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Journal of Chinese Integrative Medicine ›› 2006, Vol. 4 ›› Issue (3): 265-270.doi: 10.3736/jcim20060309

• Original Experimental Research • Previous Articles     Next Articles

Establishment of a multidrug-resistant cell line BEL-7402/5-FU of human hepatocellular carcinoma and its biological characteristics

Wei Gu, Ya-ni Zhang, Bai Li, Jie Han, Bin-bin Cheng, Chang-quan Ling   

  1. Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
  • Online:2006-05-31 Published:2006-05-15

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Objective

To establish a multidrug-resistant cell line BEL-7402/5-FU of hepatocellular carcinoma (HCC).

Methods

BEL-7402/5-FU was induced by pulse therapy combined with continuous stepwise exposure to 5-fluorouracil in vitro. MTT assay was used to determine its multidrug resistance (MDR). Biological characteristics of the BEL-7402/5-FU cell line were observed including morphological changes, cell growth curve, population doubling time, plate cloning efficiency, adherence rate, cell cycle distribution, chromosome and tumorigenicity. Accumulation amount of adriamycin (ADM) in cytoplasm was measured by flow cytometry. The protein expression of thymidylate synthase (TS) was evaluated by immuno-cytochemical method.

Results

The acquired MDR cell line of BEL-7402/5-FU was established successfully. The BEL-7402/5-FU cells showed cross-resistance to ADM, vincristine (VCR), methotrexate (MTX) and oxaliplatin (OHP), whereas still sensitive to hydroxycamptothecin (HCPT). The BEL-7402/5-FU cells tended to grow in clusters in vitro. It was found that the population doubling time of BEL-7402/5-FU cells was longer than that of its parental cells. The plate cloning efficiency and the adherence rate of BEL-7402/5-FU cells at the 2nd and 3rd hour were both lower than those of the parental cells. The distributing proportion of BEL-7402/5-FU cells in G0/G1 phase was less than that of the parental cells, whereas the distributing proportion of BEL-7402/5-FU cells in S phase was higher than that of the parental cells. The accumulation amount of ADM in cytoplasm of BEL-7402/5-FU cells was significantly lower while the expression level of TS protein of which was highly up-regulated as compared with those of the parental cells.

Conclusion

Establishment of the human HCC cell line BEL-7402/5-FU might be beneficial to the studies of 5-Fluorouracil acquired MDR mechanisms and the selection of reversal modifiers.

Key words: Hepatocellular carcinoma, Multidrug resistance, 5-fluorouracil, Cell line

CLC Number: 

  • R735.7

Figure 1

Induction of acquired resistance of human HCC BEL-7402/5-FU cells"

Table 1

Drug resistance of human HCC BEL-7402 and BEL-7402/5-FU cells to different anti-tumor drugs (ヌ±S, μmol/L)"

Cell line n IC50 (48 h)
5-FU ADM VCR OHP MTX HCPT
BEL-7402 3 23.70±9.35 1.30±0.31 0.89±0.27 43.21±12.30 60.87±29.35 10.09±1.35
BEL-7402/5-FU 3 427.60±110.50 2.75±0.58 8.83±2.54 7.72±3.65 497.25±100.41 10.52±2.62

Figure 2

Growth curve of human HCC BEL-7402/5-FU cells*P＜0.05, BEL-7402/5-FU vs BEL-7402; △P＜0.05, BEL-7402/5-FU vs BEL-7402/5-FU(1); n=12, ?x±s."

Table 2

Comparison of adherence rate between human HCC BEL-7402 and BEL-7402/5-FU cells (ヌ±S, %)"

Cell line n Adherence rate(%)
0 h 1 h 2 h 3 h 4 h 5 h 6 h
BEL-7402 4 0 43.25±5.31 86.75±4.34 92.75±11.87 93.75±11.32 95.75±11.61 95.75±11.61
BEL-7402/5-FU 4 0 35.00±4.96 61.50±2.88** 75.75±6.70* 85.25±8.18 88.50±16.29 91.00±7.78

Table 3

Comparison of cell cycle distribution between human HCC BEL-7402 and BEL-7402/5-FU cells (ヌ±S, %)"

Cell line n Cell cycle （%）
G0/G1 S G2/M
BEL-7402 3 61.38±2.85 25.54±1.17 13.07±1.69
BEL-7402/5-FU 3 48.30±1.00** 31.73±0.12* 19.95±1.05**

Figure 3

Cell cycle distribution of human HCC BEL-7402 and BEL-7402/5-FU cellsA: BEL-7402; B: BEL-7402/5-FU."

Figure 4

Accumulation of adriamycin in human HCC BEL-7402 and BEL-7402/5-FU cells"

Table 4

Comparison of TS expression between human HCC BEL-7402 and BEL-7402/5-FU cell lines （ヌ±S ）"

Cell line n Positive-area percentage (%) OD value Positive index
BEL-7402 3 7.75±2.18 14.50±1.05 1.14±0.39
BEL-7402/5-FU 3 18.23±2.32 18.67±0.52 3.41±0.47**

Figure 5

TS expression in human HCC BEL-7402 and BEL-7402/5-FU cells (Envision staining,×400)"

Figure 6

Chromosome of BEL-7402/5-FU cells （Giemsa staining, ×4 000）"

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