Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L₆)

Abstract

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/L₆) was established and the effect of curcumin on biological behavior of Hca-P/L₆ 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 the first generation of metastatic tumor cells of inguinal lymph node (Hca-P/L₁) was obtained. Then, Hca-P/L₁ was screened by the route of mouse foot pad subcutaneously → lymph node → scale-up culture in vitro → mouse foot pad subcutaneously for five times consecutively. The sensitivity of two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L₆) and two anchorage-dependent human hepatocarcinoma cell lines (SMC7721 and HepG₂) 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 15-240 μmol/L for 48 h. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 μmol/L in vitro, the effect of curcumin against cell proliferation of Hca-P and Hca-P/L₆ was observed by inverted microscope, cell growth curve and cell population doubling time; the effects of curcumin on cell cycles of Hca-P/L₆ and Hca-P were studied by flow cytometry (FCM). The results showed Hca-P/L₆ 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 30-120 μmol/L, curcumin had more inhibition on murine ascites hepatocarcinoma cell lines than on human anchorage-dependent hepatocarcinoma cell lines. At concentrations of 60-240 μmol/L, curcumin had more inhibition on Hca-P/L₆ with (the 50% inhibitory concentration) IC₅₀ of 51.48 μmol/L than on Hca-P with IC₅₀ of 90.87 μmol/L. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days, the proliferations of Hca-P/L₆ 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 15 μmol/L curcumin for 48 h, the morphous of Hca-P/L₆ 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 G₂/M phases. Hca-P/L₆ was validated to be more sensitive to curcumin than Hca-P. Hca-P/L₆ is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.

Keywords

murine ascites hepatocarcinoma cell line / metastasis / curcumin / drug sensitivity

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