Wong Charng Choon, Lim Siang Hui, Sagineedu Sreenivasa Rao, Lajis Nordin Haji, Stanslas Johnson
Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Pharmacol Res. 2016 May;107:66-78. doi: 10.1016/j.phrs.2016.02.024. Epub 2016 Mar 3.
SRJ09 (3,19-(2-bromobenzylidene)andrographolide), a semisynthetic andrographolide (AGP) derivative, was shown to induce G1 cell cycle arrest and eventually apoptosis in breast and colon cancer cell lines. The present investigation was carried out to elucidate the mechanisms cell cycle arrest and apoptosis and evaluate the in vivo antitumor activity of SRJ09. The in vitro growth inhibitory properties of compounds were assessed in colon (HCT-116) and breast (MCF-7) cancer cell lines. Immunoblotting was utilized to quantitate the protein levels in cells. The gene expressions were determined using reverse transcriptase PCR (RT-PCR). Pharmacokinetic investigation was carried out by determining SRJ09 levels in plasma of Balb/C mice using HPLC. In vivo antitumor activity was evaluated in athymic mice carrying HCT-116 colon tumor xenografts. SRJ09 displayed improved in vitro activity when compared with AGP by producing rapid cell killing effect in vitro. Its activity was not compromised in MES-SA/Dx5 multidrug resistant (MDR) cells expressing p-glycoprotein. Cells treated with SRJ09 (0.1-10μM) displayed increased p21 protein level, which corresponded with gene expression. Whereas CDK4 protein level and gene expression was suppressed. The treatment did not affect cyclin D1. Changes of these proteins paralleled G1 cell cycle arrest in both cell lines as determined by flow cytometry. Induction of apoptosis by SRJ09 in HCT-116 cells which occurred independent of p53 and bcl-2 was inhibited in the presence of caspase 8 inhibitor, implicating the extrinsic apoptotic pathway. A single dose (100mg/kg, i.p) of SRJ09 produced a plasma concentration range of 12-30.4μM. At 400mg/kg (q4dX3), it significantly retarded growth of tumor xenografts. The antitumor activity of SRJ09 is suggested mediated via the induction of p21 expression and suppression of CDK-4 expression without affecting cyclin D1 to trigger G1 arrest leading to apoptosis.
SRJ09（3,19 -（2 - 溴亚苄基）穿心莲内酯）是一种半合成的穿心莲内酯（AGP）衍生物，已证明它能诱导乳腺癌和结肠癌细胞系出现G1期细胞周期停滞并最终导致细胞凋亡。本研究旨在阐明细胞周期停滞和细胞凋亡的机制，并评估SRJ09的体内抗肿瘤活性。在结肠（HCT - 116）和乳腺（MCF - 7）癌细胞系中评估了化合物的体外生长抑制特性。利用免疫印迹法定量细胞中的蛋白质水平。使用逆转录聚合酶链反应（RT - PCR）测定基因表达。通过用高效液相色谱法测定Balb/C小鼠血浆中的SRJ09水平进行药代动力学研究。在携带HCT - 116结肠肿瘤异种移植物的无胸腺小鼠中评估体内抗肿瘤活性。与AGP相比，SRJ09在体外通过产生快速的细胞杀伤作用表现出更好的活性。其活性在表达P - 糖蛋白的MES - SA/Dx5多药耐药（MDR）细胞中未受影响。用SRJ09（0.1 - 10μM）处理的细胞显示p21蛋白水平升高，这与基因表达相对应。而CDK4蛋白水平和基因表达受到抑制。该处理不影响细胞周期蛋白D1。如通过流式细胞术所确定的，这些蛋白质的变化与两种细胞系中的G1期细胞周期停滞平行。在存在半胱天冬酶8抑制剂的情况下，SRJ09在HCT - 116细胞中诱导的不依赖于p53和bcl - 2的细胞凋亡受到抑制，这表明涉及外源性凋亡途径。单剂量（100mg/kg，腹腔注射）的SRJ09产生的血浆浓度范围为12 - 30.4μM。在400mg/kg（每4天一次，共3次）时，它显著抑制肿瘤异种移植物的生长。SRJ09的抗肿瘤活性表明是通过诱导p21表达和抑制CDK - 4表达介导的，而不影响细胞周期蛋白D1以触发G1期停滞从而导致细胞凋亡。
