Investigation Of The Mechanisms For Chemosensitization Of Arctigenin In Non-small Cell Lung Cancer

ObjectivesNon-small cell lung cancer (NSCLC) is a common malignant tumor and is the leading cause of cancer-related deaths worldwide. Despite great advances in treatment, the overall outcome is unsatisfatory, with the 5-year overall survival rate less than 20%. Combination of different anticancer agents represents an important strategy for overcoming chemoresistance and improving the survival of patients with NSCLC. Arctigenin, a dibenzylbutyrolactone lignan, is abundantly produced by Arctium lappa L and possesses anti-inflammation, antioxidant, and anti-tumor activities. Survivin is a member of the inhibitor of apoptosis protein family (IAP) that is involved in the regulation of cell survival and proliferation. A large body of evidence indicates that survivin overexpression confers tumor cell resistance to anticancer agents.. The aim of this study is to determine the effect of arctigenin on the chemosensitivity of NSCLC cells to cisplatin and to explore the role of survivin in the chemosensitization of arctigenin in NSCLC cells.Methods1. After human H460 large lung cancer cells cultured, then were treated with different concentration of arctigenin alone or in combination with cisplatin.2. For cell proliferation assay, human H460 large lung cancer cells were treated with arctigenin (1-20μM) for 72 h and tested for cell viability. For apoptosis and cell cycle analysis, H460 cells were treated with lOμM arctigenin for 48h and tested for apoptotic death and cell cycle distribution. When arctigenin was combined with cisplatin, H460 cells were treated with 10μM arctigenin alone or in combination with cisplatin at 2 or 10μM for 48h, then cell proliferation, apoptosis, and cell cycle distribution were examined.3. MTT assay was performed to determine cell viability. Annexin-v/PI double staining was done to examine apoptosis. PI staining with flow cytometry was done to determine cell cycle distribution. Western blot analysis was done to measure the protein levels of cleaved caspase-3 and PARP.4. Human full-length Survivin cDNA was amplified by polymerase chain reaction (PCR) and cloned into pcDNA3.1 (+) expression vector. The resultant survivin-expressing plasmid or empty vector was individually transfected into H460 cells using Lipofectamine 2000. Twenty-four hours after transfection, the cells were tested for survivin expression by Western blot analysis. Transfected cells were exposed to arctigenin alone or in combination with cisplatin and cell apoptosis was assessed using annexin-V/PI staining methods.5. Statistical analysis:Data are expressed as mean±standard deviation. Statistical analysis was performed using the SPSS software version 11.5. All data were analyzed by ANOVA. P<0.05 was considered statistically significant.Results1. Compared to control cells, arctigenin inhibited the proliferation of H460 cells in a dose-dependent manner(P<0.05). Arctigenin at a concentration of 10μM resulted in about 50% reduction of proliferation. The proliferation of H460 was decreased by 28% and 65% in 2 and 10μM of cisplatin, respectively. Notably,10μM of arctigenin profoundly elevated cisplatin-mediated inhibition of H460 proliferation (P< 0.01 vs. cisplatin alone-treated cells).2. After treatment with 10μM arctigenin for 48 h, the percentage of annexin-V-FITC-positive cells was increased to 9.5±2.0%, compared with 2.7± 1.2% in control(P<0.05). Western blot analysis confirmed such induction of apoptosis, and the levels of cleaved caspase-3 and PARP were markedly elevated upon arctigenin treatment(P<0.05). After a 48-h treatment, cisplatin alone at 2 and 10μM caused 8.4±1.9% and 15.6Q2.3% of H460 cells to undergo apoptosis, respectively. There was 27.2±3.1% apoptosis seen in H460 cells treated with combination of 2μM cisplatin and 10μM arctigenin(P<0.05).3. Cisplatin at the dose of 2μM did not show statistical significant effects on the cell cycle distribution. Incubation with 10μM of arctigenin or 10μM of arctigenin together with 2μM of cisplatin resulted in a G0/G1 cell cycle block, as determined by an increase of the G0/G1 population and by a corresponding reduction of the S and G2/M populations(P<0.05).4. Compared to untransfected and empty vector-transfected H460 cells, survivin-transfected cells had a 10-fold elevation in the survivin expressio(P<0.05). Low-dose cisplatin did not significantly affect the expression of survivin in H460 cells. In contrast, arctigenin significantly inhibited the expression of survivin protein(P<0.05). When cisplatin was combined with arctigenin, a greater inhibition of survivin expression was observed(P<0.05). Compared to transfection of empty vector, pre-transfection with pcDNA3.1-Survivin plasmid significantly reversed the pro-apoptotic activity of arctigenin alone or in combination with cisplatin, leading to a significant reduction in the apoptotic index(P<0.05).Conclusions1. Arctigenin exerts antiproliferative effects on H460 cells.2. Arctigenin exerts antiproliferative effects on H460 cells,which is associated with induction of apoptotic death and G0/G1 cell cycle arrest.3. Arctigenin can enhance cisplatin-mediated apoptosis in H460 cells. Therefore, arctigenin is beneficial in improving platinum-based therapy against NSCLC.4. Arctigenin is capable of inhibiting the expression of survivin in H460 cells. Arctigenin-mediated chemosensitization of H460 cells to cisplatin is associated with suppression of survivin signaling.