胞外 HMGB1 於大腸癌細胞之角色探討

Abstract

癌細胞的進程與其微環境密切相關，腫瘤組織快速生長的特性會導致腫瘤細胞容易遭遇到低氧、營養缺乏等逆境，造成細胞死亡。由於臨床上常可見到腫瘤組織的壞死現象，腫瘤微環境的壓力是有利於癌細胞存活，抑或是癌細胞先天上的生存弱點，值得深入研究。細胞壞死會釋出內部分子引起發炎反應，其中，損傷相關分子模式 (Damage-associated molecular patterns, DAMPs) 是一群內生型可引起免疫反應的誘導物。高遷移率族蛋白1 (high-mobility group box 1, HMGB1) 被認為是細胞死亡時主要釋放出來的損傷相關分子，並經常在癌症組織中存在。細胞外的 HMGB1 具有細胞激素與趨化因子等活性，故參與許多發炎相關疾病的調控；然而在癌症相關文獻中， HMGB1 卻扮演促癌與抑癌雙重角色，顯示其功能尚未完全明瞭。 在我的研究中，希望找出癌細胞周邊環境的 HMGB1 對其自身的影響。為了探討胞外 HMGB1 對癌細胞的效應，本研究利用大腸桿菌 RosettaTM (DE3) 建構 His-HMGB1 表達與純化系統，可獲得純度高之重組蛋白，同時也利用真核細胞 HEK293FT 過量表現方式取得含有HMGB1 蛋白質的細胞萃取物。實驗結果發現，胞外的 HMGB1 具有促進大腸癌細胞 NF-κB 及 Wnt 訊息的活性，然而卻會抑制癌細胞的爬行能力與球體形成能力。 HMGB1 對癌細胞的調控可能和其轉譯後修飾有關，也是造成現有文獻報導促癌、抑癌相互矛盾的原因之一，若能更加明白 HMGB1 蛋白的修飾情形，則有助於預測其功能，因而在癌症醫學上可以視其作用擬定最適合的治療方案。

The progression of cancer is profoundly affected by its microenvironment. As tumors are rapid growing mass, cells inside tumor bulk may encounter adversities such as hypoxia and nutrient deprivation, which may cause cell death. Whether stress-induced cell death is advantageous for cancer cell survival or harmful as the innate vulnerability is an issue worth studying. Clinically, necrosis is typically seen in cancer tissues. During necrosis, intracellular molecules are passively released outside,triggering inflammation. Damage-associated molecular patterns (DAMPs) are endogenous inducers of inflammation which vary in categories and functions. One of the DAMPs, high-mobility group box 1 (HMGB1) protein is thought to be the major factor released by necrotic cells. Extracellular HMGB1 possesses cytokine and chemokine activities, thus participating in inflammation-associated diseases. The research about HMGB1 in cancer, however, revealed both its pro-tumor and anti-tumor roles. Therefore, the function of HMGB1 still remains to be clarified. In this study, I aim to investigate how HMGB1 in the extracellular environment affects cancer cell properties. His-HMGB1 was produced in RosettaTM (DE3) E. coli strain and purified through affinity chromatography. HMGB1-overexpressing HEK293FT cell line was also constructed to obtain HMGB1 of eukaryotic origin. Extracellular HMGB1 was shown to activate NF-κB and Wnt activity in colorectal cancer cells; nevertheless, HMGB1 treatment may inhibit cancer cell migration and sphereforming ability. The effect of HMGB1 on cancer cells seems to has a lot to do with its post-translational modifications, which possibly leads to the pro-tumor and anti-tumor inconsistency in experimental results. The understanding of HMGB1 protein modifications would help predict its function. As a consequence, depending on the function of HMGB1 in cancer tissues, a suitable treatment strategy could be determined to combat cancer.