| Objective To study the relationship between the curative effect of interventional therapy for hepatocellular carcinoma and MDR, and establish a multidrug resistance cell line of human hepatic carcinoma by intermittent administration of high dose adriamycin (ADM) then study the mechanism of multidrug resistance and compare the difference of apoptosis rate between HepG2/ADM and HepG2 induced with ADM and Inquire it's related regulation mechanism. Material and Methods 1. Clinical trial A total of 52 HCC patients with tumor size ≥5cm were enrolled. 26 patients were treated with TACE-PAI and other 26 patients were treated with TACE 2~5 times. Biopsy was performed in the TACE group, then MDR1 and MRP mRNA was detected with reverse transcriptase polymerase chain reaction (RT-PCR) methods. 2. Human hepatocellular carcinoma cell line HepG2 was induced to multidrug resistance cell line HepG2/ADM by intermittent administration of high dose adriamycin (ADM). The multidrug resistance was detected by using MTT assay. The expressions of MDR-1 gene-coded P-glycoportein (P-gp), multidrug resistance-associated protein (MRP), and GSH / GST were examined by flow cytometry. The expressions of MDR and MRP gene were also detected using RT-PCR in both HepG2/ADM and HepG2 cell lines. 3.1 Compare the apoptosis rate with ADM (0.03mg·L-1) between HepG2/ADM and HepG2. The apoptosis rate was detected by FCM. 3.2 HepG2/ADM cells were divided into 6 groups: group 1, as control group, were cultured in 37℃, 5% CO2 condition, with no ADM administrated; group 2 were cultured in 40 ℃, 5% CO2 condition, with no ADM administrated; group 3 were cultured in 43 ℃, 5% CO2 condition, with no ADM administrated; group 4 were cultured with ADM(0.03mg·L-1) in 37℃, 5% CO2 condition; group 4 were cultured with ADM(0.03mg·L-1) in 37℃, 5% CO2 condition; group 5 were cultured with ADM(0.03mg·L-1) in 40℃, 5% CO2 condition; group 6 were cultured with ADM(0.03mg·L-1) in 43℃, 5% CO2 condition. Compare the apoptosis rate of HepG2/ADM with ADM (0.03mg·L-1) among these 6 groups. 3.3 p53, bcl-2 and bax mRNA was also detected with RT-PCR method among these 6 HepG2/ADM groups respectively. Results 1. After 3 times treatment, objective responses (CR+PR) were achieved in 16 of 26 (61.5%) patients in the TACE-PAI group compared with 8 of 26 (30.8%) in the TACE group (p<0.05). During 24 months of follow-up, the 1-, 2-year survival rate of TACE-PAI group and TACE alone group was 80.8%, 61.5%; 69.2%, 30.8% respectively. There was significant difference between the two groups (p<0.05). The positive rate of MDR1 and MRP mRNA detection in theTACE group was 73.1% (19/26) and 57.7% (15/26) respectively. The objective responses rate in positive and negative group for TACE treatment was 22.7% (5/22) and 75% (3/4) respectively. But difference was of no significant. 2. HepG2/ADM was resistant to many anti-tumor agents. The IC50 of ADM was 89.38 times higher than that of HepG2. The expression of P-gp was increased significantly to (45.1±5.1)% while that of HepG2 was only (11.3±1.2)%. There was no difference of the MRP and GSH/GST expressions between HepG2/ADM and HepG2 cells. The levels of MDR1 mRNA expression were relatively high in HepG2/ADM. 3.1 The apoptosis rate of HepG2/ADM and HepG2 were (3.5±0.5)% and (17.5±2.8)% respectively. There was significant difference between the two groups (p<0.05)。2. The apoptosis rate of groups1~6 were (2.8±0.3)%,(7.4±1.1)%,(11.3±1.9)%,(5.2±0.5)%,(18.1±1.8)%,(30.7±3.5)% respectively. Compared with chemotherapy group and thermotherapy group, the apoptosis rate of the thermochemotherapy group was the highest among these 6 groups( p<0.05). The mRNA expression detected with RT-PCR for p53, bcl-2 and bax among these 6groups were 0.21, 0.29, 0.34, 0.25, 0.43 0.48; 0.31, 0.16, 0.21, 0.13, 0.24 0.11; 0.23, 0.27, 0.29, 0.25, 0.31 and 0.35 respectively. Conclusions 1. Sequential therapy with TACE and PAI is superior to repeated TACE alone for patients with large HCC. MDR gene expression was common in HCC and it may affect the outcome of TACE treatment. 2. HepG2/ADM is a model with multidrug resistance and the levels of MDR1 mRNA expression are higher in HepG2/ADM than in HepG2. |
