Modulation of doxorubicin sensitivity by cyclosporine A in hepatocellular carcinoma cells and their doxorubicin-resistant sublines.
OBJECTIVE
Cyclosporine A (Cys) and verapamil (Ver) sensitize multidrug-resistant (MDR) cells to various anticancer drugs by interacting with membrane glycoproteins involved in the drug efflux. In the present study, we assessed the effect of Cys on the modulation of doxorubicin (DOR) sensitivity in hepatocellular carcinoma (HCC) cell lines, and their DOR-resistant sublines.


METHODS
The sensitivity to DOR and the chemosensitizing effects of Cys were assessed by using two human HCC cell lines, PLC/PRF/5 and Hep-3B, and their DOR-resistant sublines, PLC/DOR and 3B/DOR. The expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein (MRP) mRNA in these cells were detected by using a RT-PCR. The HCC cell lines grown in individual wells of 24-well plates were incubated with DOR that were sequentially diluted in culture medium in combination with 5 micromol/L Cys for 24 h. The cell viability in each well was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.


RESULTS
The mRNA of MDR1 and that of MRP were readily detectable in the HCC cell lines by RT-PCR. When 5 micromol/L Cys was added to the culture, the 50% inhibiting concentration (IC50) of DOR was reduced from 0.93 +/- 0.29 microg/mL to 0.32 +/- 0.10 microg/mL in PLC/PRF/5, and from 0.25 +/- 0.07 microg/mL to 0.09 +/- 0.04 microg/mL in Hep-3B. Furthermore, in the presence of 5 micromol/L Cys, the IC50 of DOR was reduced from 48.63 +/- 17.04 microg/mL to 0.49 +/- 0.14 microg/mL in PLC/DOR, and from 4.60 +/- 1.22 microg/mL to 0.15 +/- 0.06 microg/mL in 3B/DOR. The amounts of PCR products of MDR1 mRNA in PLC/DOR and 3B/DOR were greater than those in PLC/PRF/5 and Hep-3B, respectively.


CONCLUSIONS
In HCC, the amplification of MDR1 mRNA is probably the main mechanism underlying acquired DOR resistance. Cyclosporine is also indicated to be highly active in potentiating the anticancer activity of DOR in HCC cells and their DOR-resistant sublines.
Shiraga
K
K
First Department of Internal Medicine, Okayama University Medical School, Japan.
Sakaguchi
K
K
Senoh
T
T
Ohta
T
T
Ogawa
S
S
Sawayama
T
T
Mouri
H
H
Fujiwara
A
A
Tsuji
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000503:physiopathology*; D016572:Cyclosporine / Q000627:therapeutic use*; D004317:Doxorubicin / Q000627:therapeutic use*; D004351:Drug Resistance; D004357:Drug Synergism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000503:physiopathology*; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D012333:RNA, Messenger; D004317:Doxorubicin; D016572:Cyclosporine