The tumorigenicity and expressions of dishevelled 3 in HCT116 cells and HCT116 spherical cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

HOU Songlin , CHEN Xiaobo , LIU Daizhi , LI Xiaoqiong , ZHOU He , LI Lifa , ZHANG Guangjun ,  ZHOU Tong

1. Department of Gastrointestinal Surgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China;
2. Institute of Hepato-Biliary-Pancreas and Intestinal Disease, North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China;

Corresponding author：

ZHOU Tong, Email: zhoutong0088@163.com

Keywords：

colorectal cancer; cancer stem cells; cancer spherical cells; tumorigenicity; dishevelled 3

DOI：

10.7507/1007-9424.201707009

Video：

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Objective To explore the tumorigenicity and expressions of dishevelled 3 (DVL3) in HCT116 cells and HCT116 spherical cells. Methods Human colorectal tumor HCT116 cells were cultured in the serum-free culture medium for HCT116 spherical cells. Through the subcutaneous tumor experiment in nude mice and clone formation assay, we observed the tumor growth and colony formation ability of the two kinds of cells in vivo and in vitro. The Western blotting experiment was utilized to detect the expressions of DVL3 in these two kinds of cells. Results ① Colonyformation: the mean value of colony formation rate in the HCT116 cells group was 3.78%, and the mean value of fcolony formation rate in the HCT116 spherical cells group was 28.67%, which was higher in the HCT116 spherical cells group (t=21.16, P<0.05). ② Tumorigenicity in nude mice: 11 nude mice with tumor formation were observed in the HCT116 cells group, and the tumor formation rate was 55.0%; 18 nude mice with tumor formation were observed in the HCT116 spherical cells group, and the tumor formation rate was 90.0%, the tumor formation rate of the HCT116 spherical cells group was higher (P=0.039). The tumor volume of the HCT116 cells group was (92±31) mm³, and the tumor volume of HCT116 spherical cells group was (298±85) mm³, the tumor volume of the HCT116 spherical cells group was larger (t=9.27, P<0.05). ③ The expression of DVL3: the expression level of DVL3 in HCT116 cells was 0.12±0.05, and expression level of DVL3 in HCT116 spherical cells was 0.35±0.10, the expression level of DVL3 in HCT116 spherical cells was higher (t=4.31, P<0.05). Conclusions The HCT116 spherical cells have stronger colonization and tumorigenicity than the HCT116 cells. It has been speculatd that the high expression of DVL3 may be closely related with the stronger tumorigenicity.

Citation： HOU Songlin, CHEN Xiaobo, LIU Daizhi, LI Xiaoqiong, ZHOU He, LI Lifa, ZHANG Guangjun, ZHOU Tong. The tumorigenicity and expressions of dishevelled 3 in HCT116 cells and HCT116 spherical cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2017, 24(12): 1463-1467. doi: 10.7507/1007-9424.201707009 Copy

