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首页> 外文期刊>Cellular Physiology and Biochemistry >Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model
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Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

机译：在新型体外细胞培养模型中影响PRODH / POX依赖的促凋亡途径的细胞内脯氨酸水平操纵的功能后果。

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>Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.
机译：> 背景/目的： 研究了脯氨酸脱氢酶/脯氨酸氧化酶（PRODH / POX）依赖性细胞凋亡对脯氨酸可用性的影响。 方法： 我们生成了组成型敲除的PRODH / POX MCF-7乳腺癌细胞系（MCF-7 shPRODH / POX ）模型来分析细胞内脯氨酸水平受损的功能后果。我们在胶原蛋白生物合成中使用了脯氨酸抑制剂，2-甲氧基雌二醇（MOE），产生脯氨酸的脯氨酸酶抑制剂，雷帕霉素（Rap）和甘氨酰脯氨酸（GlyPro），脯氨酸酶的底物。胶原蛋白和DNA的生物合成通过放射分析法进行评估。使用Nucleo-Counter NC-3000测定细胞活力。蛋白质酶的活性通过比色测定法确定。通过蛋白质印迹和免疫荧光生物成像评估蛋白质的表达。通过液相色谱-质谱法分析脯氨酸的浓度。 结果： PRODH / POX敲低可降低MCF-7 shPRODH / POX 细胞的DNA和胶原蛋白的生物合成，同时增加脯氨酸酶活性和细胞内脯氨酸水平。所有研究的化合物均降低了MCF-7和MCF-7 shPRODH / POX 细胞的细胞活力。在两种细胞系中，Rap和MOE同样抑制DNA的生物合成，但是GlyPro仅在MCF-7 shPRODH / POX 和MOE + GlyPro中抑制此过程。所有这些化合物均抑制MCF-7 shPRODH / POX 细胞中胶原的生物合成，增加脯氨酸酶活性和胞质脯氨酸水平，仅在MCF-7 shPRODH / POX中有助于促存活模式的诱导。 单元格。相反，所有研究的化合物仅在MCF-7细胞中上调凋亡蛋白的表达。 结论： PRODH / POX被证实是细胞凋亡的驱动程序，并证明MCF-7 shPRODH / POX 细胞系具有很高的资格。该模型阐明了脯氨酸在PRODH / POX依赖性促凋亡途径中利用或生成脯氨酸的不同机制。

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