| BackgroundOvarian cancer is one of the most common malignant tumor in female reproductive system. Due to the ovary located in pelvic deep, early lesions not easy to find, lack of effective treatments in advanced cases, mortality rates associated with ovarian cancer remain at the top of the list of gynecologic malignant tumors. Surgery is the main method for the treatment of ovarian epithelial carcinoma, but based on the characteristics of ovarian cancer easy for pelvic and abdominal cavity metastasis, in appearance, even if the tumors confined to the primary site, it can also exist widely micrometastases, so surgery must be supplemented by chemotherapy. However, it may not only cause the loss and serious side effects of drugs, also reducing the therapeutic effect of tumor, and eventually developed into chemotherapy resistance and death, due to nonselective cytotoxicity of chemotherapy drugs. Therefore, for improving the survival rates of patients with ovarian cancer, there is an urgent need to develop novel, effective and highly specific methods for ovarian cancer treatment. Recently, targeted therapy has been gradually become a new strategy for treatment of ovarian cancer. At present, although ovarian cancer targeted therapy has made some progress in animal experiments and pre-clinical tests, but there are still many problems, including the main problem which need to be addressed is low transduction efficiency and poor cell specificity. To address the problem, we developed a novel ovarian cancer targeted treatment system TAT-OSBP-1-MKK6(E), which contains three functional domains:a) the protein transduction domain of TAT, b) the human ovarian cancer HO8910 cell-specific binding peptidel (OSBP-1), and c) the potential anti-tumor effector domain of MKK6(E). The anti-tumor efficacy and tumor-targeting efficiency of TAT-OSBP-1-MKK6(E) are evaluated by the experiments preliminarily in vitro and vivo. Our findings will provide new insight for the therapeutic approaches of ovarian cancer.MethodsIn vitro1) The prokaryotic expression vectors of pGEX-6P-3/TAT-OSBP-1-MKK6(E), pGEX-6P-3/TAT-OSBP-1, pGEX-6P-3/TAT-MKK6(E) and pGEX-6P-3/MKK6(E) were constructed and transfected into E. coli BL21(DE3). The expression of the GST fusion gene was induced with IPTG, and the GST fusion protein was purified by GST purification column. The GST fusion protein after purification was analyzed by SDS-PAGE and Western-blot and then cleaved by Prescission Protease enzyme. Protein was labeled with FITC and biotin.2) The transduction efficiency and and targeted efficacy of TAT-OSBP-1-MKK6(E) to HO8910 cells were examined by immunofluorescence assay, TAT-MKK6(E) and MKK6(E) were used as control, OSE cells were used as negative control.3) The inhibition effect of TAT-OSBP-1-MKK6(E) and TAT-OSBP-1 (negative control) on the proliferation of HO8910 cells were evaluated by CCK-8 assay, OSE cells were used as negative control.4) The transduction efficiency and apoptotic effect of TAT-OSBP-1-MKK6(E) to HO8910 cells were detected by flow cytometry, TAT-OSBP-1 and PBS were used as negative control and blank control,respectively. OSE cells were used as negative control.In vivo1)A subcutaneous xenograft model was established by s.c. injection of HO8910 cells into the arm pit on right side of eighteen female BALBC/c nude mice. Tumor-bearing mice were divided into three treatment groups that received tail vein injection of TAT-OSBP-1-MKK6(E), TAT-OSBP-1, or normal saline (NS) once every 4 days (at the 1st,5th,9th,13th and 17th day). The tumor volumes were measured with caliper once every 2 days. At the 21st day of treatment, slightly modification was made by injection of biotin-TAT-OSBP-1-MKK6(E), biotin-TAT-OSBP-1 or NS via tail vein. Two hours after injection, the xenograft tumors, ovaries and livers in the three treatment groups were harvested for subsequent experiments.2) After stained with hematoxylin-eosin(HE), pathological morphological changes in tumor xenografts were i were observed under optical microscope.3) The distribution of the biotin conjugated TAT-fusion proteins