Original Article Effects of norcantharidin on proliferation, apoptosis and migration of colon cancer

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Int J Clin Exp Med 2019;12(11):13086-13095 www.ijcem.com /ISSN:1940-5901/IJCEM0096779. Original Article Effects of norcantharidin on proliferation, apoptosis and migration of colon cancer. Zhiqiang Liao1,2, Shaolin Nie1,2. 1University of South China, Hengyang, Hunan, China; 2Department of Colorectal Surgery, Hunan Cancer Hospital, Changsha, Hunan, China. Received May 9, 2019; Accepted August 6, 2019; Epub November 15, 2019; Published November 30, 2019. Abstract: Objective: The effect and possible mechanism of norcantharidin treatment on the proliferation, apoptosis and invasion of CRC cells were studied. Methods: Colon cancer cell lines Caco2 or HT-29 were treated with nor- cantharidin chewing hormone at a concentration of 0-120 μM. The cell proliferation rate was determined by CCK-8 method, and apoptosis was detected by Hoechst-33258 staining. Transwell assay was used to assess the cell inva- sion. Western blot was used to analyze changes in apoptosis and EMT pathway molecules. Results: Norcantharidin inhibits cell proliferation and invasion and promotes apoptosis. Western blot analysis showed that norcantharidin can up-regulate the levels of Bax, cleave caspase-3 and cleave caspase-9 in a concentration-dependent manner, and down-regulate the level of Bcl-2 to affect the apoptosis of colon cancer cells. Norcantharidin can up-regulate the level of E-cadherin in a concentration-dependent manner, and down-regulate the levels of N-cadherin, Vimentin, p-ERK and integrin β6 molecules to affect colon cancer cell invasion. Conclusions: These results indicate that nor- cantharidin regulates apoptosis through the Bcl-2 signaling pathway and regulates cell invasion by affecting EMT pathway molecules, ERK phosphorylation, and integrin beta6 levels.. Keywords: Colon cancer, norcantharidin, invasion, apoptosis, mechanism. Introduction. Colon cancer is one of the most common malig- nant tumors in the digestive tract. In western countries, it is the second most common tumor type and a serious threat to human health [1]. Many epidemiological studies have shown that Western diets such as red meat may be ass- ociated with high colon cancer incidence [2]. Despite early detection and a decline in colon cancer mortality, an estimated 112,340 new cases were reported in 2007 [3-5]. The most common treatment for colon and rectal cancer is surgical resection, and then chemotherapy regimens such as 5-fluorouracil, oxaliplatin and leucovorin used for adjuvant therapy. If the patients are diagnosed early and undergoe sur- gical resection, up to 90% of them can achieve a 5-year survival rate. However, 5-year survival can be reduced to 10% in patients with distant metastasis at diagnosis [6, 7]. Patients with poor physical fitness combined with damage to the patients from surgery, and the side effects. of radiotherapy or chemotherapy have led to an increase in treatment failure [8]. Therefore, the search for anticancer drugs with low toxicity and few side effects has become a hot topic of research.. Cantharides is a frequently-used Chinese herb- al medicine in clinical practice. Cantharidin main medicinal ingredients have the effect of attacking phlegm, breaking down and dispers- ing blood [9]. It was found that cantharidin had significant inhibitory effects on multiple tumor cell types such as hepatocellular carcinoma, esophageal cancer, ovarian cancer and promy- elocytic leukemia [10]. In recent years, canthar- idin has been identified as a compound with antitumor activity [11-14]. However, it can cause serious side effects in both animal and human urinary systems [15]. Norcantharidin (NCTD), a chemical demethylation from cantharidin (CTD), overcomes urological toxicity and en- hances its antitumor activity [16]. NCTD has been approved by China’s food and drug admin-. http://www.ijcem.com. Effects of norcantharidin on colon cancer. 