH 7.5, 100 mmol/l NaCl, and 0.1% Tween 20), the membrane was probed with various antibodies and developed with enhanced chemiluminescence (Pierce, Rockford, IL, USA). The protein bands were visualized using FluorChem Q (Cell Biosciences).
The statistical significance between groups was evaluated using analysis of variance (ANOVA) and P < 0.05 was considered as statistically significant. The data are expressed as the mean ± standard deviation (SD).
miR-375 sensitized TNF-α-induced cell death in HNSCC cells
Cal27 cells were treated with TNF-α at different concentrations (5, 10, and 20 ng/ml). Changes in the cell morphology and cell death were observed using an inverted microscope. We selected 10 ng/ml as the experimental concentration, as cell death was not significant at this maximum concentration. In the combination group, the cells transfected with miR-375 mimic were treated with TNF-α (10 ng/ml) for 24 h, and more cell deaths were observed in this group than in the control. However, in the groups treated with either miR-375 mimic or TNF-α, no significant cell death was observed. The microscopic images showed obvious and direct morphological changes in different groups. The amount of cells was obviously reduced in the combined treatment group compared with the control group ( Fig. 1 A ).
The cells receiving either miR-375 mimic, or TNF-α, or both treatments were collected and examined using flow cytometry to detect the sub-G1 peak. Cal27 cells were resistant to TNF-α-induced cell death, as a low quantity of sub-G1 cells was observed with flow cytometry. A significant increase in sub-G1 cells was observed in the sample treated with the combination of TNF-α and miR-375 mimic ( P < 0.05) ( Fig. 1 B and C).
miR-375-sensitized TNF-α-induced cell death is caspase-dependent apoptosis
The apoptosis proteins were examined to determine the type of TNF-α-induced cell death sensitized through miR-375. Chromatin condensation is one of the morphological criteria used to distinguish apoptosis from other forms of cell death. Hoechst staining is typically used to identify chromatin condensation. As shown in Fig. 2 A , more cells with chromatin condensation were observed in the combination group than in any other group.
This result was confirmed through additional experiments. The caspase cascade is crucial for cell apoptosis. TNF-α induces cell apoptosis through caspase 3, and caspase 8 is a key molecule in cell apoptosis through exogenous death receptors. The proteolysis of PARP, the substrate for caspase cleavage, is a molecular marker of apoptosis. Therefore, we analyzed the expression of caspase 3, caspase 8, and PARP and their degradation products using Western blotting. We observed that the expression of caspase 3, caspase 8, and PARP was significantly reduced in the combination group, while the expression of their degradation products was increased ( Fig. 2 B).
Pre-treatment with Z-VAD effectively reduced the apoptosis of Cal27 cells in the combination group ( Fig. 2 C). The percentage of sub-G1 cells was not significantly reduced compared with the control ( P > 0.05). Western blotting showed that there were no degradation products of caspase 3, caspase 8, and PARP ( Fig. 2 B). The changes in the protein expression could be blocked using Z-VAD. The results indicated that the cell death induced through the combination of miR-375 mimic and TNF-α was completely blocked. These data indicate caspase-dependent apoptosis as the TNF-α-induced cell death sensitized through miR-375.
miR-375-induced sensitization might be associated with inhibition of NF-κB activation
The combined treatment of miR-375 mimic and TNF-α effectively suppressed the expression of NF-κB-regulated apoptosis proteins. The expression of cIAP-2 and FLIP-L was obviously reduced, while no distinct change in the expression of Bcl-xL, xIAP, and other inhibitors of apoptosis proteins was observed compared with the control, and the down-regulation increased in a time-dependent manner ( Fig. 3 ).