in nude mice was analyzed by immunohistochemical assay.4) The apoptotic effect of tumor xenografts, ovaries and liver tissues were assessed by TUNEL assays, after the treatment with TAT-OSBP-1-MKK6(E).ResultsIn vitro1) We successfully obtained TAT-OSBP-1-MKK6(E), TAT-OSBP-1,TAT-MKK6(E) and MKK6(E) recombinant protein by prokaryotic expression. The purity of FITC-labeled and biotin-conjugated fusion proteins was greater than 95%.2) Immunofluorescence experiment showed that TAT-OSBP-1-MKK6(E) could be selectively internalized into HO8910 cells, rather than OSE cells, in contrast, TAT-MKK6(E) could be equally transducted into both cell lines without cell selectivity. While MKK6(E) alone was lack of ability to penetrate the cell membranes of HO8910 and OSE cells.3) CCK-8 assay confiremed that, after the treatment with TAT-OSBP-1-MKK6(E), the survival rate of HO8910 cells was 32.05±2.45%, much lower than that of OSE cells (95.69±0.7)% or that of both cells in TAT-OSBP-1 group{(95.16±0.93)% and (93.05±0.46)%, respectively}, the difference in survival rate is statistically significant (**P< 0.01), whereas there is no statistically significant difference between the rest (P>0.05).4) Flow cytometry assay revealed that, similar to TAT-OSBP-1 group, TAT-OSBP-1-MKK6(E) can be selectively transducted into HO8910 cells, rather than OSE cells, as indicated by mean fluorescent intensities (MFI) of (95.1±2.2) and (38.1±1.1), respectively. The difference in MFI between both cell lines was statistically significant (**P< 0.01). However, TAT-OSBP-1-MKK6(E)-induced apoptosis was found exclusively in HO8910 cells., the apoptotic rate of HO8910 cells was 32.55%, much higher than that of OSE cells (4.87%), that of both cells in TAT-OSBP-1 group (6.65% and 6.73%, respectively) and that of in PBS group (6.47% and 4.32%, respectively), the difference in apoptotic rate is statistically significant (**P< 0.01), whereas there is no statistically significant difference between the rest (P>0.05).In vivo1) The TAT-OSBP-1-MKK6(E) group was associated with significantly smaller tumor volumes at day 9 compared to the two control groups (*P< 0.05). Of note, tumor volumes in the 2 control groups (TAT-OSBP-1 and NS) were similar (P> 0.05). The average of tumor weight in TAT-OSBP-1-MKK6(E) group was much lower (0.28±0.11g,**P< 0.01) than that in two control groups:TAT-OSBP-1 and normal saline, but the difference between two control groups was not significant (0.6±0.18g and 0.67±0.18g, respectively, P> 0.05).2) HE stain showed that, in TAT-OSBP-1-MKK6(E) treatment group, a large area of necrosis were distributed in the tumor specimens, with typical necrotic morphological characteristics, such as nuclear pyknosis and cellular structural disappearance. Whereas in two control groups of TAT-OSBP-1 and normal saline, most of the observed cells with typical tumor morphological features, including varied in cell size, deranged distribution, deep-dyed nucleolus, there were rare nuclear pyknosis and no obvious necrosis area.3) Immunohistochemical assay showed that TAT-OSBP-1-MKK6(E) selectively targeted human ovarian cancer HO8910 cells in nude mice, as large amounts of brown granules was found in HO8910 cells, but not in normal liver cells and almost none in normal ovarian cells in the specimens from the same mouse.4) TUNEL assay showed that TAT-OSBP-1-MKK6(E) treatment can specifically induce human ovarian cancer HO8910 cells apoptosis in nude mice, as indicated by a large number of brown-stained positive cells, which apoptotic index was (26.3±3.1)%, whereas TAT-OSBP-1-MKK6(E) did not exhibit apoptotic effect on normal ovarian and liver cells, as evidenced by a scarce presence of brown-stained positive cells.ConclusionsTAT-OSBP-1-MKK6(E) treatment can selectively target HO8910 cells or tumor xenograft, leading to significant growth inhibition and apoptosis of HO8910 cells in vitro and in vivo, with limited effects in normal cells and tissues. As such, TAT-OSBP-1-MKK6(E) may be a potential approach for ovarian cancer target therapy. |
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