13087 Int J Clin Exp Med 2019;12(11):13086-13095. istration for the treatment of liver, esophagus and stomach cancer since 1996, according to clinical studies [1, 17]. However, there are few reports about its effects on colon cancer.. In this study, the effects of norcantharidin treat- ment on proliferation, apoptosis and invasion of CRC cells and possible related mechanisms were determined by in vitro experiments and molecular mechanism analysis.. Material and methods. Human colon cancer cell lines HT29 and Caco2 were purchased form Shanghai Cell Storage Center, Chinese Academy of Sciences, Shanghai, China; H-DMEM medium and fetal bovine serum were purchased form Gibco, New York, USA; norcantharidin was obtained from the State Food and Drug Administration of China. Methyl thiazolyl tetrazolium (MTT), Propidium iodide (PI), Hoechst 33342, RNase A, Triton X-100, and dimethyl sulfoxide (DMSO) were purchased form Sigma, USA; Apoptosis Detection Kit (Annexin V-FITC/PI) were pur- chased form BD, USA; rabbit anti-human mo- noclonal antibodies against Bax, Bcl-2, cas- pase-3 and caspase-3 were purchased form Santa Cruz, USA; sheep anti-rabbit secondary antibody were purchased form Santa Cruz, USA; ECL chromogenic kit were purchased form Abcam, USA; HRP-labeled secondary antibod- ies and ECL chemiluminescent reagents were purchased from KPL, USA.. Cell culture and treatment with norcantharidin. According to the methods described in the lit- erature [18], two human colon cancer cell lines, HT29 and Caco2, were cultured and passaged in DMEM medium containing 10% fetal bovine serum, 10000 U/ml penicillin, and 10000 U/ml streptomycin. The conditions of the incubator were set to 37°C, 5% CO2, with relative humidity of 90%, and the cells were subcultured after the cells were in the logarithmic growth phase. Norcantharidin was obtained from the State Food and Drug Administration of China. Norcantharidin was dissolved in dimethyl sulf- oxide (DMSO) and the final concentration of DMSO used in the medium was 0.1%.. Cell growth inhibition test. HT29 and Caco2 cells suspended in complete medium were seeded in 96-well plates (2000. cells/well). After 24 hours of growth in the incu- bator, the medium was aspirated and 0.2 mL of complete medium containing a continuous con- centration of Caco2 was added to each well. After incubating the plates for 24 or 48 hours, 10 μl of cck-8 (Sigma) was added to each well. After incubation for 16 hours at 37°C in an incubator, the fluorescence signal was moni- tored by Spectramax M5 plate reader (Mo- lecular Devices) using 450 nm excitation and 630 nm emission wavelength. The number of viable cells in each well was proportional to the relative fluorescence unit (RFU) measured by the assay [18].. Evaluation of apoptosis by hoechst-33258 staining. CACO-2 and HT-29 cells were seeded in 12-well culture dishes (5 × 104 cells/well). After the experimental treatment, the cells were washed twice with phosphate buffer solution (PBS), and were stained with Hoechst-33258 (5 mg/ml) for 5 minutes in the dark, and then they were washed thoroughly. Karyotin was examined under a fluorescence microscope and images were captured with ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA).. Flow cytometry for detection of cell apoptosis. CACO-2 and HT-29 cells were seeded in 12-well culture dishes (5 × 104 cells/well). The cells were treated with norcantharidin (0, 20, 40, 80 μM) for 48 h and centrifuged at 2000 g per minute for 5 min. The supernatant was discard- ed, and the cells were collected. The PBS solu- tion was used to wash the cells × 2 times. The cells were centrifuged at 5 × 105 rpm. Ten μL of LAnnexin V-FITC and 5 μL of PI were added, gently mixed, and incubated at 4°C for 30 min. Flow cytometry was used to detect the cell apoptosis.. Matrigel invasion assay. In the cell invasion assay, 1 × 105 cells were placed in the upper chamber pre-coated with Matrigel. The cells in the upper chamber were resuspended in 0.2% fetal bovine serum for inoculation. Cells in the lower compartment contained 10% FBS as chemotaxis. The Transwell plate was placed in a 5% carbon dioxide incubator at 37°C for several hours, then fixed with 20% ethanol, and stained with 0.2% crystal violet for photographing.. Effects of norcantharidin on colon cancer. 