ABSTRACT
Chemotherapy is one of the major categories of medical oncology and a primary tumor treatment; however, the effectiveness of chemotherapy is restricted by drug resistance. Overcoming resistance to chemotherapy and investigating molecular targeted therapies are challenges currently faced during resistance management. Progesterone receptor membrane component 1 (PGRMC1) is an adapter protein mediating cholesterol synthesis, steroid signaling, and cytochrome p450 activation. Attention has recently focused on the role of PGRMC1 in cell survival, anti-apoptosis, and damage response. In the present study, we used knockdown and overexpression approaches in the following set of uterine sarcoma models to further evaluate the role of PGRMC1 in drug resistance: the doxorubicin-sensitive MES-SA cells and the doxorubicin-resistant MES-SA/DxR-2 µM and MES-SA/DxR-8 µM cells (with different levels of doxorubicin resistance). PGRMC1 repressed doxorubicin-induced cytotoxicity and exhibited an anti-apoptotic effect; it also promoted cell proliferation and cell cycle progression to the S phase. Of note, PGRMC1 overexpression led to the epithelial-mesenchymal transition (EMT) of the sensitive MES-SA cells, thus facilitating their migration and invasion. The combination of PGRMC1 knockdown and the P-glycoprotein inhibitor verapamil significantly decreased the viability of P-glycoprotein-overexpressing MES-SA/DxR-8 µM cells after doxorubicin treatment. Taken together, our results show that PGRMC1 contributed to chemoresistance through cell proliferation, anti-apoptosis, and EMT induction, leading to the suggestion that PGRMC1 may serve as a therapeutic target in combination with an inhibitor in different drug resistance pathways and indicating the usefulness of predictive resistance biomarkers in uterine sarcoma.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Membrane Proteins/metabolism , Receptors, Progesterone/metabolism , Sarcoma/drug therapy , Uterine Neoplasms/drug therapy , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Apoptosis , Blotting, Western , Cell Adhesion , Cell Cycle , Cell Movement , Cell Proliferation , Female , Flow Cytometry , Fluorescent Antibody Technique , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , RNA, Small Interfering/genetics , Receptors, Progesterone/antagonists & inhibitors , Receptors, Progesterone/genetics , Sarcoma/genetics , Sarcoma/pathology , Signal Transduction , Tumor Cells, Cultured , Uterine Neoplasms/genetics , Uterine Neoplasms/pathologyABSTRACT
Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.
Subject(s)
Acetyl-CoA C-Acetyltransferase/metabolism , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Malate Dehydrogenase/metabolism , Mitochondrial Proteins/metabolism , Proteome/metabolism , Acetyl-CoA C-Acetyltransferase/genetics , Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Immunoblotting , Malate Dehydrogenase/genetics , Mitochondrial Proteins/genetics , Proteome/genetics , Proteomics/methods , RNA Interference , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uterine Neoplasms/genetics , Uterine Neoplasms/metabolism , Uterine Neoplasms/pathologyABSTRACT
Drug resistance is a frequent cause of failure in cancer chemotherapy treatments. In this study, a pair of uterine sarcoma cancer lines, MES-SA, and doxorubicin-resistant partners, MES-SA/DxR-2µM cells and MES-SA/DxR-8µM cells, as a model system to investigate resistance-dependent proteome alterations and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to perform this research and the results revealed that doxorubicin-resistance altered the expression of 208 proteins in which 129 identified proteins showed dose-dependent manners in response to the levels of resistance. Further studies have used RNA interference, H2A.X phosphorylation assay, cell viability analysis, and analysis of apoptosis against reticulocalbin-1 (RCN1) proteins, to prove its potency on the formation of doxorubicin resistance as well as the attenuation of doxorubicin-associated DNA double strand breakage. To sum up, our results provide useful diagnostic markers and therapeutic candidates such as RCN1 for the treatment of doxorubicin-resistant uterine cancer.