Bangladesh Journal of Pharmacology Volume: 19; Number 2; Year 2024 Cite this article as: Wang W, He Z, Bao J. Anti-migration activity of four components from dried fruit of Forsyth- ia suspensa. Bangladesh J Pharmacol. 2024; 19: 65-68. Anti-migration activity of four components from dried fruit of Forsythia suspensa Dear Editor, Breast cancer is the most common cancer and the second leading cause of cancer mortality in women (Sung et al., 2021). Tumor metastasis is the major cause of poor prognosis and survival in cancer patients, which is responsible for about 90% of cancer deaths (Gerstberger et al., 2023). Despite tremendous advances in the treatment of primary tumors, metastatic tumors remain the most difficult cancer disease to conquer (Guan, 2015). The existing clinical drugs are not satis- fied for fighting against tumor metastasis. Therefore, there is an urgent need to develop novel drug candidates against metastatic breast cancer. Forsythia suspensa (Thunb.) Vahl. is a typical heat- clearing and detoxicating herb in traditional Chinese medicine. Its dried fruits, also known as Forsythiae Fructus, have been widely used for treating cancer since ancient China. There is a great potential to develop anti- cancer drugs from F. suspensa dried fruit-derived com- pounds. It was reported that fruit extracts had anti- proliferative activity against various cancers. The underlying mechanisms include inhibiting MAPKs/ Nrf2/HO-1 mediated anti-oxidation and anti-infla- mmation, regulating glycerophospholipid metabolism, influencing cell cycle progression and inducing apop- tosis (Bao et al., 2016a; Bao et al., 2016b; Lee et al., 2017; Zhang et al., 2019). However, the anti-metastatic activi- ty of dried fruit of F. suspensa and its bioactive com- ponents remains to be explored. In this study, the growth inhibitory effect of F. suspensa dried fruit aqueous extract was first measured on breast cancer cell line MDA-MB-231 by MTT assay (Zhang et al., 2015; Bao et al., 2016a). The aqueous extract was prepared by adding 100 g of dried herbal piece powder to 1,000 mL of distilled water to form a slurry, bringing the subsequent mixture to a boiler and simmering at 80℃ for 60 min. After the mixture cooled down, the aqueous layer was carefully decanted off of the residual solids and yielded 28.3 g lyophilized powder. The cell viability and growth inhibitory rate were calculated using the following formulas: Cell viability (%) = Test absorbance at 570 nm / Control absorbance at 570 nm × 100 Growth inhibitory rate (%) = Control cell viability – Test cell viability Breast cancer cell line MDA-MB-231 was obtained from ATCC and cultured in DMEM supplemented with 10% FBS. Cells were seeded at a density of 4 × 103 cells per well in a 96-well plate. After treating MDA-MB-231 cells with the extract for 48 hours, the numbers of viable breast cancer cells were significantly decreased in a dose-dependent manner (Figure 1A). The IC50 value of the extract was 132.1 μg/mL. When the dose of the extract was increased to 250 μg/mL, the growth inhi- bitory rate of MDA-MB-231 cells increased to 90.8%. This result demonstrated that F. suspensa dried fruit aqueous extract possessed effective anti-cancer activity against breast cancer cells. To further explore the anti-metastatic activity of F. suspensa dried fruit, the wound healing assay was app- lied to evaluate the anti-migration effect of the extract breast cancer cells (Li et al., 2022). When evaluating the anti-migration activity of a drug in vitro, the ideal condition would be that its dose would only inhibit the migration of cancer cells, with no effect on growth inhibition. Its anti-metastatic activity is not contributed by growth inhibitory effect. The anti-migration activity was assessed by migration rate (%) and inhibitory rate of migration (%) using the following formulas: Migration rate (%) = (0 hour scratch area - 48 hour scratch area) / 0 hour scratch area × 100 Migration inhibitory rate (%) = (Control migration rate - Test migration rate) / Control migration rate × 100 As shown in Figure 1B, the results showed that the extract effectively suppressed the migration of highly metastatic