Gomes Antony, Giri Biplab, Kole Labanyamoy, Saha Archita, Debnath Anindita, Gomes Aparna
Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700 009, India.
Toxicon. 2007 Nov;50(6):835-49. doi: 10.1016/j.toxicon.2007.06.018. Epub 2007 Jun 30.
In our earlier communication, it was reported that Indian toad (Bufo melanostictus) skin extract (TSE) possesses antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4), 388-400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2 cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA). Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation. The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate expression of p21(Cip1)/p27(Kip1) due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21(Cip1) and p27(Kip1) and it was likely to be linked with cell cycle arrest at G1 phase in cancer cells. From the present study, it may be suggested that the crystalline compound, BM-ANF1, was antiproliferative and apoptogenic in human leukemic and hepatoma cells.
在我们早期的通讯中，据报道印度蟾蜍（黑眶蟾蜍）皮肤提取物（TSE）在U937和K562细胞中具有抗增殖和诱导凋亡活性[Giri等人，2006年。印度蟾蜍（黑眶蟾蜍，施奈德）皮肤提取物在U937和K562细胞中的抗增殖、细胞毒性和诱导凋亡活性。毒理学48（4），388 - 400]。在本研究中，一种化合物（BM - ANF1）已通过氧化铝凝胶柱色谱从TSE中分离出来，结晶并评估其在U937、K562和HepG2细胞中的抗增殖和诱导凋亡活性。BM - ANF1对U937、K562和HepG2细胞生长产生剂量依赖性抑制。MTT法反映了其抗增殖活性，增殖细胞核抗原（PCNA）表达降低证明了这一点。流式细胞术分析表明，BM - ANF1使细胞周期停滞在G1期，并增强了U937和K562细胞中膜联蛋白 - V的结合。对U937和K562细胞的扫描电子显微镜和荧光显微镜分析揭示了该化合物的诱导凋亡性质。碱性彗星试验表明BM - ANF1产生了DNA片段化。半胱天冬酶3的剂量依赖性表达表明BM - ANF1的诱导凋亡特性是通过癌细胞中下游效应核酸酶的激活介导的。在HepG2细胞中，由于BM - ANF1处理导致p53表达增加和p21（Cip1）/p27（Kip1）适度表达，支持了BM - ANF1的诱导凋亡活性是通过p53肿瘤抑制基因表达，随后是p21（Cip1）和p27（Kip1）的表达介导的，并且它可能与癌细胞在G1期的细胞周期停滞有关。从本研究中可以推测，结晶化合物BM - ANF1在人白血病细胞和肝癌细胞中具有抗增殖和诱导凋亡作用。
