Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L<sub>6</sub>)

doi:10.1007/s11684-009-0022-9

Front Med Chin

2009, Vol. 3

Issue (2) : 119-129 https://doi.org/10.1007/s11684-009-0022-9

RESEARCH ARTICLE

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

Hongying ZHANG^1,2, Jianwu TANG², Wenting ZHU^1,2, Chunxiu HU¹, Guowang XU¹(

)

1. Department of Pathology & Forensic Medicine, Dalian Medical University, Dalian 116044, China; 2. National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

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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

Corresponding Author(s): XU Guowang,Email:xugw@dicp.ac.cn

Issue Date: 05 June 2009

Cite this article:

Hongying ZHANG,Jianwu TANG,Wenting ZHU, et al. Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L₆)[J]. Front Med Chin, 2009, 3(2): 119-129.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0022-9
https://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/119

Fig.1 Histology of transplanted tumor of medioventral line of mouse 615 inoculated by Hca-P. Tumor cells were poorly differentiated, large and multilateral in shape and varied in size with several multinucleate giant cells. Local necrosis of tumor cells was obvious.

Fig.2 Histology of the metastatic tumor of inguinal node in mouse 615 inoculated by Hca-P. Normal structure of lymph node was partially preserved and large multilateral tumor cells were distributed in lamellar areas and varied in size.

Fig.3 Hca-P/L cells cultured in medium (×100). Tumor cells grew in suspension in large round shapes and had clear cytoplasm. Some of them were in clusters and a few large cells were in the state of differentiation and proliferation.

Fig.4 Hca-P cells cultured in medium (×100). Tumor cells grew in suspension in large round shape and had clear cytoplasm. A few were in clusters and few large cells were in the state of differentiation and proliferation.

Tab.1 Lymph node spread of Hca-P and Hca-P/L in mouse 615

Fig.5 Relationship of the tumor close to the kidney with the adrenal gland in the first mouse. The tumor was adjacent and not invasive to the adrenal gland. The adrenal gland capsule was intact.

Fig.6 Histology of the tumor close to the kidney in the second mouse. There was lymphatic tissue in the tumor close to the kidney. The tumor cells were large, undifferentiated, and multilateral in shape and varied in size.

Tab.2 Values of curcumin on Hca-P/L, Hca-P, SMC7721 and HepG

Fig.7 Inhibition of curcumin on Hca-P/L, Hca-P, SMC7721 and HepG (=9). Abbreviations are the same as in Table 2.

Fig.8 Effect of curcumin on cell growth curves of Hca-P/L and Hca-P (=18)

Tab.3 Effect of curcumin on the population doubling times of Hca-P/L and Hca-P

Fig.9 Effect of curcumin on cell cycle distribution of Hca-P/L and Hca-P treated with curcumin for 48 h detected by flow cytometry. (a) Hca-P/L control croup; (b) Hca-P/L treated by curcumin group, cells were blocked in S phase; (c) Hca-P control group; (d) Hca-P treated by curcumin group, cells were blocked in S and G/M phases.

Tab.4 Comparison of cell cycle distributions of Hca-P/L and Hca-P after treatment with curcumin for 48 h

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