| Background:Gastric cancer (GC) is one of the most frequent malignant tumors,which is ranked the fourth in all the malignant tumors.However, many patients in advanced gastric cancer have lost the chance of operation. Therefore, chemotherapy has become the first choice for them. In recent years, the application of a variety of new medicine, such as5-fluorouracil, cisplatin and so on, has provided more possibilities for clinical treatment. Yet the clinical study showed that the existing chemotherapy drugs also has serious side effects with the therapeutic effect, such as neutropenia, phlebitis, and the complication caused by venous catheter, making compliance in patients with gastric cancer is poor. In recent years, the traditional Chinese medicine, a kind of auxiliary therapeutic drugs,which is more and more favor.The calculus bovis,Xihaung,has many functions, such as heat detoxification,anti-inflammatory, anti-oxidant and free radical scavenging cholagogic, and enhance immune system. It is Commonly used in throat swelling and pain, ulcers, sore tongue, and also used in furunculosis disease, such as the treatment of breast cancer, scrofula, carbuncle poison. Recent research has found the calculus bovis has effect of suppression viral replication, anti-inflammatory, gallbladder, liver function, improve the body immunity and antitumor. It is report that The cultivated calculus bovis can induce cell apoptosis and inhibit the growth of human hepatoblastoma HepG2cells.This pioneered new thinking for the research and application of the calculus bovis in the treatment of gastric cancer. So far, there is few researches the mechanism of calculus bovis on gastric cancer. Therefore, this study used calculus bovis intervention of human gastric cancer cell line, BGC-823, to observe the effect of apoptosis of BGC-823cells induced by calculus bovis, and calculus bovis combined with5-fluorouracil and cisplatin, then to provide experimental basis for further clinical application.Objective:To study the effects of growth inhibition and apoptosis of different concentration of calculus bovis, and the synergistic effects of calculus bovis combined with5-fluorouracil and cisplatin on BGC-823cells.Methods:Different concentrations of calculus bovis were given to treat BGC-823cells.Then morphological changes were observed by inverted microscope. Different concentrations of calculus bovis and calculus bovis combined with5-fluorouracil and cisplatin were given to treat BGC-823cells.Then growth inhibition of BGC-823cells was analyzed by four methyl thiazolyl tetrazolium (MTT), Flow cytometry, Hoechst33343staining and DNA gel electrophoresis was used to detect cell apoptosis.Results:1.The number was significantly reduced, the intercellular space was increased, refraction was weakened and cells exfoliated obviously of BGC-823cells after treated with different concentrations of calculus bovis.2. MTT assay indicated that each concentration group growth inhibition rate was significantly higher than the control group, BGC-823cells were treated with different concentrations of calculus bovis for24,48,72hours.The difference was significant (P<0.05). In the same concentration, with the extension of time, the growth inhibition rate was gradually increased, the difference was statistically significant (P<0.05).MTT analysis showed that5-fluorouracil (100μg/mL)、calculus bovis (400μg/mL) and cisplatin (50μg/mL) combined for48hours, the cell growth inhibition rate was0.471±0.047, the control, calculus bovis,5-fluorouracil, cisplatin, and5-fluorouracil conbined cisplatin used only groups for48hours,the cells growth inhibition rate were0±0,0.243±0.09,0.25±0.039,0.258±0.046,0.366±0.045.Compared with the control, there was difference (P<0.05); The single medication groups compared, there was no significant difference between groups (P>0.05). But the combined treatment group compared with the other groups, there was significant difference (P<0.05). The combine medication groups compared, there was significant difference between groups (P>0.05).3. BGC-823cells were treated with different concentrations of calculus bovis for24,48,72hours.Flow cytometry analysis showed that the100μg/mL calculus bovis group, compared with the control group, the difference of apoptosis rate were not significant (P>0.05).With the increase of drug concentration and action time, cell apoptosis rate was increased, the differences were statistical significance, of which200μ g/mL (P<0.05),400μg/mL,800μg/mL and1200μg/mL group (P <0.01). BGC-823cells were treated with5-fluorouracil (100μg/mL)、 calculus bovis (400μg/mL) and cisplatin (50μg/mL) combined for48hours, the apoptosis rate was38.98±2.65; the control,5-fluorouracil, cisplatin, and5-fluorouracil conbined cisplatin used only groups for48hours,the apoptosis rates were3.68±0.72,9.34±1.79,12.5±1.55,26.8±1.86. The used medication groups Compared with the control, there was significant difference (P<0.05); The combined treatment group Compared with the other groups, there was significant difference (P <0.05). The combine medication groups compared, there was significant difference between groups (P>0.05).4. BGC-823cells were treated with different concentrations of calculus bovius for24hour, Hochest33342dyeing showed the nucleolus fluorescence intensity was enhanced, the nucleus was presentation corrugated, the chromatin was condensed and the apoptotic bodies showed bright blue-stained particles.With the increase of drug concentration and the extension of action time, the apoptosis cells were increased, especially in the combination treated with5-fluorouracil (100μg/mL)、calculus bovis (400μg/mL) and cisplatin (50μg/mL) combined for48hours.5. DNA gel electrophoresis showed that treated with5-FU (100μg/mL)、calculus bovis (400μg/mL) and CDDP (50μg/mL) combined for48hours, the DNA gel present weakened.Conclusions:1. In vitro experiments indicated that a certain concentration of calculus bovis can inhibit the proliferation and induce the apoptosis of human gastric cancer cell line BGC-823.2. a certain concentration of5-fluorouracil and cisplatin used only can inhibit the proliferation of human gastric cancer cells BGC-823.3. Calculus bovis combined with5-fluorouracil, cisplatin can inhibit the proliferation and induce the apoptosis of BGC-823cells, but the apoptosis effects was stronger than5-fluorouracil, cisplatin,and5-fluorouracil combined with cisplatin groups. |
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