自噬性細胞死亡(Autophagic cell death, Ⅱ型細胞程序性死亡)和細胞凋亡(Apoptosis , I型細胞程序性死亡)是細胞死亡的兩種不同形式。他們皆為細胞反應外界的壓力所產生的變化,在某些狀況下,自噬(Autophagy)為構成細胞對外在環境的適應,但在癌症抑制上,其又構成了另一種細胞死亡途徑的關係。薑(Ginger)是亞洲國家使用已久天然食品與藥草,在薑萃取物中,薑酮醇(Gingerol)、薑烯酚(Shogaol)是主要的活性成分,其中6-shogaol具有良好之抗發炎活性、抗細菌和抗肝毒性,此外,6-shogaol能誘導肝癌細胞透過Akt-PI3K路徑促使其產生自噬、也被證明能誘導人類大腸癌細胞之凋亡。但有關6-shogaol對人類大腸直腸癌細胞HT-29的凋亡和自噬間之研究仍相當有限,因此,本研究以HT-29大腸直腸癌細胞模式探討6-shogaol對兩者死亡形式的關係。過去,本實驗室的研究已經證實,6-shogaol透過調節粒線體膜電位下降,可有效誘導HT-29凋亡。但對於6-shogaol的和caspase活性,凋亡和自噬性細胞死亡之間的發展尚不清楚。故我們假設6-shogaol抑制HT-29的細胞生長是通過自噬與凋亡。藉由檢測自噬相關的每一個階段的發展模式來討論其關係性。結果顯示,在存活率試驗中,6-shogaol在24小時內不呈現劑量效應,接著以流式細胞儀分析autophagososome和 autolysosome的活性、細胞膜外翻(Annexin-V)、caspase3/7的表現與DNA片斷化的情況,並以共軛焦顯微鏡證明autophagososome的形成,發現自噬和凋亡的發生原來是決定於不同的劑量與時間點,以18小時為分界,是兩者細胞死亡型式的交接點且並行不悖,並在低濃度(20 μM)下可以維持較長的自噬效應,高濃度下(80 μM)自噬性死亡和細胞凋亡皆大量表現。此外,6-shogaol對於細胞週期G2/M有阻滯的作用,可推測6-shogaol藉由G2/M阻滯、影響細胞的有絲分裂而促進細胞死亡。
Autophagic cell death (Type II programmed cell death) and apoptosis (Type I programmed cell death) are two distinct forms of cell death. They have a complex functional relationship in the sense that, autophagy constitutes a stress adaptation that avoids cell death (and suppresses apoptosis) and an alternative cell-death pathway. Ginger, have several phenolic alkanones among which 6-shogaol is shown to exert anti-inflammatory, anti-bacterial and anti-hepatotoxic properties. Also it has been shown to induce apoptosis in human colorectal carcinoma cells. In our previous studies, we found that 6-shogaol can induce apoptosis of HT-29 (human colorectal carcinoma cells) via modulation of mitochondrial functions. In this study, we assumed that the cell growth inhibition activity of 6-shogaol on the HT-29 is via both autophagy and apoptosis. The results showed that in the survival test, within 24 hrs treatment, effect of 6-shogaol on cell survival rate was not dose dependent. Then we used flow cytometry to analyze the autophagososome and auto- lysosome activity and did a series of apoptosis test, including Annexin-V, caspase3 / 7 activation, DNA fragment. Also, the confocal microscopy showed the formation of autophagososome which proved that the occurrence of autophagy and apoptosis was originally determined by the different doses and time points. That is, both cell death types go hand in hand and can be divided at 18 h. Autophagy can be maintained a bit longer in the low concentration (20 μM), but at high concentration (80 μM), both of autophagic death and apoptosis play significant roles. In addition, 6-shogaol which induces the G2 / M cell cycle arrest, it can be speculated that 6-shogaol affect cell mitosis and promote cell death.
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