Anti-breast cancer and toxicity studies of total secondary saponin from Anemone raddeana Rhizome on MCF-7 cells via ROS generation and PI3K/AKT/mTOR inactivation.
银莲花总次级皂苷通过 ROS 生成和 PI3K/AKT/mTOR 失活对 MCF-7 细胞的抗乳腺癌和毒性研究。
- 作者列表："Zhang D","Zhang Q","Zheng Y","Lu J
ETHNOPHARMACOLOGICAL RELEVANCE:The rhizome of Anemone raddeana Regel (A. raddeana) is a famous traditional Chinese medicine (TCM) recorded in Chinese Pharmacopoeia for the treatment of carbuncle and swelling. Carbuncle swollen is an explanation of tumor in the theory of TCM and softening and resolving hard mass effects are one of the important pharmacological activities of A. raddeana. AIM OF THE STUDY:We investigated the potential anti-breast cancer effect and toxicological properties of alkali-ethanol extract from A. raddeana, namely total secondary saponin (TSS). MATERIALS AND METHODS:Anti-proliferative effect of total saponin of A. raddeana (ATS) and TSS were tested using MTT assay. Hoechst staining, flow cytometry analysis, DCFH-DA fluorescence microscopy and western blot were carried out to evaluate the mechanisms of action of TSS. The potential anti-breast cancer activity and toxicological properties of TSS were tested in vivo. RESULTS:ATS and TSS could inhibit the proliferation of A549, HepG2, MCF-7, MDA-MB-231 and SKBr-3 cells, especially for MCF-7 cells. Flow cytometry analysis revealed that TSS (10, 12 and 15 μg/ml) could induce cell cycle arrest on G0/G1 phase and promote apoptosis of MCF-7 cells. TSS could increase Bax/Bcl-2 ratio, elevate cytochrome c levels in cytosol and activate caspase-3/9. In addition, TSS also induced ROS generation and inactivated PI3K/AKT/mTOR pathway which may involved in the mitochondrial dysfunction of MCF-7 cells. TSS showed slight toxic at the dosage of 100 and 200 mg/kg by oral administration without any toxic potential for 28 days. TSS (50, 100 and 200 mg/kg) showed significant inhibitory effect on growth of transplanted tumor in mice. At last, twenty-three C-3 monosaccharide oleanane-type triterpene saponins were tentatively identified, which may contributed to the anti-cancer activity of TSS. CONCLUSION:This study demonstrated that TSS exhibited anti-proliferative and pro-apoptosis activities on MCF-7 cells via ROS-mediated activation of mitochondrial apoptosis pathway. TSS might be used as chemotherapeutic agent for the treatment of breast cancer with relatively low toxicity.
民族药理学相关性: 银莲花根茎 (A. raddeana) 是中国药典中记载的治疗 carb肿的著名中药 (TCM)。Carb肿是中医理论对肿瘤的一种解释，软坚散结作用是 A. raddeana 的重要药理活性之一。 研究目的: 我们研究了 raddeana 碱乙醇提取物，即总次级皂苷 (TSS) 的潜在抗乳腺癌作用和毒理学特性。 材料和方法: 采用 MTT 法检测山地黄总皂苷 (ATS) 和 TSS 的抗增殖作用。通过 Hoechst 染色、流式细胞术分析、 DCFH-DA 荧光显微镜和 western blot 评价 TSS 的作用机制。体内测试了 TSS 的潜在抗乳腺癌活性和毒理学特性。 结果: ATS 和 TSS 均能抑制 A549 、 HepG2 、 MCF-7 、 MDA-MB-231 和 SKBr-3 细胞的增殖，对 MCF-7 细胞的抑制作用最强。流式细胞仪分析发现，TSS (10 、 12 和 15 μ g/ml) 可诱导细胞周期阻滞于 G0/G1 期，促进 MCF-7 细胞凋亡。TSS 可增加 Bax/Bcl-2 比值，升高胞浆细胞色素 c 水平，激活 caspase-3/9。此外，TSS 还可诱导 ROS 生成，使 PI3K/AKT/mTOR 通路失活，可能参与 MCF-7 细胞线粒体功能障碍。TSS 在 100 和 200 mg/kg 的剂量下呈轻微毒性，口服给药 28 天无任何毒性潜力。TSS (50 、 100 和 200 mg/kg) 对小鼠移植瘤的生长表现出显著的抑制作用。初步鉴定了 23 C-3 单糖齐墩果烷型三萜皂苷，可能有助于 TSS 的抗癌活性。 结论: 本研究证明 TSS 通过 ROS 介导的线粒体凋亡途径对 MCF-7 细胞具有抗增殖和促凋亡活性。TSS 有可能作为化疗药物用于乳腺癌的治疗，且毒性较低。
METHODS::The emerging significance of the bitter taste receptors (T2Rs) role in the extraoral tissues alludes to their potential role in many pathophysiological conditions. The dysregulation of T2R expression and function in disease conditions has now been demonstrated in airways diseases, neurological disorders, and in some cancers. However, the role of T2Rs in the pathophysiology of breast cancer is unexplored thus far. Previously, we demonstrated differential expression of the 25 T2Rs in breast cancer (BC) cells. Based on our previous findings we selected two T2Rs, T2R4 and T2R14 for this work. The objective of the current study is to investigate the expression of T2R4 and T2R14 in BC clinical samples and to examine their physiological role using highly metastatic BC and non-cancerous cell lines. Using approaches, which involve receptor knockdown, pharmacological activation and biochemical assays we report that (i) T2R4 and T2R14 expression patterns are dissimilar, with decreased levels of T2R4 and increased levels of T2R14 in BC clinical samples compared to non-cancerous controls. (ii) Activation of T2Rs with their respective agonist elicited physiological responses in metastatic breast cancer cells, and no responses were seen in non-tumorigenic breast epithelial cells. (iii) Agonist activation of T2Rs (irrespective of T2R subtype) induced anti-proliferative, pro-apoptotic, and anti-migratory responses in highly metastatic breast cancer cells. Taken together, our findings demonstrate that the chemosensory T2R signaling network is involved in evoking physiological responses in the metastatic breast cancer cell line.
