To the Editor: In breast cancer, it is the metastasis that makes a clear distinction between life and death.[1,2] However, effective treatment targeted against metastasis remains an unmet clinical need.[2,3] Accumulating evidence suggests that an essential step in metastasis is tumor cell acquisition of motility. Furthermore, epithelial–mesenchymal transition (EMT) initiates the early steps of tumor cell spread by endowing them with greater motility and invasiveness.[1,4] Focal adhesion kinase (FAK) controls cell mesenchymal characteristics responsible for invasiveness and adhesion, placing itself as a critical molecule in regulating EMT in various cancer cells.
Stellera chamaejasme Linn. (SCL) is a perennial herb of the Thymelaeaceae family. It has been demonstrated to be a potential anti-cancer agent in a broad spectrum of malignancies, but this well-known effect was traditionally attributed to its dose-dependent unselective cytotoxic activity. Additionally, the pharmacological properties of this extract remain incompletely understood and other possible targets of SCL have been underappreciated, which greatly limits the awareness regarding its efficacy. Therefore, to address these shortcomings, we extracted the active fractions from SCL (named ESC, that is the extract of SCL) and applied them in low doses for experiments, attempting to uncover its novel therapeutic mechanisms beyond cytotoxic effects.
Specifically, the efficacy of low-dose ESC was first identified in two BALB/c tumor mouse models (0.10 mg/kg or 1.00 mg/kg, which were 1/1000 or 1/100 of the doses commonly used in previous studies). In the subcutaneous xenograft tumor model, in line with our hypothesis, 0.10 mg/kg or 1.00 mg/kg ESC showed a minimal influence on the living conditions and body weight of mice compared with the negative control [Supplementary Figure 1A, https://links.lww.com/CM9/B270]. Moreover, the low doses of ESC mildly repressed primary tumor growth, as demonstrated by the partially reduced primary tumor weight [Supplementary Figure 1B, https://links.lww.com/CM9/B270] and volume [Supplementary Figure 1C, D, https://links.lww.com/CM9/B270].
In the mammary fat pad xenograft model, 4T1 cells stably expressing Iuciferase were inoculated into the mammary fat pad of mice. Then, the primary tumors were removed on the 42nd day and metastasis was visualized using a small animal imaging system throughout. Using this model, the microenvironmental factors of breast cancer can be effectively simulated. Besides, the specificity and quantification of metastatic evaluation can be achieved. As expected, the total photon values showed a similar trend among groups from 7 to 42 days [Figure 1A], indicating little inhibitory effects against primary tumors. In stark contrast, metastatic foci formation in ESC treated mice was significantly impaired according to small animal imaging results [Figure 1B,C]. Three weeks after the removal of primary tumors, more severe pathological changes in the lung were clearly observed in the negative control group, whereas only a few metastatic foci were observed in ESC treated mice [Figure 1D]. These results indicated that, rather than killing tumor cells directly, ESC mainly exerted anti-metastatic activity at ultra-low concentrations.
Inspired by the in vivo results, we further explored the potential association of ESC with cell motility regulation. The commonly used dosage of ESC is 20 to 100 μg/mL in vitro. In this study, we screened the safety of different ESC doses using MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, ranging from 1.56 to 25.00 μg/mL. The results clearly showed that, compared with the control cells, no significant differences in the proliferation rate were observed in the groups of selected doses for normal breast cells (MCF-10A) [Supplementary Figure 2A, https://links.lww.com/CM9/B270] and for breast cancer cells (4T1 and MDA-MB-231 cells) [Supplementary Figure 2B,C, https://links.lww.com/CM9/B270]. However, following treatment with 1.00 μg/mL ESC, the morphology of 4T1 and MDA-MB-231 cells was obviously affected, with a substantially more rounded shape and static state [Supplementary Figure 2D,E, https://links.lww.com/CM9/B270]. Transwell and Matrigel assays were performed, revealing that the percentage of migrating and invading cells was reduced in the group treated with 1.00 μg/mL ESC [Supplementary Figure 2F-I, https://links.lww.com/CM9/B270]. These data indicated that low-dose ESC suppressed tumor cell motility.
