The Effect Of Bufalin In ECA109 Cells And Esophageal Squamous Cell Carcinoma Transplantation Tumor Of Nude Mice By ERK/RSK2 Pathway

Esophageal cancer is a common gastrointestinal tumor in human, its morbidity ranks eighth and mortality ranks sixth in malignant tumors. The esphageal cancer is high of incidence in China. The morbidity of male was higher than female, the mean age of death was 63 years old in esophageal cancer patients. With the development of molecular biology research, the study showed that the development of esophageal carcinoma was involed in multi-genes, multi-factors and multi-stages. The mechanism of signal transduction pathway and anti tumor drugs became the focus of the study in recent years, which provides the direction for the clinical treatment of esophageal cancer.Ribosomal S6 kinase 2(RSK2) is a member of p90 RSK protein family, whose members are activated by extracellular signal regulated protein kinases 1 and 2(ERK1/2). Activated RSK2 affects cell proliferation, apoptosis, migration and invasion through mediating downstreams include GSK3β, BAD and Fascin-1.Bufalin is the major digoxin-like component of the traditional Chinese medicine Chansu. Its molecular formula is C24H34O4, relative molecular weight is 386.5. Our previous study results showed that bufalin could inhibit the phosphorylation of ERK in vitro. To further investigate the in vivo effect of bufalin and its influence to the downstream targets, we used the esophageal squamous cell cancer cell line ECA109, and established the esophageal squamous cell carcinoma transplantation tumor in situ by inoculating the cells in nude mice. The effects of bufalin in ECA109 cells and esophageal squamous cell cancer transplantation tumor of nude mice were detected.Objective: To investigate the effect of bufalin in ECA109 cells and esophageal squamous cell carcinoma transplantation tumor of nude mice by ERK/RSK2 pathway.Methods:1 The proliferation inhibition ratio of ECA109 cells in different concentrations of bufalin(20-100 nmol/L) groups and times was detected by CCK-8.2 The cell apoptosis index of ECA109 cells in different concentrations of bufalin(20-100 nmol/L) groups was detected by TUNEL labeling method.3 The migration distance in different concentrations of bufalin(20-100 nmol/L) groups was measured by wound healing assay(scrap migration assay), subsequently, transwell migration assay and transwell invasion assay were used to detect the anti-migration and anti-invasion effects of bufalin.4 Othotopic transplantation tumor model of esophageal carcinoma cells in nude mice was established. The weights of nude mice in model group, low dose bufalin group(BL, 0.5mg/kg), middle dose of bufalin group(BM, 1.0mg/kg), high dose of bufalin group(BH, 1.5mg/kg), PD98059 group(3.0mg/kg) and combination group(BP, bufalin+PD98059) were observed to evaluate the effect of bufalin on transplantation tumor. The volume of transplantation tumor was measured to detecte the anti-tumor effect of bufalin.5 The morphology of transplantation tumor was observed under the microscope. The cell apoptosis index of transplantation tumor was detected by TUNEL labeling method.6 The expression of ERK and RSK2 m RNA in nude mice transplanted tumor was examined by real-time quantitative PCR.7 The protein levels of ERK1/2, p-ERK1/2, RSK2, p-RSK2 and the downstream targets: GSK3β, p-GSK3β, BAD, p-BAD and Fascin-1 in nude mice transplanted tumor were examined by Western blot and Immunohistochemistry.Results:1 CCK-8 results showed that ECA109 cells treated with different concentrations of bufalin(20, 40, 60, 80, 100 nmol/L) in 12, 24, 36 hours, the inhibition rate of proliferation increased gradually, suggested that the proliferation inhibition of bufalin on ECA109 cells was in a time-dose dependent manner.2 The nucleus of apoptotic cells were stained yellow brown by TUNEL labeling method. Apoptosis index with the different concentration of bufalin(20, 40, 60, 80, 100 nmol/L) increased gradually(7.400±1.140, 13.800±1.483, 21.600±2.510, 29.400±4.037, 43.400±5.128). The apoptotic cell was in a dose-dependent manner. Compared with positive control(3.200±1.095), the diference of bufalin was statistically significant(P < 0.05).3 Wound healing assay showed that the migration distance(2.416±0.024, 2.928±0.065, 3.142±0.084, 3.566±0.097, 4.066±0.075) in different concentrations of bufalin( 20, 40, 60, 80, 100 nmol/L) was markedly than that in positive control group(1.922±0.060). Prompted bufalin can inhibit the migration of ECA109 cells. Transwell migration assay showed that the number of transmembrane cells(129.800±10.993, 91.600±10.875, 62.200±7.627, 31.300±5.143, 20.400±3.565) in bufalin intervention group(20, 40, 60, 80,100 nmol/L) was