ABSTRACT
Objective: To investigate the effect of elemene on the drug resistance of cisplatin (DDP)-resistant human lung adenocarcinoma A549/DDP cells, and its possible mechanism. Methods: The growth inhibition of A549/DDP cells treated with elemene alone or combined with different concentrations of DDP was detected by MTT assay. The morphological changes of apoptosis of A549/DDP cells treated with different concentrations of elemene (20 and 40 μg/mL) for 24 h were observed by Hoechst 33342 staing under fluorescence a microscope. The apoptosis rate of A549/DDP cells treated with different concentrations of elemene (20 and 40 μg/mL) after 24 h were detected by FCM (flow cytometry). The expressions of cytochrome C, pro-caspase-3, caspase-3 and the Bcl-2 family proteins were measured by Western blotting analysis. Results: MTT result showed that different concentrations elemene could inhibit the proliferation of A549/DDP cells in a time- and dose- dependent manner. Intriguingly, elemene plus DDP enhanced the sensitivity of A549/DDP cells to DDP and reversed the resistance of A549/DDP cells. Elemene was also a strong inducer of apoptosis in this model system, and a synergistic effect on induction of cell death was observed when the tumor cells were treated with both agents. The result showed that elemene could enhance A549/DDP cell apoptosis. Furthermore, the combination of elemene and DDP also enhanced the protein expressions of cytochrome C, caspase-3 and Bad, and reduced the protein levels of Bcl-2 and pro-caspase-3 in the A549/DDP cells. Conclusion: The reversal of the multi-drug resistance of A549/DDP cell line by elemene may result from its effect on induction of apoptosis. Elemene treatment can impaire mitochondrial membrane, release cytochrome C into cytoplasm, activate caspase-3,up-regulate pro-apoptotic protein Bad and down-regulate anti-apoptotic protein Bcl-2, and finally caused apoptosis. Copyright © 2013 by TUMOR.
ABSTRACT
Objective: To investigate the effects of gene-associated with retinoid-interferon-induced mortality 19 (GRIM -19) on chemotherapeutic sensitivity of cisplatin (DDP)-resistant human lung caner cell A549/DDP and the expressions of related genes. Methods: The recombinant plasmid PIRES-Puro2-GRIM-19-Myc and the empty vector PIRES-Puro2-Myc were transfected into A549/DDP cells by liposome transfection reagent, respectively. The expression levels of GRIM-19 protein in the A459 parental cells, A459/DDP cells, A549/DDP cells transfected with GRIM -19 gene and A549/DDP cells transfected with an empty vector were detected by Western blotting. The changes of chemotherapeutic sensitivities to multi-drugs in A549/DDP cells stably transfected with GRIM -19 gene were detected by MTT assay. The expression levels of GRIM-19, signal transducers and activators of transcription 3 (STAT3), vascular endothelial growth factor (VEGF) and P-glycoprotein (P-gp) mRNAs in the A549/DDP cells stably transfected with GRIM -19 gene were examined by real-time fluorescence quantitative-PCR (RFQ-PCR). Results: The Western blotting result showed that the expression level of GRIM-19 protein was increased in the A549/DDP cells after transfection with GRIM -19 gene. The resistance index of A549/DDP cells was 16.86±1.32 folds higher than that of the A549 parental cells. The resistance index of the A549/DDP cells after transfection with GRIM -19 gene was decreased by 3.70±0.91 folds as compared with that of the A549/DDP cells transfected with an empty vector. The expression levels of STAT3, VEGF and P-gp mRNAs in the A549/DDP cells transfected with GRIM -19 gene were decreased. Conclusion: GRIM-19 can increase the chemotherapeutic sensitivity of A549/DDP cells. This effect may be associated with the down-regulations of STAT3, VEGF and P-gp. GRIM-19 may become a potential therapeutic agent to reverse drug-resistance in A549/DDP cells. Copyright © 2012 by TUMOR.
ABSTRACT
Background and purpose: Multidrug resistance (MDR) of tumor cell is the main reason for clinical chemotherapy failure as well as cancer recurrence and metastasis. This study was to construct a lentiveral vector of RNA interference of MDR1 gene and observe its inhibitive role on the expression of MDR1 in A549 and A549/DDP cells. Methods: Oligos of base pairs for hairpin RNA targeting MDR1 were chemically synthesized. Via annealing and inserting them into the down-stream of H1 promoter of linearized pSUPER, we obtained the siRNA constructs for MDR1, which were afterwards transfected into A549, a human lung cancer cell line expressing high level MDR1, the impact of constructs was observed on the expression and interference efficiency of siRNA against MDR1. The effective sequence of siRNA targeting MDR1 gene was confirmed. Both sense and antisence oligo DNA of the targeting sequence was designed, synthesized and cloned into the PTM vector, containing a promoter and a green fluorescent protein (GFP). The resulting lentiviral vector containing MDR1 siRNA was named PTM-siMDR1 and then transfected into A549 and A549/DDP cells after being confirmed by PCR and sequencing. Results: Restriction digestion and DNA sequencing showed that the siRNA constructs for MDR1 were successfully produced and the expressed siRNA could effectively down-regnlate the expression of MDRI. PCR demonstrated that the lentivirus RNAi vector of MDR1 producing PTM-siMDR1 was constructed successfully. The chemosensitivity of A549/DDP cells to cisplatin were enhanced obviously after trartsfection. Conclusion: The lentivirus RNAi vector of MDR1 can significantly revise the resistance ofA549/ DDP cells with eisplatin after infection.