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脯氨酰内肽酶过表达诱导 MCF-7 乳腺癌细胞自噬性死亡。

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

机构信息

Department of Medicinal Chemistry, Medical University of Bialystok, Bialystok, Poland.

Department of Pharmaceutical and Biopharmaceutical Analysis, Medical University of Bialystok, Bialystok, Poland.

出版信息

Cell Physiol Biochem. 2020 Sep 12;54(5):875-887. doi: 10.33594/000000275.

DOI:10.33594/000000275

PMID:32918543

Abstract

BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells.

METHODS

The model of MCF-7 cells with prolidase overexpression (MCF-7) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry.

RESULTS

Prolidase overexpression in MCF-7 cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7 cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7 cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7 cells. The effects of studied compounds in MCF-7 cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9.

CONCLUSION

The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

摘要

背景/目的：脯氨酸脱氢酶/脯氨酸氧化酶（PRODH/POX）的脯氨酸可用性可能代表着 PRODH/POX 依赖性细胞凋亡和自噬之间的转换机制。本研究旨在评估脯氨酸酶（脯氨酸释放酶）过表达对乳腺癌 MCF-7 细胞凋亡/自噬的影响。

方法

获得了脯氨酸酶过表达（MCF-7）的 MCF-7 细胞模型。为了靶向 PRODH/POX 依赖性途径的脯氨酸，提供了甘氨酰脯氨酸（GP），并使用 2-甲氧基雌二醇（MOE）阻断脯氨酸用于胶原蛋白生物合成。使用 Nucleo-Counter NC-3000 测定细胞活力。通过比色法测定脯氨酸酶的活性。通过放射性方法测定 DNA、胶原蛋白和总蛋白质的生物合成。通过 Western blot 和免疫荧光生物成像评估蛋白质表达。通过液质联用分析脯氨酸浓度。

结果

与野生型 MCF-7 细胞相比，MCF-7 细胞中的脯氨酸酶过表达导致酶活性增加 10 倍，细胞质脯氨酸水平增加 3 倍，细胞活力和 DNA 生物合成降低。在 MCF-7 细胞中，MOE 和 GP 显著减少活细胞数量。MOE 抑制两种细胞系的 DNA 生物合成，而 GP 仅在 MCF-7 细胞中对该过程产生抑制作用。在两种细胞系中，MOE 或 MOE+GP 均抑制 DNA 和胶原蛋白生物合成。尽管 GP 在 MCF-7 细胞中刺激胶原蛋白生物合成，但它抑制了 MCF-7 细胞中的该过程。在 MCF-7 细胞中，研究化合物的作用伴随着 Atg7、LC3A/B、Beclin-1、HIF-1α 的表达增加和 PRODH/POX、活性 caspase-3 和 -9 的表达减少。