力达霉素对神经胶质瘤抑制作用及联合替莫唑胺的协同作用研究
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摘要
神经胶质瘤是最常见的原发性脑肿瘤,是高度血管化的血管生成依赖性生长的人类肿瘤。目前的报道表明神经胶质瘤的血管微环境在神经胶质瘤发生与发展、侵袭、临床诊断及治疗效果中起关健性作用。肿瘤微环境已经成为化疗的靶标之一。尽管神经胶质瘤的治疗已经取得了很多进步,中枢神经系统肿瘤患者的预后及诊断仍然很不理想,死亡率仍然很高。新的治疗策略仍然有待研究。
力达霉素(Lidamycin,LDM,C-1027)是烯二炔类抗生素家族的一个成员,它具有很强的细胞毒性、抗血管生成活性及抑制实验肿瘤生长的作用,力达霉素已经在中国进入了临床研究。为测试力达霉素对神经胶质瘤的作用及机制,我们对力达霉素作用于大鼠神经胶质瘤C6细胞和人神经胶质瘤U87细胞后的效应进行了检测,以揭示其抑制神经胶质瘤的效果。
替莫唑胺(Temozolomide,TMZ)是目前治疗恶性神经胶质瘤的有效药物,临床前研究与临床研究已经证实了替莫唑胺具有很强的抗血管生成活性及抑制肿瘤生长的作用,它能够显著提高肿瘤患者的生存率。替莫唑胺与其他类药物联合治疗神经胶质瘤已经取得了更为有效的结果,替莫唑胺新的联合治疗方法已经受到了科研工作者的高度重视。本研究探索力达霉素与替莫唑胺对神经胶质瘤的体外与体内作用,以期应用于神经胶质瘤的治疗。
1.LDM对神经胶质瘤细胞的影响及与TMZ联合的效果。
MTT分析表明,LDM(10~(-7)/mol/L～10~(14)mol/L)能够显著抑制大鼠神经胶质瘤C6细胞和人神经胶质瘤U87细胞增殖,其IC_(50)值分别为5.86×10~(-11) mol/L和2.05×10~(-11)mol/L。LDM(10~(-10) mol/L～10~(-14) mol/L)与TMZ(60μmol/L,90μmol/L,120μmol/L,150μmol/L)联合处理72小时对神经胶质瘤细胞和大鼠脑微血管内皮细胞的增殖抑制存在着协同作用。Annexin V-FITC/PI双染结合流式细胞仪结果表明LDM(0.1 nmol/L,0.5 n mol/L,1 n mol/L)以剂量依赖方式诱导细胞凋亡。LDM与TMZ联合能够显著增强神经胶质瘤C6和U87细胞的凋亡率,这与Western blot结果提示两药联合协同增强了P53凋亡途径的调节有关。
2.LDM对体外模拟肿瘤微环境的影响及与TMZ联合效果
LDM单药(0.1 nmol/L,0.5 nmol/L,1 nmol/L)能够以剂量依赖方式显著抑制大鼠脑微血管内皮细胞的侵袭。LDM(0.1 nmol/L)与TMZ(200μmol/L)联合用药后,侵袭细胞数明显的减少,与单独用药组比较,P值均小于0.001,二者存在明显的协同作用,协同指数为0.68。蛋白印迹的结果表明LDM与TMZ联合抑制大鼠脑微血管内皮细胞的侵袭与其抑制VEGFR2活性及基质金属蛋白酶MMP2和MMP9的表达有关。以不同药物组合处理大鼠神经胶质瘤C6细胞和大鼠脑微血管内皮细胞rBMEC的共培养系统72小时后,与对照组比较,大鼠脑微血管内皮细胞的迁移受到了药物处理的显著抑制,联合处理组的迁移细胞数受到更为明显的抑制,与单独用药组比较,P值均小于0.001,联合指数为0.72,ELISA方法与蛋白印迹实验结果一致表明这与大鼠神经胶质瘤C6细胞条件培养基中VEGF的表达受到药物联合处理的明显抑制有关,也与大鼠脑微血管内皮细胞VEGFR的活性和神经胶质瘤C6细胞和U87细胞VEGF的表达受到协同抑制有关。
3.LDM对大鼠脑微血管内皮细胞和体外血管生成的影响及与TMZ联合效果
