Delivery of Constitutively Active Mutant MKK6(E) With TAT-OSBP Induces Apoptosis in Human Ovarian Carcinoma HO8910 Cells.

Biologically active peptides and proteins are novel agents that show promise in the development of anticancer drugs. Their relatively low cell permeability and poor tumor selectivity, however, impede their widespread applicability. In this study, we evaluated the tumor selectivity, cellular internalization, and biological activity of a cell-permeable ovarian cancer cell-specific therapeutic protein consisting of TAT-OSBP and constitutively active MKK6(E), an upstream kinase of the p38 signaling pathway that mediates cellular apoptosis. OSBP, a 7-amino-acid peptide with high affinity for human ovarian cancer HO8910 cells, was conjugated to the cell-penetrating peptide (TAT) to form a tumor-selective peptide (TAT-OSBP), which was further conjugated with EGFP or MKK6(E). Flow cytometry and fluorescent microscopy were performed to evaluate the tumor-targeted penetration of TAT-OSBP-EGFP. The inhibitory effects of TAT-OSBP-MKK6(E) were determined by cell proliferation and apoptosis assays. The internalization efficiency of TAT-OSBP-EGFP was significantly higher than that of TAT-EGFP. TAT-OSBP-EGFP selectively penetrated HO8910 cells. TAT-OSBP-MKK6(E) fusion protein inhibited cancer cell growth to varying degrees, with the highest level of inhibition in HO8910 cells. Moreover, TAT-OSBP-MKK6(E) significantly induced apoptosis of HO8910 cells. However, there was no significant difference in apoptosis in the normal ovarian epithelial cells treated with either TAT-OSBP-MKK6(E) or TAT-MKK6(E). Our results demonstrate that TAT-OSBP-MKK6(E) is a novel artificially designed molecule, which induces apoptosis and selectively targets human ovarian carcinoma HO8910 cells. Our study provides novel insights that may aid in the development of a new generation of anticancer drugs.

Mitogen-activated Protein Kinase Kinase 6-fusion Protein (MAP2K6-FP) Potentiates the Anti-tumor effects of Paclitaxel in Ovarian Cancer.

OBJECTIVE: To investigate the antitumor effects of a mitogen-activated protein kinase (MAPK) kinase fusion protein, TAT-OSBP-MKK6E (MAP2K6-FP), and paclitaxel as single agents and in combination against HO8910 human ovarian cancer cells. METHODS: We previously synthesized a MAPK kinase-recombinant fusion protein, MAP2K6-FP, that contains three domains: a protein transduction domain TAT, a human ovarian cancer HO8910 cell-specific binding peptide (OSBP), and a potential anti-tumor effector domain MKK6 (E). The HO8910 cells were exposed to MAP2K6-FP, paclitaxel, or both for 24 h. The antiproliferative effects were determined using the Cell Counting Kit-8 assay. Antitumor synergy was determined by computing the combination index. The in vivo antitumor effects of both drugs as single agents and in combination were tested using HO8910 cells implanted subcutaneously in female BALBC/c nude mice. TUNEL assay, immunohistochemical evaluation, and western blotting were performed to investigate the mechanism of action. RESULTS: A synergistic anti-proliferative effect was observed between MAP2K6-FP and paclitaxel at multiple drug concentrations, resulting in combination indices ranging from 0.3-0.85. In vivo testing against HO8910 cells in a xenograft tumor model indicated that both drugs were effective as single agents and that MAP2K6-FP and paclitaxel in combination had a synergistic antitumor effect. The combination treatment resulted in significantly altered caspase-3, vascular endothelial growth factor (VEGF), and proliferating cell nuclear antigen expression compared to treatment with the single agents (P<0.05). CONCLUSION: Both MAP2K6-FP and paclitaxel can inhibit cell proliferation and induce apoptosis in ovarian cancer HO8910 cells. Interestingly, the combination of MAP2K6-FP and paclitaxel had a synergistic antitumor effect on HO8910 cells, which induced apoptosis by increasing caspase-3 expression and decreasing VEGF expression.

[Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo].

OBJECTIVE: To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA (shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism. METHODS: Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer. Tumor-bearing nude mice were assigned randomizely to three groups: Lenti-RhoA-sh group, Lenti- negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA, negative control lentivirus and PBS were respectively injected in the three groups. Effects of treatment were observed by tumor growth curve, tumor volume, tumor weight, and tumor inhibition rate. Xenograft tissues and liver, spleen, lung, and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase(SP)immunochemical method. The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR, immunochemistry and Western blot assay. Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and apoptotic index (AI) were counted. RESULTS: Compared with Lenti-NC group and PBS group, the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days (P < 0.01) . Tumor volume (338 ± 114) mm(3) decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group (1190 ± 332) mm(3) and PBS group (1101 ± 396) mm(3) (P < 0.01) . Tumor weight (0.23 ± 0.11) g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19) g and PBS group (0.74 ± 0.17) g (P < 0.01) . Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ± 0.06) and PBS group(1.00 ± 0.11; P < 0.01) .Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P < 0.01). TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) % compared with those in Lenti-NC group (5.2 ± 2.0) % and PBS group (6.0 ± 2.1) % (P < 0.01). CONCLUSION: Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA, inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.

