Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice.
OBJECTIVE
To establish a multidrug resistant (MDR) cell sub-line from the human hepatocarcinoma cell line (HepG2) in nude mice.


METHODS
HepG2 cell cultures were incubated with increasing concentrations of adriamycin (ADM) to develop an ADM-resistant cell subline (HepG2/ADM) with cross-resistance to other chemotherapeutic agents. Twenty male athymic BALB/c-nu/nu mice were randomized into HepG2/nude and HepG2/ADM/nude groups (10 in each group). A cell suspension (either HepG2 or HepG2/ADM) was injected subcutaneously into mice in each group. Tumor growth was recorded, and animals were sacrificed 4-5 wk after cell implantation. Tumors were prepared for histology, and viable tumor was dispersed into a single-cell suspension. The IC50 values for a number of chemotherapeutic agents were determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (MTT) assay. Rhodamine-123 retention/efflux and the level of resistance-associated proteins were determined by flow cytometry. The mRNA expression of mdr1, mrp and lrp genes was detected using reverse transcriptase polymerase chain reaction (RT-PCR) in HepG2/nude and HepG2/ADM/nude groups.


RESULTS
The appearances of HepG2/nude cells were slightly different from those of HepG2/ADM/nude cells. Similar tumor growth curves were determined in both groups. A cross-resistance to ADM, vincristine, cisplatin and 5-fluorouracil was seen in HepG2/ADM/nude group. The levels of P-glycoprotein and multidrug resistance-associated proteins were significantly increased. The mRNA expression levels of mdr1, mrp and lrp were higher in HepG2/ADM/nude cells.


CONCLUSIONS
ADM-resistant HepG2 subline in nude mice has a cross resistance to chemotherapeutic drugs. It may be used as an in vivo model to investigate the mechanisms of MDR, and explore the targeted approaches to overcoming MDR.
Zhai
Bao-Jin
BJ
Clinical Center for Tumor Therapy of 2nd Affiliated Hospital, Box 153, Institute of Ultra-sonic Engineering in Medicine, Chongqing University of Medical Sciences, 1 Medical College Road, Chongqing 400016, China.
Shao
Ze-Yong
ZY
Zhao
Chun-Liang
CL
Hu
Kai
K
Wu
Feng
F
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020112:Rhodamine 123; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics / Q000378:metabolism; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D020112:Rhodamine 123; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein