| Objective:This experiment aimed to evaluate the effect of thermoch-emotherapy on proliferation, drug-resistance and the expression of Survivin and GST-π(the gene of multidrug-resistance )in multidrugresistance human gastic adenocarcinoma cell line SGC7901/DDP in vitro, to further investigate possible mechanism of its role as reverse the drug resistance of multidrug resistance human gastic adenocarcinoma cell line SGC7901/DDP,it will provide theory evidence in thermochemotherapy for clinic.Methods:1 Multidrug resistance human gastic adenocarcinoma cell line SGC7901/DDP was incubated in culture medium in vitro,using MTT assay to detect the growth rate by hyperthermia or thermochemotherapy at different temperature groups (41℃,42℃,43℃,44℃)and in different time groups (0.5h,1h,2h), in orderto select the best temperature and time of hyperthermia.2.MTT colorimetric methods was performed to evaluate the potential cytostatic effect on human multidrug resistance gastic adenocarcinoma cell line SGC7901/DDP treated before and after with hyperthermia and the OD val-ues were computed, meanwhile ,IC50 values calculated before and after hyperthermia. To determine the reversal fold of multiple drugresistance after hyperthermia by relevant formula. 3.SGC7901/DDP treated with chemotherapy, hyperthermia and thermochemotherapy,apoptosis and distribution of cell cycle ofthe three and control grou-ps were analyzed by flow cytometry.4.Survivin,GST-πexpression level was semi-quantitately analyzedby flow cytometry and SurvivinmRNA,GST-πmRNA expression was semi-quantitately detected using RT-PCR in SGC7901/DDP cell was treated by chemotherapy,hyperthermia and thermochemothe-rapy and non-treated. Results: 1 MTT assay results:Compared with the control group, the inhibition rate of chemotherapy group was no significant difference. Hyperthermia (different temperature and time) combined chemotherapy or not both can obviously inhibit proliferation of SGC7901/DDP in vitro. The inhibition ratio of hyperthermia was stepping up as the definite temperature and time increasing, and the highest inhibition ratio was in the group treated with chemotherapy at 43℃and 2h.Compared with the control group, there was significantly statistical significance of inhibition ratio in different temperature and time groups of SGC7901/DDP cells after being treated with hyperthermia (P<0.01). The inhibition ratio of thermochemotherapy group is higher than hyperthermia grourp , the difference also has significantly statistical significance.2 After treatment with hyperthermia at 43℃for 2h of multidrug resistance human gastic adenocarcinoma cell line SGC7901/DDP, the IC50 value from 11.72μg/ml decreased to7.19μg/ml, the reversal fold of multiple drugresistance is 1.63.3 Distribution of cell cycle and apoptosis were analysised by flow cytometry, the results were: After treatment with chemotherapy,hyperthermia,thermochemotherapy,comparaed with the control group, the cell cycle of chemotherapy group was no significant difference(P>0.05).The number of cells of hyperthermia and thermochemotherapy group in G0/G1 phase increased gradually, the number of cellsin S phase decreased grudually, and G2/M phase relatively increase. That was, hyperthermia and thermochemotherapy could induce an arrest of cell cycle in G1 phase. The apoptoticpercentage increased grudually treated with chemotherapy,hyperthermia,thermochemotherapy, and the apoptotic percentage was 0.64%,2.05%,9.34% seperately, comparaed with the control group(0.56%), chemotherapy group has no significant difference(P>0.05), hyperthermia and thermochemotherapy group have statistically significant differenc(eP<0.01), and with thermochemotherapy group, the increase is most significantly. 4 FCM assay results:The expression of Survivin,GST-πprotein were examined in SGC7901/DDP cells being treated with chemotherapy,hyperthermia , thermochemotherapy. The FI-value of Survivin in SGC7901/DDP was1.011,0.736,0.629, respectively; the FI-value of GST-πin SGC7901/DDP was 1.015,0.751,0.408, respectively. Comparaed with the control group, the FI-value of Survivin and GST-πof chemotherapy group has no significant difference(P>0.05), hyperthermia and thermochemotherapy group have statistically significant difference ( P<0.01 ) , and with thermochemotherapy group, the depression is most significantly. Result show that hyperthermia,thermochemotherapy can make Survivin, GST-πprotein expression in downward trend, with thermochemotherapy group, expression the inhibited of Survivin and GST-πprotein most significant. 5 RT-PCR assay results:The expression of Survivin mRNA,GST-πmRNA were examined in SGC7901/DDP cells being treated with chemotherapy , hyperthermia ,thermochemotherapy by RT-PCR.The expression level of Survivin mRNA and GST-πmRNA from 1.005±0.03 and 0.693±0.01 reduced to 0.995±0.06,0.900±0.04,0.752±0.01 and 0.693±0.02,0.608±0.03,0.470±0.02, respectively. Comparaed with the control group, the expression of Survivin and GST-πmRNA of chemotherapy group has no significant difference(P>0.05),hyperthermia and thermochemotherapy group were significantly lower, with a very significant difference (P <0.01), and the thermochemotherapy group decreased more significantly (P <0.01 ).Conclusions: 1 This experiment confirmed that a certain temperature range, hyperthermia can inhibit the proliferation of cell lines SGC7901/DDP, especially in 43℃, 2h the inhibition ratio is the highest . 2 We confirmed that hyperthermia,thermochemotherapy induced an arrest of cell cycle in G1 phase as well as apoptosis in SGC7901/DDP cells, expressing the inducement apoptotic is one of the mechanisms of its anti-tumor.3 Hyperthermia(43℃) could emprove the sensitivity of multidrug resistance human gastic cell line SGC7901/DDP chemical agent of DDP,meanwhile hyperthermia had the reversal function on SGC7901/DDP, which may be obtained by the decrease of Survivin,GST-πmRNA and protein expression level. 4 This experiment confirmed that hyperthermia has the ability to reverse multi-drug resistance of gastric cancer cells. This provides a new theoretic way to reverse multidrug resistance for muilti-drug resistant gastric cancer integrational therapy. |
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