| Lung cancer is the most commonly diagnosed cancer worldwide, It is the leading cancer site in both cancer-related morbidity and mortality. According to World Health Organization (WHO) cancer report in 2014, an estimated 1,800,000 new cases of lung cancer were diagnosed every year and the 5-year survival rate was close to 15%. Cancer deaths in the world in 2012 are estimated to be 1,600,000 (717 398 men and 565 703 women). In china, new cases of lung cancer were estimated at 676,000, resulting in 565,000 deaths every year. Non-small cell lung cancer (NSCLC) is the prevailing type, consisting mainly (>80%) of lung cancer. Treatment for lung cancer has been greatly advanced, including improved surgical technique and a variety of new chemotherapy drugs. However, most patients with NSCLC when diagnosed show advanced stage for the lack of effective techniques for early diagnosis. Chemotherapeutic agents present a major treatment for these patients. Cisplatin (CDDP) is a component of standard treatment regimens for lung cancer. However, the resistance acquired following the sequential cycles of treatment limits the benefit of cisplatin in cancer treatment. Little is known about the exact mechanism of cisplatin resistance in lung cancer. Herein, study of the molecular mechanisms of DDP resistance will aid the clinician to oversee the resistance in advance thus improving the efficacy of lung cancer therapeutics.CXCR4 is a highly conserved seven-span transmembrane G-protein-coupled receptor that exclusively binds its ligand CXCL12, composed of 352 amino acids, the coding gene of which is located on chromosome 2q21. It's N-terminus outside the cell surface binding to the ligand, three extracellular and three intracellular loops as well as a C-terminus in the cytoplasm. CXCR4 is expressed in a broad range of tissues and plays an important role in many biological processes, such as immune cell migration, embryonic development, growth, angiogenesis, and hematopoiesis, through a cascade of signal transduction events. Since Muller investigated the functions of chemokine receptors in breast cancer in 2001, CXCR4 is highly expressed in various types of human tumors, such as breast, lung, pancreas, colon, ovary, and prostate cancers, and plays a critical role in tumorinitiation, progression and metastasis. Our previous studies have shown that CXCR4 is highly expressed in lung cancer and plays a crucial role in tumorinitiation and metastasis of lung tumor.Previously, a few studies have revealed that association between the over-expression of CXCR4 and cancer chemoresistance in some cancers. In pediatric acute lymphoblastic leukemia, CXCR4 with its expression arised can protecte from chemotherapy-induced apoptosis to enhance chemo-resistance when co-cultured with bone marrow stroma, Treatment with the CXCR4 inhibitor plerixafor diminishes stromal protection and confers chemosensitivity. Higher levels of expression of CXCR4 also have been shown significantly associated with cisplatin-based chemotherapy resistance in epithelial ovarian cancer. Although these evidences imply that CXCR4 is associated with development of drug resistance, the precise role of CXCR4 in cisplatin resistance of lung cancer remains largely unexplored.Part ?, The association between CXCR4 expression levels and cisplatin resistance in NSCLCOBJECTIVE This study aimed to examine the expression of CXCR4 in both chemosensitive and chemoresistant lung cancer tissues and cell lines.METHODS A total of 64 lung cancer specimens were collected from patients with primary NSCLC surgical resection at Renmin Hospital of Wuhan University from May 2012 to October 2015. None of the subjects had received any chemotherapy and radiotherapy prior to surgery. All of these patients were treated with platinum-based chemotherapy after surgery according to the NCCN guideline every 3 weeks for four cycles. Patients were followed up by telephone until recurrence/metastasis or until March 2016. Patients with disease progression or recurrence 12 months after surgery were defined as being chemoresistant, whereas those without recurrence or recurrence more than 12 months after surgery were defined as chemosensitive. The expression levels of CXCR4 were analyzed in A549 and A549/DDP cells (a cisplatin resistant A549 subline) by Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis and Immunofluorescence assay.RESULTS We found that the levels of CXCR4 were significantly increased in tissues from cisplatin resistance patients, compared to that in samples from cisplatin sensititive patients. We next categorized the NSCLC sensitive group into negative (40.6%), weak (28.1%), moderate (18.8%) and strong (12.5%) and resistance group into negative (15.6%), weak (34.4%), moderate (21.9%) and strong (28.1%), The staining score in resistance group was 1.63±1.07 (versus 1.03±1.06 in sensitive group). Furthermore, we examined the levels of CXCR4 expression in A549 and A549/DDP cells by relative quantitative PCR, Western blot and Immunofluorescence. both CXCR4 mRNA and protein in A549/DDP were significantly higher than that in A549 cells. These results indicate that CXCR4 may be involved in NSCLC cancer cisplatin resistance.CONCLUSIONS The expression level of CXCR4 in chemoresistant lung cancer tissue and cells was higher; CXCR4 may play an important role in the cisplatin-based chemotherapy resistance of lung cancer, indicating that CXCR4 could be the target for the treatment of lung cancer.Part ? The potential role of CXCR4 