| Glioma,the most common primary brain cancer,is highly vascularized and angiogenesis-dependent human cancers.Current reports show that the vascular microenvironment of glioma plays a central role in glioma progression,invasion, diagnosis and the effectiveness of clinic treatment.Tumor microenvironment has been recognized as a target for chemoprevention treatment.Although many aggressive therapies applied,the patients with nervous system tumors had poor and dismal prognosis with high mortality.More efficient strategies are urgent for glioma therapy.Lidamycin(LDM,also named C-1027),a member of the enediyne antitumor antibiotic family,which produced by a Streptomyces globisporus C-1027 strain isolated in China,showed extremely potent cytotoxicity,anti-angiogenic activity and tumor growth inhibition in mice.LDM has been in clinical trial in China.To determine the action of lidamycin on glioma cells and to reveal its possible mechanism,we investigated the effects of lidamycin on rat glioma C6 cell line and human glioma U87 cell line.Temozolomide(TMZ) is a very effective drug for malignant gliomas with significant survival increase.Preclinic and clinic investingations have demonstrated that TMZ exerted powerful antiangiogenic and anti-tumor effects.Despite the encouraging results,the therapeutic effects of TMZ are far less ideal.It has been well accepted that combination therapy of TMZ with some other agents are valid to glioma patients,then, the previously data from TMZ combination therapy motivated us to investigate the effects of the combination of LDM with TMZ on glioma in vitro and in vivo.1.Potent effects of LDM and combination with TMZ on glioma cellsMTT assay was performed to detect the cytotoxicity of LDM to C6 and U87 cells. The data showed that LDM has potent anti-proliferation effect on glioma cell,and the IC50 values of LDM to C6 and U87 cells were 5.86×10-11 mol/L and 2.05×10-11 mol/L, respectively.When LDM combined with TMZ,the CI values plots suggest that combinations of LDM and TMZ have synergistic effects on inhibiting C6 and U87 cell proliferation. Annexin V-FITC/PI assay was performed with flow cytometry to evaluate the proapoptotic effects of the drugs on C6 and U87 cells.The results indicated the potent and dose-dependent proapoptotic effect of LDM(0.1 nmol/L,0.5 nmol/L,1 nmol/L). The combination of LDM with TMZ resulted in a higher percentage of apoptosis in C6 and U87 cells with significant differences.Western blot showed that the synergistically regulations of P53 apoptosis pathway involved in the enhanced apoptosis.2.Effects of LDM and combination with TMZ on tumor microenvironment in vitroLDM alone(0.1 nmol/L,0.5 nmol/L,1 nmol/L) significantly inhibited invasion of rat brain microvessel endothelial cells(rBMECs) with dose-dependent manner. Compared with single drug,the invasion ratio of combination was significantly decreased(P＜0.001,vs LDM,P＜0.001,vs.TMZ).Significant synergy was presented with CI value 0.68.Meanwhile,the Western blot showed that the activity of VEGFR2 and expressions of MMP2 and MMP9 were significantly decreased in the combination, partially associated with the more efficient inhibion of rBMEC invasion.Co-culture of C6 cells and rBMEC was performed to detect glioma cell-induced rBMECs migration.The glioma C6 cell-induced rBMEC migration was significantly inhibited when C6 cells were treated with the indicated dosing regimens(LDM, 0.1nmol/L;TMZ,300μmol/L;and LDM 0.1 nmol/L+TMZ 300μmol/L,respectively) for 72h.Compared with single drug-treated condition medium,the migration ratio of combination-treated condition medium was significantly decreased(P＜0.001,vs.LDM, P＜0.001,vs.TMZ).Significant synergy was presented with CI value of 0.72.VEGF expression of C6 cells was significantly reduced(P＜0.001,vs LDM,and P＜0.001 vs TMZ) by the combination,compared with that by respective single drug.Significant synergy was observed with CI value of 0.53.Western blot of VEGF of drug-treated C6 condition medium showed similar and coincident results with ELISA assay.Meanwhile, Western blot of VEGFR for drug-treated rBMEC and VEGF for C6 cell lysate and U87 cell lysate showed that the combination treatment significantly inhibits VEGF