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2.	Greve B, Kelsch R, Spaniol K, et al. Flow cytometry in cancer stem cell analysis and separation. Cytometry A, 2012, 81(4): 284-293.
3.	龚锐, 李胜英, 霍志霞, 等. 自制免疫磁珠在前列腺肿瘤组织干细胞提取中的应用. 中国组织工程研究, 2016, 20(1): 36-41.
4.	Cariati M, Naderi A, Brown JP, et al. Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-like subpopulation within the MCF7 breast cancer cell line. Int J Cancer, 2008, 122(2): 298-304.
5.	Qi J, Lee HJ, Saquet A, et al. Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. J Biol Chem, 2017, 292(14): 5898-5908.
6.	Zhao Y, Yang ZQ, Wang Y, et al. Dishevelled-1 and dishevelled-3 affect cell invasion mainly through canonical and noncanonical Wnt pathway, respectively, and associate with poor prognosis in nonsmall cell lung cancer. Mol Carcinog, 2010, 49(8): 760-770.
7.	Kwan HT, Chan DW, Cai PC, et al. AMPK activators suppress cervical cancer cell growth through inhibition of DVL3 mediated Wnt/β-catenin signaling activity. PLoS One, 2013, 8(1): e53597.
8.	Tsui Y. Study of the roles of dishevelled-3 in stemness and cell migration in hepatocellular carcinoma. Hong Kong: The University of Hong Kong, 2013.
9.	李利发, 张井潇, 陈小波, 等. 人结直肠肿瘤干细胞致瘤能力及 Dvl2 蛋白表达的研究. 中华普通外科杂志, 2016, 31(9): 774-776.
10.	王磊, 宋顺心, 汪建平. 结直肠癌实验研究现状及展望. 中华实验外科杂志, 2013, 30(3): 429-430.
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12.	Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997, 3(7): 730-737.
13.	Zeuner A, Todaro M, Stassi G, et al. Colorectal cancer stem cells: from the crypt to the clinic. Cell Stem Cell, 2014, 15(6): 692-705.
14.	Lobba AR, Forni MF, Carreira AC, et al. Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines. Cytometry A, 2012, 81(12): 1084-1091.
15.	Mendoza FHD, Rodriguez EA. Cancer stem cells in brain tumors//Grande E, Antón A, Paricio L. Stem cells in cancer: should we believe or not? Dordrecht: Springer Netherlands, 2014: 229-243.
16.	Fan X, Liu S, Su F, et al. Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system. Urol Oncol, 2012, 30(3): 314-318.
17.	月文科, 焦锋, 刘彬, 等. 人大细胞肺癌干细胞样细胞的富集及其功能研究. 中国肺癌杂志, 2011, 14(6): 484-491.
18.	Yin BB, WU SJ, Zong HJ, et al. Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 2011, 12(4): 256-263.
19.	Kong M, Dai SQ, Li BJ, et al. Isolation of breast cancer stem cell and screening of specific polypeptide bonding to it. Chinese J Cancer Preve Treatment, 2013, 20(24):1892-1895, 1899.
20.	吕建美, 何彦津, 谢连凤, 等. 人泪腺腺样囊性癌肿瘤干细胞分离培养及特征研究. 中华眼科杂志, 2015, 51(10): 762-767.
21.	Sheng X, Li Z, Wang DL, et al. Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium. Oncol Lett, 2013, 5(3): 787-792.
22.	巩超捷, 夏璐, 张宇星, 等. 无血清悬浮法培养结肠癌 HT29 细胞系及肿瘤干细胞样亚群筛选. 内科理论与实践, 2010, 5(4): 314-318.
23.	Arab-Bafrani Z, Shahbazi-Gahrouei D, Abbasian M, et al. Culturing in serum-free culture medium on collagen type-Ⅰ-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell. Adv Biomed Res, 2016, 5(1): 59.
24.	O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 2007, 445(7123): 106-110.
25.	Song L, Li Y, He B, et al. Development of Small Molecules Targeting the Wnt Signaling Pathway in Cancer Stem Cells for the Treatment of Colorectal Cancer. Clin Colorectal Cancer, 2015, 14(3): 133-145.
26.	Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature, 2005, 434(7035): 843-850.
27.	李宁, 李建远. Dishevelled: WNT 信号通路的关键蛋白. 中外医学研究, 2012, 10(28): 148-149.
28.	Wang HY, Malbon CC. Dishevelled C-terminus: prolyl and histidinyl motifs. Acta Physiol (Oxf), 2012, 204(1): 65-73.

