Overexpression Of RIP3Sensitizes Human Breast Cancer Cells To Parthenolide-induced Apoptosis

Aim:To construct a eukaryotic expression vector containing the Receptor-interacting protein3(RIP3) gene and determine whether infection of the RIP3gene can sensitize human breast cancer cells to parthenolide. Explore the related mechanism in order to provide theoretical basis for the clinical application of parthenolide.Methods:The expression of RIP3mRNA in four breast cancer cell lines and human MCF10A mammary epithelial cells was detected using reverse transcription-polymerase chain reaction (RT-PCR). The RIP3coding sequence was amplified by polymerase chain reaction by using cDNA library derived from MCFIOA cells and the template was subcloned into mCherry vector to construct recombinant plasmids. The recombinant plasmids were infected into MCF-7and MDA-MB-231cells using lentivirus after DNA sequencing. Meanwhile the mCherry vectors was infected into MCF-7and MDA-MB-231cells as the control groups. The expression efficiency and cellular localization of mCherry-RIP3protein in MCF-7and MDA-MB-231cells were observed under fluorescence microscope and the level of RIP3expression were validated by RT-PCR and Western Blot. The MCF-7and MDA-MB-231cells were treated with various concentrations of parthenolide, cell viability was measured using the MTT assay, and intracellular reactive oxygen species (ROS) and apoptosis were measured using flow cytometry. The morphological chang was observed under microscope, and the morphological chang of cell nuclei was observed under fluorescence microscope. The change of PARP protein active segment level was detected by Western Blot. MTT assay was used to measure the changes of inhibitory effect after treated with parthenolide and NAC.Results:1. RT-PCR showed that RIP3mRNA expression was universally lacking in four breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, T47D), and was relatively high in human MCF10A mammary epithelial cells.2. The RIP3expression vector PCDH-b-mCherry-RIP3was successfully constructed and infected into MCF-7and MDA-MB-231cells. Red fluorescence located in cytoplasm was observed using a fluorescence microscope which showed high protein expression of RIP3in the gene-infected group than control group (empty vector-infected group). The level of RIP3expression were validated by RT-PCR and Western Blot.3. After treated with different concentrations of parthenolide for48hours, MTT assay showed that overexpression of RIP3made MCF-7and MDA-MB-231cells more sensitive to parthenolide-induced proliferation inhibition in vitro (P<0.05).4. Flow cytometry assay showed that when treated with parthenolide, the apoptosis rate of MCF-7and MBA-MB-231cells overexpressing RIP3were significantly higher than that of mock-vehicle groups (P<0.05).5. As the parthenolide concentration increasing, the MCF-7and MBA-MB-231cells become smaller and rounder and then disrupted/solved and dead. And a higher proportion of RIP3-MCF-7and RIP3-MBA-MB-231cells that were exposed to PTL exhibited distinct cell shrinkage and rounded shapes compared with mock-vehicle groups. After Hoechst33342staining, the nuclei of RIP3-MCF-7and MBA-MB-231cells exhibited more chromosome condensation and fragmentation.6. When treated with parthenolide, the expression of PARP protein active segment was higher in MCF-7and MBA-MB-231cells overexpressing RIP3than that in mock-vehicle groups.7. When treated with parthenolide, the intracellular reactive oxygen species (ROS) levels were higher in MCF-7and MBA-MB-231cells overexpressing RIP3than those in mock-vehicle groups. And when combined with NAC, the sensitivity of MCF-7and MBA-MB-231to parthenolide was reduced, and has no significant difference between MCF-7and MBA-MB-231cells overexpressing RIP3and mock-vehicle groups.Conclusions:1. The expression of RIP3mRNA in four breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, T47D) was universally low and was relatively high in human MCF10A mammary epithelial cells. The recombinant RIP3overexpressed plasmid was successfully constructed, and the MCF-7and MBA-MB-231cells stably expressing ectopic RIP3were obtained. The mCherry-RIP3fusion protein was located in the cytoplasm. 2. The killing ability of parthenolide to breast cancer cells is concentration dependent. Over-expressing RIP3sensitizes human breast cancer cell lines MCF-7and MDA-MB-231cells to parthenolide-induced DNA damage, proliferation inhibition and apoptosis.3. The intracellular ROS accumulation may contribute to the sensitizing effect of overexpressing RIP3to parthenolide induced MCF-7and MDA-MB-231cell death.