Inhibition of ABCG2 Sensitizes Multidrug-resistant Small Cell Lung Cancer to Chemotherapy
|School||Southern Medical University,|
|Course||Thoracic and Cardiovascular Surgery|
|Keywords||Small cell lung cancer Multi- drug-resistant ABCG2 Gene silencing|
Background: Lung cancer is the first disease to cause human cancer death, the world each year more than one million deaths due to lung cancer, of which 80% to 90% of patients with non-small cell lung cancer (non-small cell lung cancer, NSCLC), 15% -25% for small cell lung cancer (small cell lung cancer, SCLC), and the lowest degree of differentiation in the lung pathological type in small cell lung cancer, the earlier occurrence of hematogenous metastasis, so the prognosis is poor. Clinically diagnosed as small cell lung cancer patients are extremely sensitive to chemotherapy, so take chemotherapy for the treatment, but soon there will be tumor resistance, chemotherapy failure, resulting in small cell lung cancer patients with the early stage of tumor progression, relapse and metastasis, the 5-year survival rate of less than 10%. Chemotherapy resistance is one of the biggest problems of the clinical treatment of small cell lung cancer, how to reduce the resistance of small cell lung cancer chemotherapy drugs is based on clinical workers solved the key problem. Multidrug resistance is a drug-resistant form of acquired resistance to the most important, have acquired resistance to contact anticancer drugs not related to a variety of drugs in terms of function or structure. That the main tumor resistance mechanisms: (1) the pharmacological drug resistance (pharmacological resistance): refers to the resistance caused by the impact of the body of drugs, such as drugs into enhanced metabolism or activation barriers, lack of tumor blood supply drugs organization pro-poor penetration of the formation of drug haven; (2) micro-environment resistant (microenvironment resistance): refers to the survival and growth of tumor cells depends on the organ microenvironment, and the organ microenvironment by different resistance genes expression to influence the sensitivity of tumor cells to chemotherapeutic agents; (3) apoptosis resistance (apoptosis resistance): of P53, BCL-2, C-myc is involved in the control of apoptosis inhibition; (4) biochemical resistance ( biochemical resistance): refers to the complex changes in the genetic and biochemical characteristics of the tumor cells, the cells via different routes of drug resistance, which is the most widely studied ABC membrane transporter protein superfamily members, drug export pump, P- glycoprotein (P-gp), multidrug resistance associated protein (MRP), lung resistance-related protein (LRP). 1998 Doyle LA, Yang W and other breast cancer cell lines MCF-7/AdrVp, first detected in the breast cancer cell lines to a breast cancer resistance protein (the BIRADS Cancer RESistance protein, BCRP), this protein for the ATP-binding cassette transporter protein G superfamily member 2 (ATP2binding cassette super-family G member2, ABCG2), this protein chemotherapy pump drugs out of the cell, the intracellular concentration of chemotherapeutic drugs significantly reduced, so that the failure of chemotherapy, causing the tumor cells become resistant. Our previous gene chip screening results show that there are significant differences in the expression of ABCG2 in small cell lung cancer cell lines with the parental cell line. Based ABCG2 differentially expressed in small cell lung cancer cell lines with the parental cell line, we hypothesized that ABCG2 played an important role in small cell lung cancer, to explore the role of ABCG2 generated in small cell lung cancer cell resistance, solve clinical prone to drug resistance in cell lung cancer treatment provides a new theoretical basis. Objective: the experiment select small-cell lung cancer cell lines the H69 its multi-drug resistant cell line H69AR as the object of study, analysis of the morphology of the two cell lines, chemosensitivity differences. 2. Analysis of small cell lung cancer cell line H69 and its multi-drug resistant cell line H69AR cell lines at the mRNA and protein levels of ABCG2 expression differences. The high expression of ABCG2 gene silencing of ABCG2 H69AR cell lines using gene silencing technology to detect the effect observe H69AR cell lines resistant to change. Method: 1. Observed under an optical microscope small cell lung cancer cell line H69 with its multi-drug resistant cell line H69AR both morphological changes. Application of the the CCK8 assay small cell lung cancer cell lines H69 MDR-resistant cell line H69AR both with doxorubicin, etoposide, cisplatin drug sensitivity. 3. Applications of real-time quantitative PCR technology of Western-blot analysis of ABCG2 expression differences in the two cell lines. 4 real-time fluorescent quantitative PCR, the Western-blot the technology confirmed H69AR cells ABCG2 were significantly higher than the level of ABCG2-siRNA transfected H69 cells, using Liposome Lipofectamine2000 Law to enter H69AR cells, ABCG2 silent, real-time PCR , western-blot detection of gene silencing effect. ABCG2 silence 5.CCK8 assay H69AR cells to chemotherapeutic drug resistance changes. : 1.CCK8 the analysis show resistance to doxorubicin resistant cell line H69AR relative affinity of the H69 cells to cisplatin, etoposide, the phenomenon of cross-resistance, the same concentration of cisplatin in H69 cells and H69AR cells, the inhibition rate; different concentrations, acting in the same cell, the inhibition rate is also different (F = 68.543, P LT; 0.001). Resistance index of 6.05 times. The same concentration of etoposide role in H69 cells and H69AR cells, the inhibition rate is different; different concentrations, acting in the same cell, the inhibition rate is also different (F = 19.964, P LT; 0.001). Resistance index of 11.43 times. 2. Relative to H69 cells by real-time fluorescence quantitative PCR, in the the mRNA level resistant cell line H69AR in ABCG2 expression level was significantly higher (95.5-fold). Protein levels in the resistant cell line H69AR ABCG2 protein significantly increased, the difference was statistically significant (t = -4.527, P = 0.011). Designed according ABCG2 gene sequence of three groups of specific targeting of ABCG2 siRNA of ABCG2-667, of ABCG2-781, of ABCG2-979, respectively, through gene silencing techniques successfully reduced the expression levels of the resistant cell line H69AR ABCG2 and set the blank group without any intervention by the real-time fluorescence quantitative PCR ABCG2-667 group compared with the blank group of ABCG2-781 group of ABCG2-979-group ABCG2mRNA express blank group 0.525,0.875,0.620 times. Western- blot gray value analysis results show that: relative to the control group, ABCG2-667 group, ABCG2-781 group, the expression of ABCG2-979 group ABCG2 protein levels, SiRNA-blank group 0.6735 ± 0.0352,0.7227 ± 0.022,0.6821 ± 0.061 times the 667 protein grayscale correction value minimum, silence the best. 4.CCK8 law results show that, with the blank control group, negative control group, cisplatin resistant cell line after silence H69AR ABCG2-667 inhibition rate increased significantly, with a statistically significant difference (F = 151.91, P = 0.000). CCK8 method results show that, with the blank control group, negative control group, relying etoposide resistant cell line after silence H69AR ABCG2-667 inhibition rate increase, with a statistically significant difference (F = 13.652, P = 0.000). Conclusion: This study again confirmed small cell lung cancer with a multi-drug resistant characteristics, relative to small cell lung cancer parental H69 cell lines, was significantly higher in small cell lung cancer cell lines H69AR, ABCG2 level expression, gene silencing technology to reduce resistance the cells the strains H69AR the ABCG2 level cisplatin-resistant cell line by gene silencing H69AR, rely on a significant increase in etoposide resistance. Resistant human small cell lung cancer cell lines H69AR produce may be associated with increased ABCG2 expression levels are closely related. 3.ABCG2 possible as an ideal target for the reversal of small cell lung cancer resistant.