槟榔叶水提液对癌细胞的抗迁移作用及其微管靶向性的研究

Anti-migratory effects of Piper betle leaf aqueous extract on cancer cells and its microtubule targeting properties # # 槟榔叶水提液对癌细胞的抗迁移作用及其微管靶向性的研究 1 first-author †E-mail:meelee.looi@um.edu.my †E-mail:meelee.looi@um.edu.my meelee.looi@um.edu.my 2 2 2 2 3 Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia School of Biosciences, Taylor’s University, Lakeside Campus, Subang Jaya, Selangor Darul Ehsan 47500, Malaysia School of Biosciences, Taylor’s University, Lakeside Campus, Subang Jaya, Selangor Darul Ehsan 47500, Malaysia Forest Research Institute Malaysia, Kepong, Selangor Darul Ehsan 52109, Malaysia Forest Research Institute Malaysia, Kepong, Selangor Darul Ehsan 52109, Malaysia Piper betle (PB), also known as “betel” in Malay language, is a tropical Asian vine. PB leaves are commonly chewed by Asians along with betel quid. It contains phenols such as eugenol and hydroxychavicol along with chlorophyll, β-carotene, and vitamin C (Salehi et al., 2019 2019 ). Extracts from PB leaves have various medicinal properties including anticancer, antioxidant, anti-inflammatory, and antibacterial effects (Salehi et al., 2019 2019 ). Previous research has shown that PB induces cell cycle arrest at late S or G2/M phase and causes apoptosis at higher doses (Wu et al., 2014 2014 ; Guha Majumdar and Subramanian, 2019 2019 ). A combination of PB leaf extract has also been shown to enhance the cytotoxicity of the anticancer drug, 5-fluorouracil (5-FU), in cancer cells (Ng et al., 2014 2014 ). 本文研究了槟榔( Piper betle ,PB)水提取物对癌细胞转移能力的调节作用及其对微管蛋白结构和网络的影响.采用亚毒剂量对PB和5-氟尿嘧啶(5-FU)处理的癌细胞进行抗迁移研究,观察细胞迁移20小时.在所有处理组中,100 µg/mL PB处理的癌细胞均显示出最大的抗迁移作用( P =0.016).总体上,PB对癌细胞的抗迁移作用高于5-FU.通过细胞形态观察发现,PB处理的细胞表现出与标准微管抑制剂(紫杉醇)相似的细胞特征,并在PB和紫杉醇处理的癌细胞中发现M期细胞群.通过对微管结构和网络进一步研究,我们发现PB和紫杉醇处理的癌细胞表现出长期破坏的纺锤体.因此,我们认为PB具有对癌细胞的抗迁移作用,并可能改变蛋白结构和网络. Piper betle Cell migration Microtubules Tubulin network 槟榔 细胞迁移 微管 微管蛋白网络 Piper betle (PB), also known as “betel” in Malay language, is a tropical Asian vine. PB leaves are commonly chewed by Asians along with betel quid. It contains phenols such as eugenol and hydroxychavicol along with chlorophyll, β-carotene, and vitamin C (Salehi et al., 2019 2019 ). Extracts from PB leaves have various medicinal properties including anticancer, antioxidant, anti-inflammatory, and antibacterial effects (Salehi et al., 2019 2019 ). Previous research has shown that PB induces cell cycle arrest at late S or G2/M phase and causes apoptosis at higher doses (Wu et al., 2014 2014 ; Guha Majumdar and Subramanian, 2019 2019 ). A combination of PB leaf extract has also been shown to enhance the cytotoxicity of the anticancer drug, 5-fluorouracil (5-FU), in cancer cells (Ng et al., 2014 2014 ). The ability of cancer cells to disseminate to distant regions is partly the result of microtubule dynamics. Microtubules are part of the cell cytoskeleton, contribute to shape and dynamics, and are essential for cell movement and directionality (Ganguly et al., 2012 2012 ; Mukhtar et al., 2014 2014 ). As a result of rapid cancer cell division, anti-mitotic drugs that potentially target microtubule dynamics have been suggested to be the best cancer therapeutic agents (Mukhtar et al., 2014 2014 ). The potential of PB in targeting cancer cell migration was investigated in this study. The modulation effect of PB on microtubule structure and networks was also observed in comparison to a standard microtubule inhibitor, paclitaxel. The sub-toxic levels of PB and 5-FU were used to ensure no cell cytotoxicity in the cell migration analysis. After 24 h of respective treatment, values of IC 20 (20% inhibitory concentration) and IC 