Cisplatin-based chemotherapy is the standard regimen for bladder cancer patients, but its effectiveness is limited by high toxicity and the development of drug resistance. It has been reported in many studies that Cucurbitacin B has anti-carcinogenic effects by stimulating apoptosis and autophagy. Here we explored the potential role of cucurbitacin B on MB49 bladder syngeneic mouse tumor model. Single and combined doses of cucurbitacin B and cisplatin were applied to MB49 cell line and the cell viability was determined by Water‐Soluble Tetrazolium Salt‐1 (WST) method. After developing the tumor model, mice were randomly divided into four groups and then cucurbitacin B and cisplatin applied in the specified doses and time. The expression levels of apoptosis (Bcl-2, Bax, Caspase-3, cleaved Caspase-3) and autophagy proteins (Beclin-1 and LC3I, LC3II) were detected by Western Blot. Phospho-protein array analysis was performed to determine the relative levels of phosphorylation of proteins which are associated with the PI3K-Akt signaling pathway. Tumor tissues were analyzed by hematoxylin-eosin staining. In the present study, the results showed that cucurbitacin B inhibited the expression of Bcl-2 and increased the expression of Bax and cleaved Caspase 3. LC3II is markedly up-regulated in cucurbitacin B-treated cells. Cucurbitacin B reduced the phosphorylation of p27, PRAS40, and Raf-1 proteins. CuB + Cis combination synergistically decreased phosphorylation of AKT, ERK1/ERK2, mTOR, BAD levels and increased the level of AMPKα. PI3K/AKT/ mTOR pathway might be one of the targets of cucurbitacin B in MB49 bladder cancer mouse model. CuB + Cis combination reduced the tumor growth. Cucurbitacin B has no toxic effects on lung, liver, kidney, heart, and bladder. Indeed, cucurbitacin B can inhibit the tumor proliferation; induce caspase-dependent/-independent apoptosis and autophagy. Our study provided a novel perspective to research the effects of cucurbitacin B on the apoptotic and autophagic pathways in bladder cancer and a new target class for drug development.

中文翻译：

---

葫芦素B和顺铂诱导MB49小鼠膀胱癌模型中的细胞死亡途径。

基于顺铂的化疗是膀胱癌患者的标准治疗方案，但其有效性受到高毒性和耐药性发展的限制。在许多研究中已经报道，葫芦素B通过刺激细胞凋亡和自噬而具有抗癌作用。在这里，我们探讨了葫芦素B在MB49膀胱同基因小鼠肿瘤模型中的潜在作用。将单剂量和联合剂量的葫芦素B和顺铂应用于MB49细胞系，并通过水溶性四唑盐-1（WST）方法测定细胞活力。建立肿瘤模型后，将小鼠随机分为四组，然后以指定的剂量和时间应用葫芦素B和顺铂。凋亡（Bcl-2，Bax，Caspase-3，裂解的Caspase-3）和自噬蛋白（Beclin-1和LC3I，通过Western Blot检测LC3II）。进行磷酸蛋白阵列分析以确定与PI3K-Akt信号传导途径相关的蛋白的磷酸化的相对水平。通过苏木精-伊红染色分析肿瘤组织。在本研究中，结果显示葫芦素B抑制Bcl-2的表达并增加Bax的表达和Caspase 3的裂解。在葫芦素B处理的细胞中，LC3II明显上调。葫芦素B减少了p27，PRAS40和Raf-1蛋白的磷酸化。CuB + Cis组合协同降低AKT，ERK1 / ERK2，mTOR，BAD水平的磷酸化，并增加AMPKα的水平。PI3K / AKT / mTOR通路可能是葫芦素B在MB49膀胱癌小鼠模型中的靶标之一。CuB + Cis组合减少了肿瘤的生长。葫芦素B对肺，肝，肾，心脏和膀胱无毒性作用。的确，葫芦素B可以抑制肿瘤的扩散。诱导胱天蛋白酶依赖性/非依赖性凋亡和自噬。我们的研究为研究葫芦素B对膀胱癌细胞凋亡和自噬途径的影响提供了新的视角，并为药物开发提供了新的靶标类别。