Microenvironment Of Tumor Hypoxia, The Oxygen Carrier Chemotherapy Effects

Objective:The aim of current study was to investigate the potential benefit of oxygen carrier in tumor chemotherapy.The effect of oxygen carrier on the microcirculation of tumor tissues based on two established tumor models was observed and evaluated.The changes of tumor oxygenation and neovascularization after oxygen carrier administration and the possible underline mechanism were explored.Methods:(1) Cultured HeLa cells were injected into submucosa of 3-4-week-old female golden hamster cheek pouch to build the tumor model.(2) Fourty tumor burden hamsters were randomly assigned to four groups(n=10) and treated respectively:group 1: control group,hamsters were administrated with saline twice a week through a right jugular vein catheter;group 2:cisplatin group,the hamsters were administrated with cisplatin(5mg/kg b.w.) twice a week;group 3:cisplatin group plus low concentration of PEG-Hb,the hamsters were administrated with cisplatin(5mg/kg b.w.) plus PEG-Hb (0.3g/kg b.w.) twice a week.Group 4:cisplatin plus high concentration of PEG-Hb group, the hamsters were administrated with cisplatin(5mg/kg b.w.) combined with PEG-Hb (0.6g/kg b.w.) twice a week.(3) Computor assisted intravital microscopy system was used in observing and recording tumor neovascularization on 0,3,5,7 day post innoculation, then off-line analyze images to detect the changes of the microvessel tortuosity and area functional capillary density.Leukocytes movement pattern in venule was also measured on the 7th day after innoculation.(4) Immunohistochemistry of endogenous hypoxia maker (HIF-1α) and exogenous hypoxia maker(Hypoxyprobe-1) were tested in tumor tissues.(5) Serum level of soluble Intercellular adhesion molecule-1(sICAM-1),TNF-αand IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).(6) Cultured HeLa cells were subcutaneously injected to the armpits of 3-4-week-old female BALB/c nude mice to built another tumor model.(7) One month post-innoculation,the nude mice were randomly assigned to five groups(n=10) and treated respectively,group 1-4 were similar to golden hamsters' arrangement;group 5,PEG-Hb group,the nude mice were administrated with PEG-Hb(0.45g/kg b.w.) twice a week.(8) Animals were sacrificed after a month of respective treatment,tumor tissue hypoxia situation was also evaluated by endogenous hypoxia maker(HIF-1α) and exogenous hypoxia maker(Hypoxyprobe-1). Immunohistochemical staining method was used to detected the expression level of EPO,EPOR,MDR1 and CD31 in tumor tissue.(9) Western blot method was used to detected the expression of EPO/EPOR,MDR1 and HIF-1αin tumor tissue.(10) Serum level of EPO was detected by ELISA.Results:(1) In the concomitance with solid tumor development the angiogenesis is notable,tortuosity,dilation,sacculation and permeability morphological changes are the specific features of newly formed tumor vessels.In golden hamster experiment,the body weight and tumor volume on 3,5,7 day post-innoculation both were significantly decreased in group 2,3 and 4 compared with control group.(2) Microvessel tortuosity was increased post-innoculation,which was more intensive in the inner ring than the outer ring of the tumor.On the fifth day the tortuosity in group 4 was markedly reduced compared with group 2 in peritumoral area.Area capillary density was increased parallel with the tumor development,chemotherapy treatment significantly decreaed it in the inner ring surrounding tumor mass.On the fifth and seventh day the area capillary density in group 4 was markedly reduced compared with group 2.Cisplatin can induce leukocyte rolling and sticking to venule,which was not effected with PEG-Hb administration.(3) The expression of Hypoxyprobe-1 and HIF-1αwere significantly decreased in group 4 compare with group 2(P＜0.05).(4) Cisplatin can up-regulate pro-inflammatory factors(sICAM-1, TNF-α,IL-6),which was not influenced with PEG-Hb treatment.