Melatonin As Well As Artificial Oxygen Carriers For Tumor Angiogenesis And Its Mechanism Research

Objectives: Melatonin is an important natural oncostatic agent. At present there are no data available as to its possible influence on tumor angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. It is well known that vascular endothelial growth factor (VEGF) is crucial to a solid tumor's higher vascularization and development. The current study investigated the effect of melatonin on endogenous VEGF expression in three human cancer cell lines (PANC-1, HeLa and A549 cells) and to explore the underline mechanism.Methods: (1) The survival, proliferation and apoptosis effects of CoCl₂ on primary cultured three human cancer cell lines (PANC-1, HeLa and A549 cells) were detected by MTT method. (2) The survival, proliferation and apoptosis effects of MEL on primary cultured three human cancer cell lines (PANC-1, HeLa and A549 cells) were detected by MTT method. (3) RT-PCR was performed to detect the VEGF mRNA level in tumor cells treated with MEL in normoxia or hypoxia mimiced by CoCl₂. (4) Western blot and ELISA assay was performed to detect the HIF-1αand VEGF protein levels in tumor cells, when treated with MEL in normoxia or hypoxia mimiced by CoCl₂.Results: (1) 0～150μmol/L CoCl₂ could not affect the growth of of three tumor cell lines. When continuously increase the CoCl₂ concentration to 250～500μmol/L, small part of tumor cells would undergo apoptosis. (2) At near physiological concentrations (1nmol/L), melatonin had no significant effect on cellular proliferation. However, melatonin in phamacologic concentrations (1mmol/L) significantly decreased cell proliferation, and the negative effect on cells proliferation was enhanced with increasing melatonin concentrations. (3) Physiologic concentrations of melatonin have no obvious impact on the VEGF and HIF-1αexpression, whereas pharmacologic concentrations of melatonin suppress the VEGF mRNA and protein levels induced by CoCl₂. Conclusions: (1) Tumor cells were not sensitive to low concentration of CoCl₂ (100μmol/L) and CoCl₂ can mimic the hypoxia environment. (2) Pharmacologic concentration of MEL can suppress the proliferation of tumor cells and promote the apoptosis of the tumor cells. (3) Pharmacologic concentration of MEL can down-regulate the expression of VEGF induced by CoCl₂. (4) MEL had the potential to be a chemo-sensitizer due to its anti-angiogenesis effect. Objectives: (1) Observe and evaluate the effect of tumor mass to the microcirculation. (2) Study the influence of blood substitutes on the microcirculation of tumor tissues. (3) Investigate weather the blood substitutes have a chemo-sensitization effect or not. (4) Explore the underline mechanism by molecular biology Methods.Methods: (1) Cultured HeLa cells were subcutaneously injected to the armpits of3-4-week-old female BALB/c nude mice to built the tumor model. (2) After two weeks ofinoculations, the nude mice bearing tumor of 60-80mm³ were separated to five group(n=10) randomly and treated differently as below: Group 1: control group, the nude micewere administrated with saline twice a week. Group 2: cisplatin group, the nude mice wereadministrated with cisplatin (5mg/kg) twice a week. Group 3: low concentration ofPEG-Hb + cisplatin group, the nude mice were administrated with cisplatin (5mg/kg)combined with PEG-Hb (0.3g/kg) twice a week. Group 4: high concentration of PEG-Hb +cisplatin group, the nude mice were administrated with cisplatin (5mg/kg) combined withPEG-Hb (0.6mg/kg) twice a week. Group 5: PEG-Hb group: the nude mice wereadministrated with PEG-Hb (0.45 g/kg). (3) We measured tumors with calipers andcalculated volume as (legth×width²)×0.5 twice a week and draw the tumor growth curve .(4) The animals were sacrificed after a month of treatment. To assess hypoxia regions, weintraperitoneally injected mice with 60 mg/kg pimonidazol hydrochloride (Hypoxyprobe-1,Chemicon), 1h before sacrificed. (5) Immunohistochemical staining method was used todetected the expression level of HIF, VEGF and CD31 in the tumor tissue. (6) ELISAMethod was used to detect the VEGF protein level in the serum of the nude mice. (7)Total protein were extracted from the tumor tissue and Western Blot Method was used todetected the expression of HIF and VEGF protein. (8) Total RNA were extracted from thetumor tissue and RT-PCR Method were used to detected the expression of HIF and VEGF.(9) 2×10⁶ HeLa cells were injected to the check pouch of the 3-4-week-old female goldenhamster to build the tumor angiogenesis model. The hamster were separated to four group (n=10) randomly and treated differently as below: Group 1: control group, the nude mice were administrated with saline twice a week. Group 2: cisplatin group, the nude mice were administrated with cisplatin (5mg/kg) twice a week. Group 3: low concentration of PEG-Hb + cisplatin group, the nude mice were administrated with cisplatin (5mg/kg) combined with PEG-Hb (0.3 g/kg) twice a week. Group 4: high concentration of PEG-Hb + cisplatin group, the nude mice were administrated with cisplatin (5mg/kg) combined with PEG-Hb (0.6 g/kg) twice a week. We observed and recorded the mircocirculation of the check pouch by Computer assisted intravital microscopy on 0,3,5,7 days after the innoculation. The images were analyzed by computer to detect the change of the microvessel tortuosity (NMT) and functional capillary density (CD).Results: (1) PEG-Hb solution impedes the growth of tumor. Group 4 was more effective than the group 3 while there was no obviously difference between group 5 and control group. (2) Group 4 has a lower positive Immunohistochemical staining for Hypoxyprobe?-1 compared with other groups. (3) Group 4 has a lower positive Immunohistochemical staining for HIF, VEGF and CD31 compared group 1 and group 2. (4) Western Blot results shows that the expression levels of HIF and VEGF were lower than other groups. (5) RT-PCR results show that the levels of HIF RNA and VEGF RNA were lower than other groups. (6) Images analysis results shows that there were lower microvessel tortuosity and functional capillary density in group 4.Conclusions: (1) Solid tumor tissue has a hypoxia microenvironment, abnormal microvasculature and angiogenesis. (2) Combination of PEG-Hb and cisplatin was effective to enhance the antitumor effect of cisplatin. (3) PEG-Hb is effective to enhance the oxygenation of the tumor tissue. (4) PEG-Hb can downregulate the expression of VEGF and CD31 to suppress the tumor angiogenesis. (5) HIF signaling pathway plays an important role of the antiangiogenic effect of PEG-Hb.