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Objective@#To investigate the effects of c-Met inhibitor AMG-102 on the proliferation and apoptosis of laryngeal squamous carcinoma Hep-2 cells and the underlying mechanism.@*Methods@#Laryngeal squamous carcinoma cell line Hep-2 cells were treated with 2.5, 5 and 10 μmol/L AMG-102, respectively. The proliferation activities of Hep-2 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The apoptotic rate of Hep-2 cells was detected by flow cytometry analysis and Hoechst staining. The mRNA expression levels of apoptosis-related genes were detected by real-time quantitative polymerase Chain reaction (RT-qPCR), and the protein expressions of c-Met/PI3K/AKT pathway were detected by western blot.@*Results@#Compared with the control group, the proliferation rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 24 hours were (89.8±1.1)%, (79.8±1.0)% and (69.1±1.2)%, respectively; for 48 hours were (76.8±2.0)%, (60.2±1.1)% and (49.8±1.2)%, respectively; for 72 hours were (50.1±2.0)%, (41.5±1.1)% and (33.6±1.0), respectively, with significant differences (all P<0.05). The apoptotic rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours were (16.09±1.53)%, (27.51±2.02)% and (36.57±1.42)%, respectively, which were significantly higher than (3.62±0.10) % in the control group (all P<0.05). After treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours, the relative expression levels of Bcl-2 mRNA in Hep-2 cells were 0.58±0.13, 0.38±0.12 and 0.20±0.13, respectively; the relative protein expression of p-Met were 80.0±3.8, 50.6±4.2 and 28.5±1.3, respectively; the relative protein expression of p-PI3K were 87.1±0.9, 54.2±1.2 and 21.0±1.2, respectively; the relative protein expression of p-AKT were 98.7±5.6, 56.9±3.2 and 32.2±4.3, respectively; which were significantly lower than those in the control group (all P<0.05). The relative expression levels of Bax mRNA were 1.78±0.13, 2.37±0.14 and 3.05±0.13, respectively, and the relative expression levels of caspase-3 mRNA were 1.98±0.14, 2.47±0.14 and 3.15±0.13, respectively, which were significantly higher than those in the control group (all P<0.05).@*Conclusion@#c-Met inhibitor AMG-102 could inhibit the proliferation and induce apoptosis of laryngeal squamous carcinoma Hep-2 cells by regulating the c-Met/PI3K/Akt pathway.
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Objective@#To investigate the effect of c-Met inhibitor AMG-102 on proliferation and radiosensitivity in laryngeal squamous carcinoma cells.@*Methods@#The effects of AMG-102 on proliferation and radiosensitivity of laryngeal squamous carcinoma cell lines Hep-2 and KBV200 were detected by 3-(4, 5-dimethy-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and colony formation assay, respectively. The apoptosis of Hep-2 and KBV200 cells was detected by flow cytometry. The expression levels of c-Met, phospho-Met (p-Met), cleaved caspase-3 and Akt/p-Akt, Erk/p-Erk were detected by Western blot. Specific small interfering RNA targeting c-Met or plasmid of c-Met were transfected into Hep-2 and KBV200 cells to investigate the cell sensitivity to AMG-102.@*Results@#Compared with KBV200 cells, Hep-2 cells were more sensitive to AMG-102 with IC50 of 14 and 9 μmol/L, respectively. The relative expression levels of c-Met and p-Met proteins in Hep-2 cells were 194.48±0.57 and 177.76±1.53, respectively, which were significantly higher than those in KBV200 cells (171.24±1.00 and 115.37±0.56, respectively, P<0.001 for both). Exogenous hepatocyte growth factor (HGF) was added to increase the expression level of p-Met protein in KBV200 cells. The results showed that AMG-102 significantly reduced the expression of p-Met in KBV200 cells treated with HGF (P<0.001). Compared with the dimethyl sulfoxide (DMSO) group, AMG-102 treatment combined with radiotherapy significantly increased the radiosensitivity of Hep-2 cells (SER=1.28, P<0.001). However, AMG-102 had little effect on the radiosensitivity of KBV200 cells (SER=1.18, P=0.002). Compared with the 4 Gy radiotherapy alone group and the 5 μmol/L of AMG-102 alone treatment group, the apoptosis rate of Hep-2 cells in the combined treatment group was significantly increased. Meanwhile, the expression level of cleaved caspase-3 protein was also markedly increased. However, there were no significant changes in the apoptotic rate and cleaved caspase-3 expression in each treatment group of KBV200 cells. Compared with DMSO treatment group, the expression levels of p-Met, p-Akt and p-Erk were significantly decreased in the 4 Gy radiotherapy group, 5 μmol/L of AMG-102 treatment group and combined treatment group of Hep-2 cells. And the levels of p-Met, p-Akt and p-Erk in the combined treatment group were significantly lower than those in the 4 Gy radiotherapy alone group and 5 μmol/L of AMG-102 treatment alone group. By contrast, in KBV200 cells, the expression of p-Met, p-Akt and p-Erk in each group was not changed. The relative expression of p-Met in Hep-2 cells before and after radiotherapy at 30 min, 1 h, 4 h, 8 h, 24 h were 99.89±0.61, 138.62±1.00, 163.07±5.00, 87.80±1.85, 90.67±0.65 and 94.09±1.41, respectively. The level of p-Met was slightly increased after radiotherapy at 30 min and 1 h (P<0.001 for all), whereas it was significantly decreased from 4 h to 24 h after radiotherapy (P<0.05 for all). By contrast, the expression of p-Met in KBV200 cells did not change with time after radiotherapy (P>0.05). The sensitivity of Hep-2 cells to AMG-102 was decreased after silencing of c-Met, while the sensitivity of KBV200 cells to AMG-102 was not significantly changed (P>0.05). Moreover, the radiosensitivity of Hep-2 cells in c-Met knockdown group had a slightly increasing trend (SER=1.07, P=0.068). After the treatment with 10 μmol/L of AMG-102, the proliferation rate of c-Met ectopically expressed KBV200 cells was 60.05%±3.23%, It was significantly lower than that of the blank control 90.08%±1.04% and siRNA negative control (90.12%±1.01%, P<0.001). The results suggested that the overexpression of c-Met in KBV200 cells increased the radiosensitivity to AMG-102, whereas depletion of c-Met resulted in resistance to AMG-102 in Hep-2 cells. Furthermore, the radiosensitivity of KBV200 cells that overexpressed c-Met showed a decreased trend (SER=0.7, P=0.005).@*Conclusions@#c-Met inhibitor AMG-102 has a significant inhibitory effect on the proliferation of c-Met overexpressing laryngeal squamous carcinoma cells, leading to increased radiosensitivity. It suggests that molecular targeted therapy against c-Met receptor is more effective in c-Met overexpressed subtype of laryngeal squamous cell carcinoma.
