摘要
In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(Hca-P/L_(6))was established and the effect of curcumin on biological behavior of Hca-P/L_(6) was observed.Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(Hca-P)was subcutaneously inoculated into the medioventral line of a mouse 615 and thefirst generation of metastatic tumor cells of inguinal lymph node(Hca-P/L_(1))was obtained.Then,Hca-P/L_(1) was screened by the route of mouse foot pad subcutaneously!lymph node!scale-up culture in vitro!mouse foot pad subcuta-neously forfive times consecutively.The sensitivity of two murine ascites hepatocarcinoma cell lines(Hca-P and Hca-P/L_(6))and two anchorage-dependent human hepato-carcinoma cell lines(SMC7721 and HepG_(2))to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay after these cells had been pretreated by curcumin at the concentration of 15–240μmol/L for 48 h.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15μmol/L in vitro,the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted micro-scope,cell growth curve and cell population doubling time;the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied byflow cytometry(FCM).The results showed Hca-P/L_(6) spreading to the lymph nodes at multiple sites in mice was screened from Hca-P.The lymph node metastatic rate was 100%.Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner(P<0.05).At concentrations of 30–120μmol/L,curcu-min had more inhibition on murine ascites hepatocarci-noma cell lines than on human anchorage-dependent hepatocarcinoma cell lines.At concentrations of 60–240μmol/L,curcumin had more inhibition on Hca-P/L_(6) with the 50%inhibitory concentration(IC50)of 51.48μmol/L than on Hca-P with IC50 of 90.87μmol/L.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days,the proliferations of Hca-P/L_(6) and Hca-P were inhibited(P<0.05)in a time-dependent manner(P<0.01)and the population doubling time of Hca-P/L6 and Hca-P was prolonged(P<0.01),and curcumin had more inhibition on Hca-P/L6 than on Hca-P(P<0.05).After pretreatment by 15μmol/L curcumin for 48 h,the morphous of Hca-P/L_(6) was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G_(2)/M phases.Hca-P/L_(6) was validated to be more sensitive to curcumin than Hca-P.Hca-P/L_(6) is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.
基金
The study was supported by the National Natural Science Foundation of China(Grant No.90209048)and the Knowledge Innovation Program of the Chinese Academy of Sciences(Nos.KSCX2-SW-329 and KGCX2-SW-213).
