新型全方位HDAC抑制劑MPT0G236對大腸直腸癌細胞的抗癌研究

Abstract

大腸直腸癌（Colorectal Cancer, CRC）是全球最常見的癌症之一，其在全球及台灣的發病率仍然很高。雖然傳統療法如化療和手術在治療大腸直腸癌方面已取得不錯的療效，但復發率以及晚期或轉移性大腸直腸癌的死亡率依然居高不下。因此，開發和探索新的藥物治療大腸直腸癌仍然是一個重要的研究目標。 組蛋白去乙醯酶（histone deacetylases, HDACs）與組蛋白乙醯轉化酶(histone acetyltransferase, HAT) 通過對組蛋白進行乙醯化和去乙醯化，調控組蛋白與DNA的纏繞而影響核小體的結構，進而影響基因表達。這種組蛋白以及非組蛋白受質的乙醯化平衡對細胞功能具有重要影響。在腫瘤發展中，組蛋白去乙醯酶扮演著多重關鍵角色，與癌症的發生及進展密切相關。組蛋白去乙醯酶的過度表現已被證實與多種癌症的發生和預後不良有關。特別是在大腸直腸癌中，組蛋白去乙醯酶的過度表達與患者的高死亡率顯著相關。 組蛋白去乙醯酶抑制劑（HDACIs）以組蛋白去乙醯酶作為抑制目標，在多項研究中證明其在癌症治療中具有抗癌效果。至今為止，已有五種組蛋白去乙醯酶抑制劑核准上市使用在血液腫瘤的治療上。然而，這些藥物僅核准適用於血液腫瘤。其在投予在實體腫瘤模式下的療效與安全性概況尚需進一步研究。在先前研究MPT0G236一個新型全方位組蛋白去乙醯酶抑制劑再多種不同癌症細胞株中展現良好的抑制效果。為了探索具更有效針且可應用在實體腫瘤的組蛋白去乙醯酶抑制劑，本研究聚焦於MPT0G236在大腸直腸癌的抗癌效果以及其作用機制的探討。 在本研究中，我們選用了HCT-116和HT-29這兩株大腸直腸癌細胞株。經MPT0G236處理後，結果顯示MPT0G236能有效降低HCT-116和HT-29細胞的活性並抑制其增殖。同時，當MPT0G236處理正常的人臍靜脈內皮細胞（Human Umbilical Vein Endothelial Cells, HUVECs）時，其對MPT0G236的敏感性相對於大腸直腸癌細胞較低，這表明MPT0G236對癌細胞具有選擇性毒性，而對正常細胞的影響較小。進一步的實驗顯示，MPT0G236對不同亞型的組蛋白去乙醯酶具有選擇性抑制效果，尤其對第I型（包括HDAC1、HDAC2和HDAC3）以及第IIb型（HDAC6）的活性展現出顯著的抑制作用。在奈米莫爾濃度範圍內，MPT0G236即可達到百分之五十的抑制濃度，且其抑制效果優於SAHA。此外，MPT0G236亦抑制了HCT-116和HT-29細胞中的組蛋白去乙醯酶活性，導致受質α-微管蛋白與組蛋白乙醯化水平（Acety-α-Tubulin和乙醯化組蛋白）的累積，呈現出濃度依賴性，且效果顯著優於SAHA。 在我們的研究中，通過流式細胞儀和西方墨點法分析顯示，MPT0G236處理大腸直腸癌細胞後，能夠調節細胞週期相關因子的表現，如p-MPM2、p-cdc2（Y15）、cyclin B1和cdc25C，促使大腸直腸癌細胞進入分裂前期/有絲分裂期的細胞週期阻滯（G2/M arrest）。此外，研究結果還表明，MPT0G236能夠通過凋亡蛋白依賴性途徑誘導大腸直腸癌細胞進入凋亡程序，活化外源性（extrinsic）和內源性（intrinsic）途徑的凋亡蛋白酶（caspase 8、caspase 9），以及下游的凋亡蛋白酶-3和PARP的裂解。這些結果表明，MPT0G236可以通過誘導細胞凋亡和細胞週期停滯來抑制大腸直腸癌細胞的增殖。 總結來說，我們的研究證實了MPT0G236在大腸直腸癌細胞中展現出顯著的抗癌效果，為一有潛力的新型的組蛋白去乙醯酶抑制劑，可作為大腸直腸癌及其他實體腫瘤的治療的後續發展，也為大腸直腸癌治療提供了新的思路和可能性，為未來的藥物開發研究奠定了基礎。

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies, with high incidence rates both worldwide and in Taiwan. Although traditional treatments such as chemotherapy and surgery have shown satisfactory efficacy in treating CRC, the recurrence rate and mortality of advanced or metastatic CRC remain high. Therefore, developing and exploring new drug therapies for CRC continues to be an important research objective. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) regulate gene expression by modulating the acetylation and deacetylation of histones and non-histone substrates. The balance of histone and non-histone acetylation is crucial for cellular function, affecting the structural properties of histones and nucleosomes and also involved in the recruitment of oncogenic transcription factors. In tumor progression, HDACs play multiple critical roles and are closely associated with the development and progression of cancer. Overexpression of HDACs has been linked to the onset of various cancers and poor prognosis. Particularly in CRC, overexpression of HDACs is significantly correlated with high mortality rates in patients. HDAC inhibitors (HDACIs), have demonstrated anticancer effects in various of research. To date, five HDACIs have been approved for clinical use in treating hematologic malignancies. However, these drugs are only approved for hematologic cancers, and their efficacy and safety profiles in solid tumor models require further investigation. Research is still exploring more potent HDACIs for potential use in solid tumors in the future. In this study, we selected HCT-116 and HT-29 CRC cell lines. Treatment with MPT0G236 demonstrated a significant reduction in the viability and proliferation of both HCT-116 and HT-29 cells. Additionally, when MPT0G236 was applied to normal human umbilical vein endothelial cells (HUVECs), the sensitivity of these cells to MPT0G236 was relatively lower compared to CRC cells, indicating that MPT0G236 exhibits selective toxicity towards cancer cells with minimal impact on normal cells. Further experiments revealed that MPT0G236 selectively inhibits different HDAC subtypes, especially class I HDACs (including HDAC1, HDAC2, and HDAC3) and class IIb HDAC6, showing significant inhibitory effects, achieving an IC50 in the nanomolar concentration range. Its inhibitory effect was better than that of SAHA. Moreover, MPT0G236 inhibited HDAC activity in HCT-116 and HT-29 cells, leading to the accumulation of acetylated α-tubulin and acetylated histones, showing a concentration-dependent effect, with efficacy notably better than SAHA. Our research, using flow cytometry and Western blot analysis, demonstrated that MPT0G236 treatment modulates the expression of cell cycle-related factors, such as p-MPM2, p-cdc2 (Y15), cyclin B1, and cdc25C, causing CRC cells to undergo G2/M phase cell cycle arrest. Additionally, the results indicated that MPT0G236 induces apoptosis in CRC cells through a caspase-dependent pathway, activating both extrinsic and intrinsic apoptotic pathways, including activation of caspase 8, caspase 9, and caspase-3, and cleavage of PARP. These findings suggest that MPT0G236 inhibits CRC cell by inducing apoptosis and causing cell cycle arrest. In summary, our study confirms that MPT0G236 exhibits significant anticancer effects in CRC cells, representing a promising new HDAC inhibitor with potential for development as a treatment for CRC and other solid tumors. This study provides new insights and possibilities for CRC treatment and lays the foundation for future drug development research.