Ibrahim Mohamed Yousif, Hashim Najihah Mohd, Mohan Syam, Abdulla Mahmood Ameen, Kamalidehghan Behnam, Ghaderian Mostafa, Dehghan Firouzeh, Ali Landa Zeenelabdin, Arbab Ismail Adam, Yahayu Maizatulakmal, Lian Gwendoline Ee Cheng, Ahmadipour Fatemeh, Ali Hapipah Mohd
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Medical Research Centre, Jazan University, Jazan, Saudi Arabia.
Drug Des Devel Ther. 2014 Sep 27;8:1629-47. doi: 10.2147/DDDT.S66105. eCollection 2014.
Cratoxylum arborescens is an equatorial plant belonging to the family Guttiferae. In the current study, α-Mangostin (AM) was isolated and its cell death mechanism was studied. HCS was undertaken to detect the nuclear condensation, mitochondrial membrane potential, cell permeability, and the release of cytochrome c. An investigation for reactive oxygen species formation was conducted using fluorescent analysis. To determine the mechanism of cell death, human apoptosis proteome profiler assay was conducted. In addition, using immunofluorescence and immunoblotting, the levels of Bcl-2-associated X protein (Bax) and B-cell lymphoma (Bcl)-2 proteins were also tested. Caspaces such as 3/7, 8, and 9 were assessed during treatment. Using HCS and Western blot, the contribution of nuclear factor kappa-B (NF-κB) was investigated. AM had showed a selective cytotoxicity toward the cancer cells with no toxicity toward the normal cells even at 30 μg/mL, thereby indicating that AM has the attributes to induce cell death in tumor cells. The treatment of MCF-7 cells with AM prompted apoptosis with cell death-transducing signals. This regulated the mitochondrial membrane potential by down-regulation of Bcl-2 and up-regulation of Bax, thereby causing the release of cytochrome c from the mitochondria into the cytosol. The liberation of cytochrome c activated caspace-9, which, in turn, activated the downstream executioner caspace-3/7 with the cleaved poly (ADP-ribose) polymerase protein, thereby leading to apoptotic alterations. Increase of caspace 8 had showed the involvement of an extrinsic pathway. This type of apoptosis was suggested to occur through both extrinsic and intrinsic pathways and prevention of translocation of NF-κB from the cytoplasm to the nucleus. Our results revealed AM prompt apoptosis of MCF-7 cells through NF-κB, Bax/Bcl-2 and heat shock protein 70 modulation with the contribution of caspaces. Moreover, ingestion of AM at (30 and 60 mg/kg) significantly reduced tumor size in an animal model of breast cancer. Our results suggest that AM is a potentially useful agent for the treatment of breast cancer.
毛叶黄牛木是一种属于金丝桃科的赤道植物。在本研究中，分离出了α-山竹黄酮（AM）并研究了其细胞死亡机制。采用高内涵细胞成像技术（HCS）检测核浓缩、线粒体膜电位、细胞通透性以及细胞色素c的释放。使用荧光分析对活性氧的形成进行了研究。为了确定细胞死亡机制，进行了人类凋亡蛋白质组分析。此外，通过免疫荧光和免疫印迹法，还检测了Bcl-2相关X蛋白（Bax）和B细胞淋巴瘤（Bcl）-2蛋白的水平。在处理过程中评估了半胱天冬酶如3/7、8和9的活性。使用HCS和蛋白质免疫印迹法，研究了核因子κB（NF-κB）的作用。AM对癌细胞表现出选择性细胞毒性，即使在30μg/mL时对正常细胞也无毒性，这表明AM具有诱导肿瘤细胞死亡的特性。用AM处理MCF-7细胞可通过细胞死亡转导信号促使细胞凋亡。这通过下调Bcl-2和上调Bax来调节线粒体膜电位，从而导致细胞色素c从线粒体释放到细胞质中。细胞色素c的释放激活了半胱天冬酶-9，进而通过裂解的聚（ADP-核糖）聚合酶蛋白激活下游的执行性半胱天冬酶-3/7，从而导致凋亡改变。半胱天冬酶8的增加表明存在外源性途径。这种类型的凋亡被认为是通过外源性和内源性途径发生的，并且阻止了NF-κB从细胞质向细胞核的转位。我们的结果显示，AM通过NF-κB、Bax/Bcl-2和热休克蛋白70的调节以及半胱天冬酶的作用促使MCF-7细胞凋亡。此外，在乳腺癌动物模型中，摄入（30和60mg/kg）的AM可显著减小肿瘤大小。我们的结果表明，AM是一种治疗乳腺癌的潜在有效药物。
