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Abstract：
OBJECTIVE: To investigate the effects of Lathyrol on the expression of androgen receptor (AR) and sphingosine kinase 2 (SPHK2) in renal cell carcinoma (RCC) mice and to further explore the mechanism by which Lathyrol inhibits the invasion and incidence of epithelial-mesenchymal transition (EMT).
METHODS: An RCC xenograft mouse model was constructed, and the mice were randomly divided into a model group, an experiment group and a negative control group. The experiment group was intragastrically gavaged with Lathyrol solution (20 mg/kg), the model group was intragastrically gavaged with 0.9% NaCl (same volume as that used in the experiment group), and the negative control group was injected intraperitoneally with 2 mg/kg cisplatin aqueous solution. Changes in the body weight and tumor volume of the mice were recorded. Western blot (WB) was used to assess the protein expression levels of AR, p-AR, CYP17A1, PARP1, E-cadherin, N-cadherin, vimentin, α-SMA, β-catenin, and ZO-1. Protein expression levels of SPHK2, metal matrix protease 2 (MMP2), MMP9 and urokinase-type plasminogen activator (uPA) in tumor tissues were assessed by immunohistochemistry (IHC). AR expression in tumor tissues was assessed after immunofluorescence (IF) staining.
RESULTS: After 14 days of drug administration, compared with that in the model group, the tumor volumes in the negative control and experiment groups were lower; the difference in tumor volume among the model, control and experiment groups was statistically significant (P < 0.05). The differences in body weight among the three groups were not statistically significant (P > 0.05). In the model group, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively increased, the protein expression levels of E-cadherin and ZO-1 were relatively reduced (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin, and α-SMA were relatively increased (P < 0.05). In the negative control and experiment groups, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively decreased (P < 0.05), the protein expression levels of E-cadherin and ZO-1 were relatively increased (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA were relatively decreased (P < 0.05).
CONCLUSIONS: Lathyrol and cisplatin inhibit the proliferation of RCC xenografts, reduce the protein expression levels of AR, CYP17A1, SPHK2, PARP1, E-cadherin, and ZO-1 in tumor tissues (P < 0.05), and promote the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA (P < 0.05). Therefore, Lathyrol reduces RCC invasion and EMT by affecting the expression of AR and SPHK2 in RCC mice.
METHODS: An RCC xenograft mouse model was constructed, and the mice were randomly divided into a model group, an experiment group and a negative control group. The experiment group was intragastrically gavaged with Lathyrol solution (20 mg/kg), the model group was intragastrically gavaged with 0.9% NaCl (same volume as that used in the experiment group), and the negative control group was injected intraperitoneally with 2 mg/kg cisplatin aqueous solution. Changes in the body weight and tumor volume of the mice were recorded. Western blot (WB) was used to assess the protein expression levels of AR, p-AR, CYP17A1, PARP1, E-cadherin, N-cadherin, vimentin, α-SMA, β-catenin, and ZO-1. Protein expression levels of SPHK2, metal matrix protease 2 (MMP2), MMP9 and urokinase-type plasminogen activator (uPA) in tumor tissues were assessed by immunohistochemistry (IHC). AR expression in tumor tissues was assessed after immunofluorescence (IF) staining.
RESULTS: After 14 days of drug administration, compared with that in the model group, the tumor volumes in the negative control and experiment groups were lower; the difference in tumor volume among the model, control and experiment groups was statistically significant (P < 0.05). The differences in body weight among the three groups were not statistically significant (P > 0.05). In the model group, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively increased, the protein expression levels of E-cadherin and ZO-1 were relatively reduced (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin, and α-SMA were relatively increased (P < 0.05). In the negative control and experiment groups, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively decreased (P < 0.05), the protein expression levels of E-cadherin and ZO-1 were relatively increased (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA were relatively decreased (P < 0.05).
CONCLUSIONS: Lathyrol and cisplatin inhibit the proliferation of RCC xenografts, reduce the protein expression levels of AR, CYP17A1, SPHK2, PARP1, E-cadherin, and ZO-1 in tumor tissues (P < 0.05), and promote the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA (P < 0.05). Therefore, Lathyrol reduces RCC invasion and EMT by affecting the expression of AR and SPHK2 in RCC mice.
