Expression Of TRAP-1 In Breast Ductal Carcinoma And Regulation Of Mitochondrial Function

BackgroundBreast cancer, the leading death cause of women, is the most common female malignancy in the world, which accounts for 23%cancer incidence and 14% cancer mortality, respectively. The incidence and mortality of breast cancer in China were kept increasing in the last two decades, and young people are inclined to hold increasing proportion of the inflicted group. Traditional treatments include surgery, chemotherapy, and radiotherapy and so on. Comprehensive treatment strategy has achieved a great improvement and has tremendously better the prognosis of breast cancer patients. However, side effect, drug-resistance and recurrence are still major obstacles we have to overcome. Therefore, on the top list of breast cancer clinical problems are thorough investigation with regard to the mechanism of carcinogenesis, metastasis and drug-resistance, and acquirement of potential molecular targets. Mitochondrion is the powerhouse of a cell, and meanwhile is the initiator of cellular endogenous apoptosis. Mitochondrial dysfunction can cause many diseases, including cancer. Although the intimate connection of cancer and mitochondrial dysfunction has been shed light on, the underlying mechanism is still uncovered. Tumor necrosis factor receptor related protein-1 (TRAP-1) is a member of heat shock protein (HSP) family, mainly located in mitochondria, which is highly associated with carcinogenesis. Recent research indicated that TRAP-1 is significantly overexpressed in pancreas cancer, colon cancer, lung cancer, prostate cancer and so on. Our group has previously found the overexpression of TRAP-1 in breast cancer tissue, but it is still vague that this overexpression is significantly associated with the carcinogenesis, drug-resistance and prognosis of breast cancer.Material and MethodOur research includes following sections:1. Using Western blot to compare TRAP-1 expression levels in tumor tissue and adjacent normal tissue, and also compareTRAP-1 expression levels in MCF-10A, MCF-7, MDA-MB-231, and MDA-MB-436 cell lines; TRAP-1 siRNA Interference in MCF-7 cell line; TRAP-1 overexpression in MDA-MB-231 cell line.2. Transfecting pEGFP-TRAP-1 vectors to MDA-MB-231 cell line; observing the influence of TRAP-1 to mitochondrial morphology after mitochondrion stain and fixation under laser confocal microscopy; using TMRM dyeing test to observe the TRAP-1 induced disturbance of trans-membrane potential; siRNA inference of TRAP-1 in MCF-7 cell line, and comparing mitochondrion fusion/fission associated protein expressions in TRAP-1 inference and overexpression scenarios.3. Testing and comparing oxygen consumption rates (OCR), extracellular acidification rate (ECAR) and ECAR after oligomycin treatment; comparing aerobic respiration of breast tumor mitochondria in TRAP-1 overexpressed and interfered scenarios; treating MB231 TRAP-1 overexpressed cell line with hydrogen peroxide or glucose oxidase, and unveiling the influence of TRAP-1 to cellular anti-oxygenic impairment.Result:1. Western blot verified TRAP-1 was typically overexpressed in cancer tissues with comparison to tumor adjacent normal tissue.2. TRAP-l’s influence upon mitochondrial morphology:laser confocal microscopy indicated overexpression of TRAP-1 transform mitochondrial morphology from short rod-like structure to filament network structure; pS616-Drpl down-regulation and fusion protein Mfnl up-regulation were observed in TRAP-1 overexpressed MDA-MB-231 cell line, and this phenomenon was totally reversed in TRAP-1 interfered MCF-7 cell line, consistent with mitochondrial morphology change.3. TRAP-1’s influence upon mitochondrial aerobic respiration:in GFP-TRAP-1 stably transfected MDA-MB-231 cell line, TMRM signal was greatly enhanced and so was mitochondrial trans-membrane potential; OCR test showed that MDA-MB-231 cell line had a low OCR, MCF-7 cell line had a high one, and two kinds of tumor cell line both had a high rate of glycolysis;Conclusion:TRAP-1 was overexpressed in breast tumor tissues. TRAP-1 overexpression could promote mitochondrial fusion, transform mitochondrial morphological characteristics, enhance mitochondrial energy metabolism, and protect cells from extreme conditions. TRAP-1 could significantly improve the breast cancer cell’s ability to contour oxygenation impairment, which plays an important part in anti-apoptosis mechanism. TRAP-1 might be a potential target for drug-resistance investigation and treatment.