Al-Obeed Omar, El-Obeid Adila Salih, Matou-Nasri Sabine, Vaali-Mohammed Mansoor-Ali, AlHaidan Yazeid, Elwatidy Mohammed, Al Dosary Hamad, Alehaideb Zeyad, Alkhayal Khayal, Haseeb Adil, McKerrow James, Ahmad Rehan, Abdulla Maha-Hamadien
1Colorectal Research Chair, Department of Surgery, King Khalid University Hospital and College of Medicine, King Saud University, PO Box 7805 (37), Riyadh, 11472 Saudi Arabia.
2Department of Biobank, King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, Ministry of National Guard Health Affairs, PO Box 22490, Riyadh, 11426 Saudi Arabia.
Cancer Cell Int. 2020 Apr 16;20:126. doi: 10.1186/s12935-020-01206-x. eCollection 2020.
Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Evaluation of natural product-based anticancer drugs as adjuvant treatment with fewer side effects is largely unexplored research fields. Herbal melanin (HM) is an extract of the seed coats of that modulates an inflammatory response through toll-like receptor 4 (TLR4). This TLR4 receptor is also involved in the modulation of apoptosis. We therefore explored the anticancer potential of HM and specifically its effect on the molecular mechanisms underlying adenocarcinoma and metastatic colorectal cancer (mCRC) cell death in vitro.
Cell viability was evaluated using the MTT assay. Cellular reactive oxygen species (ROS), glutathione levels, and apoptotic status were assessed using fluorometric and colorimetric detection methods. HM-induced apoptotic and other signaling pathways were investigated using Western blot technology and mitochondrial transition pore assay kit. TLR4 receptor downregulation and blockade were performed using siRNA technology and neutralizing antibody, respectively.
Our results showed that HM inhibited the proliferation of the colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Furthermore, HM enhanced ROS production and decreased glutathione levels. HM-induced apoptosis was associated with mitochondrial outer membrane permeability and cytochrome c release, inhibition of the Bcl2 family proteins, and activation of caspase-3/-7. In addition, HM modulated MAPK pathways by activating the JNK pathway and by inhibiting ERK phosphorylation. TLR4 receptor downregulation enhanced HM-induced apoptosis while TLR4 receptor blockade partially alleviated HM-inhibited ERK phosphorylation.
Altogether, these findings indicate that HM exerts pro-apoptotic effects and inhibits MAPK pathway through TLR4 in mCRC and colorectal adenocarcinoma cells, suggesting HM as a promising natural-based drug for the treatment of colorectal cancer.
结直肠癌是最致命的癌症之一，需要有效且安全的化疗。评估基于天然产物的抗癌药物作为副作用较少的辅助治疗方法，在很大程度上是未被探索的研究领域。草药黑色素（HM）是从[具体植物名称未给出]种皮中提取的，它通过Toll样受体4（TLR4）调节炎症反应。该TLR4受体也参与细胞凋亡的调节。因此，我们在体外探索了HM的抗癌潜力，特别是其对腺癌和转移性结直肠癌（mCRC）细胞死亡潜在分子机制的影响。
使用MTT法评估细胞活力。采用荧光和比色检测方法评估细胞活性氧（ROS）、谷胱甘肽水平和凋亡状态。使用蛋白质印迹技术和线粒体通透性转换孔检测试剂盒研究HM诱导的凋亡及其他信号通路。分别使用小干扰RNA（siRNA）技术和中和抗体进行TLR4受体下调和阻断。
我们的结果表明，HM抑制了结直肠腺癌HT29和mCRC SW620细胞系的增殖。此外，HM增强了ROS的产生并降低了谷胱甘肽水平。HM诱导的细胞凋亡与线粒体外膜通透性和细胞色素c释放、Bcl2家族蛋白的抑制以及半胱天冬酶-3/-7的激活有关。此外，HM通过激活JNK途径和抑制ERK磷酸化来调节丝裂原活化蛋白激酶（MAPK）途径。TLR4受体下调增强了HM诱导的细胞凋亡，而TLR4受体阻断部分减轻了HM对ERK磷酸化的抑制。
总之，这些发现表明，HM在mCRC和结直肠腺癌细胞中通过TLR4发挥促凋亡作用并抑制MAPK途径，提示HM有望成为治疗结直肠癌的天然药物。