13088 Int J Clin Exp Med 2019;12(11):13086-13095. Cell migration assay. The logarithmic growth phase cells were seed- ed in a 24-well plate at 1 × 106 cells/well. After 24 hours of culture in DMEM medium, a 200 μL pipette tip was used to draw a “__” scratch on the 24-well plate. The culture medium was changed, and the exfoliated cells were removed. The cells were treated with norcantharidin (0, 20, 40, 80 μM) for 48 h, and the cells were washed with PBS × 3 times. The width of the scratch was observed under an inverted micro- scope and photographed, and it was photo- graphed in the original position for comparison to evaluate the migration ability of the cells. Migration index (%) = (initial scratch width- scratch width after healing)/initial scratch width × 100%.. Western blot. After protein separation using the different cells above, the expression of CCNB1 molecule was detected by Western blot. The Western blot method is briefly described as follows: cel- lular proteins were analyzed by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. After the PVDF membrane was blocked with 5% skim milk, anti-E-cadherin, anti-vimentin, anti-p-ERK, anti-ERK, anti-Bcl-2, anti-β6, anti-Bax, anti-capase-3 and anti-ca- pase-9 monoclonal antibodies for incubation. The GAPDH monoclonal antibody was used as an internal reference. Proteins were examined by chemiluminescence. The protein bands. obtained were quantified using Image J soft- ware and analyzed for protein levels.. Statistical analysis. All data were expressed as Mean ± standard deviation (Mean ± SD). The differences between groups were compared by using one-way ANOVA with post hoc Bonferroni pairwise comparison. P<0.05 was considered statistically significant. All statistical methods were analyzed by GraphPad Prism 6.0 and SPSS 18.0 software.. Results. Effects of norcantharidin on the growth of colon cancer cell lines Caco2 and HT29. In order to clarify the biological effects of nor- cantharidin, we selected Caco2 and HT29 cell lines to evaluate the growth of colon cancer (Figure 1). It can be seen from the figure that norcantharidincan inhibits the growth of colon cancer cell lines in a concentration-depen- dent manner. At the same time, after 48 h of culture, the inhibition rate of norcantharidin on colon cancer cells was significantly higher than that after culturing for 24 h. The result sug- gests that norcantharidin inhibits the growth of colon cancer cells in a concentration- and time- dependent manner.. Effects of norcantharidin on apoptosis of colon cancer cell lines Caco2 and HT29. In order to clarify the biological effects of nor- cantharidin on cell lines, we used Hoechst-. Figure 1. Effect of NCTD on the growth of colon cancer cell lines Caco2 and HT29. Colon cancer cell lines Caco2 (A) and HT29 (B) were treated with NCTD (0-120 μM), and cell viability was measured by MTT assay at 24 and 48 hours. *p<0.05, **p<0.01 was compared with the 0 μM solvent control group at the same time point; #p<0.05, ##p<0.01 was compared with the 10 μM solvent control group at the same time point; $p<0.05, $$p<0.01 was compared with the 20 μM solvent control group at the same time point.. Effects of norcantharidin on colon cancer. 13089 Int J Clin Exp Med 2019;12(11):13086-13095. 33258 staining and Annexin V-PI flow cytome- try to evaluate apoptosis of colon cancer cells (Figures 2, 3). Norcantharidin can promote apoptosis of colon cancer cell lines in a concen- tration-dependent manner. When the concen- tration reached 40 and 80 M, the apoptosis of cells was significantly different from that of the solvent control group. This result showed that norcantharidin can promote apoptosis of colon cancer cells in a concentration-dependent manner.. Mechanism of the effect of norcantharidin on apoptosis of colon cancer cell lines. Since the above mentioned effects of norcan- tharidin on the apoptosis of colon cancer cell lines were observed, we further analyzed its effect on apoptosis-related molecular path- ways (Figure 4). It can be seen from the figure that norcantharidin compared with the control groups can significantly up-regulate the levels of Bax, Cleaved caspase-3 and Cleaved cas- pase-9 