School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P.R. China.
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P.R. China.
Phytomedicine. 2021 Apr;84:153500. doi: 10.1016/j.phymed.2021.153500. Epub 2021 Feb 10.
A large number of breast cancer patients perishes due to metastasis instead of primary tumor, but molecular mechanisms contributing towards cancer metastasis remain poorly understood. Therefore, prompting development of novel treatment is inevitable. A vast variety of plant derived natural substance possesses several therapeutically active constituents, e.g. alkaloids, flavonoids, tannins, resins, terpenoids etc. that exhibit various pharmacological properties e.g. anti-inflammatory, anti-microbial and anti-cancer properties. Sanguinarine (SAN) alkaloid found its place among such naturally occurring substances that exerts several pharmacological activities, including anti-cancer effects.
Until now, role of SAN not only against epithelial-mesenchymal transition (EMT) but also against metastasis progression in breast cancer remains indistinct. Thus, aim of the present study was to investigate effects of SAN on EMT process and cancer metastasis in animal model.
MTT assay was performed to assess SAN effects on proliferation in breast cancer. Scratch assay was performed to evaluate effects of SAN on migration in breast cancer. Colony formation assay was performed to determine effects of SAN on colonization characteristics of breast cancer. Western blotting was performed to measure EMT regulating protein expression as well as major pathway protein expression induced against TGF-β treatment in breast cancer. Tail vein method of injecting breast cancer cells in bulb/c mice was conducted to study metastasis progression and thereafter assessing effects of SAN against metastasis in mice.
In vivo results: MTT assay performed, demonstrated dose dependent inhibition of cell proliferation in breast cancer. Scratch assay results showed, SAN played a major role as migration inhibitor in estrogen receptor positive (ER+) breast cancer. Colony forming assay results demonstrated that SAN constrains ability of breast cancer to develop into well-defined colonies. Western blotting results for EMT regulating protein expression, after TGF-β treatment showed, SAN inhibited cadherin switch in ER+ breast cancer. Moreover, expression of pathway proteins involved in EMT process after TGF-β treatment i.e. Smad, PI3K/Akt and MAP kinase were significantly masked against SAN treatment.
The appearance of metastatic nodules in lung tissues of mice model, helps to study the effects of SAN against metastasis in bulb/c mice. The obtained results have confirmed that SAN impeded lung metastasis. The macroscopic examination has confirmed metastasis inhibitory role of SAN in breast cancer. The Hematoxylin and eosin (H&E) staining results further advocate anti-metastatic characteristics of SAN, presented by fewer metastatic nodule and lesions appearance in SAN treated mice compared to untreated metastasis mice.
In summary, SAN displayed prominent anti-metastatic effects in animal model and anti-proliferation effects together with significant inhibitory potential on EMT regulating protein expression against TGF-β treatment in ER+ breast cancer. So, overall findings of our study highlighted the pre-clinical significance of SAN in animal model therefore, further studies in humans as a part of clinical trial will be needed to establish pharmacokinetics and other effects of SAN, so that it can be a potential candidate for future treatment of metastatic breast cancer (MBC).
大量乳腺癌患者因转移而不是原发性肿瘤而死亡，但导致癌症转移的分子机制仍知之甚少。因此，有必要开发新的治疗方法。大量植物衍生的天然物质具有多种治疗活性成分，例如生物碱、类黄酮、单宁、树脂、萜类等，它们表现出各种药理学特性，例如抗炎、抗菌和抗癌特性。血根碱（SAN）生物碱是天然存在的物质之一，具有多种药理学活性，包括抗癌作用。
到目前为止，SAN 不仅对上皮-间充质转化（EMT），而且对乳腺癌转移进展的作用仍不清楚。因此，本研究的目的是在动物模型中研究 SAN 对 EMT 过程和癌症转移的影响。
MTT 法检测 SAN 对乳腺癌增殖的影响。划痕试验评估 SAN 对乳腺癌迁移的影响。集落形成试验测定 SAN 对乳腺癌定植特征的影响。Western blot 法测定 EMT 调节蛋白表达及 TGF-β 诱导的主要通路蛋白表达。采用尾静脉法将乳腺癌细胞注入 bulb/c 小鼠，研究转移进展，然后评估 SAN 对小鼠转移的抑制作用。
体内结果：MTT 法检测结果表明，SAN 对乳腺癌的细胞增殖具有剂量依赖性抑制作用。划痕试验结果表明，SAN 在雌激素受体阳性（ER+）乳腺癌中作为主要的迁移抑制剂发挥作用。集落形成试验结果表明，SAN 限制了乳腺癌形成明确集落的能力。Western blot 法检测 EMT 调节蛋白表达结果显示，SAN 抑制了 ER+乳腺癌中的钙粘蛋白转换。此外，TGF-β 处理后参与 EMT 过程的通路蛋白表达，即 Smad、PI3K/Akt 和 MAP 激酶的表达明显被 SAN 处理所掩盖。
小鼠模型肺组织中转移性结节的出现有助于研究 SAN 对 bulb/c 小鼠转移的抑制作用。结果证实 SAN 抑制了肺转移。宏观检查证实了 SAN 在乳腺癌中的转移抑制作用。苏木精和伊红（H&E）染色结果进一步证明了 SAN 的抗转移特性，与未治疗的转移小鼠相比，SAN 治疗的小鼠中转移性结节和病变的出现较少。
总之，SAN 在动物模型中表现出显著的抗转移作用，以及在 ER+乳腺癌中对 EMT 调节蛋白表达的显著抑制作用，与 TGF-β 治疗的增殖抑制作用一起。因此，我们研究的总体结果强调了 SAN 在动物模型中的临床前意义，因此，需要进行人类临床试验以确定 SAN 的药代动力学和其他作用，以便使其成为未来转移性乳腺癌（MBC）治疗的潜在候选药物。
