Kumar Saurav, Das Bishnu, Maurya Garima, Dey Shreya, Gupta Parna, Sarma Jayasri Das
Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, 741246, India.
Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, 741246, India.
BMC Cancer. 2025 Feb 25;25(1):334. doi: 10.1186/s12885-025-13601-6.
The existing anticancer drugs in clinical practice show poor efficacy in cervical cancer patients and are associated with multiple side effects. Our previous study demonstrated the strong antineoplastic activity of crude extract prepared from the stem bark of Azadirachta indica (Neem) against cervical cancer. However, the active phytoconstituents of neem stem bark extract and its underlying anticancer mechanism are yet to be investigated. Thus, the present study aimed to identify the active fraction from crude neem stem bark extract to further dissect its anticancer mechanism and determine the active components.
Dichloromethane (DCM) extract from neem stem bark was prepared and fractionated using thin-layer chromatography. The fractions obtained were screened against HeLa and ME-180 cervical cancer cell lines to identify the most active fraction, which was then selected for further studies. Clonogenic assay, cell cycle analysis, apoptosis assay, and reactive oxygen species (ROS) assay were performed to determine the cytotoxicity of the active fraction. Gene expression was analyzed using real-time PCR and western blot to determine the mechanism. Additionally, the HeLa cells-derived 3D spheroid model was used to determine the antitumor efficacy of the active fraction. Electrospray ionization-mass spectrometry, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance were used to identify the phytoconstituents of the fraction.
Initial screening revealed fraction 2 (F2) as the most active fraction. Additionally, F2 showed the least cytotoxic effect on normal human fibroblast cells. Mechanistically, F2 induced cell cycle arrest and apoptosis in cervical cancer cells. F2 increased ROS levels, induced ER stress, and activated cell survival pathway. Treatment with N-acetyl cysteine revealed that F2 induced ROS-independent ER stress and apoptosis. 3D spheroid viability and growth delay experiments demonstrated the strong antitumor potential of F2. Finally, six compounds, including one flavonoid (nicotiflorin) and five limonoids, were identified in the F2 fraction.
This is the first study to identify the active fraction and its phytoconstituents from neem stem bark and demonstrate the anticancer mechanism against cervical cancer. Our study highlights the importance of investigating neem stem bark-derived limonoids and nicotiflorin as a potential source to develop new anticancer therapeutic agents.
临床实践中现有的抗癌药物对宫颈癌患者疗效不佳，且伴有多种副作用。我们之前的研究表明，印楝（Neem）茎皮粗提物对宫颈癌具有很强的抗肿瘤活性。然而，印楝茎皮提取物的活性植物成分及其潜在的抗癌机制尚待研究。因此，本研究旨在从印楝茎皮粗提物中鉴定出活性成分，进一步剖析其抗癌机制并确定活性成分。
制备印楝茎皮的二氯甲烷（DCM）提取物，并通过薄层色谱法进行分离。将得到的馏分针对HeLa和ME - 180宫颈癌细胞系进行筛选，以确定最具活性的馏分，然后选择该馏分进行进一步研究。进行克隆形成试验、细胞周期分析、凋亡试验和活性氧（ROS）试验，以确定活性馏分的细胞毒性。使用实时PCR和蛋白质免疫印迹法分析基因表达，以确定其作用机制。此外，使用源自HeLa细胞的3D球体模型来确定活性馏分的抗肿瘤疗效。采用电喷雾电离质谱、傅里叶变换红外光谱和质子核磁共振来鉴定该馏分的植物成分。
初步筛选显示馏分2（F2）是最具活性的馏分。此外，F2对正常人成纤维细胞的细胞毒性最小。从机制上讲，F2诱导宫颈癌细胞的细胞周期停滞和凋亡。F2增加了ROS水平，诱导了内质网应激，并激活了细胞存活途径。用N - 乙酰半胱氨酸处理表明，F2诱导了不依赖ROS的内质网应激和凋亡。3D球体活力和生长延迟实验证明了F2具有很强的抗肿瘤潜力。最后，在F2馏分中鉴定出六种化合物，包括一种黄酮类化合物（烟花苷）和五种柠檬苦素类化合物。
这是第一项从印楝茎皮中鉴定出活性馏分及其植物成分，并证明其对宫颈癌抗癌机制的研究。我们的研究强调了研究印楝茎皮衍生的柠檬苦素类化合物和烟花苷作为开发新型抗癌治疗药物潜在来源的重要性。