in a concentration-dependent manner, and down-regulate the level of Bcl-2 molecules. The results showed that normochelin can affect the apoptosis of colon cancer cells by affecting Bcl-2 family molecules.. Effect of norcantharidin on the invasion of colon cancer cell lines Caco2 and HT29. We further evaluated the effect of norcanthari- din on the colon cancer cell lines (Figure 5). It can be seen from the figure that norcantharidin can reduce the invasion of colon cancer cell lines in a concentration-dependent manner. When the concentrations reached 20, 40 and 80 μM, the apoptosis of the cells was signifi- cantly different from that of the solvent control group. The results showed that norcantharidin can reduce the invasion of colon cancer cells in a concentration-dependent manner.. Effect of norcantharidin on the migration of colon cancer cell lines Caco2 and HT29. In order to clarify the biological effects of nor- cantharidinon cell lines, we evaluated the effect of norcantharidin on the migration of colon cancer cell lines (Figure 6). It can be seen from the figure that norcantharidin can reduce the migration of colon cancer cells in a concen- tration-dependent manner. At the beginning, the scratch width of all groups was basically equal. When the concentrations reached 20, 40 and 80 μM, norcantharidin significantly promoted the migration of colon cancer SW-. Figure 2. Effect of NCTD on apoptosis of colon cancer cell lines Caco2 and HT29 (Hoechst-33258 cell staining method). Colon cancer cell lines Caco2 (A) and HT29 (B) were treated with NCTD (0-80 μM), and apoptosis was detected by Hoechst-33258 cell staining at 24 h. *p<0.05, **p<0.01 was compared with the 0 μM solvent control group at the same time point; #p<0.05 was compared with the 20 μM solvent control group at the same time point; $p<0.05 was compared with the 40 μM solvent control group at the same time point.. Effects of norcantharidin on colon cancer. 13090 Int J Clin Exp Med 2019;12(11):13086-13095. 480 cells. This was significantly different from 0 μM concentration (P<0.01). The results showed that norcantharidin can reduce the invasion of colon cancer cells in a concentra- tion-dependent manner.. Mechanism of the effect of norcantharidin on the Invasion of colon cancer cell lines. Since the above-mentioned inhibitory effect of norcantharidin on colon cancer cell line inva- sion was observed, we further analyzed the lev- els of EMT pathway molecule, ERK phosphory- lation and integrin β6 (Figure 7). From the results, NCTD can up-regulate the level of. E-cadherin in a concentration-dependent man- ner and down-regulate the levels of N-cadherin, Vimentin, p-ERK and integrin β6 molecules; the difference was significantly different from that of the solvent control group. The results showed that norcantharidin can affect the inva- sion of colon cancer cells by affecting the levels of EMT pathway molecules, ERK phosphoryla- tion and integrin β6.. Discussion. In the present study, we studied the treatment of colon cancer cells Caco2 or HT-29 with nor- cantharidin at a concentration of 0-80 μM. Cell proliferation was measured by CCK-8 method,. Figure 3. Effect of NCTD on apoptosis of colon cancer cell lines Caco2 and HT29 (Annexin V-PI flow cytometry). (A) The apoptosis of colon cancer cells lines Caco2 (A) and HT29 (B) treated with NCTD (0-80 μM) after 24 h by Annexin V-PI flow cytometry; (B) The histogram of apoptosis rate comparison in Caco2; (C) The histogram of apoptosis rate comparison in HT29. *p<0.05, **p<0.01 was compared with the 0 μM solvent control group at the same time point; #p<0.05 was compared with the 20 μM solvent control group at the same time point; $p<0.05 was compared with the 40 μM solvent control group at the same time point.. Effects of norcantharidin on colon cancer. 