| Background:Ovarian cancer is the leading cause of gynecological cancer mortality. Its treatment usually consists of optimal cytoreductive surgery and platinum-based combination chemotherapies. Although the platinum compounds such as cisplatin and carboplatin are first line anti-cancer drugs, development of resistance to the platinum-based therapies has emerged as a major obstacle in the treatment of ovarian cancers. As a result, the 5-year survival rate for advanced ovarian cancer (stage III and IV) is only about 20%-30%. At present, the anti-cancer activity of platinum has been deeply investigated, but mechanisms that underlie platinum resistance are poorly understood, especially platinum resistance forecast. Early on, studies started to focus on genetic and molecular mechanisms that mediate resistance. Recently, the role of microenvironment, especially, of extraccllular matrix in platinum resistance is being explored and novel targets arc being identified.We have been focusing on platinum resistance of ovarian cancer since 2003. Five new proteins (annexin A3, Destrin, IDHc, GSTO1-1, and Cofilin 1) which are correlated with platinum resistance were picked out through proteomics technology. Among them, the up-regulated annexin A3 are coexpressing in all platinum resistant ovarian cancer cells, and we chose it as our target protein to study on platinum resistance. Annexin A3 is up-regulated in ovarian cancer tissues of clinical platinum resistant patients through immunohistochemistry, and overexpressed annexin A3 could decrease disease-free period. In nu-nu mice, cisplatin could not inhibit tumor growth if annexin A3 is up-regulated in ovarian cancer cells. Besides, high level annexin A3 decreases Pt concentration and Pt-DNA combining weight in ovarian cancer cells, and increases cell resistance to cisplatin rather than taxol and epirubicin. Overexpressed annexin A3 also down-regulates P53 level. Based on above researches, here we studied on whether annexin A3 could become a biomarker to prognosticate platinum resistance in clinic. Western blotting was used to detect secreted annexin A3 in culture medium of ovarian cancer cells, and analyze correlation between secretory volume and cell sensitive to platinum. Serum annexin A3 expression was measured using ELISA. Secretory mechanism of annexin A3 in ovarian cancer cells was investigated by using transmission electron microscopy (TEM) and immunoelectron microscopy (IEM). Based on the secretory pathway of annexin A3 in ovarian cancer cells, platinum resistance molecular mechanism was explained based on correlations among secreted annexin A3, exosome and Pt efflux.Methods:1. Platinum sensitive and resistant ovarian cancer cells were stable transfected with sense and anti-sense annexin A3 respectively. Immunofluorescence (IF) was used to detected annexin A3 location and expression. Western blotting was used to detect secreted annexin A3 in culture medium of ovarian cancer cells and analyze correlation between secretory volume and cell sensitive to platinum. Sera from 50 ovarian cancer patients were collected and annexin A3 expression were evaluated through ELISA.2. Annexin A3 was down-regulated by RNAi. Ultramicrostructures of all ovarian cancer cells were observed by using transmission electron microscopy (TEM) and annexin A3 distribution in cytoplasma was investigated by immunoelectron microscopy (IEM). Exosome was purified from culture medium by using two different methods, and optima method was chosen to analyze the relationship between annexin A3 and exosome secretory volume.3. Pt concentration in exosome, Pt uptake, and Pt efflux were evaluated through inductively coupled plasma mass spectrometry (ICP-MS) respectively. For observing the effluence of annexin A3 to Pt absorption rate, after stimulation with 10?M CDDP, all cells, including platinum sensitive cells. platinum resistant cells, transfectant with sense annexin A3, and cells with annexin A3 siRNA, were collected at different time to detect intracellular Pt concentration. For detecting Pt efflux rate, after stimulation with 10?M CDDP for 36 h, culture medium was replaced with fresh DMEM, then DMEM was collected at differenti time to evaluate the Pt concentraion. Exosome was isolated from culture medium by using differential centrifugation, and Pt concentration in exosome was determinated through ICP-MS.4. Among the 50 ovarian cancer patieints which have been introduced in Part 1,30 patients were drug resistant and the rest were drug sensitive. Sera of 30 normal women were collected as control. Serum annexin A3 expression was evaluated using ELISA. The differences were analyzed through Mann-Whitney U test. Kaplan-Meier survival curve was used to determine the relationship between serum annexin A3 and disease free time.Results1. After stable infection with sense or anti-sense annexin A3 plasmids. annexin A3 level in ovarian cancer