長期胰島素處理對非小細胞肺癌細胞株產生吉非替尼（Gefitinib）抗藥性之影響

Abstract

肺癌是目前全世界主要致死癌症之一，而其中有超過90％的肺癌患者是屬於非小細胞肺癌（non-small cell lung cancer，NSCLC）。與歐美族群不同，亞洲人種的NSCLC主要是由EGFR（epidemal growth factor receptor）基因的突變造成。 在Tyrosine kinase inhibitor (TKI)類的用藥中，Gefitinib是當前治療肺癌的臨床第一線標靶用藥。然而，許多病患在治療一至兩年內會出現抗藥性。Gefitinib抗藥性形成的可能原因多樣，其中類胰島素生長因子受器（insulin-like growth factor-I receptor，IGF1R）路徑的活化是TKI治療法產生抗藥性的一個重要因素，其機制尚未完全明瞭，而許多的臨床病例也指出第二型糖尿病（Type 2 diabetes mellitus）與NSCLC的致死率高度相關，此可能是高胰島素血及高血糖可能在Gefitinib抗藥性的病程發展中扮演重要的角色。 為進一步探討高胰島素血誘發的Gefitinib抗藥性之分子機制，我們以HCC827及HCC4006兩株NSCLC細胞株建立細胞模式之離體試驗。其中以MTS細胞活性分析檢測得知兩者皆對Gefitinib具有敏感性，而加入Gefitinib培養24小時後，HCC827及HCC4006之IC50濃度分別為4.43 μM與39.42 μM。將長效型胰島素（Glargine）及Gefitinib同時加入培養基後，HCC4006細胞隨著Glargine劑量上升，細胞存活率回升，亦即Glargine誘發了細胞對Gefitinib之抗藥性。在西方點墨法檢測結果中，可見在處理Gefitinib並同時加入Glargine會增加IGFR及AKT磷酸化比例；相反的ERK磷酸化表現量卻未增加，由此可推斷胰島素可能會通過PI3K/AKT之路徑誘發Gefitinib抗藥性產生，並使得細胞存活率增加，而不是透過MAPK/ERK路徑傳遞相關訊號。 總之，本研究為了解胰島素在Gefitinib抗藥性中所扮演的角色，我們已經成功建立離體的細胞模型以模擬糖尿病患體內持續維持高胰島素水平，藉以研究高胰島素誘發之Gefitinib抗藥性機制，胰島素可能會透過INSR或IGFR及其下游PI3K/AKT路徑誘發Gefitinib抗藥性產生，由於原本被抑制的路徑被胰島素重新激活，癌細胞得以繼續生存。此研究提供一個新的觀點，可供同時患有糖尿病及非小細胞肺癌之病患療法參考，然而是否有其他因素共同影響Gefitinib抗藥性，仍需進一步的試驗去證實。

Lung cancer is currently the leading cause of cancer-related mortality worldwide. Among all oncogenes, human epidermal growth factor receptor (EGFR/HER) has been recognized as the major cause of Non-small cell lung carcinoma (NSCLC) in Asia. Therefore, blocking EGFR family pathways with EGFR TK inhibitors (TKIs) can suppress tumor cell proliferation and initiate apoptosis. Gefitinib, the EGFR tyrosine kinase inhibitor (EGFR-TKI), is the most common anti-NSCLC drug in the Asia. However, previous clinical evidences showed that patients received gefitinib treatment would suffer drug resistance, suggesting that gefitinib treatment induces activation of the IGF-IR pathway and its downstream signaling components. The molecular mechanism of gefitinib resistance is still unclear. Increasing evidence showed that type 2 diabetes mellitus (T2DM), which is characterized with hyperinsulinemia and hyperglycemia, is associated with the risk of NSCLC mortality. Thus, hyperinsulinemia and hyperglycemia might play a role in gefitinib resistance development. To investigate the molecular mechanisms involved in hyperinsulinemia induced gefitinib resistance in NSCLC, we established an in vitro cell model using an EGFR NSCLC cell lines, HCC827 and HCC4006. The results of MTS cell viability assay showed that HCC827 and HCC4006 were sensitive to gefitinib. After treated for 24 hours, the IC50 was approximately 4.43 μM and 39.42 μM for HCC827 and HCC4006, respectively. Glargine we used in experiments is a long-acting insulin analogue to mimic long term hyperinsulinemia. Glargine (0 nM to 100 nM) co-treated with gefitinib dose-dependently induced gefitinib resistance in HCC4006 cells. In our Western blotting results, glargine increased phosphorylation of IGFR and AKT, but not ERK suggesting that glargine might go through PI3K/AKT pathway to make cell survival under gefitinib treatment, instead of MAPK/ERK pathway which would directly affect DNA transcription in nucleus. In summary, we have established an applicable in vitro model for studying the molecular mechanisms of hyperinsulinemia induced gefitinib resistance in NSCLC. Glargine might decrease gefitinib drug efficiency by upregulating IGFR downstream pathway, PI3K/AKT. Due to the upregulation of this pathway, NSCLC cell survival could be induced. We provided a new view of NSCLC targeted therapy when patients are both suffering from T2DM and NSCLC. However, further research is needed to verify the association between hyperinsulinemia and gefitinib resistance.