| Esophageal cancer is one of the most common gastrointestinal malignant tumors, China is the esophageal cancer high-risk area, the most common pathological histology is squamous cell cancer. Despite clinical treatment of esophageal cancer continuously improve and perfect, its mortality rate is still high. Through the study of esophageal cancer for years, we find that the occurring and development of esophageal cancer is a complex process which involves of poly genes, many factors and multi-stage. Moreover, it is related with abnormal activation of signal transduction pathways. In consequence, to study the related activation of signal transduction pathways and the effect of antitumor drugs on the esophageal cancer, is still the focal point of esophageal cancer treatment research.The abnormal-activation of Akt/mTOR signaling pathways existied in the course of tumor have been confirmed by many reports. mTOR pathway not only adjusts protein synthesis in cells, but also closely related with cell survival, growth and proliferation. As a kind of serine/threonine kinase,mTOR is composed of 2549 amino acids and participate in the regulation of a lot of important cell function. P70S6K and 4EBP1 are two major molecules in downstream of mTOR. They undertake most of the mTOR signaling functions.When mTOR signaling pathway is activated, it makes P70S6K and 4E-BP1 phosphorylation. If we inhibite the mTOR signaling, P70S6K and 4E-BP1 will dephosphorylation and inhibite the translation processes, ultimately induce cell apoptosis and make the stagnation of the cell cycle.Rapamycin(RAPA), a specific inhibitor of mTOR, belongs to macro lide drugs which has good anti-cancer effects. Researches showed that it was effective on prostate cancer, breast cancer, pancreatic cancer and o ther malignant tumors. However, the exact mechanism of whether rapamycin can inhibit the proliferation of esophageal squamous cancer and ind uce apoptosis remains unclear.We used the esophageal squamous cell cancer cell line TE13 in this experiment to observe the effects of rapamycin on mTOR pathway and cell apoptosis, in order to reveal the mechanisms of the occurring and development of esophageal cancer. It will help the clinical treatment of esophageal cancer.Objective:To investigate the effects of rapamycin on esophageal cancer TE13 cell apoptosis and the related mechanisms.Methods:1 The effect of different concentrations of rapamycin TE13 proliferation of cells was determined by MTT test.2 The protein expressions of p-P70S6K and p-4EBP1 in TE13 cell were examined by Western blot after treated with different concentrations of rapamycin.3 The protein expressions of c IAP-1 and BAD in TE13 cell were detected by Western blot after putting in 100 nmol/l rapamycin 2hours, 6hours, 12 hours,24hours, 36 hours and 48 hours.4 The apoptotic rate and the cell cycle analysis were detected after adding different concentrations of rapamycin with Flow cytometry by PI staining method.5 The cell apoptosis index was examined by TUNEL labeling method.6 Statistical processing.Results:1 MTT resultsAfter treated with 20, 40, 80, 160, 320, 640 nmol/L rapamycin for 24 h,the inhibition rates of TE13 cells were 7.513 ± 0.162%, 14.004 ± 0.714%,16.089±1.650%, 22.235±0.459%, 40.134±0.501%, 52.423±0.673%, it was significantly higher than control group, the different of them was statistically significant(P <0.05). It suggested that rapamycin could inhibit the growth of TE13 cell in a dose-dependent manner.2 The protein expressions of p-P70S6K and p-4EBP1 in TE13 cell wereexamined by Western blot after treated with 20, 40, 80, 160nmol/l rapamycin for 24 h.(1) The expression of p-P70S6K was gradually reduced(0.714±0.045,0.606±0.023, 0.552±0.020, 0.332±0.022, P<0.05). The different of them was statistically significant.(2) The expression of p-4EBP1 was gradually reduced(0.952±0.085,0.866±0.068, 0.692±0.059, 0.462±0.019, P < 0.05). The difference was statistically significant.3 We used Western Blot to detect the protein expressions of c IAP-1 and BAD in TE13 cell after adding 100nmol/l rapamycin for 2h, 6h, 12 h, 24 h, 36 h and 48 h.(1)After adding rapamycin, the expression of c IAP-1 was dropped off along with the time(0.842±0.013, 0.717±0.017, 0.645±0.021, 0.514±0.014,0.469±0.026, 0.218±0.018, P < 0.05). The difference was statistically significant.(2) After adding rapamycin, the expression of BAD was increased as time goes on(0.465±0.024, 0.596±0.042, 0.655±0.023, 0.883±0.019,0.958±0.015, 1.157±0.013, P<0.05). The different of them was statistically significant.4 The flow cytometry results showed that:After adding rapamycin 0, 20, 40, 60, 80 and 160 nmol/l for 24 h, the apoptotic rate was gradually increased(4.12±0.327%, 5.67±0.206%,8.46±0.198%, 10.97±0.440%, 18.42±0.551%, 23.60±0.754%, P<0.05). The TE13 cell cycle appeared obviously G0/G1 phase retardation and the cell percentage of G0/G1 was increased from 9.8±1.819% to 61.5±2.394%. The different of them was statistically significant.5 The TUNEL staining pointed that: the positive cell nuclei was with brownish yellow, but the normal cell nucleus was not dyed. After adding rapamycin 0, 40, 60, 80 and 160nmol/l for 24 h, the apoptotic index was gradually increased(5.67±1.435%, 7.97±1.433%, 35.79±2.886%,51.89±2.778%, P<0.05). The different of them was statistically significant.Conclusions:1 Rapamycin could inhibit the growth of TE13 cells and induce esophageal cancer cell TE13 cell apoptosis in dose dependent manner.Rapamycin could induce lower expression of c IAP-1 and higher expression of BAD in time dependent manner.2 Rapamycin induces TE13 cell apoptosis by inhibiting mTOR downstream substrates(4E-BP1 and p70S6K), and then inhibit the growth of esophageal cancer cell. |
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