探討 paracaspase 抑制劑毒殺活化型瀰漫性大B 細胞 淋巴瘤生長的機制

Abstract

瀰漫性大 B 細胞淋巴瘤(DLBCL)，根據基因表現可以分為兩大族群。分別為活化型細胞(Activated B-cell like DLBCL, ABC-DLBCL) 與生長中心型細胞(Germinal center B-cell like DLBCL, GCB-DLBCL)。持續活化的NF-κB 路徑活性是維持活化型細胞生存的必要條件。在先前文獻已被報導，CBM (CARMA1-BCL10-MALT1) 複合物中的CARMA1 蛋白突變是造成NF-κB 路徑活化的原因之一。而MALT1 蛋白在活化CBM 複合物與NF-κB 訊息傳遞路徑扮演相當重要的角色。多個研究文獻發現，MALT1 蛋白酶活性(protease activity) 的抑制劑可以有效地殺死活化型細胞，但是相關機制目前仍不清楚。在本篇文章中，將會觀察活化型與生長中心型細胞處理MALT1 抑制劑後，對於NF-κB 路徑的影響。藉此來探討抑制劑殺死活化型細胞的相關機制。由結果中可以觀察到，MALT1 與BCL10的切割產物以及p-IκBα 與IL-6 的產生均只出現在活化型細胞株HBL-1 與OCI-Ly3。這代表著，NF-κB 路徑在活化型細胞中就已經活化了。接著使用MTT 方法測得MALT1 抑制劑(MI-2 z-VRPR-FMK) 以及 IKK 路徑抑制劑(BMS-345541 Bay-11-7085) 在 BJAB 與OCI-Ly3 細胞的IC50 分別為1.54μM、259.7μM、4.22μM 與0.93μM 以及0.58μM、13.76μM、1.45μM 與0.33μM。由此結果得知BAJB 細胞對於這四種NF-κB 路徑抑制劑的耐受性都比OCI-Ly3 細胞來的好。當生長中心型的BJAB 細胞以TPA/Ionomycin 方法刺激NF-κB 路徑活化後，再與活化型OCI-Ly3細胞一樣，被MI-2 藥物處理，都可觀察到BCL10 切割產物消失、IκBα 與 p-IκBα以及IL-6 核醣核酸表現量都下降。若OCI-Ly3 細胞與BJAB 細胞在MALT1knockdown 的處理下，同樣先需使用TPA/Ionomycin 刺激BJAB 細胞的NF-κB路徑活化後，則也可觀察到BCL10 切割產物消失以及IL-6 核醣核酸表現量下降，但是IκBα 與 p-IκBα 表現量並無改變。因此由實驗結果得知，MI-2 藥物處理細胞或是knockdown MALT1 後，確實會使NF-κB 訊息傳遞路徑受到抑制。而我們實驗室認為MI-2 藥物毒殺細胞的可能機制是，MALT1 蛋白酶活性受到抑制後，此蛋白產生dominant negative effect 所導致的現象。

Diffuse large B cell lymphoma (DLBCL) can be categorized into two subtypes based on their gene expression patterns. One is activated B-cell like diffuse large B cell lymphoma (ABC type, ABC-DLBCL), the other is germinal center B-cell like diffuse large B cell lymphoma (GCB type, GCB-DLBCL). The NF-κB signaling pathway was constitutively activated in ABC-type DLBCL cells. The activated NF-κB signaling was found to be required for the survival of ABC-type DLBCL cells. Mutation in CARMA1 protein, a recruitment member of CBM (CARMA1-BCL10-MALT1) complex, was previously reported to be involved in the activation of the NF-κB signaling pathway. MALT1, a paracaspase, played an important role in the active CBM complex formation and the activated NF-κB signaling. Several studies reported that proteolytic inhibitors of MALT1 selectively killed the ABC-type DLBCL cells. However, detailed mechanisms mediating the cytotoxic effect of MALT1 inhibitors in ABC-type DLBCL cells were not clear. In this study, molecules in the NF-κB signaling pathway were examined in both ABC- and GCB-type DLBCL cells treated with MALT1 inhibitors to elucidate the possible mechanism(s) mediating the cytotoxic effect in ABC-type DLBCL cells. DLBCL cells with MALT1 knockdown were also included for comparison.The cleavage products of MALT1 and BCL10 , the expression of p-IκBα and IL-6 were present in lysates of HBL1 and OCI-Ly3 cells. The NF-κB signaling pathway was indeed, as reported, activated in these two ABC-type DLBCL cells. MTT assay was utilized to measure IC50 to several cytotoxic agents including two reported MALT1 inhibitors (MI-2,z-VRPR-FMK), two known IKK inhibitors (BMS-345541,Bay-11-7085). BJAB, GCB-type DLBCL cells, was relatively resistant to these NF-κB signaling inhibitors as compared to OCI-Ly3, ABC-type DLBCL cells. TPA/Ionomycin were utilized to stimulate NF-κB signaling in BJAB cells. Pretreatment with MI-2 diminished the appearance of BCL10 cleavage products. Expression levels of p-IκBα and IL-6 were also reduced. Same results were observed in MI-2 treated OCI-Ly3 cells. MALT1 proteolytic activity and IL-6 expression were dampened in TPA/Ionomycin stimulated BJAB cells with MALT1 knockdown as reported. However, expression levels of IκBα and p-IκBα were not affected. Same results were observed in MALT1 knockdown OCI-Ly3 cells. The possible mechanism suggested to be the dominant negative effect of uncleaved MALT1 protein mediating the cytotoxic effect of MALT1 inhibitors in ABC-type DLBCL cells, causing cells to death.