Shikonin inhibits migration and invasion of triple-negative breast cancer cells by suppressing epithelial-mesenchymal transition via miR-17-5p/PTEN/Akt pathway

Abstract

Background: Triple-negative breast cancer(TNBC) is a great threat to global women’s health due to its high metastatic potential. Epithelial-to-mesenchymal transition (EMT) is considered as a key event in the process of metastasis. So the pharmacological targeting of EMT might be a promising strategy in improving the therapeutic efficacy of TNBC. Here, we investigated the effect of shikonin exerting on EMT and consequently the metastasis of TNBC cells and its underlying mechanism.Methods: The invasive and migratory capacities of MDA-MB-231 cells were tested using transwell invasion and wound healing assay. MiR-17-5p expression was examined by qRT-PCR. MiR-17-5p targeted genes were predicted with different bioinformatic algorithms from four databases (TargetScan, miRanda, PITA and picTar) and further screened by KEGG pathway enrichment analysis. The differential expressions of predicted genes and their correlations with miR-17-5p were identifed in breast cancer patients based on The Cancer Genome Atlas (TCGA) database. The interaction between PTEN and miR-17-5p was analyzed by luciferase reporter assay. The overexpression vector and small interfering RNA were constructed to investigate the role PTEN played in matastasis and EMT regulation. The expressions of EMT markers, Akt and p-Akt were evaluated by western blot.Results: Shikonin inhibited the migration and invasion of MDA-MB-231 cells by suppressing EMT. Shikonin suppressed the expression of miR-17-5p, which was upregulated in breast cancer and promoted cancer cell migration, invasion and EMT. The 3’-untranslated region of PTEN was found to be direct binding target of miR-17-5p. PTEN expression increased or decreased in breast cancer cells transfected with miR-17-5p inhibitors or mimics respectively. PTEN functioned as a suppressor both in the metastasis and EMT of TNBC cells. Overexpression or knockdown of PTEN reduced or increased the Akt and p-Akt expression respectively.Conclusions: Shikonin inhibits migration and invasion of TNBC cells by suppressing EMT via miR-17-5p/PTEN/Akt pathway. This suggests shikonin as a promising therapeutic agent to counteract metastasis in the TNBC patients.

Shikonin inhibits the proliferation of cervical cancer cells via FAK/AKT/GSK3β signalling

“…Cells were cultured in serum-free DMEM medium overnight for shikonin treatment. The concentration of shikonin used was 2.5 µM, according to the concentration-related experiments and previously published studies (27,30). Following treatment, all cells were incubated with cell lysis buffer (cat.…”

Pharmacological and analytical aspects of alkannin/shikonin and their derivatives: An update from 2008 to 2022

“…It significantly reduces the expression of miR-17-5p which leads to activation of tumor suppressor gene (PTEN). However, overexpression of PTEN downregulates the Akt expression thereby inhibiting metastasis ( Bao et al, 2020 ).…”

“…Increased Ca 2+ levels leading to ER stress Colon cancer Han et al, 2019 Murine mammary cancer (4 T1) and human breast cancer cells (MDA-MB-231) Orthotopic model of murine mammary cancer cells. Activation of p38 and JNK signaling pathways; Increase caspase3/7 activity; Inhibits proliferation, migration and invasion ability of cells Breast cancer Xu et al, 2019 Human breast cancer cells (MDA-MB-231) Reduced expression of miR-17-5p and upregulation of PTEN expression with decreased levels of Akt and p-Akt leads to EMT suppression Triple negative breast cancer Bao et al, 2020 Human ovarian cancer cell (SKOV3) Downregulation of Bcl-2, AKT and PI3K whereas upregulation of Bax, Caspase-3 and Caspase-9 Ovarian cancer Zhang et al, 2020 Human melanoma (A375) and normal human liver-derived cells (MIHA) Zebrafish Tumor Model Reduced expression of STAT3 dimer. Decreased levels of Bcl-2, Mcl-1, MMP-9 and MMP-2 Malignant melanoma Cao et al, 2020 Human colorectal adenocarcinoma (SW620 and HCT116 cell lines) Subcutaneous tumor mouse model Induce autophagosome formation via LC3 cleavage; Upregulate expression and promote galectin-1 dimerization Colorectal carcinoma Zhang et al, 2020 Hepatocellular carcinoma cells lines (LM3, SMMC-7721, Huh-7, and HepG2) and a normal liver cell line (LO2) Downregulates the expression of PKM2; Increased expression of Bax, cyto C, cleaved Caspase-9, and cleaved Caspase-3, and decreased expression of Bcl-2 Hepatocellular Carcinoma Liu et al, 2020 Hepatocellular carcinoma cell lines (Huh-7 and HepG2) Modulation of the SMAD7/TGF‐β signaling pathway through regulation of miR‐106b Hepatocellular carcinoma Li & Zeng, 2020 Shikonin, acetyl shikonin, and β , β -dimethyl acryl shikonin Human breast cancer cells (MDA-MB-231) and Murine mammary cancer (4T1).…”