significantly less than the positive control group(146.200±11.923). The difference was statistically significant(P < 0.05). Transwell invasion assay showed that the number of transmatrigel cells in bufalin intervention groups(20, 40, 60, 80, 100 nmol/L) were 86.500±10.967, 74.300±4.398, 55.100±7.109, 38.100±6.008, 28.400±3.658, respectively, they were significantly less than the positive control group(98.400±5.835). The difference was statistically significant( P < 0.05).4 After injected the liquid of cells, all nude mice growed tumors. No obvious abnormality of the nude mice mental state, activity and weight in each group(model, BL, BM, BH, PD98059, BP). The size of nude mice tumor in each group were 1.758±0.181cm3, 1.680±0.150cm3, 1.285±0.134cm3, 0.873 ±0.095cm3, 0.815±0.108cm3, 0.530±0.104cm3, respectively. Compared with control group, the difference of BM, BH, PD98059 and BP groups were statistically significant( P < 0.05). It suggested that bufalin could inhibit the growth of esophageal cancer transplantation tumor.5 The morphology of nude mice orthotopic transplanted tumor: The tumors in each groups were observed on HE stained sections by the microscope. The cells were all low differentiated squamous cell carcinoma cells. The heteromorphisms were obvious and dark. Nucleolus were prominent. Bridges and keratosis were hardly observed between carcinoma cells. The degree of tumor necrosis gradually increased in each group. The BP group was most obvious.6 The cell apoptosis index of transplantation tumor in model, BL, BM, BH, PD98059 and BP groups were 6.030±0.622%, 11.020±0.694%, 19.070±0.907%, 25.060±1.397%, 20.020±1.197%, 17.070±0.738%, respectively. BH group was the highest. Compared with control group, the differences of BM, BH, PD98059 and BP groups were statistically significant( P < 0.05).7 Real-time quantitative PCR results showed:(1) The △CT of ERK m RNA in model, BL, BM, BH, PD98059 and BP groups were 0.270± 0.084, 0.293±0.081, 0.596±0.224, 0.857±0.183, 0.868±0.187, 1.313±0.282, respectively.(2) The △CT of RSK m RNA in model, BL, BM, BH, PD98059 and BP groups were 0.340±0.062, 0.337±0.071, 0.642±0.226, 0.915 ± 0.170, 0.923±0.176, 1.413±0.269, respectively. The relative expression of ERK and RSK2 m RNA gradually decreased. The differences of BM, BH, PD98059 and BP groups were both statistically significant(P<0.05) compared with control group.8 Western blot results showed:(1) The protein level of ERK1/2, RSK2 and BAD in each groups were no significant difference(P>0.05).(2)The protein level of p-ERK in model, BL, BM, BH, PD98059 and BP groups were 0.721±0.094, 0.695±0.095, 0.555±0.080, 0.388±0.052, 0.341±0.060, 0.235± 0.056, respectively. The protein level of p-RSK2 in model, BL, BM, BH, PD98059 and BP groups were 0.613±0.085, 0.612±0.084, 0.427±0.089, 0.305±0.056, 0.258±0.051, 0.158±0.058, respectively. The protein level of p-ERK1/2 and p-RSK2 gradually decreased. The difference of BM, BH, PD98059 and BP groups were statistically significant(P< 0.05) compared with control group.(3) The protein level of GSK3β increased gradually, the protein level of p-GSK3β, p-BAD and Fascin-1 decreased gradually, the difference of BM, BH, PD98059 and BP groups were statistically significant(P<0.05) compared with control group.9 Immunohistochemistry results:(1) The protein level of ERK1/2, RSK2 and BAD in each group were no significant differences(P>0.05).(2) The immunoreactivity score medians of p-ERK in each group(model group, BL group, BM group, BH group, PD98059 group, BP) were 8, 8, 6, 4, 5 and 3. The immuno- reactivity score medians of p-RSK in each groups(model group, BL group, BM group, BH group, PD98059 group, BP) were 8, 8, 5, 3, 3 and 1. The protein level of p-ERK1/2 and p-RSK2 gradually decreased. The difference of BM, BH, PD98059 and BP groups were statistically significant(P < 0.05) compared with control group;(3) The protein level of GSK3β increased gradually, while the protein level of p-GSK3β, p-BAD and Fascin-1 decreased gradually, the difference of BM, BH, PD98059 and BP groups were statistically significant( P < 0.05) compared with control group.Conclusions:1 Bufalin can inhibite the proliferation and promote the apoptosis of ECA109 cells.2 Bufalin can inhibit the invasion and migration of ECA109 cells.3 Bufalin have significant inhibitory effect on the esophageal squamous cell transplantation tumor in nude mice.4 Bufalin can inhibite the growth of transplantation tumor in nude mice by downregulating the level of ERK1/2 and RSK2 phosphorylation.5 Bufalin may inhibite the proliferation of tumor through Inhibiting the inactivation of GSK3β, promote apoptosis through downregulation of p-BAD and inhibite invasion and migration through downregulation of Fascin-1.