30 (30% inhibitory concentration) recorded for PB on human lung adenocarcinoma (A549) cells were 20 and 100 μg/mL, respectively, whereas IC 20 and IC 30 for 5-FU on A549 cells were determined at 4.0 and 12.5 μmol/L, respectively. The anti-migration effects of PB and 5-FU were observed real-time on A549 cells for 20 h using xCELLigence (ACEA Biosciences, USA). A high cell index (CI) value indicates a positive migration effect as more cells adhere to the gold microelectrodes, causing high impedance. The negative control group (cells with serum-free media) showed CI values less than 0.5 (Fig. 1 1 ). This implies that cells did not migrate throughout the experiment. However, after 20 h, CI was found to exceed 3.0 for the positive control group (cells in media with chemo-attractant). Data showed that A549 cells migrated from the upper chamber (cells in serum-free media) to the lower one (media containing fetal bovine serum (FBS)) in response to the FBS chemo-attractant. Significant differences in CI values were also found between the negative and positive control groups ( P < 0.05). 5-FU: 5-fluorouracil; PB: Piper betle . * P < 0.05, compared with the positive control group; # P < 0.05, compared with the negative control group After 5 h of respective treatments, no significant differences in CI values were seen between groups ( F =3.107, P =0.060). However, significant differences were observed between treatment groups after 10 h ( F =6.114, P= 0.008), 15 h ( F =10.984, P= 0.001), and 20 h ( F =10.555, P =0.001). Indeed, after 10 h, cells in the positive control wells migrated towards the chemo-attractant, leading to high CI values (Fig. 1 1 ). Data showed that low concentrations of PB (20.0 μg/mL) and 5-FU (4.0 and 12.5 μmol/L) had minimal effects in inhibiting cell migration; cells treated with 100 μg/mL of PB had significant anti-migration effects ( P =0.034). This result implies that 100 μg/mL PB inhibited cell migration from the upper chamber to the lower one. 5-FU has potential in obstructing cancer cell migration as previously shown (Shakibaei et al., 2015 2015 ). PB exerted greater anti-migratory effect compared with 5-FU (Fig. 1 1 ). We further observed cell morphology, structure, and the tubulin network in PB-, paclitaxel-, and 5-FU-treated human colorectal adenocarcinoma cells (HT29). We observed apoptotic cells in HT29 treated with high-dose 5-FU (Fig. 2e 2e ) and noted a population of round cells in paclitaxel-and PB-treated cancer cells (Figs. 2c and 2g 2c and 2g ). Round cells are a feature of the M phase as previously reported (Théry and Bornens, 2008 2008 ; Cadart et al., 2014 2014 ). Thus, based on our morphological observations of PB-treated cancer cells (round shaped), we hypothesize that the possible mechanism of cell death triggered by PB is likely via microtubule polymerization. We therefore then included paclitaxel for comparison purposes and found both PB-and paclitaxel-treated cancer cells have similar morphologies. Paclitaxel is a microtubule inhibitor that can cause M phase cell-cycle arrest (Perez, 2009 2009 ); it stimulates polymerization which prevents or suppresses, microtubule disassembly, leading to cell-cycle arrest in the G2/M phase and ultimately apoptosis (Perez, 2009 2009 ). We therefore further investigated microtubule structure in respective treatment groups; results showed that PB had a similar long distorted spindle appearance compared with paclitaxel-treated cells (Figs. 2d and 2h 2d and 2h ). Distorted spindles are a defect on microtubules and can prevent cells from dividing (Ganguly et al., 2015 2015 ). We proposed that microtubule network disruption might represent