Ananda Sadagopan Suresh Kumar, Mohebali Nooshin, Looi Chung Yeng, Hasanpourghadi Mohadeseh, Pandurangan Ashok Kumar, Arya Aditya, Karimian Hamed, Mustafa Mohd Rais
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia.
Department of Biochemistry, Central Leather Research Institute, Council of Scientific and Industrial Research (CSIR), Adyar, Chennai, 600 020, India.
J Exp Clin Cancer Res. 2015 Dec 8;34:147. doi: 10.1186/s13046-015-0266-y.
Natural compounds have been demonstrated to lower breast cancer risk and sensitize tumor cells to anticancer therapies. Recently, we demonstrated that vernodalin (the active constituent of the medicinal herb Centratherum anthelminticum seeds) induces apoptosis in breast cancer cell-lines. The aim of this work was to gain an insight into the underlying anticancer mechanism of vernodalin using in vitro and in vivo model.
Vernodalin was isolated through the bioassay guided fractionation from the seeds. The protein expression of p-Akt, PI3K, FOXO3a, Bim, p27kip1, cyclinD1, and cyclinE was examined by the Western blot analysis. Immunoprecipitation assays were performed to analyse Akt kinase activity. Small interfering RNA (siRNA) was used to study the role of FOXO3a upregulation and their targets during vernodalin treatment. Immunofluorescence, subcellular localisation of FOXO3a by Western blot was performed to analyse FOXO3a localisation in nucleus of breast cancer cells. Immunohistochemical analysis of PCNA, Ki67, p27kip1, FOXO3a and p-FOXO3a in the LA7-induced mammary gland tumor model was performed.
Our results showed that vernodalin regulates cancer cell apoptosis through activation of FOXO transcription factors and its downstream targets (Bim, p27Kip1, p21Waf1/cip1, cyclin D1, cyclin E) as examined by Western blots. Furthermore, we showed that FOXO3a/PI3K-Akt played a significant role in vernodalin induced apoptosis in breast cancer cells. Immunoprecipitation assays showed Akt kinase activity was downregulated. Immunofluorescence, subcellular fractionation and Western blot showed FOXO3a accumulation in the nucleus of breast cancer cells after vernodalin treatment. Silencing of FOXO3a protected breast cancer cells against vernodalin induced apoptosis. The anti-tumor action of vernodalin was further confirmed by examining cell proliferative markers, PCNA and Ki67 in the LA7-induced mammary gland tumor model. We also corroborated our findings in vivo by showing upregulation of p27Kip1, FOXO3a and decrease in the p-FOXO3a level in vernodalin-treated breast tumor tissue.
Our results suggest that PI3K-Akt/FOXOa pathway is a critical mediator of vernodalin-induced cytotoxicity and this compound could be further developed as a potential chemopreventive or chemotherapeutic agent for breast cancer therapy.
天然化合物已被证明可降低乳腺癌风险，并使肿瘤细胞对抗癌疗法敏感。最近，我们证明了斑鸠菊素（药用植物驱虫斑鸠菊种子的活性成分）可诱导乳腺癌细胞系凋亡。这项工作的目的是使用体外和体内模型深入了解斑鸠菊素潜在的抗癌机制。
通过生物测定引导的分级分离从种子中分离出斑鸠菊素。通过蛋白质印迹分析检测p-Akt、PI3K、FOXO3a、Bim、p27kip1、细胞周期蛋白D1和细胞周期蛋白E的蛋白表达。进行免疫沉淀试验以分析Akt激酶活性。使用小干扰RNA（siRNA）研究FOXO3a上调及其在斑鸠菊素治疗期间的靶标的作用。通过蛋白质印迹进行免疫荧光、FOXO3a的亚细胞定位，以分析FOXO3a在乳腺癌细胞核中的定位。对LA7诱导的乳腺肿瘤模型中的PCNA、Ki67、p27kip1、FOXO3a和p-FOXO3a进行免疫组织化学分析。
我们的结果表明，如蛋白质印迹检测所示，斑鸠菊素通过激活FOXO转录因子及其下游靶标（Bim、p27Kip1、p21Waf1/cip1、细胞周期蛋白D1、细胞周期蛋白E）来调节癌细胞凋亡。此外，我们表明FOXO3a/PI3K-Akt在斑鸠菊素诱导的乳腺癌细胞凋亡中起重要作用。免疫沉淀试验表明Akt激酶活性下调。免疫荧光、亚细胞分级分离和蛋白质印迹显示斑鸠菊素处理后FOXO3a在乳腺癌细胞核中积累。FOXO3a的沉默保护乳腺癌细胞免受斑鸠菊素诱导的凋亡。通过检测LA7诱导的乳腺肿瘤模型中的细胞增殖标志物PCNA和Ki67，进一步证实了斑鸠菊素的抗肿瘤作用。我们还通过显示斑鸠菊素处理的乳腺肿瘤组织中p27Kip1、FOXO3a的上调和p-FOXO3a水平的降低，在体内证实了我们的发现。
我们的结果表明PI3K-Akt/FOXOa途径是斑鸠菊素诱导的细胞毒性的关键介质，这种化合物可进一步开发为乳腺癌治疗的潜在化学预防或化学治疗剂。