13091 Int J Clin Exp Med 2019;12(11):13086-13095. and apoptosis was measured by using Ho- echst-33258 staining and flow cytometry ev- aluation. Transwell assay was used to assess the invasion. Cell scratch assay was used to evaluate cell migration. Western blot analysis of apoptosis and changes in EMT pathway mol- ecules was performed to determine the effect of norcantharidin treatment on CRC cell prolif-. eration, the influence of apoptosis and invasion and its possible mechanism. Our results showed that norcantharidin inhibited cell prolif- eration, invasion and migration, and promoted apoptosis. Western blot analysis showed that norcantharidin can up-regulate the level of E-cadherin in a concentration-dependent man- ner, and down-regulate the levels of N-cadhe-. Figure 4. NCTD effects on apoptosis-related molecules of colon cancer cell lines Caco2 and HT29. (A) The expres- sion of Bax, Bcl-2, Cleaved caspase-3, and Cleaved caspase-9 in colon cancer cells treated with NCTD (0-80 μM) de- tected by Western blotting after 24 h; (B) The histogram of protein expression of Bax, Bcl-2, Cleaved caspase-3, and Cleaved caspase-9 in Caco2; (C) The histogram of protein expression of Bax, Bcl-2, Cleaved caspase-3, and Cleaved caspase-9 in HT29. *p<0.05, **p<0.01 was compared with the 0 μM solvent control group at the same time point; #p<0.05 was compared with the 20 μM solvent control group at the same time point; $p<0.05 was compared with the 40 μM solvent control group at the same time point.. Effects of norcantharidin on colon cancer. 13092 Int J Clin Exp Med 2019;12(11):13086-13095. Figure 5. The effect of NCTD on the invasion of colon cancer cell lines Caco2 and HT29. Transwell method was used to evaluate the effect of NCTD (0-80 μM) on the invasion of colon cancer cell lines Caco2 and HT29. Cell invasion was detected by cell crystal violet staining at 48 h collection chamber. *p<0.05, **p<0.01 was compared with the 0 μM solvent control group at the same time point. #p<0.05 was compared with the 20 μM solvent control group at the same time point; $p<0.05 was compared with the 40 μM solvent control group at the same time point.. Figure 6. Effect of norcantharidin on the migration of colon cancer cell lines Caco2 and HT29. Norcanthapyr re- duced the migration of colon cancer cells in a concentration-dependent manner. At the beginning, the scratch width of all groups was basically equal. When the concentrations reached 20, 40 and 80 μM, norcantharidin significantly promoted the migration of colon cancer SW480 cells.. Effects of norcantharidin on colon cancer. 13093 Int J Clin Exp Med 2019;12(11):13086-13095. rin, Vimentin, p-ERK and integrin β6 molecules to affect colon cancer cell invasion and migration.. Numerous studies have shown that NCTD has the ability to induce cell death in a variety of cancer cells, such as colorectal cancer and lung cancer cells [19]. The anti-cancer activity of NCTD is dependent on its ability to activate the caspase signaling pathway, leading to apop- tosis accompanied by a decrease [20] in the Bcl-2/Bax ratio [19, 21]. The results of this. study showed that norcantharidin can up-regu- late the levels of Bax, Cleaved caspase-3 and Cleaved caspase-9 in a concentration-depen- dent manner, and down-regulate the level of Bcl-2 molecules. This suggests that the rele- vant Bcl-2 pathway molecule is involved in the apoptosis effect of norcantharidinon colon can- cer cells.. EMT is the process by which the cell phenotype changes from epithelial cells to mesenchymal cells. EMT process, which plays an important. Figure 7. The effect of NCTD on the molecular pathways involved in the invasion of colon cancer cell lines Caco2 and HT29. Colon cancer cell lines Caco2 and HT29 were treated with NCTD (0-120 μM), and EMT pathway molecules, ERK phosphorylation and integrin β6 levels were analyzed by Western blot at 24 hours. It can be seen from the results that NCTD can up-regulate the level of E-cadherin in a concentration-dependent manner and down-regulate the levels of N-cadherin, Vimentin, p-ERK and integrin β6 molecules. *p<0.05 was compared with the 0 μM solvent control group at the same time point; **p<0.01 was compared with the 0 μM solvent control group at the same time point.. Effects of norcantharidin on colon cancer. 