the possible mechanism by which PB triggers cancer cell death. (a, b) Untreated; (c, d) 10 μg/mL paclitaxel; (e, f) 130 μmol/L 5-FU; (g, h) 200 μg/mL PB. Cell morphologies were observed under 400×magnification. Red arrows indicate apoptotic cells, while green arrows indicate M phase cells. Microtubule networks were observed under 1000×magnification. Tubulin was stained with anti-tubulin antibody at 1:1000 dilution followed by goat anti-mouse IgG conjugated with DyLight 488 at 1:1000 dilution (green signal). Nuclei were counterstained with DAPI (blue signal). Paclitaxel-and PB-treated HT29 cells have long spindles (yellow arrows). 5-FU: 5-fluorouracil; PB: Piper betle ; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride It has previously been reported that microtubule-targeting drugs inhibit cell motility, triggering both anti-migratory and anti-angiogenic effects (Belotti et al., 1996 1996 ; Hayot et al., 2006 2006 ; Ganguly et al., 2015 2015 ). Microtubules are an integral part of the cytoskeletal system of all eukaryotic cells. Indeed, because of their dynamic behavior, these structures play an important role in numerous biological processes including mitosis, cellular motility, and cytoplasmic transport. Cancer cell death can be induced via minor alterations of microtubule dynamics that engage the spindle checkpoint and cell cycle arrest (Blajeski et al., 2002 2002 ). As previously reported, different concentrations of microtubule-targeting drugs exert varied effects on cancer cells (Hayot et al., 2006 2006 ; Ganguly et al., 2015 2015 ; Gandalovičová et al., 2017 2017 ). A high drug concentration is needed to cause microtubules defects (Ganguly et al., 2015 2015 ), while a low concentration is capable of inhibiting cell migration (Hayot et al., 2006 2006 ). In this study, PB in higher concentrations (200 μg/mL) triggered a microtubule polymerization response, while lower concentrations (20 and 100 μg/mL) inhibited cell migration. We have demonstrated the potential use of PB as a microtubule-targeting agent which also exhibits anti-migratory effects on cancer cells. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References D Belotti , V Vergani , T Drudis , 等 . The microtubule-affecting drug paclitaxel has antiangiogenic activity . Clin Cancer Res , 1996 . 2 ( 11 ): 1843 - 1849 . 9816139 . The microtubule-affecting drug paclitaxel has antiangiogenic activity AL Blajeski , VA Phan , TJ Kottke , 等 . G 1 and G 2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells . J Clin Invest , 2002 . 110 ( 1 ): 91 - 99 . 12093892 DOI: 10.1172/JCI13275 http://doi.org/10.1172/JCI13275 . G1 and G2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells C Cadart , E Zlotek-Zlotkiewicz , M le Berre , 等 . Exploring the function of cell shape and size during mitosis . Dev Cell , 2014 . 29 ( 2 ): 159 - 169 . 24780736 DOI: 10.1016/j.devcel.2014.04.009 http://doi.org/10.1016/j.devcel.2014.04.009 . Exploring the function of cell shape and size during mitosis A Gandalovičová , D Rosel , M Fernandes , 等 . Migrastatics–anti-metastatic and anti-invasion drugs: promises and challenges . Trends Cancer , 2017 . 3 ( 6 ): 391 - 406 . 28670628 DOI: 10.1016/j.trecan.2017.04.008 http://doi.org/10.1016/j.trecan.2017.04.008 . Migrastatics–anti-metastatic and anti-invasion drugs: promises and challenges A Ganguly , HL Yang , R Sharma , 等 . The role of microtubules and their dynamics in cell migration . J Biol Chem , 2012 . 287 ( 52 ): 43359 - 43369 . 23135278 DOI: 10.1074/jbc.M112.423905 http://doi.org/10.1074/jbc.M112.423905 . The role of microtubules and their dynamics in cell migration A Ganguly , F Cabral , HL Yang , 等 . Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells . Oncoscience , 2015 . 2 ( 6 ): 585 - 595 . 26244166 . Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells A Guha Majumdar , M Subramanian . Hydroxychavicol from Piper betle induces apoptosis, cell cycle arrest, and inhibits epithelial-mesenchymal transition in pancreatic cancer cells . Biochem Pharmacol , 2019 . 166 274 - 291 . 31154000 DOI: 10.1016/j.bcp.2019.05.025 http://doi.org/10.1016/j.bcp.2019.05.025 . Hydroxychavicol from Piper betle induces apoptosis, cell cycle arrest, and inhibits epithelial-mesenchymal transition in pancreatic cancer cells C Hayot , O Debeir , P van Ham , 等 . Characterization of the activities of actin-affecting drugs on tumor cell migration . Toxicol Appl Pharmacol , 2006 . 211 ( 1 ): 30 - 40 . 16005926 DOI: 10.1016/j.taap.2005.06.006 http://doi.org/10.1016/j.taap.2005.06.006 . Characterization of the activities of actin-affecting drugs on tumor cell migration E Mukhtar , VM Adhami , H Mukhtar . Targeting microtubules by natural agents for cancer therapy . Mol Cancer Ther , 2014 . 13 ( 2 ): 275 - 284 . 24435445 DOI: 10.1158/1535-7163.MCT-13-0791 http://doi.org/10.1158/1535-7163.MCT-13-0791 . Targeting microtubules by natural agents for cancer therapy PL Ng , NF Rajab , SM Then , 等 . Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2014 . 15 ( 8 ): 692 - 700 . DOI: 10.1631/jzus.B1300303 http://doi.org/10.1631/jzus.B1300303 . Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells EA Perez . Microtubule inhibitors: differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance . Mol Cancer Ther , 2009 . 8 ( 8 ): 2086 - 2095 . 19671735 DOI: 10.1158/1535-7163.Mct-09-0366 http://doi.org/10.1158/1535-7163.Mct-09-0366 . Microtubule inhibitors: differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance B Salehi , ZA Zakaria , R Gyawali , 等 . Piper species: a comprehensive review on their phytochemistry, biological activities and applications . Molecules , 2019 . 24 ( 7 ): 1364 DOI: 10.3390/molecules24071364 http://doi.org/10.3390/molecules24071364 . Piper species: a comprehensive review on their phytochemistry, biological activities and applications M Shakibaei , P Kraehe , B Popper , 等 . Curcumin potentiates antitumor activity of 5-fluorouracil in a 3D alginate tumor microenvironment of colorectal cancer . BMC Cancer, 15:250 , 2015 . DOI: 10.1186/s12885-015-1291-0 http://doi.org/10.1186/s12885-015-1291-0 . Curcumin potentiates antitumor activity of 5-fluorouracil in a 3D alginate tumor microenvironment of colorectal cancer M Théry , M Bornens . Get round and stiff for mitosis . HFSP J , 2008 . 2 ( 2 ): 65 - 71 . 19404473 DOI: 10.2976/1.2895661 http://doi.org/10.2976/1.2895661 . Get round and stiff for mitosis PF Wu , HC Tseng , CC Chyau , 等 . Piper betle leaf extracts induced human hepatocellular carcinoma HEP3B cell death via MAPKS regulating the p73 pathway in vitro and in vivo . Food Funct , 2014 . 5 ( 12 ): 3320 - 3328 . 25371988 DOI: 10.1039/C4FO00810C http://doi.org/10.1039/C4FO00810C . Piper betle leaf extracts induced human hepatocellular carcinoma HEP3B cell death via MAPKS regulating the p73 pathway in vitro and in vivo We thank Taylor’s University and Forest Research Institute Malaysia (FRIM), Malaysia, for the laboratory facilities. Compliance with ethics guidelines Mee Lee LOOI, Alwyn Khai Howe WONG, Shelly Anne GNAPRAGASAN, Anis Zafirah JAPRI, Aiysvariyah RAJEDADRAM, and Kar Yong PIN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B2000278 Project supported by the Taylor’s Research Grant Scheme (No. TRGS/MFS/2/2013/SBS/003) 2020-05-27 2020-07-16 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 21