实验目的

本课题通过结构修饰合成22种灵芝酸A衍生物，利用体内外模型探讨其抗宫颈癌作用，并阐明其可能的作用机制，为发现副作用更低、更有效的抗宫颈癌药物提供科学依据。

实验方法

对灵芝酸A进行还原修饰、酰胺化修饰和酯化修饰合成灵芝酸A衍生物。采用CCK-8法进行抗肿瘤活性筛选，细胞克隆实验测定抗肿瘤活性最强的溴苯胺酯衍生物GaAD19对Hela细胞集落形成的影响，流式AnnexinV/ PI双染、Western blot、TUNEL和免疫组化检测细胞凋亡。

流式细胞术检测GaAD19对Hela细胞周期分布的影响，q-PCR检测GaAD19对Hela细胞周期调节蛋白mRNA水平的影响。

通过靶点预测分析和分子对接预测GaAD19作用于Hela细胞的信号通路，采用Western blot检测GaAD19对Hela细胞信号通路蛋白和通路上游基因MAP2K4和MAP2K7的表达的影响，q-PCR检测GaAD19对Hela细胞JNK通路膜蛋白受体表达的影响，加入JNK激动剂Anisomycin和抑制剂SP600125研究GaAD19对Hela细胞增殖、凋亡和周期的调节作用。

通过构建U14裸鼠移植瘤模型，研究GaAD19的体内抑瘤活性，Western blot检测肿瘤组织蛋白的表达，HE染色法对肝、肾、脾进行病理学检查，以明确GaAD19体内抗肿瘤作用的作用机制。

研究结果

合成22种灵芝酸A衍生物

A进行还原修饰、酰胺化修饰和酯化修饰合成22种灵芝酸A衍生物。

GaAD19诱导宫颈癌Hela细胞凋亡

结果显示，GaAD19对Hela细胞具有明显的抑制活性，对正常细胞系的抑制作用较弱。细胞克隆实验表明GaAD19对Hela细胞的克隆形成具有显著的抑制作用。在抗凋亡方面，GaAD19能够上调促凋亡蛋白Cleaved Caspase 9、Cleaved PARP-1和Bax的表达，下调抗凋亡蛋白Bcl-2的表达。流式细胞术、TUNEL和免疫组化的结果表明，GaAD19可以剂量依赖性的引起Hela细胞凋亡。

GaAD19诱导宫颈癌Hela细胞周期阻滞

流式细胞术结果显示，GaAD19阻断了细胞周期从G₁期向S期的转变。同时，q-PCR结果显示GaAD19能够显著下调Hela细胞周期调节蛋白CDK1，CDK2，CDK4，CDK6，CDK7，CDK8，ccne1，PCLAF和Ube2c的mRNA水平。

GaAD19抑制宫颈癌Hela细胞的JNK通路

通过靶点预测分析发现MAPK8是GaAD19作用于Hela细胞的一个核心基因。将GaAD19与JNK通路进行分子对接得到的最低结合能为-12.31 kcal/mol。Western blot结果表明，GaAD19剂量依赖性的阻止JNK通路及其上游基因的激活，对ERK，p38，AMPKα1-S485，AMPKα1-S496，AMPKβ1-S108和Akt通路蛋白的表达无显著差异。q-PCR结果显示，GaAD19主要作用于JNK通路生长因子、细胞应激和G_12/13偶联受体膜蛋白受体，下调了Crk，CrkL，Shc，GRB2，Sos，Ras，Rac，cdc42，Gα12和Gβ的mRNA水平。加入JNK激动剂Anisomycin和抑制剂SP600125后可以分别减弱和增强GaAD19对Hela细胞增殖、凋亡和周期的影响。

GaAD19抑制U14裸鼠异体移植瘤的生长

体内实验结果显示，GaAD19对U14裸鼠异体移植瘤具有良好的抑制作用，而且对裸鼠体重以及肝脏、肾脏、脾脏等没有明显的毒性作用。此外，GaAD19明显减少肿瘤组织中p-JNK蛋白的表达，与体外实验及结果一致。

结论

GaAD19能够显著抑制Hela细胞的增殖，促进Hela细胞凋亡，以及诱导Hela细胞的G₁/S期阻滞。此外，GaAD19能够显著抑制U14裸鼠异体移植瘤的生长。其机制均与抑制JNK信号通路相关。

Aim:

In this study, we synthesized 22 kinds of ganoderic acid A derivatives through structural modification, explored their anti-cervical cancer effects by using in vitro and in vivo models, and clarifies its possible mechanism, so as to provide scientific basis for the discovery of more effective anti-cervical cancer drugs with lower side effects.