LDM对大鼠脑微血管内皮细胞的增殖有强烈的抑制作用,其IC_(50)值为1.06×10~(-11)mol/L,同时也存在着剂量依赖性诱导内皮细胞凋亡作用。LDM与TMZ联合后可增强细胞毒作用及诱导细胞凋亡作用。LDM可以阻断内皮细胞形成的小管,并随着剂量加大,破坏小管的能力越强。LDM 0.1 nmol/L与TMZ 200μmol/L联合处理内皮细胞,形成的管状结构要比LDM 0.1 nmol/L组少,P＜0.01,与TMZ 200μmol/L相比较,P＜0.001,二者的联合指数为0.75。LDM 0.1 nmol/L或TMZ 200μmol/L作用于大鼠动脉环后,都显示了明显的抑制作用,二者联合后能够更为明显的抑制动脉环发芽,与单独用药组比较P值均小于0.001,联合指数为0.8。
4.LDM与TMZ联合对人神经胶质瘤U87裸鼠移植瘤生长抑制
LDM 0.025mg/kg和0.05mg/kg能够显著抑制人神经胶质瘤U87裸鼠移植瘤的生长,TMZ也能够显著抑制肿瘤人神经胶质瘤的生长,与对照组相比较,P值均小于0.001。各药物处理组对动物体重都没有明显的影响。肿瘤生长曲线和瘤重量分析结果显示,LDM与TMZ单药能够产生抑制肿瘤生长的效果,LDM 0.025mg/kg和0.05mg/kg分别与TMZ 0.5mg/kg联合能够更为显著的抑制肿瘤的生长,联合指数小于1,表明二者能够协同抑制神经胶质瘤的生长。
经原位TUNEL检测,LDM单药能够诱导细胞凋亡,且随着剂量加大,凋亡率也增加。LDM与TMZ 0.5 mg/kg联合后可明显的增加凋亡率,与单独用药组比较P值均小于0.001,LDM 0.025mg/kg和0.05mg/kg与TMZ联合的协同指数分别为0.75和0.77,表明二者在体内协同诱导细胞凋亡。
CD31的免疫组织化学分析表明,LDM单药、TMZ单药及二者联合都能够降低人神经胶质瘤U87裸鼠移植瘤血管密度,与对照组相比较均存在显著性差异。二者联合能够更为显著的抑制肿瘤组织的血管生成,LDM 0.025mg/kg和0.05mg/kg分别与TMZ 0.5mg/kg联合后,其抑制肿瘤血管生成的协同指数分别为0.88和0.78,表明二者能够协同抑制肿瘤血管生成。同时,VEGF的免疫组织化学分析表明,LDM与TMZ联合对肿瘤组织中VEGF的表达也产生协同抑制作用,LDM 0.025mg/kg与TMZ0.5mg/kg的协同指数为0.93;LDM 0.05mg/kg与TMZ 0.5mg/kg抑制VEGF表达的协同指数为0.42。
本研究工作采用MTT法分析了药物对神经胶质瘤细胞和脑微血管内皮细胞增殖的影响,采用Annexin V-FITC/PI法检测了药物处理后细胞凋亡,同时,为了更全面的反映神经胶质瘤血管微环境,从肿瘤旁分泌网络考虑,采用共培养系统观察了药物对大鼠神经胶质瘤C6细胞和大鼠脑微血管内皮细胞相互作用的影响,包括细胞迁移和VEGF的分泌。以大鼠脑微血管内皮细胞的小管形成实验和大鼠动脉环实验作为体外血管生成的模型,来分析药物对血管生成的影响。体内实验中,在分析了力达霉素与替莫唑胺联合对肿瘤生长的影响,同时也分析了二者联合对细胞凋亡和微血管密度及VEGF表达的影响。我们的工作证明:力达霉素强烈抑制神经胶质瘤的细胞增殖和诱导神经胶质瘤细胞凋亡,并抑制血管生成,同时力达霉素可以与替莫唑胺联合作用,协同抑制神经胶质瘤的生长,增加细胞凋亡诱导及增加对VEGF表达的抑制。
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 cells
MTT 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 IC_(50) 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 vitro
LDM 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 IC_(50) 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.
