| Purpose:Breast cancer is one of the most common malignant tumors in women.However,drug resistance is a major challenge in the treatment of breast cancer.Although the association between glutamine metabolism and tamoxifen resistance is well established,the relationship between proline metabolism and PRODH action and chemotherapeutic drug resistance in breast cancer remains to be investigated.PRODH has been identified as a mitochondrial tumor suppressor.Therefore,we will explore the expression of PRODH in tamoxifen-resistant breast cancer cells and its effect on the malignant progression of breast cancer through a series of experiments in vivo and in vitro,and further explore its potential impact,in order to provide a potential new method to overcome the tamoxifen-resistant breast cancer cells.Methods:1.To investigate the expression of PRODH in tamoxifen resistant breast cancer cells.Firstly,using the concentration gradient method,breast cancer tamoxifen resistant cell lines MCF-7/Tam R and T47D/Tam R are established;Real-time PCR and Western blotting were used to detect the expression of PRODH in the two cell lines;Finally,the proline concentration in the two cell lines was determined using a proline kit.2.To investigate the effect of PRODH on tamoxifen resistance in breast cancer cells.Firstly,a breast cancer cell line stably and highly expressing PRODH was constructed by plasmid transfection,and the construction of PRODH was verified by Western blot;Condly,the change of proline in the breast cancer cell line stably and highly expressing PRODH is detected by a proline kit;Then,MTT assay and soft agar colony formation assay were used to detect the effect of resistant cell lines on tamoxifen sensitivity and colony formation;Finally,in vivo experiments,the stable high expression of PRODH resistant cell line was injected into nude mice to establish xenograft tumor model,in the presence of tamoxifen,to observe the effect of PRODH on tumor growth in vivo.3.To explore the effect of specific knockdown of PRODH on tamoxifen resistance in breast cancer cells.The plasmid was transfected to construct specific knockdown PRODH gene in breast cancer cells,and the expression of PRODH gene was verified by Western blot;Next,the proline level in the breast cancer cells in which the PRODH gene was specifically knocked down was measured using a proline kit;Finally,MTT assay and soft agar colony formation assay were used to detect the effect of breast cancer cells with specific knockdown of PRODH on tamoxifen resistance and colony formation.4.To investigate the effect of PRODH on iron death in tamoxifen resistant breast cancer cells.Firstly,the expression of Glutathione peroxidase 4(GPX4),a marker of ferroptosis,was detected by Real-time PCR and Western blotting;The effect of high expression of PRODH on the expression of GPX4 was observed by Western blot;Next,the effects of stable high expression of PRODH on the levels of glutathione(GSH)and reactive oxygen species(ROS)in breast cancer resistant cell lines were determined by glutathione kit and flow cytometry.5.To explore the effect of PRODH on tamoxifen resistance of breast cancer cells after adding ferrostatin-1(Fer-1).Firstly,the breast cancer resistant cell line stably expressing PRODH was treated with ferroptosis inhibitor,and the change of PRODH on GPX4 expression was observed by Western blotting;Next,changes in the levels of GSH and ROS in PRODH antagonistic cell lines after addition of Fer-1 were determined by glutathione kit and flow cytometry;Then the effect of PRODH on the relative survival rate and colony formation was observed by MTT assay and soft agar cloning assay;Finally,the effect of PRODH on tumor size in nude mice after adding Fer-1 was observed in vivo.Results:1.The two breast cancer resistant cell lines were successfully established by MTT assay.The results of Western blot and real-time quantitative PCR showed that the expression of PRODH was down-regulated at m RNA and protein levels in the two resistant cell lines compared with the parental cells.Proline kit results showed that proline concentrations were elevated in both resistant cell lines compared to control cells.2.The expression of PRODH was up-regulated and the concentration of proline was decreased in the two resistant cell lines with stable high expression of PRODH.The results of MTT assay and soft agar colony formation assay showed that stable high expression of PRODH enhanced the sensitivity of drug-resistant cells to tamoxifen and reduced the colony formation ability of cells.In vivo results showed that tamoxifen treatment further enhanced the inhibitory effect of PRODH on tumor growth,showing the same results as in vitro experiments.3.The expression of PRODH was down-regulated and the concentration of proline was increased in breast cancer cell lines with specific knockdown of PRODH.Specific knockdown of PRODH enhances the resistance of breast cancer cells to tamoxifen and enhances the clonogenic capacity of breast cancer cells.4.GPX4,a marker of ferroptosis,was up-regulated in both resistant cells compared to the parental cells.In two tamoxifen resistant breast cancer cell lines,stable high expression of PRODH down-regulated GPX4 expression.The kit results showed that GSH concentrations were increased and ROS levels were decreased in both resistant cell lines compared to the parent cells.More importantly,re-expression of PRODH reversed these changes in both tamoxifen-resistant cells.5.Fer-1 treatment attenuated the inhibitory effect of PRODH on GPX4 expression in both resistant cell lines;In addition,Fer-1 treatment attenuated the effects of PRODH on the levels of ROS and GSH in both resistant cells;Compared with the control cells,the survival rate of the cells increased and the colony formation of the cells was enhanced after adding Fer-1.In vivo,treatment with the iron-death inhibitor Fer-1 reversed the inhibitory effect of PRODH on tumor growth.Conclusion:In this study,we investigated the effect of PRODH on tamoxifen resistance in breast cancer cells.PRODH expression was down-regulated in tamoxifen resistant breast cancer cells.In vitro and in vivo,re-expression of PRODH sensitizes resistant cells to tamoxifen.Knockout of PRODH specifically makes breast cancer cells resistant to tamoxifen.We show that ferroptosis is suppressed in tamoxifen-resistant breast cancer cells,and that overexpression of PRODH restores ferroptosis in tamoxifen-resistant breast cancer cells.Treatment with ferroptosis inhibitors suggests that PRODH modulates tamoxifen resistance by modulating ferroptosis in tamoxifen-resistant cells.This study suggests that PRODH offers a potential new approach to overcoming tamoxifen resistance in breast cancer cells. |
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