SCLC is an aggressive cancer of neuroendocrine origin that progresses rapidly, often with endocrine abnormalities or carcinoid syndrome, and approximately two-thirds of patients have metastases by the time of diagnosis [22]. Lung cancer metastasis is the process by which a primary malignant tumor in the lung is seeded far away from its primary site through multiple pathways [13]. Despite a high response rate to the initial chemotherapy and radiotherapy, most patients present with advanced disease and rapidly develop resistance with distant metastases [17, 23]. Therefore, new effective drugs for the treatment of SCLC with low toxicity are urgently required. In present study, Tan IIA was found to inhibit the migration and proliferation of SCLC, which provides a new avenue for the development of treatments for SCLC.
Tan IIA is the main active component derived from Salvia miltiorrhiza. Previous studies have shown that Tan IIA exerts anti-tumor effects [24] and inhibits the growth of various tumor cell lines including liver, pancreatic and colorectal cancer [12]. However, the effects of Tan IIA on SCLC and the potential anti-metastatic mechanisms are not known. One study suggests that EMT is involved in the metastatic process of both NSCLC and SCLC [25]. During EMT, epithelial cells shed their connections to neighboring cells, changing from apical-basal polarity to anterior-posterior polarity, and taking on the properties of migrating mesenchymal cells. Adhesion is promoted in cells undergoing EMT by repression of epithelial genes, leading to their separation from adjacent cells. The classical epithelial marker E-calmodulin is a key component of adherent junctions and is the most prominent target for inhibition of EMT [26]. E-calmodulin has been shown to impair the strength of cellular adhesion and promote cellular motility [27]. In contrast, vimentin, an abundant cytoplasmic intermediate filament protein, is also involved in cancer metastasis by stabilizing cell connections [28]. In our study, we demonstrated that Tan IIA inhibited tumor growth and suppressed cell migration by increasing E-cadherin expression and reducing vimentin expression. We also demonstrated that Tan IIA significantly reduced the cell migration in wound healing assays and Transwell migration assays. Furthermore, the same concentrations of Tan IIA did not affect the migration ability of normal lung cells lines and did not trigger cell apoptosis, suggesting that Tan IIA has relatively low toxicity.
In addition, BP analysis also showed that the DEGs downregulated by Tan IIA are indeed enriched in the cell adhesion and inflammation categories, which are associated with the process of EMT. Furthermore, KEGG pathway enrichment analysis showed that these DEGs are closely associated with the PI3K/Akt pathway. PI3K is a major intracellular signaling pathway responsible for a variety of key cellular processes that are aberrantly activated in cancer and contribute to tumorigenesis and progression [29]. The PI3K/Akt pathway can be aberrantly activated through a variety of mechanisms. Once activated, Akt phosphorylates many substrates including mammalian target of rapamycin (mTOR), which functions as a downstream effector of Akt to activate many proteins and promote cancer progression [30]. Some herbal medicines can inhibit tumors by modulating the PI3K/Akt signaling pathway. For example, Shikonin inhibits the migration and invasion of glioblastoma cells by targeting PI3K/Akt [31]. Furthermore, Licochalcone A, which is extracted from the root of Glycyrrhiza inflata inhibits cell proliferation, migration, and invasion by suppressing the PI3K/Akt signaling pathway in oral squamous cell carcinoma [32]. Previous studies also demonstrated PI3K/Akt pathway activation induces E-cadherin expression and inhibits vimentin expression to suppress cancer cell proliferation and migration [33, 34]. In our study, we observed that Tan IIA indeed downregulated the expression of PI3K and p-Akt in an effect that was mimicked by LY294002 as a common inhibitor of PI3K. Due to its association with increased cell proliferation and survival, elevated expression of PI3K-related proteins is considered a hallmark of cancer [35]. PI3K transduces extracellular signals from G protein-coupled receptors (GPCRs) to activate multiple downstream signaling proteins, including Akt (also known as protein kinase B). Akt phosphorylates multiple downstream effectors, including mTOR and glycogen synthase kinase 3(GSK-3), which contribute to cancer cell growth, proliferation, and survival [30]. Previous studies had confirmed that Tan IIA affects malignant cell growth by inhibiting the PI3K-Akt-mTOR pathway [17, 36, 37]. Although the expression of mTOR was not detected in present study, we speculate that the mechanism by which Tan IIA inhibits SCLC metastasis is associated with mTOR suppression, therefore, this hypothesis remains to be tested.
Using an in vivo xenografted tumor model in mice, we showed that the tumor volume and weight were both reduced after administration of Tan IIA without obvious cytotoxic effects based on the results of serum biochemical analysis and body weight measurements. Although the number of mice in each group is limited, we also observed that this dosage of Tan IIA has low toxicity in mice, which was consistent with the results observed in vitro. Also in accordance with the in vitro results, we found that the expression of E-cadherin was increased by Tan IIA, while the expression of PI3K and p-Akt was suppressed in vivo.