| Ovarian cancer, the most common gynecologic malignancy, is the fifth leading cause of death from cancer in women and the leading cause of death from gynecological cancer. The evolution of surgical techniques, chemotherapy, radiotherapy and adjuvant therapy through multiple clinical trials over the past three decades has resulted in improvements in ovarian cancer treatment and increase in 5-year survival rate. However, the high incidence of platinum- resistance following first-line platinum-paclitaxel doublet treatment makes ovarian cancer incurable.Epidemiologic and experimental animal studies have shown that chronic stress promotes tumor development and progression and the sympathetic transmitter, noradrenaline, has been considered as an important mediator. Thus far, the majority of research on the deleterious effects of stress has focused on the neuroendocrine regulation of the immune response. In both animal and human studies, chronic stress has been shown to decrease cellular immune parameters, such as natural killer cell cytotoxicity and T-cell responses to mitogen stimulation. Recently, there is growing evidence confirming that alterations in neuroendocrine dynamics due to chronic stress can cause alterations in tumor pathogenesis.Several report demonstrated that noradrenaline could act on tumor cells directly and promote tumor growth and angiogenesis through up-regulation the expression of VEGF, bFGF and MMP-9. Further more, both propranolol and ADRB2 siRNA could completely block noradrenaline-induced effects. However, its molecular mechanisms had not been uncovered.As the translation initiation of VEGF, bFGF and MMP-9 depend on eIF4F complex, the ERK-Mnk1 pathway and Akt-mTOR pathway could regulate eIF4F activities by phosphorylation of eIF4E or 4E-BPs, and noradrenaline could activate both the ERK-Mnk1 pathway and Akt-mTOR pathway, we postulate that noradrenaline promote tumor growth and angiogenesis through positive regulation of eIF4F complex. Meanwhile, eIF4F complex controls the translation initiation of survivin, an anti-apoptotic protein which plays key roles in determination of chemosensitivity. Therefore, we suppose that noradrenaline could induce chemoresistance by up-regulation the translation of survivin. At last, we prepared a novel fusion protein composed of protein transduction domain and phosphorylation-defective truncated 4E-BP2, and investigated whether it could enhance the chemosensitivity of ovarian cancer cells by disruption of eIF4F assembly. Methods: Wound healing assay was carried out to detect the effect of norepinephrine on SKOV3 cell migration. Apoptosis rate and cell cycle phase distribution of SKOV3 cells were measured by flow cytometry (FCM). The effect of norepinephrine on cell apoptosis and chemosensitivity were examined by Annexin V/PI staining and DAPI staining. The expression levels of Akt,p-mTOR,p-p70S6K,p-4E-BP1,p-ERK1,p-Mnk1,p-eIF4E,HIF-1α,c-myc,ODC,MDM2 and survivin was analyzed by Western blot and the secretion of VEGF was examined by ELISA assay.After design and construction a prokaryotic expression vector pVIP4- t4EBP2, a novel fusion protein PTD4-EGFP-t4EBP2 was highly expressed in E.coli ER2566 and purified. The effect of PTD4-EGFP-t4EBP2 on SKOV3 cell proliferation was evaluated by MTT assay. Annexin-V/PI staining was used to evaluate the effect of PTD4-EGFP-t4EBP2 on cisplain sensitivity. The effects of PTD4-EGFP-t4EBP2 on VEGF secretion and survivin expression was examined by ELISA assay and Western blot, respectively.Results: After treatment of SKOV3 cells with noradrenaline, the mean remaining width at 3h, 6h and 12h was 98.7±2.2%, 95.4±3.0% and 92.7±2.2% of the original wound width, but the mean remaining width in the control group was 93.0±2.4%,80.7±3.7% and 69.8±2.6%. Noradrenaline decrease the proliferation index of SKOV3 cells to 39.6±1.3% and 41.0±1.6% at 3h and 6h (P<0.001), but at 12h and 24h, the proliferation index was increased to 70.4±3.8% and 63.9±4.8%(P<0.001). Cell growth curve demonstrated that the tumor cells proliferate very quickly between 12 and 24 hours. Annexin-V/PI staining demonstrated that at early phase, pretreatment SKOV3 cells with noradrenaline significantly promote cisplatin-induced apoptosis, but after 9 hours pretreatment, the tumor cells are resistant to cisplatin-induced death. Western Blot demonstrated that noradrenaline significantly increase the phosphorylation levels of ERK1, Mnk1 and eIF4E, suggested that ERK-Mnk1 pathway was activated. Meanwhile, phosphorylation levels of Akt, mTOR, p70S6K and 4E-BP1 were also elevated, suggested that Akt-mTOR pathway was another downstream signal pathway that was activated by noradrenaline. And HIF-1α, c-myc, ODC, MDM2 and survivin, whose translation initiation has been proved to depend on the activity of eIF4F complex were all up-regulated.DNA sequencing confirmed that prokaryotic expression vector pET28a- PTD4 and pVIP4 were successfully constructed. Truncated 4E-BP2 cDNA was amplified by RT-PCR using total RNA from SKOV3 cellls as template and subsequently cloned into pVIP4, double endonuclease cutting, PCR with specific primers and DNA sequencing suggested that pVIP4-t4EBP2 was successfully. After transforming pVIP4-t4EBP2 into E.coli ER2566 competent cells, recombinant protein PTD4-EGFP-t4EBP2 was highly expressed and prepared. Under fluorescence microscope, we find that fusion protein PTD4-EGFP-t4EBP2 could enter SKOV3 cells with high efficiency, and both the secretion of VEGF and the expression of survivin were down-regulated. After transduction of SKOV3 cells with PTD4-EGFP-t4EBP2 for 24 or 48 hours, the inhibition rate was 16.9±4.4% and 47.8±6.2%, respectively. And transduction of SKOV3 cells with PTD4-EGFP-t4EBP2 for 24 hours, the percent of early apoptotic cells of SKOV3 cells was elevated from 3.3±1.0% to 14.2±1.2%, and the percent of apoptotic cells induced by cisplatin was elevated from 16.3±1.8% to 45.2±3.3% (P<0.001).Conclusion: Though noradrenaline enhances the cisplatin sensitivity of SKOV3 cells at early phase, the tumor cells are resistant to apoptosis after 9 hours. Also, noradrenaline promotes the proliferation of SKOV3 cells after 9 hours, but an obvious inhibition could be detected at 3 hours or 6 hours. Moreover, noradrenaline could activate both ERK-Mnk1 pathway and Akt-mTOR pathway which were demonstrated by up-regulation the phosphorylation levels of ERK, Mnk1 and eIF4E, or the phosphorylation levels of Akt, mTOR, p70S6K and 4E-BP1. And noradrenaline could up-regulate the expression of c-myc, ODC, MDM2, survivin and HIF-1αand enhance VEGF secretion, all of which are involved in promoting tumor growth and angiogenesis, confirmed that eIF4F complex plays key roles in tumor progression. The recombinant fusion protein PTD4-EGFP-t4EBP2 significantly inhibits SKOV3 cell proliferation, VEGF secretion and sensitizes the tumor cells to cisplatin through survivin expression.
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