METHODS::RAD50 is commonly depleted in basal-like breast cancer with concomitant absence of INPP4B and several tumor suppressors such as BRCA1 and TP53. Our previous study revealed that INPP4B and RAD50 interact and such an interaction is associated with breast cancer survival at the transcriptional, translational and genomic levels. In the present study, we explored single nucleotide polymorphisms (SNPs) of these two genes that have synergistic effects on breast cancer survival to decipher mechanisms driving their interactions at the genetic level. The Cox's proportional hazards model was used to test whether SNPs of these two genes are interactively associated with breast cancer survival, following expression quantitative trait loci (eQTL) analysis and functional investigations. Our study revealed two disease-associating blocks, each encompassing five and two non-linkage disequilibrium linked SNPs of INPP4B and RAD50, respectively. Concomitant presence of any rare homozygote from each disease-associating block is synergistically prognostic of poor breast cancer survival. Such synergy is mediated via bypassing pathways controlling cell proliferation and DNA damage repair, which are represented by INPP4B and RAD50. Our study provided genetic evidence of interactions between INPP4B and RAD50, and deepened our understandings on the orchestrated genetic machinery governing tumor progression.
METHODS:BACKGROUND:Increased usage of genomic risk assessment assays suggests increased reliance on data provided by these assays to guide therapy decisions. The current study aimed to assess the change in treatment decision and physician confidence based on the 70-gene risk of recurrence signature (70-GS, MammaPrint) and the 80-gene molecular subtype signature (80-GS, BluePrint) in early stage breast cancer patients. METHODS:IMPACt, a prospective, case-only study, enrolled 452 patients between November 2015 and August 2017. The primary objective population included 358 patients with stage I-II, hormone receptor-positive, HER2-negative breast cancer. The recommended treatment plan and physician confidence were captured before and after receiving results for 70-GS and 80-GS. Treatment was started after obtaining results. The distribution of 70-GS High Risk (HR) and Low Risk (LR) patients was evaluated, in addition to the distribution of 80-GS compared to IHC status. RESULTS:The 70-GS classified 62.5% (n = 224/358) of patients as LR and 37.5% (n = 134/358) as HR. Treatment decisions were changed for 24.0% (n = 86/358) of patients after receiving 70-GS and 80-GS results. Of the LR patients initially prescribed CT, 71.0% (44/62) had CT removed from their treatment recommendation. Of the HR patients not initially prescribed CT, 65.1% (41/63) had CT added. After receiving 70-GS results, CT was included in 83.6% (n = 112/134) of 70-GS HR patient treatment plans, and 91.5% (n = 205/224) of 70-GS LR patient treatment plans did not include CT. For patients who disagreed with the treatment recommended by their physicians, most (94.1%, n = 16/17) elected not to receive CT when it was recommended. For patients whose physician-recommended treatment plan was discordant with 70-GS results, discordance was significantly associated with age and lymph node status. CONCLUSIONS:The IMPACt trial showed that treatment plans were 88.5% (n = 317/358) in agreement with 70-GS results, indicating that physicians make treatment decisions in clinical practice based on the 70-GS result. In clinically high risk, 70-GS Low Risk patients, there was a 60.0% reduction in treatment recommendations that include CT. Additionally, physicians reported having greater confidence in treatment decisions for their patients in 72% (n = 258/358) of cases after receiving 70-GS results. TRIAL REGISTRATION:"Measuring the Impact of MammaPrint on Adjuvant and Neoadjuvant Treatment in Breast Cancer Patients: A Prospective Registry" (NCT02670577) retrospectively registered on Jan 27, 2016.