[Study on sensitivity to chemotherapy by combination therapy with tumor necrosis factor-alpha and bromocriptine in the multidrug resistant subcell line of HepG2].
OBJECTIVE
To investigate the change of chemosensitivity of hepatocarcinoma cell line (HepG(2)/ADM) after treated by bromocriptine (BCT) combination with human tumor necrosis factor-alpha (TNF-alpha).


METHODS
Firstly, TNF-alpha gene was transfected into HepG(2)/ADM cell line by liposome to establish a cell model expressing the TNF-alpha protein stably. All experiments were divided into four groups and named blank control group (group A), drug resistant group HepG(2)/ADM (group B), TNF-alpha gene group HepG(2)/ADM/TNF (group C) and BCT group (group D) respectively. And group D came from group C treated with BCT simultaneously. MTT assay was tested to detect the sensitivity to ADM of each group and Rhodamine 123 (Rh123) was applied to test the function of P-gp by flow cytometric analysis (FCM). MDR associated genes and proteins and PKC-alpha protein were detected by immunohistochemistry (IHC), Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) methods, respectively. The expression and the apoptosis rate of Bcl-2 in the hepatocarcinoma cells were detected by FCM.


RESULTS
There was significant difference between group C and D in the rate of reversing resistance and the intracellular Rho123 accumulation (P < 0.01). MDR1 mRNA and P-gp protein expression in group C and D were low similar to that in group A, but no difference could be found among them (P > 0.05). As we found that PKC-alpha protein expression was downregulated in group D but Bcl-2 protein expression was downregulated in group C, and there was significant difference compared to other groups. The apoptosis rate of hepatocarcinoma cells was much higher in group D than that in group C (P < 0.01) with FCM, but similar to group A (P > 0.05).


CONCLUSIONS
Synergistic effect of BCT and TNF-alpha on reversing hepatocellular carcinoma multidrug resistance could enhance the susceptibility of HepG(2)/ADM cells to cytotoxic drugs.
Ding
Lei
L
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.
Chen
Xiao-ping
XP
Zhang
Zhi-wei
ZW
Jing
Kai
K
Cao
Bin
B
Zhu
Peng
P
Li
Jie
J
Zhang
De-yong
DY
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001971:Bromocriptine / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D018432:Drug Resistance, Multiple / Q000187:drug effects* / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D001971:Bromocriptine