Activation of caspase-8 is not esse

Activation of caspase-8 is not essential for cell death in human colon cancer cells
Our previous studies showed that DRE treatment rapidly and efficiently activates the extrinsic pathway of apoptosis in leukemia, melanoma and pancreatic cancer cells and this process is dependent on the activation of caspase-8 as the cells with a dominant-negative Fas-Associated Death Domain (Dn-FADD) are unresponsive to the DRE treatment [12]. Here we examined whether similar activation of the extrinsic apoptotic pathway takes place in DRE-treated colorectal cancer cells. Using substrates specific fluorescent-labeled inhibitors of caspases (FLICA; DEVD – Caspase-3; IETD – Caspase-8), the activation of these caspases was monitored in HT-29 and NCM460 cells following the DRE treatment between 15 minutes to 48 hours. The results obtained by image-based cytometry showed a 68% increase in caspase-8 activity with a corresponding 30% increase in propidium iodide staining in HT-29 cells treated with 2.5 mg/ml DRE for an hour. Furthermore, this response was specific to HT-29 cancer cells, as NCM460 normal cells remained unaffected by the same treatment. The NCM460 cells did not activate caspase-8 and did not uptake propidium iodide (Figure ​(Figure6A).6A). As mitochondrial membrane destabilization was observed in colon cancer cells treated with DRE (Figure 5A – 5B), we wanted to confirm the link between caspase-8 activation and the loss of mitochondrial membrane potential. Following treatment, the cells were lysed and probed for the truncated pro-apoptotic protein, Bid. Bid is known to translocate to the mitochondria following cleavage and activation by activated caspase-8. This translocation to the mitochondria has been shown to play a role in the destabilization of the mitochondrial membrane [13]. DRE treatment led to the truncation of Bid in HT-29 cells selectively, with no increase in Bid truncation in NCM460 cells (Figure ​(Figure6B6B).

結果 (繁體中文) 1:

Activation of caspase-8 is not essential for cell death in human colon cancer cells
Our previous studies showed that DRE treatment rapidly and efficiently activates the extrinsic pathway of apoptosis in leukemia, melanoma and pancreatic cancer cells and this process is dependent on the activation of caspase-8 as the cells with a dominant-negative Fas-Associated Death Domain (Dn-FADD) are unresponsive to the DRE treatment [12]. Here we examined whether similar activation of the extrinsic apoptotic pathway takes place in DRE-treated colorectal cancer cells. Using substrates specific fluorescent-labeled inhibitors of caspases (FLICA; DEVD – Caspase-3; IETD – Caspase-8), the activation of these caspases was monitored in HT-29 and NCM460 cells following the DRE treatment between 15 minutes to 48 hours. The results obtained by image-based cytometry showed a 68% increase in caspase-8 activity with a corresponding 30% increase in propidium iodide staining in HT-29 cells treated with 2.5 mg/ml DRE for an hour. Furthermore, this response was specific to HT-29 cancer cells, as NCM460 normal cells remained unaffected by the same treatment. The NCM460 cells did not activate caspase-8 and did not uptake propidium iodide (Figure ​(Figure6A).6A). As mitochondrial membrane destabilization was observed in colon cancer cells treated with DRE (Figure 5A – 5B), we wanted to confirm the link between caspase-8 activation and the loss of mitochondrial membrane potential. Following treatment, the cells were lysed and probed for the truncated pro-apoptotic protein, Bid. Bid is known to translocate to the mitochondria following cleavage and activation by activated caspase-8. This translocation to the mitochondria has been shown to play a role in the destabilization of the mitochondrial membrane [13]. DRE treatment led to the truncation of Bid in HT-29 cells selectively, with no increase in Bid truncation in NCM460 cells (Figure ​(Figure6B6B).

結果 (繁體中文) 2:

卡巴塞-8的活化對於人類結腸癌細胞的細胞死亡不是必要的
我們先前的研究表明，DRE治療快速有效地啟動白血病、黑色素瘤和胰腺癌細胞中凋亡的外在通路，這個過程取決於caspase-8的活化，因為細胞具有主導陰性Fas關聯死亡域（Dn-FADD）對DRE治療無回應[12]。在這裡，我們檢查了在DRE治療的結腸直腸癌細胞中是否也發生類似的外在凋亡通路的活化。使用基質特定的螢光標記抑制劑的卡巴塞（FLICA;DEVD = 卡斯帕塞-3;IETD – Caspase-8），在DRE治療15分鐘至48小時後，在HT-29和NCM460細胞中監測這些卡巴塞的活化。基於圖像的細胞測定結果顯示，在HT-29細胞中，用2.5mg/ml DRE處理一小時，碘化鈉染色的碘化鈉染色增加68%，其中碘化鈉染色增加68%。此外，這種反應是特定于HT-29癌細胞，因為NCM460正常細胞仍然不受相同的治療。NCM460細胞未啟動卡帕西-8，且未攝入碘化鈉（圖6A）。由於在使用DRE治療的結腸癌細胞中觀察到線粒體膜不穩定（圖5A = 5B），我們希望確認casae-8活化與線粒體膜電位損失之間的聯繫。治療後，細胞被解傷，並探查截斷的親凋亡蛋白，Bid。投標已知在裂解後轉移到線粒體，並啟動了caspase-8。這種對線粒體的易位已被證明線上粒體膜的不穩定中發揮了作用[13]。DRE治療導致HT-29細胞中投標的選擇性截斷，NCM460細胞的Bid截斷沒有增加（圖6B6B）。

結果 (繁體中文) 3:

caspase-8的啟動對結腸癌細胞死亡的影響
我們以前的研究表明，dre治療能快速有效地啟動白血病、黑色素瘤和胰腺癌細胞凋亡的外源性途徑，這一過程依賴於caspase-8的啟動，因為具有顯性負fas相關死亡結構域（dn-fadd）的細胞對dre治療無反應[12]。在這裡，我們研究了在dre治療的結直腸癌細胞中是否有類似的外源性凋亡途徑的啟動。使用底物特异性螢光標記的caspase抑制劑（flica；devd–caspase-3；ietd–caspase-8），在dre處理15分鐘至48小時後，在ht-29和ncm460細胞中監測這些caspase的啟動。影像細胞儀檢測結果顯示，2.5mg/ml dre作用1h後，ht-29細胞caspase-8活性新增68%，碘化丙啶染色新增30%。此外，這種反應是針對ht-29癌細胞的，因為ncm460正常細胞不受相同治療的影響。NCM460細胞不啟動caspase-8，也不攝取碘化丙啶（圖6a）。由於在用dre治療的結腸癌細胞中觀察到線粒體膜失穩（圖5a-5b），我們想確認caspase-8啟動與線粒體膜電位喪失之間的聯系。治療後，對細胞進行裂解並檢測截短的促凋亡蛋白bid。已知bid在被啟動的caspase-8切割和啟動後轉移到線粒體。這種向線粒體的易位已經被證明線上粒體膜的失穩中起作用[13]。dre處理導致ht-29細胞的bid選擇性截斷，而ncm460細胞的bid截斷沒有新增（圖6b6b）。