The Experimental Study On The Reverse Of Multidrug Resistance Of Lung Cancer Using Arsenic Trioxide

Background and Objective: The drug-resistance and metastasis in early stages of human lung cancer have significant effect on chemotherapy of human lung cancer and survival rat of patients with lung carcinoma. The objective of this study is to observe the effects of arsenic trioxide (As₂O₃) and DDP alone or in combination on reverse of resistance in HTB-56/DDP cell lines and apoptosis-inducing effects of arsenic trioxide in vitro and vivo. To detect the changes of MRP, LRP, P-gp, bcl-2, et al of transplanted tumor in BAL/C mice after treatment with arsenic trioxide(As₂O₃), DDP or in combined, and explore its possible mechanisms of multidrug resistance, thereby providing rational clinical application.Materials and Methods: The HTB-56, HTB-56/DDP cell lines were grown in RPMI-1640 medium supplemented with 10% calf serum, 100 units/ml penicillin, 100μg/ml streptomycin, incubated at 37℃ in humidified atmosphere of 5%CO₂ and 95%O₂.The sensitivity of HTB-56, HTB-56/DDP cells to DDP, As₂O₃ and in combination was tested by means of counting living cells using microcopy through trypan blue staining. The growth inhibiting and IC50 of cells were measured by MTT. The morphological changes of apoptotic cells were detected by transmission electron microscopy. The transplantable multidrug resistance lung cancer cell line HTB-56/DDP model was established in BALB/C mice by subcutaneous implantation, which were divided 4 groups including group A(control), group B(As₂O₃), group C(DDP), group D(combined). After growth of 6 weeks with intraperitoneal injection with normal sodium, As₂O₃, DDP and DDP combined with low dosage of As₂O₃. Then we killed the mice, collected blood, weighed the body of mice and weight of tumor. Flow cytometry(FCM) was used to detect the changes of P-gpexpression and apoptosis rate after different drug treatment. RT-PCR assay was used to determine the expression level of MRP and LRP-mRNA . The expressions of p53, bcl-2, bax were detected by immunocytochemistry method. All of which are related with multidrug resistance(MDR).Results: Both HTB-56 and HTB-56/DDP can be inhibited by As2O3. Compared with two groups with no statistically difference(/?>0.05). However, the resistance of HTB-56/DDP cell line to DDP was 14.65 times than that of HTB-56. The resistance of HTB-56/DDP cell line to DDP combined with AS2O3 of low-concentration was 6.35. The times of reverse of resistance were 3.5. Both cells growth curve showed that inhibition appeared after 48 hours of administration of AS2O3 and inhibiting rates by AS2O3 were significantly different in dose-dependent and time-dependent manners (p<0.05). The inhibition increased (p<0.05) as AS2O3 concentration increased. The IC50 values of both cell lines to AS2O3 were similar( p>0.05). AS2O3 could reinforce the sensitivity of HTB-56/DDP cells to DDP and induce apoptosis of cell lines. The typical morphological changes of apoptotic cells were observed by transmission electron microscopy. AS2O3 had some effect on inhibition of tumor growth and combined with DDP can significantly inhibit tumor growth and weight in BALB/C mice, with the weight of tumor treated by AS2O3 was lower than that of group A and group D (p<0.01). The average weights of mice treated by higher dosage of AS2O3 were lower than that of group A (p<0.05), which mean there were also had some side effects on the growth of mice. 0.25mg/kg AS2O3 could effectively reverse the resistance of HTB-56/DDP cell to DDP in vivo, which had no side effect on mice than that of group D (p>0.05). FCM results showed that the expression of P-gp after using DDP was relatively higher than that of using other drugs. There was significantly difference between them after administration of As2O3 and DDP (p<0.05). The rate of apoptosis of group B and group C was significantly increased compared with group A and group D. RT-PCR was used to detect the MRP, LRP-mRNA. The expression of MRP-mRNA was significantly higher both in group B and group C than that in group D and group A (p<0.05). However, the expression of LRP-mRNA was same in four groups (/?>0.05). The results also indicate that the expression of bcl-2 and p53 was both significantly lower in group B and group Cthan that of group D and group A (p<0.01), while the expression of bax preotein was higher in group B and group C than that of group D and group A(p<0.05).Conclusions: AS2O3 is an effective anti-tumor drug and an effective drug for reversal of HTB-56/DDP to DDP. As2O3 can also effectively reverse the resistance of HTB-56/DDP cells in BALB/C to DDP and induce apoptosis of HTB-56/DDP cells in vitro and vivo. High dosage of AS2O3 has some side effect on mice. AS2O3 can up-regulate the expression of bax and down-regulate expression of P53, bcl-2, MRP-mRNA and LRP-mRNA, which are concerned with MDR and apoptosis and the growth of lung cancer.