Up-regulation of apoptosis by gonadotrophin-releasing hormone agonist in cultures of endometrial cells from women with symptomatic myomas.

BACKGROUND: The aim of the present study was to evaluate the effect of gonadotrophin-releasing hormone agonist (GnRH-a), which is widely used in the medical treatment of symptomatic myomas, on the rate of endometrial cell apoptosis in cultures from women with symptomatic myomas. METHODS: The study included 36 women with symptomatic myomas without endometrial hyperplasia or endometrial carcinoma, and 22 controls. Endometrial biopsy specimens were obtained from all subjects. Levels of apoptosis were examined in epithelial endometrial cell cultures before and after incubation with GnRH-a (triptorelin). The percentage of apoptotic cells was evaluated using the terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling assay and flow cytometry was used to evaluate Annexin V levels. RESULTS: Levels of spontaneous apoptosis were significantly lower in endometrial cultures from patients with symptomatic myomas than in those from control subjects (P < 0.01). Concentrations as low as 10(-7) M GnRH-a enhanced apoptosis in endometrial cultures from patients with symptomatic myomas (3.48% +/- 0.27% apoptotic cells in untreated samples and 25.45 +/- 0.95% in cells treated with 10(-7) M GnRH-a; P <0.01). The percentage of apoptotic cells also increased when cultures from control women were treated with GnRH-a (8.10 +/- 0.18% in untreated samples and 15.29 +/- 2.30% in treated samples; P <0.01). Levels of apoptosis were dependent on both dose of GnRH-a and time of treatment. CONCLUSIONS: GnRH-a stimulates apoptosis in endometrial cells from patients with symptomatic myomas and this could, at least in part, account for the therapeutic action of GnRH-a.

Efficacy of gonadotropin-releasing hormone agonist and an extended-interval dosing regimen in the treatment of patients with adenomyosis and endometriosis.

AIMS: To determine the effects of gonadotropin-releasing hormone agonist (GnRH-a) and an extended-interval dosing regimen in the treatment of patients with adenomyosis and endometriosis. METHODS: This was a prospective observational study in the setting of a hospital outpatient clinic. Seventy women suffering from adenomyosis and endometriosis were randomly divided into 2 groups: extended-interval dosing (experimental group) and conventional dosing (control group). METHODS: Patients in the experimental group received a 4-dose regimen (triptorelin 3.75 mg by intramuscular injection every 6 weeks for a total of 4 doses). The patients in the control group received a conventional regimen (1 injection every 4 weeks for a total of 6 doses). The main outcome measures were relief and recurrence of dysmenorrhea and related climacteric symptoms, reduction of uterine volume, and serum levels of 17-beta-oestradiol (E(2)), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). RESULTS: The reliving rate of dysmenorrhea was 100% in patients treated with both the new regimen and the convention regimen after 6 months. The uterine volume was reduced 37.6% and 39.2%, respectively. And the levels of LH, FSH and E(2) were decreased significantly (p < 0.001). The E(2 )levels were reduced to the postmenopausal level. The hormone profile of the experimental group was similar to that of the control group (p > 0.05). CONCLUSION: The use of the extended-interval dosing regimen of triptorelin depot in patients with adenomyosis or endometriosis results in a consistent hypo-oestrogenised state, which is similar to that achieved by the conventional regimen. The new regimen reduces the cost of treatment.

Effect of first, second, and third trimester placental factors on CD4, CCR5, and CXCR4 expression in human peripheral blood lymphocytes.

OBJECTIVE: To investigate effect of first, second, and third trimester placental factors (PF) on CD4, CCR5, and CXCR4 expression in human peripheral blood lymphocytes (PBLs), and to explore their influence on human immunodeficiency virus (HIV) vertical transmission. METHODS: Human peripheral blood mononuclear cells (PBMCs) were treated with first, second, and third trimester PF (concentration 25%) respectively for 24 hours. The expression of CD4, CCR5, and CXCR4 in PBLs, and the percentages of CCR5(+), CXCR4(+)ìand CCR5(+)CXCR4(+) cells in peripheral blood CD4(+) lymphocytes were determined with flow cytometry. RESULTS: All trimester PFs reduced CCR5 expression in PBLs. The efficiency of the first trimester PF was higher than that of the second and third trimester PF. The percentage of CCR5(+) cells in peripheral blood CD4(+) lymphocytes of PF groups was significantly lower than that of the control group, and the percentage of CCR5(+) cells in peripheral blood CD4(+) lymphocytes of the first trimester PF group was significantly lower than that of the second and third trimester group. The percentages of CCR5(+)CXCR4(+) cells in peripheral blood CD4(+) lymphocytes of PF groups were significantly decreased as compared with the control group, and the percentage of CCR5(+)CXCR4(+) cells in peripheral blood CD4(+) lymphocytes of the first trimester PF group was significantly lower than that of the third trimester PF group. CONCLUSION: PF can reduce the expression of CCR5 in human PBLs and peripheral blood CD4(+) lymphocytes, indicating that PF might reduce R5 virus infection via preventing HIV entry, and might play an important role in reducing R5 virus intrauterine infection.