in the response of NSCLC to chemotherapyOBJECTIVE To explore the effect (expression and implication) of CXCR4 silence induced by siRNA on the cisplatin resistance of lung cancer, and investigate whether CXCR4 plays an important role in cisplatin-based chemotherapy resistance of lung cancer.METHODS We constructed recombinant siRNA-CXCR4 plasmids and transfected them into A549 and A549/DDP cells (a cisplatin resistant A549 subline) in vitro. Reverse transcription polymerase chain reaction (RT-PCR) and and western blotting were used to measure the expression of CXCR4 RNA and protein, respectively. We further investigated whether knockdown of CXCR4 could result in increasing cisplatin sensitivity of the NSCLC cells by MTT assays; The CCK-8 assay was applied to further confirm the role of CXCR4 knocking down in A549 and A549/DDP cells. Flow cytometry was used to detect cell apoptosis of A549 and A549/DDP cells by RNA interference induced.RESULTS Both A549 and A549/DDP si-CXCR4 group cells expressed lower levels of CXCR4 mRNA and protein than A549 and A549/DDP group cells (p<0.0001). As the results of MTT assays:the half-maximal inhibitory concentration (IC50) at 48h was 6.665±0.215?g/ml and 4.373±0.077?g/ml for A549 and si-CXCR4 A549 cells, respectively (P<0.0001),14.837±0.099?g/ml and 9.969±0.318?g/ml for A549/DDP and si-CXCR4 A549/DDP cells, respectively (P<0.0001), Both A549 and A549/DDP si-CXCR4 cells were more sensitive to the cisplatin treatment than A549 and A549/DDP cells,indicating that CXCR4 knockdown increases sensitivity to cisplatin. In addition, cisplatin effectively inhibited CXCR4 expression in both cells in a dose-dependent manner.As shown by flow cytometry analysis,A549 and A549/DDP transfected with negative control siRNA showed about 11.16% and 4.93% apoptotic cells, respectively, whereas the apoptotic cells were about 20.62% and 6.64% in CXCR4 knocking down group cells, respectively. The results showed that knockdown of CXCR4 with small interfering RNA also contribute to the recovery of cisplatin-induced apoptosis in A549 and A549/DDP cells. The CCK-8 assay was applied to further confirm the role of CXCR4 knocking down in A549 and A549/DDP cells. Si-CXCR4 significantly suppressed cell proliferation in A549 and A549/DDP from 24 h to 72 h (P<0.0001).CONCLUSIONS These findings suggest CXCR4 silence induced by siRNA can increases sensitivity to cisplatin of lung cancer.Part ?. CXCR4 promote cisplatin resistance through up-regulation of CYP1B1OBJECTIVE In the present study, we demonstrated that CXCR4 was significantly highly expressed in cisplatin-resistant NSCLC patients and A549/DDP cell line; we also reported that CXCR4 inhibition by siRNA could reverse chemoresistance and retard tumor growth. But the precise role of CXCR4 in cisplatin resistance of lung cancer remains largely unexplored.METHODS We performed the correlation of CXCR4 with cytochrome P450 related-molecules by bioinformatics. the association between CYP1B1 and cisplatin resistance in NSCLC, the expression of CYP1B1 in 32 NSCLC and 32 NSCLC with cisplatin-resistance tissues was examined by immunohistochemistry. Western blotting was used to measure the expression of CYP1B1 protein when the expression of CXCR4 in A549/DDP cells was blocked by siRNA. Reverse transcription polymerase chain reaction (RT-PCR) and and western blotting were used to measure the expression of CYP1B1 RNA and protein after transfected of siRNA-CYP1B1 respectively. We further investigated whether knockdown of CYP1B1 could result in increasing survival rate of the NSCLC cells by CCK-8 assay. Flow cytometry was used to detect cell apoptosis of A549 and A549/DDP cells by RNA interference induced.RESULTS We just found CXCR4 have positive correlation with CYP1B1 (r=0.616, p<.0001) and (r=0.538, p<0.0001) in GSE30219 and GSE41271 respectively. While the CYP1B1 level was weak or not detected in most of the sensitive group, and weak or strong staining in resistant group, then we categorized the NSCLC sensitive group into negative (25.0%), weak (37.5%), moderate (28.1%) and strong (9.4%) and resistant group into negative (12.6%), weak (25.0%), moderate (21.9%) and strong (40.6%). The staining score in resistant group was 1.69±1.19 (versus 1.08±0.97 in sensitive group).Further, we analyzed CYP1B1 expression with the clinical outcomes of 64 NSCLC, and found the expression of CYP1B1 correlated with disease free survival, strong staining of CYP1B1 showed the poor survival than moderate, weak and negative respectively (Fig 5D). Finally, we confirmed that CXCR4 have positive correlation with CYP1B1 (r=0.44, P<0.001) in NSCLC patients. The results of RT-PCR and and western blotting showed that CYP1B1 mRNA and protein level were significantly down-regulated due to knockdown of CXCR4. Transfection with CYP1B1 siRNA resulted in a dramatic reduction in endogenous levels of CYP1B1 in A549/DDP by Western Blot. Knockdown of CYP1B1 significantly decreased the survival rate of A549/DDP cells, but didn't decrease the survival rate of A549 cells. Meanwhile, A549 and A549/DDP transfected with negative control siRNA showed about 2.0% and 0.7% apoptotic cells, respectively, whereas the apoptotic cells were about 7.5% and 2.0% in CYP1B1 silencing group cells, respectively. The apoptosis significantly increased in A549 and A549/DDP after inhibiting CYP1B1 expression.CONCLUSIONS CXCR4 may through regulated the expression of CYPIBI to promote cisplatin resistance... |
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