在Wnt訊息路徑中,β-catenin為訊息傳遞路徑中重要的調節蛋白,其穩定性與入核現象被視為路徑活化的關鍵。Wnt訊息路徑所調控下游基因之表現與細胞存活、細胞侵襲和細胞抗藥性有密切關係。本實驗將探討在肺癌細胞是否藉由小孢子靈芝免疫調節蛋白(Fungal immunomodulatory protein- Ganoderma microsporum,GMI) 抑制Wnt訊息路徑之β-catenin表現量。經西方墨點法發現,GMI可有效抑制β-catenin蛋白表現量,並由核質分離法可發現GMI會抑制H1355細胞核和與細胞質的β-catenin蛋白量。以TOPFLASH/FOPFLASH reporter assay,GMI具有抑制β-catenin/Tcf 轉錄活性。RT-PCR與Real-time PCR結果發現,GMI具降低β-catenin下游基因cyclin-D1與survivin表現。透過CCK-8方法與流式細胞儀偵測GMI抑制肺癌細胞之細胞存活現象並促使細胞死亡。以Wnt3a刺激細胞可增加β-catenin蛋白而減少cleaved caspase 7,反之,將β-catenin基因關閉則可增加cleaved caspase 7表現。分別以MG132或LLnL (proteasome inhibitor) 或SB216763 (p-GSK3β抑制劑)處理細胞可抑制 GMI將β-catenin降解。在非小細胞肺癌中,具EGFR突變細胞具有高表現的β-catenin蛋白量。在EGFR雙重突變L858R/T790M細胞H1975對於GMI敏感,GMI可有效減少β-catenin,而以ICG-001 (Wnt/β-catenin/TCF 基因轉錄抑制劑,且專一結合於element-binding protein (CBP)) 可增加細胞死亡。本研究結果顯示,GMI可抑制β-catenin的表現進而抑制Wnt/β-catenin路徑與癌細胞增生,在EGFR雙重L858R/T790M突變細胞H1975也具有明顯的抑制效果,因此未來將進一步分析GMI抑制β-catenin後的下游基因表現與癌症增生的關係。
β-catenin play a central role in Wnt signaling pathway. Aberrant activation of Wnt/β-catenin signaling downstream genes, such as survivin, c-myc, cyclin D involved in cell resistant, cell survival and cell invasion. In this study, we investigated the effects of GMI, a recombinant fungal immunomodulatory protein from Ganoderma microsporum on β-catenin/Tcf signaling, apoptosis induction and β-catenin/Tcf transcription activity in lung cancer. Lung cancer cells were treated with GMI for cell viability and cell apoptosis measured by CCK-8 assay and flow cytometry analysis. Nuclear and cytoplasmic β-catenin protein level were inhibited by GMI analyzed on Western blot. In addition, proteasome inhibitors MG132 and LLnL or p-GSK3β inhibitors SB216763 prevented the degradation of β-catenin mediated by GMI. Stimulation of Wnt3a induced β-catenin and reduced cleaved caspase 7. Knockdown of β-catenin gene expression elevated cleaved caspase 7. GMI decreased β-catenin/Tcf transcription activity by TOPFLASH/FOPFLASH reporter assay. Survivin and cyclin D1 have been down-regulated by GMI on RT-PCR and real time PCR. Furthermore, NSCLC patients with epidermal growth factor receptor gene mutant had high expression of β-catenin. Our findings indicate that GMI can decrease β-catenin expression and induces cell death in H1975 (EGFR L858R/T790M mutate) lung cancer cells. GMI might be therapeutic agent targeting to EGFR-double mutation H1975 lung cancer cells. In conclusion, our data demonstrated that GMI reduced β-catenin and inhibits the Wnt signaling pathway in non-small cell lung cancer (NSCLC).
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