力达霉素(Lidamycin,LDM,C-1027)是烯二炔类抗生素家族的一个成员,它具有很强的细胞毒性、抗血管生成活性及抑制实验肿瘤生长的作用,力达霉素已经在中国进入了临床研究。为测试力达霉素对神经胶质瘤的作用及机制,我们对力达霉素作用于大鼠神经胶质瘤C6细胞和人神经胶质瘤U87细胞后的效应进行了检测,以揭示其抑制神经胶质瘤的效果。
替莫唑胺(Temozolomide,TMZ)是目前治疗恶性神经胶质瘤的有效药物,临床前研究与临床研究已经证实了替莫唑胺具有很强的抗血管生成活性及抑制肿瘤生长的作用,它能够显著提高肿瘤患者的生存率。替莫唑胺与其他类药物联合治疗神经胶质瘤已经取得了更为有效的结果,替莫唑胺新的联合治疗方法已经受到了科研工作者的高度重视。本研究探索力达霉素与替莫唑胺对神经胶质瘤的体外与体内作用,以期应用于神经胶质瘤的治疗。
1.LDM对神经胶质瘤细胞的影响及与TMZ联合的效果。
MTT分析表明,LDM(10~(-7)/mol/L～10~(14)mol/L)能够显著抑制大鼠神经胶质瘤C6细胞和人神经胶质瘤U87细胞增殖,其IC_(50)值分别为5.86×10~(-11) mol/L和2.05×10~(-11)mol/L。LDM(10~(-10) mol/L～10~(-14) mol/L)与TMZ(60μmol/L,90μmol/L,120μmol/L,150μmol/L)联合处理72小时对神经胶质瘤细胞和大鼠脑微血管内皮细胞的增殖抑制存在着协同作用。Annexin V-FITC/PI双染结合流式细胞仪结果表明LDM(0.1 nmol/L,0.5 n mol/L,1 n mol/L)以剂量依赖方式诱导细胞凋亡。LDM与TMZ联合能够显著增强神经胶质瘤C6和U87细胞的凋亡率,这与Western blot结果提示两药联合协同增强了P53凋亡途径的调节有关。
2.LDM对体外模拟肿瘤微环境的影响及与TMZ联合效果
LDM单药(0.1 nmol/L,0.5 nmol/L,1 nmol/L)能够以剂量依赖方式显著抑制大鼠脑微血管内皮细胞的侵袭。LDM(0.1 nmol/L)与TMZ(200μmol/L)联合用药后,侵袭细胞数明显的减少,与单独用药组比较,P值均小于0.001,二者存在明显的协同作用,协同指数为0.68。蛋白印迹的结果表明LDM与TMZ联合抑制大鼠脑微血管内皮细胞的侵袭与其抑制VEGFR2活性及基质金属蛋白酶MMP2和MMP9的表达有关。以不同药物组合处理大鼠神经胶质瘤C6细胞和大鼠脑微血管内皮细胞rBMEC的共培养系统72小时后,与对照组比较,大鼠脑微血管内皮细胞的迁移受到了药物处理的显著抑制,联合处理组的迁移细胞数受到更为明显的抑制,与单独用药组比较,P值均小于0.001,联合指数为0.72,ELISA方法与蛋白印迹实验结果一致表明这与大鼠神经胶质瘤C6细胞条件培养基中VEGF的表达受到药物联合处理的明显抑制有关,也与大鼠脑微血管内皮细胞VEGFR的活性和神经胶质瘤C6细胞和U87细胞VEGF的表达受到协同抑制有关。
3.LDM对大鼠脑微血管内皮细胞和体外血管生成的影响及与TMZ联合效果
LDM对大鼠脑微血管内皮细胞的增殖有强烈的抑制作用,其IC_(50)值为1.06×10~(-11)mol/L,同时也存在着剂量依赖性诱导内皮细胞凋亡作用。LDM与TMZ联合后可增强细胞毒作用及诱导细胞凋亡作用。LDM可以阻断内皮细胞形成的小管,并随着剂量加大,破坏小管的能力越强。LDM 0.1 nmol/L与TMZ 200μmol/L联合处理内皮细胞,形成的管状结构要比LDM 0.1 nmol/L组少,P＜0.01,与TMZ 200μmol/L相比较,P＜0.001,二者的联合指数为0.75。LDM 0.1 nmol/L或TMZ 200μmol/L作用于大鼠动脉环后,都显示了明显的抑制作用,二者联合后能够更为明显的抑制动脉环发芽,与单独用药组比较P值均小于0.001,联合指数为0.8。
