Unfolding the apoptotic mechanism of antioxidant enriched-leaves of Tabebuia pallida (lindl.) miers in EAC cells and mouse model
Unfolding the apoptotic mechanism of antioxidant enriched-leaves of Tabebuia pallida (lindl.) miers in EAC cells and mouse model
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DOI：
10.1016/j.jep.2021.114297
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发表时间：
2021-06-28
影响因子：
5.4
通讯作者：
Alam, A. H. M. Khurshid
中科院分区：
文献类型：
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作者：
Rahman, Md Mahbubur;Reza, A. S. M. Ali;Alam, A. H. M. Khurshid
民族药理学相关性：苍白药（Lindl.）苍叶草（T. pallida）是一种著名的加勒比本土药用植物。苍白树的叶子和树皮以草药或药茶的形式被用作传统药物，以治疗癌症，发烧和疼痛。此外，苍白树叶提取物还具有抗肿瘤活性。然而，白桦叶（TPL）、茎皮（TPSB）、根皮（TPRB）和花（TPF）的化学特征和抗癌活性机制尚不清楚。研究目的：采用Ehrlich腹水癌（EAC）培养细胞和EAC小鼠模型，探讨苍蝇子对细胞凋亡的调控作用。采用LC-ESI-MS/MS对苍桦提取物进行成分分析。材料和方法：用80%甲醇提取TPL、TPSB、TPRB和TPF的干燥和粉末。利用体外培养的EAC细胞和携带EAC的小鼠，通过评估细胞毒性、肿瘤细胞生长抑制、小鼠寿命变化、血液学和生化指标来研究其抗癌活性。显微镜下观察细胞凋亡，RT-PCR检测细胞凋亡相关基因（Bcl-2、Bcl-xL、NF - kappa-B、PARP-1、p53、Bax、caspase-3和8）的表达。采用LC-ESI-MS对活性提取物中的主要化合物进行鉴定。采用计算机辅助分析方法，从该植物的10个分离化合物中筛选出具有抗氧化和抗癌活性的最佳植物成分。结果：在EAC小鼠中，与未治疗的对照组相比，TPL提取物表现出最高的癌细胞毒性，显著抑制肿瘤细胞生长（p < 0.001），减少腹水（p < 0.01），延长寿命（p < 0.001），正常化血液参数（RBC/WBC计数），增加超氧化物歧化酶和过氧化氢酶水平。与未处理的EAC细胞相比，tpl处理的EAC细胞表现出膜泡、染色质凝聚和核断裂以及caspase-3活化的凋亡特征。此外，膜联蛋白V-FITC和碘化丙啶信号在TPL处理下显著增强，提示细胞凋亡诱导。TPL处理后的促凋亡和抗凋亡信号显示p53、Bax和PARP-1表达上调，NF kappa-B、Bcl-2和Bcl-xL表达下调，表明TPL使促凋亡和抗凋亡基因的平衡向细胞死亡方向转移。TPL的LC-ESI-MS数据显示，糖苷、lapachol和槲皮素抗氧化剂及其衍生物的混合物与癌细胞靶点显著相关。该化合物天竺葵苷-3- o -葡萄糖苷被发现在计算机辅助模型中最有效。结论：苍白草TPL提取物具有明显的抗肿瘤活性。肿瘤抑制机制是由于抗氧化酶和半胱天冬酶的激活诱导细胞凋亡，并通过促进细胞死亡的促凋亡和抗凋亡基因平衡的改变介导。
Ethnopharmacological relevance: Tabebuia pallida (Lindl.) Miers (T. pallida) is a well-known native Caribbean medicinal plant. The leaves and barks of T. pallida are used as traditional medicine in the form of herbal or medicinal tea to manage cancer, fever, and pain. Moreover, extracts from the leaves of T. pallida showed anticancer activity. However, the chemical profile and mechanism of anticancer activity of T. pallida leaves (TPL), stem bark (TPSB), root bark (TPRB) and flowers (TPF) remain unexplored.Aim of the study: The present study was designed to explore the regulation of apoptosis by T. pallida using Ehrlich Ascites Carcinoma (EAC) cultured cells and an EAC mouse model. LC-ESI-MS/MS was used for compositional analysis of T. pallida extracts.Materials and methods: Dried and powdered TPL, TPSB, TPRB and TPF were extracted with 80% methanol. Using cultured EAC cells and EAC-bearing mice with and without these extracts, anticancer activities were studied by assessing cytotoxicity and tumor cell growth inhibition, changes in life span of mice, and hematological and biochemical parameters. Apoptosis was analyzed by microscopy and expression of selected apoptosis-related genes (Bcl-2, Bcl-xL, NF kappa-B, PARP-1, p53, Bax, caspase-3 and-8) using RT-PCR. LC-ESI-MS analysis was performed to identify the major compounds from active extracts. Computer aided analyses was undertaken to sort out the best-fit phytoconstituent of total ten isolated compounds of this plant for antioxidant and anticancer activity.Results: In EAC mice compared with untreated controls, the TPL extract exhibited the highest cancer cell toxicity with significant tumor cell growth inhibition (p < 0.001), reduced ascites by body weight (p < 0.01), increased the life span (p < 0.001), normalized blood parameters (RBC/WBC counts), and increased the levels of superoxide dismutase and catalase. TPL-treated EAC cells showed increased apoptotic characteristics of membrane blebbing, chromatin condensation and nuclear fragmentation, and caspase-3 activation, compared with untreated EAC cells. Moreover, annexin V-FITC and propidium iodide signals were greatly enhanced in response to TPL treatment, indicating apoptosis induction. Pro-and anti-apoptotic signaling after TPL treatment demonstrated up-regulated p53, Bax and PARP-1, and down-regulated NF kappa-B, Bcl-2 and Bcl-xL expression, suggesting that TPL shifts the balance of pro-and anti-apoptotic genes towards cell death. LC-ESI-MS data of TPL showed a mixture of glycosides, lapachol, and quercetin antioxidant and its derivatives that were significantly linked to cancer cell targets. The compound, pelargonidin-3-O-glucoside was found to be most effective in computer aided models.Conclusions: In conclusion, the TPL extract of T. pallida possesses significant anticancer activity. The tumor suppressive mechanism is due to apoptosis induced by activation of antioxidant enzymes and caspases and mediated by a change in the balance of pro-and anti-apoptotic genes that promotes cell death.