[Everolimus inhibits malignant phenotype of erdafitinib-resistant bladder cancer cells through the p70S6K/PI3K/MAPK signaling pathway].

内容摘要

To investigate the regulatory effect of everolimus on biological behaviors of rdafitinib-resistant bladder cancer cells and the underlying mechanism. An erdafitinib-resistant RT-112-RS cell model was established by continuous induction of RT-112 cells with erdafitinib at the optimized concentration (5 &#x3bc;mol/L) for 16 weeks. The resistant cells were treated with PDGF-BB alone or in combination with 0.01, 0.1, or 1 &#x3bc;mol/L everolimus, and the cytotoxicity (IC50) of everolimus was determined with CCK-8 assay. The changes in cell migration and invasion were evaluated using scratch wound healing assay and Transwell assays. The expressions of the genes of interest in the cells treated with everolimus (0.001- 1 &#x3bc;mol/L) were detected using RT-qPCR, and cell apoptosis was analyzed with flow cytometry. Western blotting was performed to assess phosphorylation levels of the key proteins in the mTORC1/p70S6K, PI3K/Akt, and Raf1/MEK/ERK pathways. Everolimus within the concentration range of 0.001-0.1 &#x3bc;mol/L dose-dependently inhibited RT-112-RS cell proliferation. RT-112-RS cells showed significantly enhanced migration and invasion compared with their parental RT-112 cells. Treatment with 1 &#x3bc;mol/L everolimus significantly suppressed p70S6K phosphorylation but did not result in an enhanced inhibition of cell proliferation effect. Everolimus at 1 &#x3bc;mol/L significantly activated the Raf1/MEK/ERK pathway without affecting PI3K/Akt (Thr308) or mTORC2/Akt (Ser473) phosphorylation. Treatment of the cells with the MEK inhibitor U-0126 obviously reversed everolimus-induced ERK1/2 activation, while the PI3K inhibitor LY294002 only partially reduced ERK1/2 activation in everolimus-treated cells. Everolimus inhibits RT-112-RS cell proliferation by suppressing p70S6K activity, and at high concentrations, everolimus can result in activation of the PI3K-Raf1/MEK/ERK pathway possibly by relieving negative feedback inhibition to partially offset its inhibitory effect on cell proliferation. &#x76ee;&#x7684;: &#x63a2;&#x8ba8;&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x901a;&#x8fc7;&#x8c03;&#x63a7;p70S6K/PI3K/MAPK&#x4fe1;&#x53f7;&#x901a;&#x8def;&#x5bf9;&#x8180;&#x80f1;&#x764c;&#x8010;&#x836f;&#x7ec6;&#x80de;RT-112-RS&#x751f;&#x7269;&#x5b66;&#x884c;&#x4e3a;&#x7684;&#x5f71;&#x54cd;&#x3002;&#x65b9;&#x6cd5;: &#x91c7;&#x7528;&#x68af;&#x5ea6;&#x6cd5;&#x7b5b;&#x9009;&#x5384;&#x8fbe;&#x66ff;&#x5c3c;&#x6d53;&#x5ea6;&#xff0c;&#x9009;&#x53d6;5 &#x3bc;mol/L&#x4f5c;&#x4e3a;&#x7ec8;&#x6d53;&#x5ea6;&#x5e76;&#x6301;&#x7eed;&#x8bf1;&#x5bfc;16&#x5468;&#x4ee5;&#x5efa;&#x7acb;&#x5384;&#x8fbe;&#x66ff;&#x5c3c;&#x8010;&#x836f;&#x7684;RT-112-RS&#x7ec6;&#x80de;&#x6a21;&#x578b;&#x3002;&#x8bbe;&#x5bf9;&#x7167;&#x7ec4;&#xff08;RT-112-RS&#x5e38;&#x89c4;&#x57f9;&#x517b;&#xff09;&#x3001;PDGF&#x7ec4;&#xff08;+PDGF-BB&#xff09;&#x3001;PDGF+0.01 &#x3bc;mol/L&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x7ec4;&#x3001;PDGF+0.1 &#x3bc;mol/L&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x7ec4;&#x53ca;PDGF+1 &#x3bc;mol/L&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x7ec4;&#x3002;CCK-8&#x6cd5;&#x6d4b;&#x5b9a;&#x7ec6;&#x80de;&#x6bd2;&#x6027;&#x5e76;&#x8ba1;&#x7b97;IC50;&#x5212;&#x75d5;&#x548c;Transwell&#x5b9e;&#x9a8c;&#x8bc4;&#x4f30;&#x8fc1;&#x79fb;&#x4e0e;&#x4fb5;&#x88ad;&#x80fd;&#x529b;;RT-qPCR&#x68c0;&#x6d4b;0.001~1 &#x3bc;mol/L&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x5904;&#x7406;&#x4e0b;&#x76f8;&#x5173;&#x57fa;&#x56e0;&#x8868;&#x8fbe;;&#x6d41;&#x5f0f;&#x7ec6;&#x80de;&#x672f;&#x5206;&#x6790;&#x51cb;&#x4ea1;&#x7387;;Western blotting&#x68c0;&#x6d4b;mTORC1/p70S6K&#x3001;PI3K/Akt&#x53ca;Raf1/MEK/ERK&#x901a;&#x8def;&#x5173;&#x952e;&#x86cb;&#x767d;&#x78f7;&#x9178;&#x5316;&#x6c34;&#x5e73;&#x3002;&#x7ed3;&#x679c;: &#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x5728;0.001~0.1 &#x3bc;mol/L&#x8303;&#x56f4;&#x5185;&#x5448;&#x5242;&#x91cf;&#x4f9d;&#x8d56;&#x6027;&#x6291;&#x5236;RT-112-RS&#x589e;&#x6b96;&#xff08;P<0.05&#xff09;&#x3002;&#x76f8;&#x6bd4;RT-112&#x7ec6;&#x80de;&#xff0c;RT-112-RS&#x8fc1;&#x79fb;&#x4e0e;&#x4fb5;&#x88ad;&#x80fd;&#x529b;&#x589e;&#x5f3a;&#xff08;819.83&#xb1;71.39 vs 1579&#xb1;136.30&#xff0c;P<0.05&#xff09;&#x3002;1 &#x3bc;mol/L&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x663e;&#x8457;&#x6291;&#x5236;p70S6K&#x78f7;&#x9178;&#x5316;&#xff08;P<0.05&#xff09;&#xff0c;&#x4f46;&#x672a;&#x8fdb;&#x4e00;&#x6b65;&#x589e;&#x5f3a;&#x6297;&#x589e;&#x6b96;&#x6548;&#x5e94;&#xff0c;&#x5e76;&#x7279;&#x5f02;&#x6027;&#x6fc0;&#x6d3b;Raf1/MEK/ERK&#x901a;&#x8def;&#xff0c;&#x5bf9;PI3K/Akt&#xff08;Thr308&#xff09;&#x6216;mTORC2/Akt&#xff08;Ser473&#xff09;&#x65e0;&#x660e;&#x663e;&#x5f71;&#x54cd;&#x3002;MEK&#x6291;&#x5236;&#x5242;U-0126&#x53ef;&#x9006;&#x8f6c;&#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x8bf1;&#x5bfc;&#x7684;ERK1/2&#x6d3b;&#x5316;&#xff08;5.52&#xb1;0.40 vs 0.74&#xb1;0.07&#xff0c;P<0.05&#xff09;&#xff0c;PI3K&#x6291;&#x5236;&#x5242;LY294002&#x5219;&#x51cf;&#x5f31;ERK1/2&#x6fc0;&#x6d3b;&#xff08;7.58&#xb1;1.54 vs 11.2&#xb1;0.24&#xff0c;P<0.05&#xff09;&#x3002;&#x7ed3;&#x8bba;: &#x4f9d;&#x7ef4;&#x83ab;&#x53f8;&#x901a;&#x8fc7;&#x6291;&#x5236;p70S6K&#x6d3b;&#x6027;&#x6291;&#x5236;RT-112-RS&#x589e;&#x6b96;&#x3002;&#x800c;&#x9ad8;&#x6d53;&#x5ea6;&#x65f6;&#x53ef;&#x80fd;&#x56e0;&#x51cf;&#x5f31;&#x8d1f;&#x53cd;&#x9988;&#x6291;&#x5236;&#x800c;&#x6fc0;&#x6d3b;PI3K-Raf1/MEK/ERK&#x901a;&#x8def;&#xff0c;&#x4ece;&#x800c;&#x90e8;&#x5206;&#x62b5;&#x6d88;&#x5176;&#x6297;&#x589e;&#x6b96;&#x4f5c;&#x7528;&#x3002;.