Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, 510405, Guangdong, People's Republic of China.
J Ethnopharmacol. 2022 Aug 10;294:115353. doi: 10.1016/j.jep.2022.115353. Epub 2022 May 6.
Marsdenia tenacissima is a medicinal plant, used as a raw material for cancer treatment in China. In our previous studies, 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (MT2), the main steroid aglycone isolated from M. tenacissima, was found to significantly enhance the antitumor activity of paclitaxel (PTX) in vivo. However, it is unclear whether MT2 reverses multidrug resistance (MDR) in tumors.
To determine the role and mechanism of MT2 in reversing tumor MDR.
MDR cell line HeLa/Tax was established from the human cervical carcinoma cell line HeLa by long-term exposure to subtoxic concentrations of PTX and was used to evaluate the ability of MT2 to restore chemosensitivity of cells both in vitro and in a nude mouse model. The expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) was determined using western blotting and immunohistochemistry. The substrate transport function was assessed using an MDR function assay kit. The binding modes of MT2 and P-gp were determined using the conformation-sensitive anti-P-gp antibodies. The permeability and transport properties of MT2 were analyzed in Caco-2 cell monolayers.
Compared to parental cells, HeLa/Tax cells overexpress P-gp and MRP2 and are approximately 100-360 fold more resistant to the anticancer drugs PTX, docetaxel, and vinblastine. MT2 at 5 or 10 μmol/L significantly increased the sensitivity of HeLa/Tax to these three anticancer drugs (18-56-fold decrease in IC value) and suppressed the expression of P-gp and MRP2. Knockdown of P-gp with small interfering RNA partially reversed MT2-induced sensitivity to PTX in HeLa/Tax cells. Moreover, MT2 directly inhibited P-gp-mediated substrate transport while interacting with membrane P-gp in non-substrate ways. MT2 was highly permeable and could not be transported in the Caco-2 cell monolayers. In nude mice bearing HeLa/Tax xenografts, the combination treatment with MT2 and PTX exerted a synergistic inhibitory effect on the growth of tumors and the expression of P-gp and MRP2 without increasing toxicity.
MT2 is a potential agent for reversing MDR. It impedes membrane drug efflux pumps by suppressing P-gp and MRP2 expression, and directly inhibiting the transport function of P-gp.
密花藤是一种药用植物，在中国被用作治疗癌症的原料。在我们之前的研究中，从密花藤中分离得到的主要甾体苷元 11α-O-2-甲基丁酰基-12β-O-齐墩果酸-tenacigenin B（MT2），被发现能显著增强紫杉醇（PTX）在体内的抗肿瘤活性。然而，目前尚不清楚 MT2 是否能逆转肿瘤的多药耐药性（MDR）。
确定 MT2 在逆转肿瘤 MDR 中的作用和机制。
通过长期暴露于亚毒性浓度的 PTX，从人宫颈癌细胞系 HeLa 中建立多药耐药细胞系 HeLa/Tax，并用于评估 MT2 在体外和裸鼠模型中恢复细胞化学敏感性的能力。采用 Western blot 和免疫组化法测定 P-糖蛋白（P-gp）和多药耐药相关蛋白 2（MRP2）的表达。采用 MDR 功能测定试剂盒评估底物转运功能。采用构象敏感抗 P-gp 抗体测定 MT2 与 P-gp 的结合模式。在 Caco-2 细胞单层中分析 MT2 的通透性和转运特性。
与亲本细胞相比，HeLa/Tax 细胞过度表达 P-gp 和 MRP2，对抗癌药物紫杉醇、多西他赛和长春碱的耐药性约为 100-360 倍。5 或 10μmol/L 的 MT2 显著增加了 HeLa/Tax 对这三种抗癌药物的敏感性（IC 值降低 18-56 倍），并抑制了 P-gp 和 MRP2 的表达。用小干扰 RNA 敲低 P-gp 可部分逆转 MT2 诱导的 HeLa/Tax 细胞对 PTX 的敏感性。此外，MT2 直接抑制 P-gp 介导的底物转运，同时以非底物方式与膜 P-gp 相互作用。MT2 具有高通透性，不能在 Caco-2 细胞单层中转运。在荷 HeLa/Tax 异种移植瘤的裸鼠中，MT2 与 PTX 联合治疗对肿瘤生长和 P-gp、MRP2 的表达具有协同抑制作用，而不增加毒性。
MT2 是一种有潜力的逆转 MDR 的药物。它通过抑制 P-gp 和 MRP2 的表达来阻碍膜药物外排泵，并直接抑制 P-gp 的转运功能。