1. Sandoval M, Harris L. Cancer stem cells. Oncology (Williston Park), 2014, 28(12): 1110-1111.
2. Greve B, Kelsch R, Spaniol K, et al. Flow cytometry in cancer stem cell analysis and separation. Cytometry A, 2012, 81(4): 284-293.
3. 龚锐, 李胜英, 霍志霞, 等. 自制免疫磁珠在前列腺肿瘤组织干细胞提取中的应用. 中国组织工程研究, 2016, 20(1): 36-41.
4. Cariati M, Naderi A, Brown JP, et al. Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-like subpopulation within the MCF7 breast cancer cell line. Int J Cancer, 2008, 122(2): 298-304.
5. Qi J, Lee HJ, Saquet A, et al. Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. J Biol Chem, 2017, 292(14): 5898-5908.
6. Zhao Y, Yang ZQ, Wang Y, et al. Dishevelled-1 and dishevelled-3 affect cell invasion mainly through canonical and noncanonical Wnt pathway, respectively, and associate with poor prognosis in nonsmall cell lung cancer. Mol Carcinog, 2010, 49(8): 760-770.
7. Kwan HT, Chan DW, Cai PC, et al. AMPK activators suppress cervical cancer cell growth through inhibition of DVL3 mediated Wnt/β-catenin signaling activity. PLoS One, 2013, 8(1): e53597.
8. Tsui Y. Study of the roles of dishevelled-3 in stemness and cell migration in hepatocellular carcinoma. Hong Kong: The University of Hong Kong, 2013.
9. 李利发, 张井潇, 陈小波, 等. 人结直肠肿瘤干细胞致瘤能力及 Dvl2 蛋白表达的研究. 中华普通外科杂志, 2016, 31(9): 774-776.
10. 王磊, 宋顺心, 汪建平. 结直肠癌实验研究现状及展望. 中华实验外科杂志, 2013, 30(3): 429-430.
11. Nangia-Makker P, Yu Y, Farhana L, et al. Colorectal cancer stem cells//Babashah S. Cancer stem cells: emerging concepts and future perspectives in translational oncology. Switzerland: Springer International Publishing, 2015: 227-245.
12. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997, 3(7): 730-737.
13. Zeuner A, Todaro M, Stassi G, et al. Colorectal cancer stem cells: from the crypt to the clinic. Cell Stem Cell, 2014, 15(6): 692-705.
14. Lobba AR, Forni MF, Carreira AC, et al. Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines. Cytometry A, 2012, 81(12): 1084-1091.
15. Mendoza FHD, Rodriguez EA. Cancer stem cells in brain tumors//Grande E, Antón A, Paricio L. Stem cells in cancer: should we believe or not? Dordrecht: Springer Netherlands, 2014: 229-243.
16. Fan X, Liu S, Su F, et al. Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system. Urol Oncol, 2012, 30(3): 314-318.
17. 月文科, 焦锋, 刘彬, 等. 人大细胞肺癌干细胞样细胞的富集及其功能研究. 中国肺癌杂志, 2011, 14(6): 484-491.
18. Yin BB, WU SJ, Zong HJ, et al. Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 2011, 12(4): 256-263.
19. Kong M, Dai SQ, Li BJ, et al. Isolation of breast cancer stem cell and screening of specific polypeptide bonding to it. Chinese J Cancer Preve Treatment, 2013, 20(24):1892-1895, 1899.
20. 吕建美, 何彦津, 谢连凤, 等. 人泪腺腺样囊性癌肿瘤干细胞分离培养及特征研究. 中华眼科杂志, 2015, 51(10): 762-767.
21. Sheng X, Li Z, Wang DL, et al. Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium. Oncol Lett, 2013, 5(3): 787-792.
22. 巩超捷, 夏璐, 张宇星, 等. 无血清悬浮法培养结肠癌 HT29 细胞系及肿瘤干细胞样亚群筛选. 内科理论与实践, 2010, 5(4): 314-318.
23. Arab-Bafrani Z, Shahbazi-Gahrouei D, Abbasian M, et al. Culturing in serum-free culture medium on collagen type-Ⅰ-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell. Adv Biomed Res, 2016, 5(1): 59.
24. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 2007, 445(7123): 106-110.
25. Song L, Li Y, He B, et al. Development of Small Molecules Targeting the Wnt Signaling Pathway in Cancer Stem Cells for the Treatment of Colorectal Cancer. Clin Colorectal Cancer, 2015, 14(3): 133-145.
26. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature, 2005, 434(7035): 843-850.
27. 李宁, 李建远. Dishevelled: WNT 信号通路的关键蛋白. 中外医学研究, 2012, 10(28): 148-149.
28. Wang HY, Malbon CC. Dishevelled C-terminus: prolyl and histidinyl motifs. Acta Physiol (Oxf), 2012, 204(1): 65-73.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

The tumorigenicity and expressions of dishevelled 3 in HCT116 cells and HCT116 spherical cells

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