Molecularly, cell motility is determined by EMT, which alters gene expression patterns, including the downregulation of epithelial markers, such as E-cadherin (CDH1), and upregulation of mesenchymal markers, such as vimentin (VIM).[1,4] In our study, a polymerase chain reaction was used to detect the mRNA transcription levels of related molecules in 4T1 and MDA-MB-231 cells treated with 0.25 to 4.00 μg/mL of ESC. As expected, ESC-treated cells exhibited significantly increased expression of the tumor suppressor CDH1. In contrast, the expression of the mesenchymal marker VIM and EMT-initiating factor Snail1 was downregulated [Supplementary Figure 3A,B, https://links.lww.com/CM9/B270]. Therefore, ESC may act as a tumor suppressor by regulating EMT-associated gene expression, providing a molecular basis for interfering with cellular invasiveness.
Considering the central role of transforming growth factor-beta (TGF-β) in the regulation of EMT and breast cancer motility, we further established a TGF-β1 stimulated model in vitro. Microscopic observation showed that treatment with TGF-β1 induced mesenchymal-like changes in breast cancer cells, including elongated fibroblast-like characteristics and an increased length and number of pseudopodia. However, this phenotypic transition was markedly reversed by 1.00 μg/mL ESC [Supplementary Figures 3C and 4A, https://links.lww.com/CM9/B270]. The epithelial characteristics, such as a cobblestone-like morphology and enhanced cell-cell adhesion, were successfully restored, suggesting that ESC may function as an EMT blocker in response to TGF-β1 stimulation in vitro.
Given that FAK may regulate EMT through p38 mitogen-activated protein kinase (P38), we hypothesized that there might be a potential connection between ESC and FAK. As expected, molecular analysis showed that, following low-dose ESC inhibition, FAK phosphorylation was decreased, along with increased expression of CDH1 and decreased expression of VIM and phosphorylated-P38 [Supplementary Figure 4B, https://links.lww.com/CM9/B270]. These results suggested that, through the phosphorylation inhibition of FAK, ESC can neutralize the pro-metastatic effect of EMT.
Despite the tremendous technological progression in anti-malignant diagnosis and treatment, metastatic intervention represents the bottleneck severely restricting the effectiveness of cancer therapies. Identifying candidates with a promising toxicity/efficacy ratio remains one of the most attractive tasks for global scientists. Accordingly, our results suggested that SCL may be a promising therapeutic candidate for targeting metastasis through its EMT-inhibitory effect.
In addition, our study shows potential implications in the following aspects: (1) Independent of its tumor toxicity, we identified for the first time the therapeutic value of ultra-low-dose SCL, which may provide crucial evidence for a breakthrough in its reasonable application in cancer treatment. (2) In addition to the effectiveness validation, we further revealed the potential regulatory association between ESC and the FAK signaling pathway, which warrants further exploration. (3) Technologically, the experimental platform applied in our study, which mainly consisted of tumor cell breast fat pad in situ transplantation combined with specific and dynamic metastasis evaluation in vivo, provided a reliable strategy for the research and development of anti-metastatic drugs.
SCL needs to be further investigated through the following in-depth study. First, focusing on FAK signaling transduction, the exact mechanistic study of SCL in the inhibition of breast cancer metastasis requires further detailed elucidation. Second, the purified chemical compounds isolated from ESC should be another focus for future studies.
Conflicts of interest
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5. Kan XX, Li Q, Chen X, Wang YJ, Li YJ, Yang Q, et al. A novel cell cycle blocker extracted from Stellera chamaejasme L. inhibits the proliferation of hepatocarcinoma cells. Oncol Rep 2016;35:3480–3488. doi: 10.3892/or.2016.4742.