PSD-007光动力疗法对荷人鼻咽癌裸鼠移植瘤的抑制作用及机制研究

Inhibitory effect and mechanism of PSD-007 photodynamic therapy on human nasopharyngeal carcinoma transplanted tumors in nude mice

目的探讨癌光啉(PSD-007)光动力疗法(PDT)对荷人鼻咽癌裸鼠移植瘤的抑制作用及机制。方法建立50只荷人鼻咽癌裸鼠移植瘤模型,采用随机数字表法随机分为5组:对照组(A组)、单纯PSD-007组(B组)、单纯光照组(C组)、局部注射PSD-007+光照组(D组)、腹腔注射PSD-007+光照组(E组),每组10只。治疗7 d后,测量各组裸鼠瘤体质量、肿瘤体积并计算肿瘤抑制率;HE染色检测各组裸鼠肿瘤的组织病理学变化;蛋白质印迹法检测肿瘤组织自噬相关基因Beclin 1、微管相关蛋白1轻链3-β(LC3)和磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路相关蛋白表达;实时荧光定量PCR检测肿瘤组织LC3、Beclin 1 mRNA表达。结果治疗结束后,A组、B组、C组、D组和E组荷人鼻咽癌裸鼠瘤体质量分别为(2.05±0.18)g、(2.02±0.20)g、(2.04±0.15)g、(0.43±0.11)g、(0.94±0.12)g,肿瘤体积分别为(1.11±0.13)cm3、(1.18±0.16)cm3、(1.13±0.14)cm3、(0.51±0.07)cm3、(0.65±0.10)cm3,5组间差异均具有统计学意义(F=236.749,P<0.001;F=62.418,P<0.001);与A组、B组和C组相比,D组和E组裸鼠瘤体质量、肿瘤体积均显著降低,差异均具有统计学意义(均P<0.001);与E组相比,D组裸鼠瘤体质量、肿瘤体积均显著降低(P<0.001;P=0.023)。B组、C组、D组、E组的肿瘤抑制率分别为(1.07±0.11)%、(0.55±0.06)%、(79.11±0.06)%、(54.05±0.08)%,4组间差异有统计学意义(F=235.987,P<0.001),与B组、C组和E组相比,D组抑瘤效果最明显(均P<0.05)。HE染色结果表明,与A组、B组和C组相比,D组和E组均出现较大肿瘤坏死区,坏死区周围可见大量炎性细胞浸润,残留肿瘤细胞出现空泡样变性,胞核固缩明显;D组肿瘤坏死程度高于E组。5组裸鼠瘤体中PI3K蛋白相对表达量分别为1.01±0.06、1.00±0.05、1.01±0.05、0.23±0.02、0.48±0.04,p-Akt/Akt蛋白相对表达量分别为0.66±0.06、0.65±0.05、0.64±0.05、0.06±0.02、0.17±0.02,p-mTOR/mTOR蛋白相对表达量分别为1.06±0.06、1.01±0.06、1.01±0.06、0.30±0.02、0.45±0.04,LC3Ⅱ/LC3Ⅰ蛋白相对表达量比值分别为0.85±0.05、0.83±0.05、0.83±0.06、0.22±0.02、0.41±0.04,Beclin 1蛋白相对表达量分别为0.66±0.06、0.64±0.05、0.64±0.06、1.67±0.07、1.02±0.05,LC3 mRNA相对表达量分别为0.98±0.29、0.92±0.25、1.02±0.26、3.76±0.28、2.38±0.28,Beclin 1 mRNA相对表达量分别为1.11±0.40、1.19±0.29、1.16±0.24、6.84±0.54、2.94±0.48,差异均具有统计学意义(F=190.160,P<0.001;F=160.014,P<0.001;F=160.183,P<0.001;F=119.964,P<0.001;F=186.257,P<0.001;F=211.089,P<0.001;F=374.835,P<0.001),与A组、B组和C组相比,D组和E组裸鼠瘤体中PI3K、p-Akt/Akt和p-mTOR/mTOR蛋白相对表达量及LC3Ⅱ/LC3Ⅰ蛋白相对表达量比值均显著降低,LC3 mRNA相对表达量、Beclin 1蛋白及mRNA相对表达量均显著升高(均P<0.001);与D组相比,E组裸鼠瘤体中PI3K、p-Akt/Akt和p-mTOR/mTOR蛋白相对表达量及LC3Ⅱ/LC3Ⅰ蛋白相对表达量比值均显著升高,而Beclin 1蛋白相对表达量及LC3、Beclin 1 mRNA相对表达量均降低(均P<0.05)。结论PSD-007 PDT对荷人鼻咽癌裸鼠移植瘤具有抑制作用,可能与PSD-007 PDT抑制PI3K/Akt/mTOR信号通路的活化及促进自噬有关;局部注射PSD-007 PDT效果更显著。