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BACKGROUND: Tumor necrosis factor-associated protein 1 (TRAP1) is a heat-shock protein 90-related mitochondrial chaperone. Accumulative evidence has demonstrated that TRAP1 overexpression is closely related to carcinogenesis. However, the exact function and mechanism of TRAP1 in the occurrence of laryngeal carcinoma remains unclear. OBJECTIVE: To investigate whether RNA interference can inhibit TRAP1 overexpression and to explore its effects on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. METHODS: CD133+CD44+ laryngeal carcinoma stem cells were sorted from human laryngeal carcinoma Hep-2 cellsusing immunomagnetic beads. The shRNA sequence of TRAP1 was designed and synthesized and CD133+CD44+ laryngeal carcinoma stem cells were transfected with LipofectamineTM 2000. Cell counting kit-8 assay, colony formation assay and flow cytometry were used to investigate the effects of interference of TRAP1 expression on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. Spectrophotometric method was used to detect the activity of caspase-3, -8 and -9. RESULTS AND CONCLUSION: TRAP1 mRNA and protein expression levels were significantly decreased in TRAP1 shRNA-transfected CD133+CD44+ laryngeal carcinoma stem cells (P < 0.01). Compared with the blank control and negative control groups, the growth and colony formation of CD133+CD44+ laryngeal carcinoma stem cells were significantly inhibited in the TRAP1 shRNA-transfected group (P < 0.05). Apoptosis of CD133+CD44+ laryngeal carcinoma stem cells was significantly inhibited in the TRAP1 shRNA-transfected group as compared with the blank control and negative control groups (P < 0.05). TRAP1 shRNA-mediated cell apoptosis was associated with the activation of caspase-3, -8 and -9. These results suggest that RNA interference targeting inhibition of TRAP1 suppresses cell growth but promotes apoptosis in CD133+CD44+ aryngeal carcinoma stem cells. TRAP1 is likely to be a gene target for treatment of laryngeal carcinoma.
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Objective To observe changes of erythropoietin (EPO) and rheology in moderate and severe obstructive sleep apnea hypopnea syndrome (OSAHS) patients after the noninvasive positive pressure ventilation (NPPV) treatment. Methods Healthy adults were selected as control group (n=40) while moderate to severe OSAHS patients were selected as OSAHS group. OSAHS group was underwent NPPV treatment then, Levels of sleep apnea hypopnea index (AHI), sleep mean minimum oxygen saturation (LSaO2) and serum erythropoietin (EPO) were assessed, routine blood test and hemodynam-ic indexes were also checked before treatment and 1 and 3 months after treatment in both groups. Results In both groups serum EPO, blood, blood rheology indexes, AHI, LSaO2 were analysised at each time point by ANOVA repeated measures, all of which show significant different between groups and between each time points within the same group. Indexes in OSAHS group improved with prolonged treatment , but they are in the normal range in the control. Conclusion OSAHS pa-tients with NPPV therapy can significantly relieve hypoxia, reduce serum EPO level and blood viscosity. So NPPV has impor-tant clinical significance in prevention and treatment of OSAHS.
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Objective To investigate the effect of lysyl oxidase (LOX) gene expression on proliferation, invasion and radiotherapy sensitivity in laryngeal cancer Hep-2 cells. Methods Hep-2 cells were divided into control group (normal cultured), negative control group (transfection reagent) and transfection group (LOX siRNA transfected). The expressions of LOX, Ki-67, PCNA, MMP-2 and MMP-9 mRNA were detected by real time-PCR and Western blot methods. The cell sur-vival rate and apoptosis irradiated by different doses of X-rays (0, 3, 6, 9, 12, 15 and 18 Gy) were detected by MTT and flow cytometry (FCM). Results The expression levels of LOX, Ki-67, PCNA, MMP-2 and MMP-9 protein and mRNA were sig-nificantly lower in Hep-2 cells after transfection than those of control group and negative control group (P<0.05). The cell survival rate was significantly inhibited 24 hours after irradiation (12, 15 and 18 Gy) in a dose-dependent manner (P<0.05). The apoptotic rate of transfection cells combined with radiotherapy was significantly higher than that of control and the nega-tive control groups (%:79.11 ± 1.26 vs 5.01 ± 1.02, 4.95 ± 1.12, 43.21 ± 2.1,P<0.05). Conclusion The expression of LOX can be down-regulated after LOX siRNA transfection, inhibiting proliferation and enhancing radiosensitivity, which may be related to the down-regulation of Ki-67, PCNA, MMP-2 and MMP-9 protein expression.