expression and VEGFR activity.3.Effects of LDM and combination with TMZ on rBMEC and angiogenesis in vitro.By MTT assay,LDM showed potent anti-proliferation effect on rBMEC,and the IC50 values was 1.06×10-11 mol/L.When LDM combined with TMZ,the CI values plots suggest that combinations of LDM and TMZ have synergistic effects on inhibiting rBMEC proliferation.Annexin V-FITC/PI assay indicated the potent and dose-dependent proapoptotic effect of LDM to rBMEC.The combination of LDM with TMZ enhanced the apoptosis of rBMECs,the apoptotic ratio of combination is significantly higher than that of LDM(P＜0.01) or TMZ(P＜0.01) alone.In tube formation assay for rBMEC,the intact tubes were counted and analyzed. LDM disrupted tube formation by dose-dependent manner.Tube formation in combination treatment(LDM 0.1 nmol/L+TMZ 200μmol/L) was significantly decreased,as compared with single drug(P＜0.01 vs LDM;P＜0.001 vs TMZ),and significant synergism was obtained with CI value of 0.75.The rat aortic ring was incubated with medium containing drugs.The single drug administration of LDM(0.1 nmol/L) or TMZ(200μmol/L) showed inhibition on the sprouting of rat aortic ring. When administrated in combination of LDM with TMZ,the sprouting of aortic ring was inhibited more significantly than the single drug treatments(P＜0.001,vs LDM,and P＜0.001,vs TMZ).Synergistic effect was confirmed with CI value of 0.8.4.Synergistic Effects of Combination Therapy on Tumor Growth of Glioma U87 Xenograft In Nude Mice.As the tumor growth curves indicated,LDM given alone inhibited the growth of tumors in nude mice with dose-dependent manner.TMZ treatment also exhibited significant growth inhibition(P＜0.001,vs control).The combinations of LDM with TMZ exerted more significantly synergistic anti-tumor effects than the agents given alone.No statistically significant body weight changes between the animal groups. Similar results were obtained from tumor weights histogram.The combination therapies CI values＜1 indicates synergism of the two drugs.As measured by the TUNEL method, LDM given alone increased the apoptosis ratio of tumors in nude mice with dose-dependent manner.Greater degrees of apoptosis were observed in the groups that received combinations of LDM and TMZ.Combination of LDM 0.05mg/kg with TMZ obtained greater apoptosis index than that of LDM 0.025 mg/kg with TMZ.The CI values were 0.75 and 0.77,respectively.Immunohistological examination showed that the combination of LDM and TMZ significantly and synergistically decreased the microvessel density in U87 xenograft,as compared with single drug treated groups.The CI values were 0.88 for combination of LDM 0.025mg/kg with TMZ and 0.78 for combination of LDM 0.05/kg with TMZ,respectively.Meanwhile,the combination of LDM and TMZ also synergistically inhibited VEGF expression in tumor.The CI values were 0.93 for combination of LDM 0.025mg/kg with TMZ and 0.42 for combination of LDM 0.05/kg with TMZ,respectively. In conclusion,in our work,MTT assay and Annexin V-FITC/PI were performed to investigate the effects of lidamycin plus temozolomide on cell proliferation and apoptosis. Meanwhile,to more proximately mirror the glioma vascular microenvironment,a co-culture system of rat brain microvessel endothelial cells with rat glioma C6 cells was applied to examine endothelial cell migration,invasion and VEGF secretion. Angiogenesis associated endothelial events were also investigated with tube formation and rat aortic ring sprouting assay.Expressions of VEGF and MMPs were explored with Western blot.Finally,we examined the synergistically inhibitory effects of enediyne agent lidamycin(C-1027) plus TMZ on the growth of human glioma xenograft,the associated apoptosis and glioma vascular microenvironment in vivo.Our work demonstrates that the high potency of lidamycin in the inhibition of glioma cell proliferation and the induction of apoptosis in association with antiangiogenesis in vitro and in vivo;and notably,the synergism of lidamycin plus temozolomide on glioma suppression.Study results imply the involvements of enhanced apoptosis and decreased VEGF-induced angiogenesis in glioma microenvironment.
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