cancer cell line MDA-MB-231 by 31.6 and 33.4% at concentrations of 15.6 and 31.2 μg/mL, respec- tively. This result demonstrated the anti-metastatic acti- vity of F. suspensa dried fruit against breast cancer cells. Forsythoside A, forsythoside I, forsythoside H, and iso- forsythiaside are representative components of F. sus- pensa dried fruit, which are considered to be potential bioactive compounds (Yang et al., 2022). Forsythoside A and forsythoside I have been found with cytotoxic activity against gastric cancer, breast cancer, and mela- noma (Nie et al., 2022). However, their anti-metastatic activity has not been explored. In this study, the anti- metastatic effect of forsythoside A, forsythoside I, forsythoside H, and isoforsythiaside (Chengdu Must Bio-Technology Co., Ltd., China) on breast cancer cell A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2024; 19: 65-68 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v19i2.72632 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor line MDA-MB-231 was explored. The cytotoxic concentrations of forsythoside A, forsy- thoside I, forsythoside H and isoforsythiaside were first determined, and selected drug doses without growth inhibitory effect were used to conduct their anti- migration activity. The results showed that forsythoside A, forsythoside I, forsythoside H, and isoforsythiaside at concentrations of 2.5, 5, 10, 20, and 30 μg/mL all had no growth inhibitory effects on MDA-MB-231 cells (Figure 2). So, these doses were used to perform later anti-migration activity assessment. The wound healing assay was again applied to determine the anti-migration activity of forsythoside A, forsythoside I, forsythoside H and isoforsythiaside on MDA-MB-231 cancer cells. The results showed that forsythoside A, forsythoside I, forsythoside H, and iso- forsythiaside significantly suppressed migration of cancer cells by 13.2, 4.5, 13.2, and 20.0% at the concen- tration of 20 µg/mL, respectively (Figure 3A,B). When their dose was increased to 30 µg/mL, their migration 66 Bangladesh J Pharmacol 2024; 19: 65-68 - 7.8 15.6 31.2 62.5 125 250 500 1000 - 7.8 15.6 31.2 - 7.8 15.6 31.2 % C e ll v ia b ili ty 120 80 40 0 Figure 1: Cell viability (A) and migration inhibitory effect (B, C) of F. suspensa dried fruit aqueous extract on MDA-MB-231 cell lines incubated for 48 hours. Migration inhibitory effect was assessed by wound healing assay Figure 2: Growth inhibitory effect of forsythoside A, forsythoside I, forsythoside H and isoforsythiaside. (A) Chemical structures of forsythoside A, forsythoside I, forsythoside H and isoforsythiaside. (B) Growth inhibitory effect of FA, FI, FH and IFA on MDA -MB- 231 cells. Cells were incubated with indicated compounds for 24, 48 and 72 hours A B C 60 40 20 0 % In h ib it o ry r a te o f m ig ra ti o n A Forsythoside A Forsythoside I Forsythoside H Isoforsythoside 72 Hours 24 Hours 48 Hours B % C e ll v ia b ili ty 150 100 50 0 % C e ll v ia b ili ty 150 100 50 0 - 2.5 5 10 20 30 - 2.5 5 10 20 30 Concentration (µg/mL) Concentration (µg/mL) Forsythoside A Forsythoside I Forsythoside H Isoforsythoside Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) Figure 3: Migration inhibitory effect of forsythoside A, forsythoside I, forsythoside H and isoforsythiaside. (A) Wound healing assay to evaluate anti-migration activity of compounds at doses of 0, 2.5, 5, 10, 20 and 30 µg/mL. Cells were incubated with indi- cated compounds for 48 hours. (B) The statistics of inhibitory rate of migration for indicated compounds assessed by wound heal- ing assay. (C) Transwell assay to evaluate anti-migration activity of forsythoside H and isoforsythiaside at doses of 0, 10, 20 and 30 µg/mL. Cells were incubated with forsythoside H and isoforsythiaside for 8 hours. (D) The statistics of inhibitory rate of migra- tion for indicated compounds assessed by transwell assay A B C - 2.5 5 10 20 30 µg/mL - 2.5 5 10 20 30 - 2.5 5 10 20 30 - 2.5 5 10 20 30 - 2.5 5 10 20 30 30 20 10 0 -10 % In h ib it o ry r a te o f m ig ra ti o n 40 30 20 10 0 Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) Forsythoside A Forsythoside I Forsythoside H Isoforsythoside