13094 Int J Clin Exp Med 2019;12(11):13086-13095. role in regulating the invasion of malignant epi- thelial tumor cells [22], has been widely accept- ed as a key step in distant tumor metastasis. During EMT, expression of epithelin markers such as E-cadherin is reduced, and expression of mesenchymal protein markers such as vimentin and N-cadherin is up-regulated [23].. Integrin is a transmembrane glycoprotein receptor composed of non-covalently bound alpha and beta subunits and belongs to a fam- ily of cell surface adhesion molecules. avβ6 is a specific integrin isoform that is expressed only in epithelial cells and its main ligand is fibronec- tin (FN). In normal epithelial cells, the expres- sion of αvβ6 is rare and difficult to detect. But it is significantly increased in injury and/or inflam- mation as well as epithelial tumors (gastric cancer, colon cancer and others) [24]. De novo expression of integrin αvβ6 has been shown to regulate several behaviors of colon cancer cells, including cell adhesion and fibronectin diffusion, proliferation in collagen gels, tumor growth, cell invasion and metastasis, and apop- tosis [25]. It is known that direct ligation between αvβ6 and ERK2 can increase the phosphorylation level of ERK2 in colorectal cancer cells, and activated ERK1/2 can increase Ets-1 transcriptional activity by form- ing a signaling complex with Ets-1. This en- hances transcriptional activation of genes that bind to the promoter region and induce involve- ment in malignancy [26]. The results of this study showed that NCTD can up-regulate the level of E-cadherin in a concentration-depen- dent manner and down-regulate the levels of N-cadherin, Vimentin, p-ERK and integrin β6 molecules. This result shows that norcanthari- din can affect the invasion of colon cancer cells by affecting the levels of EMT pathway mole- cules, ERK phosphorylation and integrin β6.. In summary, norcantharidin regulates apopto- sis through the Bcl-2 signaling pathway and regulates invasion by affecting EMT pathway molecules, ERK phosphorylation, and integrin β6levels. This study demonstrates that norcan- tharidin can affect the biological behavior of colon cancer cells, which may provide a basis for the development of therapeutic strategies for CRC.. Disclosure of conflict of interest. None.. Address correspondence to: Shaolin Nie, Univer- sity of South China, Hengyang, Hunan, China; De- partment of Colorectal Surgery, Hunan Cancer Hos- pital, No.283, Tongxinpo Road, Changsha 410006, Hunan, China. Tel: +86-13786151230; E-mail: nies- la@163.com. References. [1] Goss PE, Strasser-Weippl K, Lee-Bychkovsky BL, Fan L, Li J, Chavarri-Guerra Y, Liedke PE, Pramesh CS, Badovinac-Crnjevic T, Sheikine Y, Chen Z, Qiao YL, Shao Z, Wu YL, Fan D, Chow LW, Wang J, Zhang Q, Yu S, Shen G, He J, Puru- shotham A, Sullivan R, Badwe R, Banavali SD, Nair R, Kumar L, Parikh P, Subramanian S, Chaturvedi P, Iyer S, Shastri SS, Digumarti R, Soto-Perez-de-Celis E, Adilbay D, Semiglazov V, Orlov S, Kaidarova D, Tsimafeyeu I, Tatishchev S, Danishevskiy KD, Hurlbert M, Vail C, St Louis J and Chan A. Challenges to effective cancer control in China, India, and Russia. Lancet On- col 2014; 15: 489-538.. [2] Haggar FA and Boushey RP. Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg 2009; 22: 191-197.. [3] Kang JI, Hong JY, Choi JS and Lee SK. Columbi- anadin inhibits cell proliferation by inducing apoptosis and necroptosis in HCT116 colon cancer cells. Biomol Ther (Seoul) 2016; 24: 320-327.. [4] Alexander DD, Weed DL, Miller PE and Mo- hamed MA. Red meat and colorectal cancer: a quantitative update on the state of the epide- miologic science. J Am Coll Nutr 2015; 34: 521-543.. [5] Vieira AR, Abar L, Chan DSM, Vingeliene S, Po- lemiti E, Stevens C, Greenwood D and Norat T. Foods and beverages and colorectal cancer risk: a systematic review and meta-analysis of cohort studies, an update of the evidence of the WCRF-AICR continuous update project. Ann Oncol 2017; 28: 1788-1802.. [6] Kuipers EJ and Spaander MC. Colorectal can- cer screening by colonoscopy, CT-colonogra- phy, or fecal immunochemical test. J Natl Can- cer Inst 2015; 108.. [7] Ristau J, Staffa J, Schrotz-King P, Gigic B, Ma- kar KW, Hoffmeister M, Brenner H, Ulrich A, Schneider M, Ulrich CM and Habermann N. Suitability of circulating miRNAs as potential prognostic markers in colorectal cancer. Can- cer Epidemiol Biomarkers Prev 2014; 23: 2632-2637.. [8] Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Lerro C, Fede- wa S, Lin C, Leach C, Cannady RS, Cho H, Scop- pa S, Hachey M, Kirch R, Jemal A and Ward E.. mailto:niesla@163.com mailto:niesla@163.com. Effects of norcantharidin on colon cancer. 