Interleukin-4對於HT-29大腸癌細胞生長與轉移之影響

Abstract

Interlukin-4 (IL-4) 是一種由T helper 2 細胞所分泌的細胞激素，調控T helper 2 細胞分化與增生。過去文獻發現IL-4能夠促進癌細胞的生長，包括乳癌、前列腺癌等癌細胞，並且影響癌細胞的生長以及轉移能力。本論文我們利用MTT試驗發現，在缺乏血清的情況下，IL-4促進HT-29大腸癌細胞的生長。利用群落生成分析IL-4對於細胞生長的影響，結果顯示IL-4能增加HT-29細胞群落生長的數目。遷移與侵入在癌細胞的轉移中扮演重要角色，wound healing試驗的結果顯示，IL-4會促進HT-29細胞遷移的能力，並且促進上皮間質轉化相關基因表現。此外，我們也發現IL-4調控了HT-29大腸癌細胞許多黏液素以及促發炎細胞激素基因的表現。先前我們的研究發現在HT-29細胞中，IL-4會誘導類固醇荷爾蒙生成基因HSD3B1的大量表現，並能促進HT-29細胞產生具生物活性的糖類皮質素。已知糖類皮質素會促進T細胞的凋亡。我們將老鼠脾臟細胞培養於HT-29細胞培養液中，並利用流式細胞儀進行細胞凋亡分析，發現經IL-4處理的HT-29細胞培養液培能促進T細胞的凋亡，說明IL-4可能經由HSD3B1的表現，促進HT-29糖類皮質素產生而促進T細胞的凋亡。進一步研究表觀遺傳學是否調控IL-4誘導的HSD3B1表現，我們發現廣效型組蛋白去乙醯化蛋白酶 (HDAC)抑制劑能有效降低IL-4所誘導之HSD3B1表現，甲基轉移酶抑制劑則沒有明顯效應，說明組蛋白去乙醯化蛋白酶的活性對於IL-4所誘導之HSD3B1表現可能是重要的。將細胞中的HDAC1、HDAC2或HDAC3 進行knockdown後，能夠抑制IL-4所誘導之HSD3B1表現，說明HDAC對於IL-4所誘導HSD3B1表現是重要的。然而，探討其可能的作用機制，實驗結果顯示HDAC抑制劑不會改變HDAC1、HDAC2、HDAC3以及HDAC4的表現量，不會影響IL-4兩個重要訊息傳遞路徑STAT6與AKT之活化，也不會影響IL-4所誘導之STAT6與HSD3B1啟動子的結合。綜合以上結果顯示，IL-4會促進大腸癌細胞HT-29的生長與遷移，以及促進HSD3B1的表現產生糖類皮質素而影響免疫反應。

Interleukin-4 (IL-4) is a cytokine secreted by T helper 2 cells which controls the differentiation and proliferation of T helper 2 cells. It has been demonstrated that IL-4 promotes cancer cell growth and metastasis, including breast cancer and prostate cancer. Here we showed that IL-4 stimulated HT-29 colon cancer cells growth under serum free condition measured by MTT assay. Besides, colony formation assay was performed to investigate the effects of IL-4 on the clonogenicity of HT-29 cells. Results showed that IL-4 promoted the ability of colony formation. Migration and invasion play a critical role in cancer metastasis. We further showed that IL-4 promoted the migration ability measured by wound healing assay and upregulated epithelial-mesenchymal transition-associated genes expression in HT-29 cells. We also found that IL-4 altered the expression of mucin genes and pro-inflammatory cytokines. Our previous study found that IL-4 induced steroidogenic gene HSD3B1 expression and stimulated bioactive glucocorticoids (GCs) production in HT-29 cells. It is known that GCs promote T cells apoptosis. Using flow cytometry analysis, we found that conditioned medium from HT-29 cells treated with IL-4 induced murine splenic T cells apoptosis, suggesting that IL-4 stimulated the production of GCs and promoted the T cells apoptosis, probably through the induction of HSD3B1 expression. To further investigate whether the epigenetic modifications are involved in IL-4-induced HSD3B1 expression. We found that IL-4-induced HSD3B1 expression was abolished by several HDAC inhibitors; however, the methyltransferase inhibitor had no significant effects. We further found HDAC1, HDAC2, or HDAC3 knockdown attenuated IL-4-induced HSD3B1 protein expression, indicating that these HDACs are critical for IL-4-induced HSD3B1 expression in HT-29 cells. IL-4 had no significant effects on HDAC1, HDAC2, HDAC3, and HDAC4 proteins expression, the phosphorylation of IL-4 downstream signaling STAT6 and AKT, and the ability of IL-4-induced STAT6 binding to HSD3B1 promoter. In summary, our study indicates that IL-4 promotes HT-29 cancer cell growth, migration, and the immunoregulatory GCs synthesis via HSD3B1 induction.