Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

中文翻译：

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活性氧的产生和线粒体功能障碍，通过环二肽Cyclo（-Pro-Tyr）启动人肝细胞癌HepG2细胞的凋亡。

考虑到其重要的生物学和药理活性，环状二肽越来越重要。这项研究的目的是调查从海洋海绵Callyspongia fistularis共生细菌短小芽孢杆菌AMK1鉴定出的二肽Cyclo（-Pro-Tyr）（DP）的抗癌活性及其在肝癌HepG2细胞系中的潜在凋亡机制。进行MTT分析以证明DP在HepG2细胞和小鼠成纤维细胞McCoy细胞中的细胞毒作用。最初，使用碘化丙啶（PI）和a啶橙/溴化乙锭（AO / EB）双重染色鉴定DP的凋亡诱导活性，然后通过DNA片段化分析和凋亡相关标记Bax，Bcl-2的蛋白质印迹分析进行确认，细胞色素c，caspase-3和裂解的聚（ADP-核糖）聚合酶（PARP）。进行了若丹明123染色以观察DP对线粒体膜电位（MMP）的影响，并进行DCFH-DA（二氯二氢荧光素二乙酸酯）染色以测量细胞内活性氧（ROS）的水平。MTT结果表明，DP在HepG2细胞中启动了剂量依赖性细胞毒性，但在以指定浓度用DP处理的小鼠成纤维细胞McCoy细胞中却没有明显的毒性。DP诱导凋亡，其通过PI和AO / EB双重染色以及DNA片段化的凋亡小体的出现得以证实。DP显着提高了Bax / Bcl-2比率，破坏了线粒体膜电位（MMP），增强了线粒体细胞色素c的释放，增加了caspase-3的活化，PARP的裂解和细胞内活性氧（ROS）水平的提高。除此以外，DP成功抑制PI3K，AKT的磷酸化并增加PTEN表达。这些结果表明DP可能通过线粒体功能障碍启动凋亡并下调HepG2细胞中的PI3K / Akt信号通路而对正常成纤维细胞无毒性作用，从而具有抗癌作用。因此，DP可被开发为治疗肝细胞癌的潜在替代治疗剂。