Mechanism of Inhibitory Effect of Ouabain on the Proliferation and Invasion of Human Colorectal Cancer Multidrug-Resistant Cells
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摘要:
目的 探讨结直肠癌耐药细胞增殖及侵袭力与Na+, K+-ATP酶活性及其β1亚单位和P糖蛋白(P-gp)表达的关系, 及哇巴因增加化疗敏感性的可能机制。 方法 以人结直肠癌亲本细胞(SW480)和耐奥沙利铂细胞(SW480/OxR)为研究对象, 采用MTS法、Transwell小室、生化酶学、实时定量PCR(Real time quantitative, RT-PCR)及流式细胞技术等方法比较SW480细胞与SW480/OxR细胞的增殖及侵袭力、Na+, K+-ATP酶活性及其β1亚单位和P-gp表达的差异, 观察哇巴因对SW480/OxR细胞上述指标的影响。 结果 与SW480细胞比较, SW480/OxR细胞增殖活力无明显变化(P > 0.05), 而细胞侵袭力却显著增加, Na+, K+-ATP酶活性下降, β1亚单位表达下调, P-gp表达上调(P均 < 0.01);哇巴因能显著抑制SW480/OxR细胞增殖活力, 减弱其侵袭力, 下调SW480/OxR细胞P-gp蛋白表达, 上调SW480/OxR细胞β1亚单位蛋白表达, 增加SW480/OxR细胞Na+, K+-ATP酶活性(P均 < 0.01)。 结论 Na+, K+-ATP酶活性下降可能源于β1亚单位表达下调, 并参与结直肠癌的耐药; 哇巴因能部分逆转结直肠癌耐药细胞MDR, 可能与增加Na+, K+-ATP酶活性及下调P-gp表达有关。 Abstract:Objective This work aimed to explore the effects of Na+/K+-ATPase activity and the expression of itsβ1-subunit and P-glycoprotein(P-gp) on the proliferation and invasion of human colorectal cancer parental cells(SW480) and oxaliplatin-resistant cells(SW480/OxR).The molecular mechanisms of ouabain for reversing the multidrug resistance(MDR) of human colorectal cancer oxaliplatin-resistant cells were also examined. Methods SW480 and SW480/OxR cells derived from the same patient were treated with or without ouabain at the physiological concentration of 1 nM.The SW480 and SW480/OxR cell proliferation capacity was assessed by the MTS assay.The invasion capacity was deteremined using a Transwell chamber.The Na+/K+-ATPase activity was measured by biochemical and enzymological techniques.The expression of theβ1-subunit and P-gp of Na+/K+-ATPase was determined by real-time quantitative PCR, Western blotting, and flow cytometry. Results The capacity of invasion significantly increased in the SW480/OxR cells compared with the SW480 cells(P < 0.01).There was no difference between the SW480 and SW480/OxR cell proliferation capacities(P > 0.05).The Na +/K+-ATPase activity significantly decreased in SW480/OxR cells compared with SW480 cells(P < 0.01).The expression of the Na +/K+-ATPaseβ1-subunit in mRNA and protein levels was lower in SW480/OxR cells than in SW480 cells(P < 0.01).However, the expression of P-gp in mRNA and protein levels was higher in SW480/OxR cells than in SW480 cells(P < 0.01).Interestingly, ouabain at the physiological concentration of 1 nM significantly enhanced the activity of Na +/K+-ATPase in SW480/OxR cells(P < 0.05).The expression of theβ1-subunit was also upregulated and that of P-gp was downregulated at the protein level, thereby inducing a decrease in the capacity of SW480/OxR cell growth inhibition and invasion.Nevertheless, after ouabain treatment for 48 h, the Na +/K+-ATPase activity andβ1-subunit protein expression level in SW480/OxR cells were still lower than those in SW480 cells(P < 0.01). Conclusion Decreased Na +/K+-ATPase activity can be attributed to the downregulation of Na+/K+-ATPaseβ1-subunit expression and may cause the MDR of human colorectal cancer cells.Ouabain could partly reverse the MDR of such cells, which can be attributed to increased Na+/K+-ATPase activity and P-gp expression downregulation. -
Key words:
- Colorectal cancer /
- Na+/K+-ATPase /
- P-glycoprotein /
- Multidrug resistance of chemotherapy /
- Ouabain
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图 1 化疗药物对结直肠癌细胞增殖能力的影响
*与SW480对照组比较，P < 0.01
Figure 1. Effects of oxaliplatin and 5-fluorouracil on the proliferation of SW480 and SW480OxR cells at 48 h
图 2 结直肠癌细胞P糖蛋白表达散点图
SSC：侧散射；a：同型对照，b：SW480细胞，c：SW480/OxR细胞
Figure 2. Scatter diagram of the cells that expressed P-gp in SW480 and SW480/ OxR cells
图 3 结直肠癌细胞及哇巴因干预耐药细胞后Na+，K+-ATP酶β1亚单位表达
1：SW480细胞；2：SW480/OxR细胞；3：SW480/OxR细胞+哇巴因
Figure 3. Protein-level expression of the Na+/K+-ATPase β1-subunit in SW480 and SW480/OxR cells with or without ouabain treatment
图 4 哇巴因对结直肠癌细胞P糖蛋白表达的影响
Figure 4. Effects of ouabain on the expression of P-gp in SW480 and SW480/OxR cells
图 5 哇巴因对SW480/OxR细胞Na+，K+-ATP酶活性作用
*与SW480/OxR比较，P < 0.05；**与SW480比较，P < 0.05
Figure 5. Na+/K+-ATPase activity of SW480/OxR cells treated with ouabain
表 1 荧光实时定量PCR引物序列
Table 1. Primer sequence used in real-time quantitative PCR
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