Shikonins as natural products from traditional Chinese medicinal plants: their biosynthesis, genetic regulation, structuralmodifications, and pharmaceutical functions

“…基于阿卡宁的结构修饰 相比于紫草宁而言，针对其对映异构体阿卡宁的结构修饰工作相对较少，主 要是我国学者报道了一些研究进展(图 7) 。为了进一步提高阿卡宁对肿瘤细胞 作用的选择性，Wang 等 [100,101] 对阿卡宁的萘醌环和支链羟基同时进行修饰，得 到了阿卡宁衍生物(4a) ，随后又获得阿卡宁肟(4b) 。为了进一步研究紫草宁/ 阿卡宁肟 DMAKO 的抗肿瘤活性靶点，Huang 等 [108] 又针对阿卡宁肟进行了一系 列乙酰化和泛素化修饰(4c)。 He 等 [93] 为了提高阿卡宁对端粒酶的结合能，对阿 卡宁的支链羟基进行糖基化修饰获得了阿卡宁糖苷(4d)。 [109,110] 或途径抑制乳腺癌、肺癌、肝癌、结肠癌、 胶质瘤等肿瘤的生长(参见表 3)。 以下分别针对全球发病率排序靠前的癌症--乳腺癌、肺癌、结肠直癌、胃癌、肝癌、甲状腺癌、胰腺癌、血癌、黑色素瘤 及骨肉瘤，简述近年来有关紫草宁抑制各类肿瘤增殖的研究进展。 4.1.1. 紫草宁对乳腺癌的抗癌作用 在抗乳腺癌增殖方面 ，研究者 [111] 发现紫草宁可以通过调节 miR-17-5p/ Phosphatase and tensin homolog(PTEN)/ protein kinase B(Akt)通路或抑制基 质金属蛋白酶(matrix metallopeptidase, MMP)2/9 [112] 的活化，阻止乳腺癌细胞 迁移和侵袭； 紫草宁还可以诱导小鼠乳腺癌细胞经 p38 依赖性途径发生凋亡 [113] ； 可诱导雌激素受体 (estrogen receptor, ER) 阳性细胞 MCF-7 发生坏死和凋亡 [114] ； 还可通过抑制丙酮酸激酶(pyruvate kinase, PK)M2 的表达，增强紫杉醇对三阴 性乳腺癌的治疗效果 [115] [116][117][118] 。另有研究报道，紫草宁以 PI3K/Akt 信号通路为靶点，能够克服阿 法替尼治疗 NSCLC 的耐药性 [119] 。 Yeh 等研究表明紫草宁通过 p53 介导的细胞凋 亡途径对 A549 肺癌细胞具有潜在的和剂量相关的细胞毒作用，为临床上治疗肺 癌提供了一种有前途的辅助化疗候选药物 [120] 。在基础的机制研究方面，紫草宁 能激活活性氧(reactive oxygen species, ROS)介导的内质网应激反应，从而使 NSCLC 凋亡 [121] 。另外，紫草宁通过抑制整合素 1 表达和抑制细胞外信号调节激 酶(Extracellular signal regulated kinase, ERK)1/2 信号通路活性，减弱 NSCLC 对细胞外基质的粘附和转移能力 [122][123][124] [125] 。另有研究发现紫草 宁也可通过调节 p53， 核因子 E2 相关因子 2 (nuclear factor E2-related factor 2, Nrf2) 以及 ROS 依赖性途径诱导细胞凋亡 [126] 。细胞增殖是通过细胞周期来完成的，因 此细胞周期的每个检验点对于细胞正常增殖非常重要， 许多抗癌药物也是通过阻 滞细胞周期发挥其活性的。研究发现 [127] 紫草宁可通过抑制缺氧诱导因子阻滞结 肠癌细胞周期，从而抑制细胞增殖。在细胞自噬方面，紫草宁可靶向调节 galectin-1 和 c-Jun N-terminal kinase(JNK)信号通路或抑制 Yes 相关蛋白 (Yes-associated protein, YAP)表达，诱导结肠癌自噬 [128] 。此外，紫草宁还表现 出显著的抗胃癌活性，例如紫草宁通过抑制早期生长反应 1(Egr1)介导 p21 基 因表达诱导人胃癌细胞周期阻滞 [129] ；还可通过 toll 样受体 2(toll-like receptor 2, TLR2)-和核因子 kappa-B (nuclear factor kappa-B, NF-κB)介导的信号途径， 抑制胃癌细胞侵袭和迁移 [130][131][132] 。 4.1.4. 紫草宁对甲状腺癌和胰腺癌的抗癌作用 紫草宁抑制甲状腺癌和胰腺癌增殖的功能主要是通过诱导细胞凋亡和自噬 以及抑制细胞迁移和侵袭而实现。其中，抗甲状腺癌机制是通过降低 DNA 甲基 转移酶 1(DNMT1)或失活 PI3K/AKT 信号通路活性，抑制甲状腺癌细胞的生 长、 迁移和侵袭 [133,…”