Forsythoside A Forsythoside I Forsythoside H Isoforsythoside D Forsythoside H Isoforsythoside - 10 20 30 µg/mL - 10 20 30 - 10 20 30 M ig ra ti o n c e ll n u m b e r % o f c o n tr o l Concentration (µg/mL) Concentration (µg/mL) Forsythoside H Isoforsythoside 120 80 40 0 150 100 50 0 Bangladesh J Pharmacol 2024; 19: 65-68 67 inhibitory rate increased to 16.8, 17.9, 16.3, and 25.7%, respectively (Figure 3A,B). In general, forsythoside A, forsythoside I, forsythoside H, and isoforsythiaside suppressed cancer cell migration in a dose-dependent manner. Isoforsythiaside and forsythoside H exhibited the best migration inhibitory effect at doses of 20 and 30 µg/mL compared to forsythoside A and forsythoside I. The transwell assay was next used to further evaluate anti-migration activity of forsythoside H and isoforsy- thiaside (Zaki et al., 2019). MDA-MB-231 cells were seeded in the upper chamber of transwell inserts con- taining serum-free medium and exposed to varying concentrations of forsythoside H and isoforsythiaside. The transwell inserts were placed into 24-well plates and 500 μL culture medium containing 10% FBS was added to each lower chamber. The membrane pore size between upper and lower chambers was 8.0 µm, so that invasive cancer cells could migrate from upper cham- bers to lower chambers. After drug incubation for 12 hours, the migrated cells were fixed by 4% parafor- maldehyde for 30 min, then stained the cells with 0.1% crystal violet for 30 min. Migrated cells were visualized by microscope as shown in Figure 3C. The results of the transwell assay revealed that forsythoside H and isofor- sythiaside at concentrations of 20 and 30 µg/mL exhibi- ted effective migration inhibitory activity on MDA-MB- 231 cancer cells (Figure 3C,D), which is consistent with previous findings in wound healing assay (Figure 3A,B). To sum up, the anti-cancer growth and anti-migration activities of F. suspensa dried fruit aqueous extract against breast cancer cells were studied, of which the anti-migration activities of F. suspensa dried fruit was reported. Forsythoside H and isoforsythiaside were identified as important anti-metastatic compounds in F. suspensa dried fruit responsible for its anti-metastatic activity, which was evidenced by wound healing assay and transwell assay. Forsythoside H and isoforsythia- side demonstrated effective migration inhibitory activity on MDA-MB-231 cancer cells at doses 20 and 30 µg/mL. From these results, forsythoside H and isofor- sythiaside could be potential drug candidates for deve- loping novel anti-metastatic treatments. The anti-metastatic activities of F. suspensa dried fruit aqueous extract and its bioactive compounds were only assessed by in vitro assays using wound healing and transwell approaches, further validation studies on evaluating their anti-metastatic activities in animals, such as mice, were deserved in the future. Financial support: This work was supported by National Natural Science Foundation of China (No. 82104057), Hainan Provincial Natural Science Foundation of China (No. 822RC649), Young Elite Scientists Sponsorship Program by Hainan Association for Science and Technology (No. QCQTXM202210) and Hainan Nanhai New Star Project (No. 202309009). Ethical issue: The development, acquisition, authentication, cryopreservation, and transfer of cell lines between laborato- ries were followed according to the guidelines published in British Journal of Cancer, 2014. Conflict of interest: The authors declare no competing inter- ests. Acknowledgment: The authors thank members of the Bao laboratory for helpful advice and discussion. Wanrong Wang, Zongmao He and Jiaolin Bao Key Laboratory of Tropical Biological Resources of Ministry of Educa- tion, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou, 570228, China. Corresponding author: Jiaolin Bao email: baojiaolin@hainanu.edu.cn References Bao J, Ding R, Zou L, Zhang C, Wang K, Liu F, Li P, Chen M, Wan JB, Su H, Wang Y. Forsythiae fructus inhibits B16 melanoma growth involving MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation. Am J Chin Med. 2016; 44: 1043-61. 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