Document Type : Original Article(s)

Authors

1 Cancer and Stem Cell Research Center, Maltepe University, Istanbul, Turkey

2 Department of Histology and Embryology, Faculty of Medicine, Maltepe University, Istanbul, Turkey

Abstract

Background: The importance of extracellular matrix (ECM) components in the progression of hepatocellular carcinoma (HCC) has been shown in many studies. Although restoring or activating apoptosis in tumors is an active area of cancer research, little is known regarding the effects of collagen type I, the main ECM component in the liver, on apoptosis of HCC cells. Here, we investigated the apoptotic profiles of HCC cells in a microenvironment with collagen type I.
Method: In this in vitro study, we assessed the effects of collagen type I on HepG2 cells in pre-confluent and confluent states. We determined the mRNA levels of 25 genes, which are the key players of apoptosis. Flow cytometry-based apoptosis detection was performed by use of Annexin V/PI staining. Confocal laser scanning microscopy was used to assess P53 immunofluorescence in the cells.
Results: The microenvironment with collagen type I and the confluency state of HepG2 cells affected the expression of 13 genes involved in apoptosis. We observed no significant change in the number of cells undergoing apoptosis depending on the confluency state or the presence of collagen type I. P53 immunofluorescence demonstrated no significant changes.
Conclusion: We propose an apoptotic balance concerning overall cell survival, which might be caused by the counteraction of positive and negative mediators of apoptosis. This study might provide data for the involvement of collagen type I in apoptotic responses of HCC and contribute to a better understanding of cancer microenvironment.

Keywords

How to cite this article:

Cakil YD, Akbulut Z, Maras H, Kayali DG, Aktas RG. Collagen type I induces a balance in the expression of anti- and pro-apoptotic genes in hepatocellular carcinoma cells. Middle East J Cancer. 2022;13(1):89-98. doi: 10. 30476/mejc.2021.85875.1312.