摘要：
目的：观察Lathyrol对肾细胞癌（RCC）小鼠雄激素受体（AR）和鞘氨醇激酶2（SPHK2）表达的影响，进一步探讨Lathyrol抑制上皮间质转化（EMT）侵袭和发生的机制。
方法：构建RCC异种移植小鼠模型，将小鼠随机分为模型组,实验组和阴性对照组。实验组用Lathyrol溶液（20mg/kg）胃内灌胃，模型组灌胃0.9%NaCl(与实验组相同体积),阴性对照组腹腔注射2mg/kg顺铂水溶液。记录小鼠体重和肿瘤体积的变化。Westernblot（WB）用于评估AR的蛋白表达水平，p-AR,CYP17A1,PARP1,E-cadherin,N-钙黏着蛋白,波形蛋白,α-SMA,β-连环蛋白，ZO-1SPHK2，金属基质蛋白酶2（MMP2）的蛋白质表达水平，通过免疫组织化学（IHC）评估肿瘤组织中的MMP9和尿激酶型纤溶酶原激活物（uPA）。免疫荧光（IF）染色后评估肿瘤组织中的AR表达。
结果：给药14天后，与模型组相比，阴性对照组和实验组的肿瘤体积较低；模型之间的肿瘤体积差异，对照组与实验组比较差异有统计学意义(P<0.05)。三组间体重差异无统计学意义(P>0.05)。在模型组中，AR的蛋白质表达水平，p-AR,CYP17A1、SPHK2和PARP1相对升高，E-cadherin和ZO-1蛋白表达水平相对降低(P<0.05),和N-钙黏着蛋白的表达水平，β-连环蛋白，波形蛋白,α-SMA相对升高(P<0.05)。在阴性对照组和实验组中，AR的蛋白质表达水平，p-AR,CYP17A1、SPHK2和PARP1相对降低(P<0.05),E-cadherin和ZO-1蛋白表达水平相对升高(P<0.05),和N-钙黏着蛋白的表达水平，β-连环蛋白，波形蛋白和α-SMA相对降低(P<0.05)。
结论：Lathyrol和顺铂抑制肾癌移植瘤的增殖，降低AR的蛋白质表达水平，CYP17A1,SPHK2,PARP1,E-cadherin,ZO-1在肿瘤组织中的表达(P<0.05),并促进N-cadherin的蛋白质表达水平，β-连环蛋白，波形蛋白和α-SMA(P<0.05)。因此,Lathyrol通过影响RCC小鼠AR和SPHK2的表达来降低RCC侵袭和EMT。
方法：构建RCC异种移植小鼠模型，将小鼠随机分为模型组,实验组和阴性对照组。实验组用Lathyrol溶液（20mg/kg）胃内灌胃，模型组灌胃0.9%NaCl(与实验组相同体积),阴性对照组腹腔注射2mg/kg顺铂水溶液。记录小鼠体重和肿瘤体积的变化。Westernblot（WB）用于评估AR的蛋白表达水平，p-AR,CYP17A1,PARP1,E-cadherin,N-钙黏着蛋白,波形蛋白,α-SMA,β-连环蛋白，ZO-1SPHK2，金属基质蛋白酶2（MMP2）的蛋白质表达水平，通过免疫组织化学（IHC）评估肿瘤组织中的MMP9和尿激酶型纤溶酶原激活物（uPA）。免疫荧光（IF）染色后评估肿瘤组织中的AR表达。
结果：给药14天后，与模型组相比，阴性对照组和实验组的肿瘤体积较低；模型之间的肿瘤体积差异，对照组与实验组比较差异有统计学意义(P<0.05)。三组间体重差异无统计学意义(P>0.05)。在模型组中，AR的蛋白质表达水平，p-AR,CYP17A1、SPHK2和PARP1相对升高，E-cadherin和ZO-1蛋白表达水平相对降低(P<0.05),和N-钙黏着蛋白的表达水平，β-连环蛋白，波形蛋白,α-SMA相对升高(P<0.05)。在阴性对照组和实验组中，AR的蛋白质表达水平，p-AR,CYP17A1、SPHK2和PARP1相对降低(P<0.05),E-cadherin和ZO-1蛋白表达水平相对升高(P<0.05),和N-钙黏着蛋白的表达水平，β-连环蛋白，波形蛋白和α-SMA相对降低(P<0.05)。
结论：Lathyrol和顺铂抑制肾癌移植瘤的增殖，降低AR的蛋白质表达水平，CYP17A1,SPHK2,PARP1,E-cadherin,ZO-1在肿瘤组织中的表达(P<0.05),并促进N-cadherin的蛋白质表达水平，β-连环蛋白，波形蛋白和α-SMA(P<0.05)。因此,Lathyrol通过影响RCC小鼠AR和SPHK2的表达来降低RCC侵袭和EMT。
