Abstract：
Cucurbitacin B (CuB) is a compound found in plants like Cucurbitaceae that has shown promise in fighting cancer, particularly in lung cancer. However, the specific impact of CuB on ferroptosis and how it works in lung cancer cells has not been fully understood. Our research has discovered that CuB can effectively slow down the growth of non-small cell lung cancer (NSCLC) cells. Even in small amounts, it was able to inhibit the growth of various NSCLC cell lines. This inhibitory effect was reversed when ferroptosis inhibitors DFO, Lip-1 and Fer-1 were introduced. CuB was found to increase the levels of reactive oxygen species (ROS), lipid ROS, MDA, and ferrous ions within H358 lung cancer cells, leading to a decrease in GSH, mitochondrial membrane potential (MMP) and changes in ferroptosis-related proteins in a dose-dependent manner. These findings were also confirmed in A549 lung cancer cells. In A549 cells, different concentrations of CuB induced the accumulation of intracellular lipid ROS, ferrous ions and changes in ferroptosis-related indicators in a concentration-dependent manner. Meanwhile, the cytotoxic effect induced by CuB in A549 cells was counteracted by ferroptosis inhibitors DFO and Fer-1. Through network pharmacology, we identified potential targets related to ferroptosis in NSCLC cells treated with CuB, with STAT3 targets showing high scores. Further experiments using molecular docking and cell thermal shift assay (CETSA) revealed that CuB interacts with the STAT3 protein. Western blot and immunofluorescence staining demonstrated that CuB inhibits the phosphorylation of STAT3 (P-STAT3) in H358 cells. Silencing STAT3 enhanced CuB-induced accumulation of lipid ROS and iron ions, as well as the expression of ferroptosis-related proteins. On the other hand, overexpression of STAT3 reversed the effects of CuB-induced ferroptosis. The results indicate that CuB has the capability to suppress STAT3 activation, resulting in ferroptosis, and could be a promising treatment choice for NSCLC.
摘要：
葫芦素B（CuB）是在南瓜科植物中发现的一种化合物，在对抗癌症方面显示出希望，尤其是肺癌。然而,CuB对铁凋亡的具体影响及其在肺癌细胞中的作用尚未完全了解.我们的研究发现，CuB可以有效减缓非小细胞肺癌（NSCLC）细胞的生长。即使是少量的，它能够抑制各种NSCLC细胞系的生长。当铁凋亡抑制剂DFO时，这种抑制作用被逆转，介绍了Lip-1和Fer-1。发现CuB会增加活性氧（ROS）的水平，脂质ROS，MDA,和H358肺癌细胞中的亚铁离子，导致GSH的减少，线粒体膜电位（MMP）和铁凋亡相关蛋白的变化呈剂量依赖性。这些发现也在A549肺癌细胞中得到证实。在A549细胞中，不同浓度的CuB诱导细胞内脂质ROS的积累,亚铁离子和铁沉积相关指标的变化呈浓度依赖性。同时,CuB在A549细胞中诱导的细胞毒性作用被铁凋亡抑制剂DFO和Fer-1抵消。通过网络药理学，我们确定了用CuB处理的NSCLC细胞中与铁凋亡相关的潜在靶标，STAT3目标显示高分。使用分子对接和细胞热移位测定(CETSA)的进一步实验揭示CuB与STAT3蛋白相互作用。Westernblot和免疫荧光染色显示CuB抑制H358细胞STAT3(P-STAT3)的磷酸化。沉默STAT3增强了CuB诱导的脂质ROS和铁离子的积累，以及铁凋亡相关蛋白的表达。另一方面,STAT3的过表达逆转了CuB诱导的铁凋亡的作用。结果表明，CuB具有抑制STAT3激活的能力，导致铁性凋亡，并且可能是NSCLC的有希望的治疗选择。