cells were up or down-regualted stably. Annexin A3 is a kind of plasmosins, and the level changes occur in cytoplasma. Annexin A3 exists in culture medium of ovarian cancer cells in vitro. Cells with high level annexin A3 could secret much more proteins into culture medium. Besides, high level annexin A3 was detected in culture medium of cisplatin resistant cells. Serum annexin A3 could also be measured with ELISA, but the level is very low, and distribution is unnormal.2. According to the results of Signal-P software, annexin A3 has no signal peptide. So the protein couldn't secret outside through ER-Golgi pathway. Under transmission electron microscope, much more multivesicular bodies (MVBs) are viewed in annexin A3 overexpressed cells. After interference with annexin A3 siRNA, the number of multivesicular decreased significantly. These MVBs contain particles, and have a tendency to fuse with celluar membrane. Colloidal gold was used to mark annexin A3 protein. Under immunoelectron microscope, annexin A3 distributes in MVBs and on its surface. Exosomes isolated from culture supernatant of SKOV3/Cis are in the range of 40-100 nm in diameter and exhibit the typical cup-shaped morphology. Annexin A3 positive exosomes are observed under IEM. Exosome was fixed quantity by protein level. Each million cisplatin resistant cells (SKOV3/Cis) secret 3.985±1.194?g exosomes in 24 h, while only 0.525±0.214±g exosomes are secreted from cisplatin sensitive cells (SKOV3) (P=0.02<0.05). After stable transfection with sense annexin A3 plasmid, SKOV3 cells secret 2.971±0.35?g exosomes in 24 h (P=0.001<0.01). SKOV3/Cis cells secret only 1.447±0.899?g exosomes in 24 after annexin A3 was down-regulated by anti-sense annexin A3 (P=0.004<0.01). The same tendency is observed in A2780 and its cisplatin resistant subline.3. Pt concentration in cells and culture medium were detected. Annexin A3 could not affect Pt uptake speed in 8 h after treatment with cisplatin. However, overexpressed annexin A3 increases Pt efflux speed. Pt could be detected in exosome. Through exosome, SKOV3/Cis could out put more Pt (P=0.047), and Pt efflux volume decreases when annexin A3 level is down-regulated (P=0.028). Oppositely, SKOV3 cells release more Pt (P=0.004) after sense annexin A3 plasmid transfection.4. The average level of annexin A3 in normal women sera is 0.8590±0.0744 ng/ml (n=30), and serum annexin A3 mean concentration in ovarian cancer patients is 1.6898±2.6563 ng/ml (n=50). There is significant difference between ovarian cancer patients and normal women (P<0.0001).50 ovarian cancer patients were divided into two groups,30 in drug resistant group and 20 in drug sensitive group. The mean serum annexin A3 level in the two groups are 2.1145±3.3833 ng/ml and 1.0528±0.1178 ng/ml respectively. The P value is 0.0009. Furthermore, among the resistant patients,19 out of 30 have serum annexin A3 level higher than 1.13 ng/ml, whereas only 4 out of 20 platinum-sensitive patients have serum annexin A3 level above this level. Analysis of all the 50 patients found that high-level annexin A3 (over 1.13 ng/ml) also significantly decrease the disease-free time (P=0.009<0.05).Discussion1. Annexin A3 distributes mainly in cytoplasm of ovarian caner. Ovarian cancer cells could secrete annexin A3 in vitro. And the secretory volume is associated with cell sensitivity to cisplatin and annexin A3 concentration in cells. Annexin A3 in sera could be detected with ELISA. All these demonstrate that annexin A3 is a kind of secretory proteins.2. Annexin A3 secretes outside through MVBs rather than ER-Golgi pathway. Examination of these ovarian cancer cells using TEM and IEM revealed that overexpression of annexin A3 results in the formation of more MVBs-like vesicles. Some of the vesicles contain annexin A3 inside and appear fusing with cell membrane. Besides, annexin A3 is associated with exosomes released from the platinum-resistant ovarian cancer cells.3. Annexin A3 secretes through exosome. And annexin A3 concentraion in cells determines exosome volume. So annexin A3 might regulate drug resistance through exosmoe. Exosomes bring out Pt. Overexpressed annexin A3 releases more Pt through exosome. Besides. Overexpressed annexin A3 increased Pt efflux speed. However, annexin A3 could not affect Pt uptake speed in 8 h after treatment with cisplatin. Above all, annexin A3 regulates platinum resistance through exosome.4. Clinical research with small samples showed that serum annexin A3 concentration of ovarian cancer patients is higher than which of normal women. Platinum resistant patients could express higher level annexin A3 in sera than sensitive patients. Because all sera was collected before the first chemotherapy, so high serum annexin A3 could prognosticate platinum resistance in clinic with both over 50% sensitivity and specificity. |
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