透過生物資訊學探討Ferroptosis在人類大腸直腸癌細胞中植化素Withaferin A合併鉑金類抗癌藥物的角色

Abstract

在大腸直腸癌（Colorectal cancer，CRC）中，鉑金類藥物Cisplatin（Cis）的藥物不敏感與副作用的特性已成為十分嚴重問題。鐵依賴型細胞死亡（Ferroptosis）是透過增加細胞內游離鐵堆積並促進脂質過氧化物生成所導致的新型態細胞死亡機制。透過生物資訊學分析結果發現Cis藥物阻抗的CRC患者體內較藥物敏感的患者具有鐵代謝失調的問題。CRC組織較其他癌症組織有堆積較多二價鐵離子的潛力，並且CRC組織相較於正常組織有累積較多游離鐵與促進脂質過氧化物形成的特性，因此透過二價游離鐵促進細胞活性氧物質生成與降低抗氧化能力來誘導Ferroptosis將有機會改善CRC較低的無復發存活率。此外，相較於HCT116，Ferroptosis促進劑RSL3在HT-29中能引起更高的生長抑制作用，並且可以被Ferroptosis抑制劑Ferrostatin-1和Deferoxamine逆轉細胞生長抑制。同時HT-29中有較低的出鐵蛋白表現，代表細胞中可透過增加細胞內游離鐵來促進Ferroptosis。生物資訊學分析結果發現南非醉茄的酯類成分Withaferin A（WA）具有誘導Ferroptosis的潛力，並且在CRC中WA較Cis有較佳的藥物敏感性。合併Cis與WA能夠促進HT-29進行Ferroptosis相關生長抑制、脂質過氧化物累積、游離鐵累積與降低GPX4蛋白表現。此外，合併Cis與WA能增加shGFP HT-29的細胞生長抑制與促進ferritin與LC3B蛋白共位的情形，而這些結果在加入合併藥物的shATG5 HT-29會被抑制，代表Cis與WA可以增加細胞進行ferrtinophagy。綜合以上結果，本研究利用生物資訊學與實驗數據證實在CRC中植化素WA合併Cis能產生協同作用並促進Ferroptosis。

Drug resistance and side effect in Cisplatin (Cis) had become a serious problem in colorectal cancer (CRC). Ferroptosis is an iron-dependent regulated cell death caused by toxic lipid peroxidation. Through bioinformatics analysis, we discovered that Cis-resistant CRC patients exhibit iron metabolism disorders compared to the Cis-sensitive patients. Besides, CRC tissues, in contrast to other cancer tissues and normal tissues near by the CRC, tended to accumulate Fe2+ and promote lipid peroxidation. Therefore, inducing ferroptosis by increasing intracellular Fe2+ and reducing antioxidant capacity may improve the low relapse-free survival rate in CRC. Compared to HCT116, ferroptosis inducer RSL3 induced a higher growth inhibition in HT-29, and the growth inhibition was reduced by ferroptosis inhibitor Ferrostatin-1 and Deferoxamine. This observation is associated with a lower ferroportin in HT-29, indicating that increasing labile iron pool can promote Ferroptosis in HT-29. By analyzing the transcriptomics data, natural compound Withaferin A (WA) derived from Withania somnifera has the potential to induce Ferroptosis, and WA exhibits better drug sensitivity in CRC compared to Cis. Combination of Cis and WA-induced Ferroptosis in HT-29 was characterized by increasing growth inhibition and repressing GPX4 protein expression, also increasing lipid peroxides and cellular Fe2+ accumulation. Furthermore, the combination of Cis and WA increased growth inhibition and promoted the colocalization of ferritin and LC3B in shGFP HT-29 cells, while these effects were suppressed in Cis and WA treated shATG 5 HT-29 cells, indicating that Cis and WA enhanced ferritinophagy in HT-29 cells. Collectively, our unique integrated screening results and experimental data support the combination of natural compound WA and Cisplatin in producing synergistic effects and inducing ferroptosis in CRC.