1.Ingber DE. Can cancer be reversed by engineering the tumor microenvironment? Semin Cancer Biol. 2008;18(5):356-64. doi:10.1016/j.semcancer.2008. 03.016.
2.TahmasebiBirgani M, Carloni V. Tumor microenvironment, a paradigm in hepatocellular carcinoma progression and therapy. Int J Mol Sci. 2017;18(2):405. doi:10.3390/ijms18020405.
3.Xu S, Xu H, Wang W, Li S, Li H, Li T, et al. The role of collagen in cancer: from bench to bedside. J Transl Med. 2019;17(1):309. doi:10.1186/s12967-019-2058-1.
4.Schrader J, Gordon-Walker TT, Aucott RL, van Deemter M, Quaas A, Walsh S, et al. Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells. Hepatology. 2011; 53(4):1192-205. doi: 10.1002/hep. 24108.
5.Zhang R, Ma M, Dong G, Yao RR, Li JH, Zheng QD, et al. Increased matrix stiffness promotes tumor progression of residual hepatocellular carcinoma after insufficient heat treatment. Cancer Sci. 2017; 108(9): 1778-86. doi:10.1111/cas.13322.
6.Carloni V, Luong TV, Rombouts K. Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever. Liver Int. 2014;34(6):834-43. doi:10.1111/liv.12465.
7.Zhu GG, Risteli L, Mäkinen M, Risteli J, Kauppila A, Stenbäck F. Immunohistochemical study of type I collagen and type I pN-collagen in benign and malignant ovarian neoplasms. Cancer. 1995;75(4):1010-7. doi: 10.1002/1097-0142(1995021 5)75:4<1010::aid-cncr2820750417>3.0.co;2-o.
8.Huijbers IJ, Iravani M, Popov S, Robertson D, Al-Sarraj S, Jones C, et al. A role for fibrillar collagen deposition and the collagen internalization receptor endo180 in glioma invasion. PLoS One. 2010; 5(3):e9808. doi: 10.1371/journal.pone.0009808.
9.Armstrong T, Packham G, Murphy LB, Bateman AC, Conti JA, Fine DR, et al. Type I collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma. Clin Cancer Res. 2004;10(21):7427-37. doi: 10.1158/1078-0432.CCR-03-0825.
10.Senthebane DA, Jonker T, Rowe A, Thomford NE, Munro D, Dandara C, et al. The role of tumor microenvironment in chemoresistance: 3D extracellular matrices as accomplices. Int J Mol Sci. 2018;19(10). doi: 10.3390/ijms19102861.
11.Liu S, Liao G, Li G. Regulatory effects of COL1A1 on apoptosis induced by radiation in cervical cancer cells. Cancer Cell Int. 2017;17:73. doi: 10.1186/ s12935-017-0443-5.
12.Chowdhury SR, Muneyuki Y, Takezawa Y, Kino-oka M, Saito A, Sawa Y, et al. Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts. J Biosci Bioeng. 2010; 109(3):310-3. doi: 10.1016/j.jbiosc.2009.09.042.
13.Sivertsson L, Ek M, Darnell M, Edebert I, Ingelman-Sundberg M, Neve EP. CYP3A4 catalytic activity is induced in confluent Huh7 hepatoma cells. Drug Metab Dispos. 2010;38(6):995-1002. doi: 10.1124/ dmd.110.032367.
14.Fadeev RS, Chekanov AV, Dolgikh NV, Akatov VS. Increase in resistance of A431 cancer cells to TRAIL-induced apoptosis in confluent cultures. Biophysics. 2012; 57(4):491-5. doi: 10.1134/S0006350912040045.
15.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402-8. doi: 10.1006/meth.2001.1262.
16. Saeki K, Yuo A, Kato M, Miyazono K, Yazaki Y, Takaku F. Cell density-dependent apoptosis in HL-60 cells, which is mediated by an unknown soluble factor, is inhibited by transforming growth factor beta1 and overexpression of Bcl-2. J Biol Chem. 1997; 272(32): 20003-10. doi: 10.1074/jbc.272.32.20003.
17. Perugorria MJ, Castillo J, Latasa MU, Goñi S, Segura V, Sangro B, et al. Wilms' tumor 1 gene expression in hepatocellular carcinoma promotes cell dedifferentiation and resistance to chemotherapy. Cancer Res. 2009; 69(4):1358-67. doi: 10.1158/0008-5472.CAN-08-2545.
18. Sera T, Hiasa Y, Mashiba T, Tokumoto Y, Hirooka M, Konishi I, et al. Wilms' tumour 1 gene expression is increased in hepatocellular carcinoma and associated with poor prognosis. Eur J Cancer. 2008; 44(4):600-8. doi: 10.1016/j.ejca.2008.01.008.
19. Chen SR, Chen M, Wang XN, Zhang J, Wen Q, Ji SY, et al. The Wilms tumor gene, Wt1, maintains testicular cord integrity by regulating the expression of Col4a1 and Col4a2. Biol Reprod. 2013; 88(3):56. doi: 10.1095/biolreprod.112.105379.16.
20. Egeblad M, Rasch MG, Weaver VM. Dynamic interplay between the collagen scaffold and tumor evolution. Curr Opin Cell Biol. 2010; 22(5):697-706. doi: 10.1016/j.ceb.2010.08.015.
21.Zheng X, Liu W, Xiang J, Liu P, Ke M, Wang B, et al. Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin beta1/FAK signaling pathway in nonalcoholic fatty liver. Oncotarget. 2017; 8(56): 95586-95. doi: 10.18632/oncotarget.21525.
22.Zhang R, Ma M, Lin XH, Liu HH, Chen J, Chen J, et al. Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment. BMC Cancer. 2018; 18(1): 901. doi: 10.1186/s12885-018-4820-9.
23.Duncan MB. Extracellular matrix transcriptome dynamics in hepatocellular carcinoma. Matrix Biol. 2013;32(7-8):393-8. 10.1016/j.matbio.2013.05.003.
24.Jewer M, Findlay SD, Postovit LM. Post-transcriptional regulation in cancer progression: Microenvironmental control of alternative splicing and translation. J Cell Commun Signal. 2012;6(4):233-48. doi: 10. 1007/s12079-012-0179-x.
25.Garcia EJ, Lawson D, Cotsonis G, Cohen C. Hepatocellular carcinoma and markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance. Appl Immunohistochem Mol Morphol. 2002;10(3): 210-7. doi: 10.1097/00129039-200209000-00004.
26.Li L, Zhao GD, Shi Z, Qi LL, Zhou LY, Fu ZX. The Ras/Raf/MEK/ERK signaling pathway and its role in the occurrence and development of HCC. Oncol Lett. 2016; 12(5):3045-50. doi: 10.3892/ol.2016.5110.
27.Fabregat I. Dysregulation of apoptosis in hepatocellular carcinoma cells. World J Gastroenterol. 2009;15(5). doi: 10.3748/wjg.15.513.
28.He Y, Chen J, Ren J, Wang G, Cai G. Type I collagen inhibits hydroxyl radical-induced apoptosis. J Biochem. 2002;132(3):373-9. doi: 10.1093/oxfordjournals. jbchem.a003232.
29.Naci D, Berrazouane S, Barabé F, Aoudjit F. Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation. Sci Rep. 2019; 9(1):19455. doi: 10.1038/s41598-019-55934-w.
30.Affo S, Yu LX, Schwabe RF. The role of cancer-associated fibroblasts and fibrosis in liver cancer. Annu Rev Pathol. 2017; 12:153-86. doi: 10.1146/annurev-pathol-052016-100322.