| Part 1 The effect and mechanism of hypoxia inducible factor-1 alpha inhibitor berberine hydrochloride on the radiation sensitivity of hypoxic esophageal squamous cell carcinoma cellsObjective:To observe the inhibition of berberine hydrochloride(berberine) effect of hypoxia inducible factor 1 alpha (HIF-la) and its downstream target gene vascμlar endothelial growth factor (VEGF) in hypoxia esophageal squamous cell carcinoma(ESCC) cells, and detect the effects and the mechanism of berberine in various ESCC cell lines to radiation sensitivity in vitro and in vivo.Method:Choose two kinds of esophageal squamous cell carcinoma cell line ECA109, TE13 in our study, In different concentration gradient and time point of berberine treated tumor cells by methyl thiazolyl tetrazolium colorimetric assay (MTT assay) observed the effect of berberine on proliferation of ESCC cells. Using immunofluorescence technique to observe the expression of ECA109 HIF-la cells before and after hypoxia treated, and observe the expression of HIF-la concentration of berberine used after application; Western blot detected the expression of HIF-la and VEGF after treatment of berberine were given; We set four groups:normoxic; hypoxia; hypoxia combined with low concentration berberine and positive reference group of cisplatin, which all received 6MV-X-ray irradiation, then we observed the effect of berberine on hypoxia radiosensitivity in ESCC cells of the plate clone formation; AnnexinV-PI staining were detected by flow cytometry to detect cell apoptosis rate of cells; nude mouse ECA109 transplanted tumor model, we established the control group, radiation alone group, intraperitoneal injection of berberine group, intraperitoneal injection of berberine combined with irradiation group. All the mice in irradiation group received a 8Gy single dose radiotherapy in tumor site,14 days after radiotherapy, nude mice were executed, the tumor size was measured, and the expression of HIF-1 and VEGF in the tumor were detected by immunohistochemistry.Resμlts:Berberine inhibited ESCC cells-ECA109 and TE13 growth in an obvious time and dose dependence; by clicking on the mμltiple target model fitting curve showed different concentrations of berberine pretreatment in hypoxia ESCC cells, compared to the irradiation alone group, can significantly increase the radiation sensitivity of hypoxic cells. Compared with the irradiation group, berberine increased apoptosis rate significantly in dose-dependent manner; berberine inhibited the expression of HIF-1α and VEGF in hypoxic ESCC cells in an obvious dose dependence. Berberine can significantly inhibit the growth of ECA109 cells transplanted tumor model in nude mice after radiotherapy. The immunohistochemistry also confirmed that berberine can reduce the expression of HIF-1α and VEGF in the transplanted tumor.Conclusion:Our resμIts showed that berberine increased radiosensitivity of ESCC cells and xenografts, and this was associated with the inhibition of HIF-1α and VEGF expression. These data suggest that berberine may be a potential radiotherapy sensitization drugs due to its significant anti-hypoxia activity.Part2:Melittin enhances radiosensitivity of hypoxia nasopharyngeal carcinoma cell lines CNE-2 by suppressing HIF-1αObjective:To observe the inhibition of melittin effect of hypoxia inducible factor 1 alpha (HIF-1α) and its downstream target gene vascμ lar endothelial growth factor (VEGF) in hypoxia nasopharyngeal carcinoma(NPC) cell line, CNE-2 cells. We detected the effects and the mechanism of melittin in CNE-2 cells to radiation sensitivity in vitro and in vivo.Method:We used CCK-8 assay to bserved the effect of melittin on proliferation of CNE-2 cells in different concentration gradient and time point. Western blot detected the expression of HIF-1α and VEGF after treatment of melittin were given; We set four groups:normoxic; hypoxia; hypoxia combined with melittin and normoxia with melittin, which all received 6MV-X-ray irradiation, then we observed the effect of melittin on hypoxia radiosensitivity in CNE-2 cells of the plate clone formation; AnnexinV-PI staining were detected by flow cytometry to detect cell apoptosis rate of cells; nude mouse CNE-2 transplanted tumor model, we established the control group, radiation alone group, intraperitoneal injection of melittin group, intraperitoneal injection of melittin combined with irradiation group. All the mice in irradiation group received a 6Gy single dose radiotherapy in tumor site,14 days after radiotherapy, nude mice were executed, the tumor size was measured.ResμIts:Melittin inhibited CNE-2 cells growth in an obvious time and dose dependence; by clicking on the mμltiple target model fitting curve showed different concentrations of melittin pretreatment in hypoxia CNE-2 cells, compared to the irradiation alone group, can significantly increase the radiation sensitivity of hypoxic NPC cells. Compared with the irradiation group, melittin increased apoptosis rate significantly in dose-dependent manner; melittin inhibited the expression of HIF-la and VEGF in hypoxic CNE-2 cells in an obvious dose dependence, melittin can significantly inhibit the growth of CNE-2 cells transplanted tumor model in nude mice after radiotherapy.Conclusion:These data suggest that melittin enhances radiosensitivity of NPC cells under hypoxia condition, and this is associated with the suppression of HIF-1α expression. Melittin appears to be a potential radiotherapy sensitization agent due to its significant anti hypoxia activity.
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