Methods:

Through reduction modification, amidation modification and esterification modification, ganoderic acid A derivatives were synthesized. The anti-tumor activity was screened by CCK-8 method. The effect of GaAD19, a bromoaniline ester derivative with the strongest anti-tumor activity, on Hela cell colony formation was determined by cell cloning assay. Apoptosis was detected by AnnexinV/ PI staining, Western blot, TUNEL and immunohistochemistry.

The effect of GaAD19 on the cell cycle distribution of Hela cells was detected by flow cytometry, and the effect of GaAD19 on the mRNA level of Hela cell cycle regulator was detected by q-PCR.

Target prediction analysis and molecular docking were used to predict the signal pathway of Hela cells acted by GaAD19, and Western blot was used to detect the effects of GaAD19 on the expression of signal pathway proteins and upstream genes MAP2K4 and MAP2K7 of Hela cells. q-PCR was used to detect the effects of GaAD19 on the expression of JNK pathway membrane protein receptors in Hela cells. JNK agonist Anisomycin and inhibitor SP600125 were added to study the regulating effects of GaAD19 on proliferation, apoptosis and cell cycle of Hela cells.

The in vivo antitumor activity of GaAD19 was studied through constructing U14 transplanted tumor model in nude mouse. The expression of tumor tissue protein was detected by Western blot. Liver, kidney and spleen were examined by HE staining to clarify the mechanism of anti-tumor effect of GaAD19 in vivo.

Results:

Synthesize 22 ganoderic acid A derivatives

Through reduction modification, amidation modification and esterification modification, 22 ganoderic acid A derivatives were synthesized.

GaAD19 induced apoptosis of cervical cancer Hela cells

The results of CCK-8 showed that GaAD19 had obvious inhibitory activity on Hela cells, but had weak inhibitory effect on normal cell lines. Cell cloning assay showed that GaAD19 had significant inhibitory effect on the clonal formation of Hela cells. In terms of anti-apoptosis, GaAD19 could up-regulate the expression of pro-apoptotic proteins, Cleaved Caspase 9, Cleaved PARP-1 and Bax, and down-regulate the expression of anti-apoptotic protein Bcl-2. The results of flow cytometry, TUNEL and immunohistochemistry showed that GaAD19 induced Hela cell apoptosis in a dose-dependent manner.

GaAD19 induced cell cycle arrest of cervical cancer Hela cells

Flow cytometry showed that GaAD19 blocked the cell cycle transition from G₁ phase to S phase. Meanwhile, q-PCR results showed that GaAD19 could significantly down-regulate the mRNA levels of Hela cell cycle regulatory proteins CDK1, CDK2, CDK4, CDK6, CDK7, CDK8, ccne1, PCLAF and Ube2c.

GaAD19 inhibited the JNK pathway in cervical cancer Hela cells

Through target prediction analysis, it was found that MAPK8 is a hub gene of GaAD19 acting on Hela cells. The lowest binding energy obtained by molecular docking of GaAD19 with JNK pathway is -12.31 kcal/mol. Western blot results showed that the GaAD19 dose-dependent inhibited the activation of JNK pathway and its upstream genes, but had no significant differences in the expression of ERK, p38, AMPKα1-S485, AMPKα1-S496, AMPKβ1-S108 and Akt pathway proteins. q-PCR results showed that GaAD19 mainly acted on JNK pathway growth factors, cell stress and G_12/13-coupled receptors membrane protein receptors, down-regulating mRNA levels of Crk, CrkL, Shc, GRB2, Sos, Ras, Rac, cdc42, Gα12 and Gβ. The addition of JNK agonist Anisomycin and inhibitor SP600125 could weaken and enhance the effects of GaAD19 on Hela cell proliferation, apoptosis and cell cycle, respectively.

GaAD19 inhibited the growth of U14 xenografts in nude mouse

The results of in vivo experiments showed that GaAD19 had a good inhibitory effect on U14 xenografts in nude mouse, and had no obvious toxic effect on body weight, liver, kidney and spleen of nude mouse. In addition, GaAD19 significantly reduced the expression of p-JNK protein in tumor tissues, which was consistent with the results of in vitro experiments.

Conclusions:

GaAD19 can significantly inhibit the proliferation of Hela cells, promote apoptosis of Hela cells, and induce the arrest of G₁/S phase of Hela cells. In addition, GaAD19 significantly inhibited the growth of U14 xenografts in nude mouse. The mechanism is related to the inhibition of JNK signal pathway.