13095 Int J Clin Exp Med 2019;12(11):13086-13095. Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 2012; 62: 220-241.. [9] Moed L, Shwayder TA and Chang MW. Can- tharidin revisited: a blistering defense of an ancient medicine. Arch Dermatol 2001; 137: 1357-1360.. [10] Pon D, Wang Z, Le KN and Chow MS. Harness- ing traditional Chinese medicine to improve cancer therapy: issues for future development. Ther Deliv 2010; 1: 335-344.. [11] Li CC, Yu FS, Fan MJ, Chen YY, Lien JC, Chou YC, Lu HF, Tang NY, Peng SF, Huang WW and Chung JG. Anticancer effects of cantharidin in A431 human skin cancer (epidermoid carcino- ma) cells in vitro and in vivo. Environ Toxicol 2017; 32: 723-738.. [12] Le AP, Zhang LL, Liu W and Shi YF. Cantharidin inhibits cell proliferation and induces apopto- sis through G2/M phase cell cycle arrest in he- patocellular carcinoma stem cells. Oncol Rep 2016; 35: 2970-2976.. [13] Shen M, Wu MY, Chen LP, Zhi Q, Gong FR, Chen K, Li DM, Wu Y, Tao M and Li W. Cantharidin represses invasion of pancreatic cancer cells through accelerated degradation of MMP2 mRNA. Sci Rep 2015; 5: 11836.. [14] Hsiao YP, Tsai CH, Wu PP, Hsu SC, Liu HC, Huang YP, Yang JH and Chung JG. Cantharidin induces G2/M phase arrest by inhibition of Cdc25c and Cyclin A and triggers apoptosis through reactive oxygen species and the mito- chondriadependent pathways of A375.S2 hu- man melanoma cells. Int J Oncol 2014; 45: 2393-2402.. [15] Cotovio P, Silva C, Guedes Marques M, Ferrer F, Costa F, Carreira A and Campos M. Acute kid- ney injury by cantharidin poisoning following a silly bet on an ugly beetle. Clin Kidney J 2013; 6: 201-203.. [16] Hsieh CH, Chao KS, Liao HF and Chen YJ. Nor- cantharidin, derivative of cantharidin, for can- cer stem cells. Evid Based Complement Alter- nat Med 2013; 2013: 838651.. [17] El-Tantawy NL. Helminthes and insects: mala- dies or therapies. Parasitol Res 2015; 114: 359-377.. [18] Wu S, Wu F and Jiang Z. Effect of HOXA6 on the proliferation, apoptosis, migration and in- vasion of colorectal cancer cells. Int J Oncol 2018; 52: 2093-2100.. [19] Xie J, Zhang Y, Hu X, Lv R, Xiao D, Jiang L and Bao Q. Norcantharidin inhibits Wnt signal path- way via promoter demethylation of WIF-1 in hu- man non-small cell lung cancer. Med Oncol 2015; 32: 145.. [20] Chen YN, Chen JC, Yin SC, Wang GS, Tsauer W, Hsu SF and Hsu SL. Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells. Int J Can- cer 2002; 100: 158-165.. [21] Yang PY, Chen MF, Tsai CH, Hu DN, Chang FR and Wu YC. Involvement of caspase and MAPK activities in norcantharidin-induced colorectal cancer cell apoptosis. Toxicol In Vitro 2010; 24: 766-775.. [22] McConkey DJ, Choi W, Marquis L, Martin F, Wil- liams MB, Shah J, Svatek R, Das A, Adam L, Kamat A, Siefker-Radtke A and Dinney C. Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer. Cancer Metastasis Rev 2009; 28: 335- 344.. [23] De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, Gespach C, Bracke M and Berx G. Molecular and pathological signa- tures of epithelial-mesenchymal transitions at the cancer invasion front. Histochem Cell Biol 2008; 130: 481-494.. [24] Nieberler M, Reuning U, Reichart F, Notni J, Wester HJ, Schwaiger M, Weinmuller M, Rader A, Steiger K and Kessler H. Exploring the role of RGD-recognizing integrins in cancer. Cancers (Basel) 2017; 9.. [25] Liu S, Wang J, Niu W, Liu E, Wang J, Peng C, Lin P, Wang B, Khan AQ, Gao H, Liang B, Shahbaz M and Niu J. The beta6-integrin-ERK/MAP ki- nase pathway contributes to chemo resistance in colon cancer. Cancer Lett 2013; 328: 325- 334.. [26] Peng C, Li Z, Niu Z, Niu W, Xu Z, Gao H, Niu W, Wang J, He Z, Gao C, Lin P, Agrez M, Zhang Z and Niu J. Norcantharidin suppresses colon cancer cell epithelial-mesenchymal transition by inhibiting the alphavbeta6-ERK-Ets1 signal- ing pathway. Sci Rep 2016; 6: 20500.

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