Liver Cancer
Copyright ©The Author(s) 2002. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 15, 2002; 8(4): 644-649
Published online Aug 15, 2002. doi: 10.3748/wjg.v8.i4.644
Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R
Jia-Yin Yang, Hua-You Luo, Qi-Yuan Lin, Zi-Ming Liu, Lu-Nan Yan, Ping Lin, Jie Zhang, Shong Lei
Jia-Yin Yang, Hua-You Luo, Qi-Yuan Lin, Zi-Ming Liu, Lu-Nan Yan, Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610044, Sichuan Province, China
Ping Lin, Cancer Research Institution, West China Hospital, Sichuan University, 610044, Sichuan Province, China
Jie Zhang, Department of Confocal Laser Scanning Microscopy, West China Hospital, Sichuan University, 610044, Sichuan Province, China
Shong Lei, Cancer Biotechnological Treatment Center, West China Hospital, Sichuan University, 610044, Sichuan Province, China
Supported by the grant from National Nature Science Foundation of China, No.39770723
Correspondence to: Dr. Jia-Yin Yang, Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610044, Sichuan Province, China. yjy7429@hotmail.com
Telephone: +86-571-87033324 Fax: +86-571-87236570
Received: March 25, 2002
Revised: April 5, 2002
Accepted: April 9, 2002
Published online: August 15, 2002
Abstract

AIM: To investigate the correlation between subcellular daunorubicin distribution and the multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R.

METHODS: The multidrug resistant cell line SMMC-7721/R, a human hepatocellular carcinoma cell line, was established. Antisense oligonucleotides (AS-ODN) were used to obtain different multidrug resistance phenotypes by inhibiting the expression of mdr1 gene and/or multidrug resistance-related protein gene (mrp) using Lipofectamine as delivery agent. Expression of mdr1 and mrp genes was evaluated by RT-PCR and Western blotting. Intracellular daunorubicin (DNR) concentration was measured by flow cytometry. Subcellular DNR distribution was analyzed by confocal laser scanning microscopy. Adriamycin (ADM) and DNR sensitivity was examined by MTT method.

RESULTS: Low level expression of mdr1 and mrp mRNAs and no expression of P-Glycoprotein (P-gp) and multidrug resistance-related protein (P190) were detected in parental sensitive cells SMMC-7721/S, but over-expression of these two genes was observed in drug-resistant cell SMMC-7721/R. The expression of mdr1 and mrp genes in SMMC-7721/R cells was down-regulated to the level in the SMMC-7721/S cells by AS-ODN. Intracellular DNR concentration in SMMC-7721/S cells was 10 times higher than that in SMMC-7721/R cells. In SMMC7721/S cells intracellular DNR distributed evenly in the nucleus and cytoplasm, while in SMMC-7721/R cells DNR distributed in a punctate pattern in the cytoplasm and was reduced in the nucleus. DNR concentration in SMMC-7721/R cells co-transfected with AS-ODNs targeting to mdr1 and mrp mRNAs recovered to 25 percent of that in SMMC7721/S cells. Intracellular DNR distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cell, and the cells resistance index (RI) to DNR and ADM decreased at most from 88.0 and 116.0 to 4.0 and 2.3, respectively. Co-Transfection of two AS-ODNs showed a stronger synergistic effect than separate transfection.

CONCLUSIONS: P-gp and P190 are two members mediating MDR in cell line SMMC7721/R. Intracellular drug concentration increase and subcellular distribution change are two important factors in multidrug resistance (MDR) formation. The second factor, drugs transport by P-gp and P190 from cell nucleus to organell in cytoplasm, may play a more important role.

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