Influence of hypoxia-inducible factor-1 alpha on the susceptibility of oral squamous cell carcinomas cell lines against chemotherapeutic drugs and radiation / 中国组织工程研究

ABSTRACT

BACKGROUND: The transcription factor, hypoxia-inducible factor-1al pha (HIF-1α), is the key regulator that controls the hypoxic response of mammalian cells. However, the role of HIF-1α in the therapeutic efficacy of chemo-radiotherapy in oral squamous cell carcinomas (OSC) is poorly understood. OBJECTIVE: To investigate the effect of HIF-α on the susceptibility of OSC cell lines against chemotherapeutic drugs and radiation. DESIGN: AN observational comparative experiment. SETTING: Beijing Chaoyang Hospital Affiliated to Capital Medical University. MATERIALS: OSC-2, OSC-4, OSC-5, and OSC-6 cell lines were estab lished from oral squamous cell carcinoma (The cell lines were from the De partment of Oral Oncology, Kochi Medical School, Japan). METHODS: The experiments were completed in Beijing Chaoyang Hospi tal affiliated to Capital Medical University from September 2004 to August 2006. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin. ① Total cell lysates were extracted from untreated OSC cells and those treated with 30 Gy γ-rays, 100 μmol/L cis-dichlorodiamine plat inum (CDDP), or 100 μmol/L 5-fluorouracil (5-FU). The expressions of HIF-1α protein and mRNA were determined with western blotting analysis and real-time polymerase chain reaction (PCR). ② Inhibition of cell prolif eration after different interventions: OSC cells were seeded in 96-well mi croplates (1.0×104) and cultured for 48 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 mmol/L 5-FU, and then the inhibition of cell proliferation was detected with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ③ Apoptosis after different interventions: OSC cell lines were cultured for 24 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 μmol/L 5-FU. The cells were then stained with annexin V-FITC and propidium iodide, and the numbers of apoptotic cells were analyzed by a FACScan cytometer (only for the OSC cells treated with γ-rays and CDDP). ④ Plasmid construction and transfection: Human HIF 1α cDNA was cloned into the pcDNA3.1/V5-His TOPO expression vector. OSC-2 cells were temporarily transfected with HIF-1α cDNA. OSC-5 cells were temporarily transfected with HIF-1α siRNA (Sense sequence is 5' CUGAUGACCAGCAACUUGAtt 3'). The blank control group was also set, the inhibition of cell proliferation was observed, and the apoptosis was ana lyzed (only in the CDDP-treated group) by using the methods mentioned above. MAIN OUTCOME MEASURES: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines; ② Inhibition of proliferation and the apoptosis of OSC cell lines after different interventions; ③ Overexpression and knockdown of HIF-1α by expression vector and siRNA; ④ Influence of overexpression and knockdown of HIF-1α on the susceptibility of OSC cell lines against γ-rays and chemotherapeutic drugs. RESULTS: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines: The relative expression levels of HIF-1α mRNA were 1.0, 2.2, 4.3 and 4.0 in OSC-2, OSC-4, OSC-5 and OSC-6 cells, respectively. Simi larly, the expression of HIF-1α total cell lysates proteins and nuclear proteins were much lower in OSC-2 and OSC-4 cells than in OSC-5 and OSC6 cells. ② Inhibition of proliferation and the apoptosis in OSC cell lines after different interventions: After the treatment with g-rays, CDDP, and 5-FU, the cell numbers decreased obviously to (39.5±3.2)%, (39.2±1.2)%,and(47.9±3.6)% in OSC-2 cells, (53.9±6.6)%, (54.3±1.4)%, (54.8±3.8)%in OSC-4 cells. Those decreased to (74.1±3.8)%, (76.5±9.1)%, (69.6±7.7)%in OSC-5 cells and (71.4±7.4)%, (84.4±8.8)%, (82.0±4.5)% in OSC-6 cells.After the OSC cells were treated of with CDDP, the numbers of apoptotic cells were (50.9±1.3)%, (67.3±2.2)%, (12.2±0.8)% and (38.6±0.9)% in OSC-2, OSC-4, OSC-5 and OSC-6 cells. G-rays induced the apoptosis were (21.2±1.1)%, (14.6±0.9)%, (9.7±1.0)% and (10.4±0.8)% in OSC-2, OSC-4,OSC-5 and OSC-6 cells. ③ Inhibition of proliferation and the apoptosis in the transfected cell after different interventions: The cell numbers of HIF-1 α-transfected OSC-2 cells treated with γ-rays, CDDP and 5-FU were much more than those in the control group (t=-4.693 ,-8.617,-6.721, P ＜ 0.01),whereas the cell numbers of OSC-5 cells transfected with siRNA were obviously fewer than those in the control group (t=5.800, 5.595, 4.253, P ＜ 0.05-0.01). After the incubation of HIF-1α-transfected OSC-2 cells with CDDP for 24 hours, apoptosis was detected in (34.0±1.9)% of the cells, which was lower than that in the control group [(49.6±3.4)%, t=6.937, P ＜ 0.01]. After the incubation of HIF-1α siRNA transfected OSC-5 cells with CDDP for 24hours, apoptosis was detected in (27.7±2.3)% of the HIF-1α siRNA transfected OSC-5 cells, which was obviously higher than that in the blank control group [(11.4±2.1)%, t=-8.941, P ＜ 0.01].CONCLUSION: The expression level of HIF-1α correlates negatively with the susceptibility of OSC cells against chemotherapeutic drugs and radiation. The down-regulation of HIF-1α expression enhances the susceptibility of OSC cells against chemotherapeutic drugs and radiation. Therefore, the down-regulation of HIF-1α may be a potentially effective strategy for cancer treatment.