RESUMEN
BACKGROUND: Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Recent studies have revealed an association between autophagy and drug resistance. METHODS: We previously synthesized a MAPK kinase-recombinant fusion protein, MAP2K6-FP, that contains 3 domains: a protein transduction domain TAT, a human ovarian cancer HO8910 cell-specific binding peptide, and a potential antitumor effector domain MKK6(E). In this study, we investigated the effect of MAP2K6-FP on HO8910 cells treated with paclitaxel. RESULTS: The IC50 (concentration by which 50% cell growth was inhibited) was 20 µM for paclitaxel alone, 1.5 µg/mL for MAP2K6-FP alone, and 0.3 µg/mL for MAP2K6-FP and 15 µM for paclitaxel if combined, respectively. In addition, immunohistochemistry assay demonstrated that tumor tissues from ovarian cancer patients showed higher expression of LC-3, the autophagy-related protein, compared with normal ovarian tissues. MAP2K6-FP (0, 2.5, 5, 10, 20, and 40 µg/mL) dose-dependently increased the LC-3 expression in HO8910 cells. Immunofluorescence assay showed that paclitaxel alone increased the expression of LC-3 in HO8910 cells, which was further enhanced by the combination with MAP2K6-FP. Downregulation of LC-3 expression using LC-3 small interfering RNA inhibited the cytotoxicity effect of MAP2K6-FP. Furthermore, either MAP2K6-FP alone or in combination with paclitaxel increased the ratio of expressions of Beclin-1/Bcl-2, another autophagy-related markers, compared with paclitaxel alone. CONCLUSIONS: MAP2K6-FP enhanced the sensitiveness of paclitaxel for ovarian cancer via inducing autophagy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , MAP Quinasa Quinasa 6/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Proteínas Recombinantes de Fusión/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Productos del Gen tat/genética , Productos del Gen tat/farmacología , Humanos , MAP Quinasa Quinasa 6/genética , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Dominios Proteicos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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.
Asunto(s)
Carcinoma/tratamiento farmacológico , Productos del Gen tat/farmacología , MAP Quinasa Quinasa 6/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Femenino , Productos del Gen tat/genética , Proteínas Fluorescentes Verdes , Humanos , MAP Quinasa Quinasa 6/genética , Mutación , Receptores de Esteroides/genéticaRESUMEN
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.