Objective To explore the inhibitory effect and mechanism of photocarcinorin(PSD-007)photodynamic therapy(PDT)on human nasopharyngeal carcinoma transplanted tumors in nude mice.Methods A total of 50 transplanted tumor nude mice models of human nasopharyngeal carcinoma were established and randomly divided into 5 groups:control group(group A),simple PSD-007 group(group B),simple light group(group C),local injection of PSD-007+light group(group D),intraperitoneal injection of PSD-007+light group(group E)using the method of random number table,10 mice in each group.After 7 days of treatment,the tumor mass and tumor volume of nude mice in each group were measured and the tumor inhibition rate was calculated.HE staining was used to detect the histopathological changes of tumors in nude mice.Western blotting was used to detect the expressions of autophagy-related gene Beclin 1,microtubules associated protein 1 light chain 3-β(LC3)and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway related proteins.Real-time fluorescent quantitative PCR was used to detect LC3,Beclin 1 mRNA expressions in tumor tissues.Results After treatment,the tumor mass of nude mice bearing human nasopharyngeal carcinoma in group A,B,C,D and E were(2.05±0.18)g,(2.02±0.20)g,(2.04±0.15)g,(0.43±0.11)g and(0.94±0.12)g,and the tumor volumes were(1.11±0.13)cm3,(1.18±0.16)cm3,(1.13±0.14)cm3,(0.51±0.07)cm3and(0.65±0.10)cm3,and there were statistically significant differences among the 5 groups(F=236.749,P<0.001;F=62.418,P<0.001).Compared with group A,B and C,the tumor mass and tumor volumes of nude mice in group D and E were significantly reduced,and there were statistically significant differences(all P<0.001);compared with group E,the tumor mass and tumor volume of nude mice in group D were significantly reduced(P<0.001;P=0.023).The tumor inhibition rates of group B,C,D and E were(1.07±0.11)%,(0.55±0.06)%,(79.11±0.06)%and(54.05±0.08)%,with a statistically significant difference(F=235.987,P<0.001),compared with group B,C and E,group D had the most obvious tumor suppressing effect(all P<0.05).HE staining results showed that compared with group A,B and C,group D and E had larger tumor necrosis areas,more inflammatory cell infiltration,more vacuolar degeneration,and obvious nuclear shrinkage.The tumor necrosis degree in group D was higher than that in group E.The relative expressions of PI3K protein of group A,B,C,D and E were 1.01±0.06,1.00±0.05,1.01±0.05,0.23±0.02,0.48±0.04,p-Akt/Akt protein relative expressions were 0.66±0.06,0.65±0.05,0.64±0.05,0.06±0.02,0.17±0.02,p-mTOR/mTOR protein relative expressions were 1.06±0.06,1.01±0.06,1.01±0.06,0.30±0.02,0.45±0.04.The protein relative expression ratios of LC3Ⅱ/LC3Ⅰwere 0.85±0.05,0.83±0.05,0.83±0.06,0.22±0.02,0.41±0.04,and Beclin 1 protein relative expressions were 0.66±0.06,0.64±0.05,0.64±0.06,1.67±0.07,1.02±0.05,LC3 mRNA relative expressions were 0.98±0.29,0.92±0.25,1.02±0.26,3.76±0.28,2.38±0.28,and Beclin 1 mRNA relative expressions of were 1.11±0.40,1.19±0.29,1.16±0.24,6.84±0.54,2.94±0.48.There were statistically significant differences(F=190.160,P<0.001;F=160.014,P<0.001;F=160.183,P<0.001;F=119.964,P<0.001;F=186.257,P<0.001;F=211.089,P<0.001;F=374.835,P<0.001).Compared with group A,B and C,PI3K,p-Akt/Akt and p-mTOR/mTOR protein relative expression levels and protein relative expression ratios of LC3Ⅱ/LC3Ⅰin nude mice tumors of group D and E were significantly reduced,and LC3 mRNA,Beclin 1 protein and mRNA relative expression levels were significantly increased,with statistically significant differences(all P<0.001);PI3K,p-Akt/Akt and p-mTOR/mTOR protein relative expression levels and protein relative expression ratio of LC3Ⅱ/LC3Ⅰin nude mice tumors of group E were significantly higher than those of group D,while Beclin 1 protein relative expression levels,LC3 and Beclin 1 mRNA relative expression levels were reduced(all P<0.05).Conclusion PSD-007 PDT has an inhibitory effect on human nasopharyngeal carcinoma transplanted tumors in nude mice,which may be related to the inhibition of PI3K/Akt/mTOR signaling pathway activation and the promotion of autophagy.Compared with intraperitoneal injection,local injection of PSD-007 PDT is more effective.