Background and objective: Lung cancer is the leading cause of cancer-related deaths worldwide. Approximately 15% of all histological types consist of small cell lung cancer (SCLC). Chemotherapy is one of the major treatment method. Though the current first-line standard chemotherapy regimen for SCLC is active in most SCLC cases, however the disease recurs shortly after the first successful treatment with multi-drug resistance (MDR) phenotype. Our previously study showed that SPHK1 was associated with MDR in SCLC. The aim of this study is to investigate the role of sphingosine kinase 1 (SPHK1) showed in small cell lung multi-drug resistance.
Methods: Firstly, the analysis of QRT-PCR and Western blot were used to study differential expression of SPHK1 from mRNA and protein levels in both the H69 and H69AR cell lines. Then, Downregulation of SPHK1 by transfection with siRNA in H69AR. Moreover, the sensitivities of cells to chemotherapy drugs such as ADM, DDP, VP-16 were detected by CCK8 assay. The change of cell cycle and apoptosis rate were detected by flow cytometry. Meanwhile, expression of SPHK1 in clinical specimens were detected by QT-PCR and immunohistochemistry. Relation of SPHK1 expression with clinicopathological features and prognosis of patients was studied.
Results: The expression of SPHK1 was significantly decreased in H69AR cells that in the H69 cells. The sensitivities of H69AR cells to chemotherapy drugs were increased when up-regulated the expression of SPHK1, enforced SPHK1 expression increased cell apoptosis and the cell cycle arrest in G0/G1 phase in H69AR cells, the expression of SPHK1 was not associated with gender, age, but significantly correlated with clinical stage, chemosensitivity and overall survival (P<0.05).
Conclusions: Our results suggest that SPHK1 is involved in the regulation of small cell lung cancer multi-drug resistance, SPHK1 may be as potentialtarget gene to evaluate the chemosensitivity and clinical prognostic for SCLC.
背景与目的 小细胞肺癌约占全部肺癌的15%，化疗是其主要的治疗方法之一，虽然早期对一线化疗方案敏感，但极易出现多药耐药而导致治疗失败。前期基因芯片发现SPHK1与小细胞肺癌的耐药性相关，本研究进一步探讨SPHK1在小细胞肺癌多药耐药中的作用。方法 首先通过QRT-PCR和Western blot从基因和蛋白水平检测化疗敏感细胞株H69及多药耐药细胞株H69AR中SPHK1的差异表达；转染siRNA下调H69AR细胞中的SPHK1的表达，通过CCK8检测细胞对各种化疗药物（ADM, DDP, VP-16）的敏感性变化，流式细胞仪检测细胞周期及凋亡的变化。同时收集小细胞肺癌化疗前组织和血液标本，将其分为化疗敏感组和耐药组，QRT-PCR检测小细胞肺癌患者血液标本中SPHK1的表达，免疫组化法检测小细胞肺癌患者组织标本中SPHK1的表达，分析SPHK1与小细胞肺癌患者预后相关性。结果 SPHK1在耐药细胞H69AR中的表达明显高于H69，下调H69AR中SPHK1的表达能够增加细胞对化疗药物的敏感性，促进细胞的凋亡，细胞周期发生G0/G1期阻滞，SPHK1在小细胞肺癌耐药患者中的表达较敏感患者明显增加，SPHK1的表达与患者的性别、年龄无关，与疾病的分期、对化疗的敏感性及生存时间密切相关，差异具有统计学意义（P<0.05）。结论 SPHK1参与调节小细胞肺癌多药耐药，SPHK1可作为评估小细胞肺癌化疗敏感性及临床预后的潜在靶基因。