(6) In BALB/c nude mice study,chemotherapy combined with PEG-Hb administration can enhance its tumor inhibition effect,after treatment the tumor volume was decreased sharply in group 4.(7) The lowest hypoxia marker expression was detected in group 4.EPO/EPOR,MDR1 and CD31 were decreased in group 4 compare with group2(P＜0.05).EPO level was positive correlation with HIF-1αexpression(P=0.021 r=0.76).(8) Serum level of EPO was decreased in group 4 compare with group2(P＜0.05).Conclusion:(1)The tumor model established in golden hamster cheek pouch is a ideal model for microvascular observation.It is favorable in tumor newly formed vessels observation and evaluation of drugs influence on tumor microcirculation.(2)Tissue hypoxia and angiogenesis are concomitant with neoplasma,high concentration of PEG-Hb combined with cisplatin can be benefit to tumor tissue oxygenation,subsequently contribute to anti-angiogenesis and tumor microvessels normalization.(3) There are no significant difference in body weight and tumor volume changes when cisplatin combined with or without blood substitute in a short time period.(4) Cisplatin treatment involves in endothelium injury and inflammatory reaction,adding blood substitute could not depress inflammation.(5)After one month treatment nude mice tumor volume inhibited obviously in group 4,blood substitue concentration is an important factor in assistant tumor chemotherapy.(6) EPO/EPOR and MDR1 which are modulated by HIF-1αmight participate in the process of blood substitue chemotherapy sensitizasion. Objective:To investigate the effect of cobalt chloride on tumor cells proliferation and apoptosis in vitro,to explore the reasonable strategy of cobalt chemical mimic hypoxia.Method:Two tumor cell lines(A549 and HeLa) were cultured in vitro,After the cells were exposed to CoCl₂(0.05～2mmol/L) for different time period(4～48h),cells viability,proliferation and apoptosis were maesured by MTT and FCM methods.HIF-1αand related apoptosis proteins expression were detected by Western blot.Result:At concentration of 2001μmol/L within 24hr cell viability was weakly changed. However,higher dose or prolonged challenge of CoCl₂ significantly decreased cell survival rate(p＜0.05).It is clearly showed that most of the damaged cells were early apoptosis population after CoCl₂(200μmol/L) incubation within 24hr,contrary to a higher concentration of CoCl₂(800umol/L) most of the damaged cells were late apoptosis and necrosis parts.Moreover,A549 cell apoptosis rate was significantly higher than HeLa cell (39%vs29%).Western blot analysis revealed that CoCl₂(200μmol/L) exposure implicated in up-regulating the expression of HIF-1α,Bax,p53 and down-regulating the expression of Bcl-2 in A549 cell within 24hr,Similar results were observed in HeLa cell,except p53.A low level of p53 protein expression was detected with or without CoCl₂ in HeLa cell.But higher concentration(～400μM) or prolonged CoCl₂ challenge changed the tendency of HIF-1αexpression in the two lines as well as Bcl-2,Bax.Therefore,this feature illustrated that prolonged or higher dose of cobalt exposure was not positive to expected chemical hypoxia effect(HIF-1αexpression).Conclusion:Dose and time-dependent effect of cobalt chloride on the tumor cells proliferation and apoptosis,most of the damaged cells were early apoptosis population after cobalt(200μM) incubation within 24hr,higher dose of CoCl₂ caught late apoptosis and necrosis.P53 did not involved in the process of HeLa cell's apoptosis.The apoptosis effect of CoCl₂ is tumor cell specific.In a limited range of cobalt concentration HIF-1αexpression posseses positive relation with cobalt.But prolonged or higher dose of cobalt exposure was not positive to expected chemical hypoxia effect(HIF-1αexpression).The concentration around 200μM within 24hr period time may be a reasonable choice in chemical hypoxia mimicry.The concentration and duration of cobalt chloride should be taken into consideration in chemical mimic hypoxia.