Kaur Manjinder, Agarwal Chapla, Singh Rana P, Guan Xiangming, Dwivedi Chandradhar, Agarwal Rajesh
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Carcinogenesis. 2005 Feb;26(2):369-80. doi: 10.1093/carcin/bgh325. Epub 2004 Nov 4.
alpha-Santalol, an active component of sandalwood oil, has been studied in detail in recent years for its skin cancer preventive efficacy in murine models of skin carcinogenesis; however, the mechanism of its efficacy is not defined. Two major biological events responsible for the clonal expansion of transformed/initiated cells into tumors are uncontrolled growth and loss of apoptotic death. Accordingly, in the present study, employing human epidermoid carcinoma A431 cells, we assessed whether alpha-santalol causes cell growth inhibition and/or cell death by apoptosis. Treatment of cells with alpha-santalol at concentrations of 25-75 microM resulted in a concentration- and a time-dependent decrease in cell number, which was largely due to cell death. Fluorescence-activated cell sorting analysis of Annexin V/propidium iodide (PI) stained cells revealed that alpha-santalol induces a strong apoptosis as early as 3 h post-treatment, which increases further in a concentration- and a time-dependent manner up to 12 h. Mechanistic studies showed an involvement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage through activation of upstream caspase-8 and -9. Further, the treatment of cells with alpha-santalol also led to disruption of the mitochondrial membrane potential and cytochrome c release into the cytosol, thereby implicating the involvement of the mitochondrial pathway. Pre-treatment of cells with caspase-8 or -9 inhibitor, pan caspase inhibitor or cycloheximide totally blocked alpha-santalol-caused caspase-3 activity and cleavage, but only partially reversed apoptotic cell death. This suggests involvement of both caspase-dependent and -independent pathways, at least under caspase inhibiting conditions, in alpha-santalol-caused apoptosis. Together, this study for the first time identifies the apoptotic effect of alpha-santalol, and defines the mechanism of apoptotic cascade activated by this agent in A431 cells, which might be contributing to its overall cancer preventive efficacy in mouse skin cancer models.
α-檀香醇是檀香油的一种活性成分，近年来在皮肤癌发生的小鼠模型中，对其皮肤癌预防功效进行了详细研究；然而，其功效机制尚未明确。导致转化/起始细胞克隆性扩增形成肿瘤的两个主要生物学事件是生长失控和凋亡性死亡缺失。因此，在本研究中，我们使用人表皮样癌A431细胞，评估α-檀香醇是否通过凋亡导致细胞生长抑制和/或细胞死亡。用浓度为25 - 75微摩尔的α-檀香醇处理细胞，导致细胞数量呈浓度和时间依赖性减少，这主要是由于细胞死亡。对膜联蛋白V/碘化丙啶（PI）染色细胞进行荧光激活细胞分选分析表明，α-檀香醇早在处理后3小时就诱导强烈凋亡，这种凋亡在浓度和时间依赖性方式下进一步增加，直至12小时。机制研究表明，通过上游半胱天冬酶-8和-9的激活，参与了半胱天冬酶-3的激活和聚（ADP-核糖）聚合酶的切割。此外，用α-檀香醇处理细胞还导致线粒体膜电位破坏和细胞色素c释放到细胞质中，从而暗示线粒体途径的参与。用半胱天冬酶-8或-9抑制剂、泛半胱天冬酶抑制剂或环己酰亚胺预处理细胞，完全阻断了α-檀香醇引起的半胱天冬酶-3活性和切割，但仅部分逆转凋亡性细胞死亡。这表明至少在半胱天冬酶抑制条件下，半胱天冬酶依赖性和非依赖性途径都参与了α-檀香醇引起的凋亡。总之，本研究首次确定了α-檀香醇的凋亡作用，并确定了该药物在A431细胞中激活凋亡级联反应的机制，这可能有助于其在小鼠皮肤癌模型中的总体癌症预防功效。
