| Objective: The cancer occurrence and development involves more than one kind of factors and the multi-steps processes, the most prominent manifestation is uncontrolled proliferation of tumor cell, which is related with disorders of apoptosis regulation. Heat shock proteins (HSPs) are ubiquitous in organisms, with high conservative in organic evolution and expression in physiological condition or stress response. Many studies found that HSP is over-expressed in most tumors. HSPs have a dual function depending on their intracellular or extracellular location. Intracellular HSPs regulate apoptosis of tumor cells by various mechanisms. Extracellular HSPs can elicit an anti-tumor immune response modulated either by the adaptive or innate immune system. Recently, the studys on roles of HSP27 and HSP70 in apoptosis of tumor cells and anti-tumor immunity and prognosis of patients with carcinomas have been hot topics for researchers on oncology, while the results were controversial. The national researches have demonstrated that patients with low expressions of HSP70 in esophageal squamous cell carcinoma have a poor prognosis. Nakajima and his colleagues reported that patients with high expressions of HSP70 in esophageal squamous cell carcinoma have a beneficial prognosis. But He Yong found that over-expressions of HSP70 in laryngeal carcinoma was a sign of poor prognosis. Patients with high expressions of HSP27 in squamous cell carcinoma, such as esophageal carcinoma and oral cancer, have a beneficial prognosis, while patients with high expressions of HSP27 in adenocarcinoma, such as gastric cercinoma,breast cancer, prostate carcinoma and ovarian cancer, have a poor prognosis. The controversial results may prompt the effects of HSP27 and HSP70 on apoptosis of squamous cell carcinoma and adenocarcinoma cells may be different. To investigate the difference of the effects of HSP27 and HSP70 on apoptosis of squamous cell carcinoma and adenocarcinoma cells, this study focused on the influence of HSP27 and HSP70 antisense oligonueleotide to cultured human esophageal cancer TE-13 cells and stomach cancer MGC-803 cells proliferation and apoptosis.Methods: The human esophageal cancer TE-13 cells and stomach cancer MGC-803 cells were cultured in vitro and were separately cultured in different medium conditions. The two tumor cell lines were randomly divided into blank control group and experimental group. The experimental group was separately added with different concentrations of HSP27 and HSP70 antisense oligonueleotide with the final concentrations of 5 ,10 and 15μmol/L. The control group was added medium conditions without HSP27 and HSP70 antisense oligonueleotide. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the two tumor cell lines proliferation after the two tumor cell lines were treated at different intervals (24 h, 48 h and 72 h) respectively. Flow cytometry (FCM) was used to analyze the cell cycle and cell apoptosis after the two tumor cell lines were treated for 48 hours.Results:1. Effects of HSP27 and HSP70 antisense oligonueleotide on TE-13 cells proliferationThe proliferation of TE-13 cells was significantly inhibited by 15μmol/L of HSP27 and HSP70 antisense oligonucleotide for 24 h, as well as 5,10,15μmol/L of HSP27 or 15μmol/L of HSP 70 antisense oligonucleotide for 48 h and 72 h incubation. The inhibitory fraction of TE-13 cells exposed to HSP27 and HSP70 antisense oligonucleotide 24 h was 13.68% and 10.02%, and for 48 h was 29.16% and 20.84%, and for 72 h was 15.24% and 22.46%, respectively. The effect of HSP70 antisense oligonucleotide was dose-and time-dependent, while HSP27 antisense oligonucleotide was not dose-and time-dependent.2. Effects of HSP27 and HSP70 antisense oligonueleotide on MGC-803 cells proliferation The proliferation of MGC-803 cells was significantly inhibited by 5,10,15μmol/L of HSP27 and HSP70 antisense oligonucleotide for 24 h, as well as 15μmol/L of HSP27 or 5,10,15μmol/L of HSP70 antisense oligonucleotide for 48 h, as well as 10, 15μmol/L of HSP27 or 5,10,15μmol/L of HSP70 antisense oligonucleotide for 72 h incubation. The inhibitory fraction of MGC-803 cells exposed to HSP27 and HSP70 antisense oligonucleotide for 24 h was 16% and 17.15%, and for 48 h was 27.09% and 21.26%; and for 72 h was 23.16%and 23.55%, respectively. The effect of HSP27 antisense oligonucleotide was dose-dependent, while HSP70 antisense oligonucleotide was time-dependent.3. Changes by HSP27 and HSP70 antisense oligonucleotide in TE-13 cell cycleThe cell cycle phase of TE-13 cells had changed, when exposed to HSP27 and HSP70 antisense oligonucleotide at concentration of 5, 10, 15μmol/L for 48 h. The proportion of cells in the G0/G1-, G2/M-phase of cell cycle did not alter, and S-phase of cells increased after sustained incubation of TE-13 cells with HSP27 and HSP70 antisense oligonucleotide (10 and 15μmol/L). The results showed that HSP27 and HSP70 antisense oligonucleotide inhibited the cell proliferation by changing the distribution of cell cycle phase via S-phase delay.4. Changes by HSP27 and HSP70 antisense oligonucleotide in MGC-803 cell cycleThe cell cycle phase of MGC-803 cells changed when exposed to HSP27 and HSP70 antisense oligonucleotide for 48 h at the concentration of 5, 10, 15μmol/L. The proportion of cells in the G0/G1 phase of cell cycle did not alter, S-phase of cells increased, but G2/M-phase of cell cycle was decreased after sustained incubation of MGC-803 cells with HSP27 and HSP70 antisense oligonucleotide (15μmol/L). These data suggested that HSP27 and HSP70 antisense oligonucleotide inhibited the MGC-803 cell proliferation via S-phase delay.5. Apoptosis of TE-13 cells induced by HSP27 or HSP70 antisense oligonueleotideHSP27 or HSP70 antisense oligonucleotide (5,10,15μmol/L)could induce the apoptosis of TE-13 cells. The apoptotic rate in groups treated with HSP27 or HSP70 antisense oligonucleotide was markedly higher than that in control group. The apoptotic rate of TE-13 cells exposed to HSP27 and HSP70 antisense oligonucleotide at the concentration of 5μmol/ml for 48h was(8.56±2.95)% and(12.76±0.49)%, and 10μmol/ml was(11.01±2.46)% and(11.38±2.18)%, and 15μmol/ml was(10.52±1.86)% and(13.21±1.67)%, respectively. The apoptotic rate of MGC-803 cells was failed, so the results were not adopted.Conclusions:1. HSP27 or HSP70 antisense oligonueleotide can inhibit the human esophageal cancer TE-13 cells proliferation in vitro depending on the concentration and the time. The effect of HSP70 antisense oligonucleotide was dose-and time-dependent, while HSP27 antisense oligonucleotide was not dose-and time-dependent.2. HSP27 or HSP70 antisense oligonueleotide can inhibit the stomach cancer MGC-803 cells proliferation in vitro. The effect of HSP27 antisense oligonucleotide was dose-dependent, while HSP70 antisense oligonucleotide was time-dependent.3. HSP27 or HSP70 antisense oligonueleotide produced an inhibitory effect on the two tumor cell lines, namely, TE-13 cells and MGC-803 cells, and changed their distribution of cell cycle phase, and induced TE-13 cells apoptosis.4. The effects of HSP27 and HSP70 on inhibition of cultured human squamous cell carcinoma and adenocarcinoma cells are basically the same.
|
PDF Full Text Request