4.LDM与TMZ联合对人神经胶质瘤U87裸鼠移植瘤生长抑制
LDM 0.025mg/kg和0.05mg/kg能够显著抑制人神经胶质瘤U87裸鼠移植瘤的生长,TMZ也能够显著抑制肿瘤人神经胶质瘤的生长,与对照组相比较,P值均小于0.001。各药物处理组对动物体重都没有明显的影响。肿瘤生长曲线和瘤重量分析结果显示,LDM与TMZ单药能够产生抑制肿瘤生长的效果,LDM 0.025mg/kg和0.05mg/kg分别与TMZ 0.5mg/kg联合能够更为显著的抑制肿瘤的生长,联合指数小于1,表明二者能够协同抑制神经胶质瘤的生长。
经原位TUNEL检测,LDM单药能够诱导细胞凋亡,且随着剂量加大,凋亡率也增加。LDM与TMZ 0.5 mg/kg联合后可明显的增加凋亡率,与单独用药组比较P值均小于0.001,LDM 0.025mg/kg和0.05mg/kg与TMZ联合的协同指数分别为0.75和0.77,表明二者在体内协同诱导细胞凋亡。
CD31的免疫组织化学分析表明,LDM单药、TMZ单药及二者联合都能够降低人神经胶质瘤U87裸鼠移植瘤血管密度,与对照组相比较均存在显著性差异。二者联合能够更为显著的抑制肿瘤组织的血管生成,LDM 0.025mg/kg和0.05mg/kg分别与TMZ 0.5mg/kg联合后,其抑制肿瘤血管生成的协同指数分别为0.88和0.78,表明二者能够协同抑制肿瘤血管生成。同时,VEGF的免疫组织化学分析表明,LDM与TMZ联合对肿瘤组织中VEGF的表达也产生协同抑制作用,LDM 0.025mg/kg与TMZ0.5mg/kg的协同指数为0.93;LDM 0.05mg/kg与TMZ 0.5mg/kg抑制VEGF表达的协同指数为0.42。
本研究工作采用MTT法分析了药物对神经胶质瘤细胞和脑微血管内皮细胞增殖的影响,采用Annexin V-FITC/PI法检测了药物处理后细胞凋亡,同时,为了更全面的反映神经胶质瘤血管微环境,从肿瘤旁分泌网络考虑,采用共培养系统观察了药物对大鼠神经胶质瘤C6细胞和大鼠脑微血管内皮细胞相互作用的影响,包括细胞迁移和VEGF的分泌。以大鼠脑微血管内皮细胞的小管形成实验和大鼠动脉环实验作为体外血管生成的模型,来分析药物对血管生成的影响。体内实验中,在分析了力达霉素与替莫唑胺联合对肿瘤生长的影响,同时也分析了二者联合对细胞凋亡和微血管密度及VEGF表达的影响。我们的工作证明:力达霉素强烈抑制神经胶质瘤的细胞增殖和诱导神经胶质瘤细胞凋亡,并抑制血管生成,同时力达霉素可以与替莫唑胺联合作用,协同抑制神经胶质瘤的生长,增加细胞凋亡诱导及增加对VEGF表达的抑制。
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 cells
MTT 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 IC_(50) 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 vitro
LDM 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 IC_(50) 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.
引文
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