Research Article

Functional characterization of HIC, a P2Y1 agonist, as a p53 stabilizer for prostate cancer cell death induction

Molecular Signaling Lab, Faculty of Medicine & Health Technology, Tampere University, Finland

BioMeditech & Tays Cancer Center, Tampere University Hospital, PO Box 553, 33101, Tampere, Finland

Search for more papers by this author

Akshaya Murugesan

Molecular Signaling Lab, Faculty of Medicine & Health Technology, Tampere University, Finland

BioMeditech & Tays Cancer Center, Tampere University Hospital, PO Box 553, 33101, Tampere, Finland

Department of Biotechnology, Lady Doak College, Thallakulam, Madurai, 625002, India

Search for more papers by this author

Nuno R Candeias

Faculty of Engineering & Natural Sciences, Tampere University, 33101, Tampere, Finland

LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal

Search for more papers by this author

Olli Yli-Harja

Computational Systems Biology Group, Faculty of Medicine & Health Technology, Tampere University, PO Box 553, 33101, Tampere, Finland

Institute for Systems Biology, 1441N 34th Street, Seattle, WA 98103-8904, USA

Search for more papers by this author

Meenakshisundaram Kandhavelu

*Author for correspondence:

E-mail Address: meenakshisundaram.kandhavelu@tuni.fi

https://orcid.org/0000-0002-4986-055X

Molecular Signaling Lab, Faculty of Medicine & Health Technology, Tampere University, Finland

BioMeditech & Tays Cancer Center, Tampere University Hospital, PO Box 553, 33101, Tampere, Finland

Search for more papers by this author

Published Online:10 Sep 2021https://doi.org/10.4155/fmc-2021-0159

View article

Abstract

Background: (1-(2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile (HIC), an agonist of the P2Y1 receptor (P2Y1R), induces cell death in prostate cancer cells. However, the molecular mechanism behind the inhibition of HIC in prostate cancer remains elusive. Methods & results: Here, to outline the inhibitory role of HIC on prostate cancer cells, PC-3 and DU145 cell lines were treated with the respective IC₅₀ concentrations, which reduced cell proliferation, adherence properties and spheroid formation. HIC was able to arrest the cell cycle at G1/S phase and also induced apoptosis and DNA damage, validated by gene expression profiling. HIC inhibited the prostate cancer cells’ migration and invasion, revealing its antimetastatic ability. P2Y1R-targeted HIC affects p53, MAPK and NF-κB protein expression, thereby improving the p53 stabilization essential for G1/S arrest and cell death. Conclusion: These findings provide an insight on the potential use of HIC, which remains the mainstay treatment for prostate cancer.

Graphical abstract

Keywords:

References

1. Attard G, Parker C, Eeles RA et al. Prostate cancer. Lancet 387(10013), 70–82 (2016).
MedlineGoogle Scholar
2. Litwin MS, Tan HJ. The diagnosis and treatment of prostate cancer: a review. JAMA 317(24), 2532–2542 (2017).
MedlineGoogle Scholar
3. Marucci G, Santinelli C, Buccioni M et al. Anticancer activity study of A3 adenosine receptor agonists. Life Sci. 205, 155–163 (2018).
Medline CASGoogle Scholar
4. Yu S, Sun L, Jiao Y, Lee LTO. The role of G protein-coupled receptor kinases in cancer. Int. J. Biol. Sci. 14(2), 189–203 (2018).
Medline CASGoogle Scholar
5. Saengsawang W, Rasenick MM. G protein-coupled receptors. Encycl. Cell Biol. 3, 51–55 (2016).
Google Scholar
6. Janssens R, Communi D, Pirotton S, Samson M, Parmentier M, Boeynaems JM. Cloning and tissue distribution of the human P2Y1 receptor. Biochem. Biophys. Res. Commun. 221(3), 588–593 (1996).
Medline CASGoogle Scholar
7. Wei Q, Costanzi S, Liu QZ, Gao ZG, Jacobson KA. Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells. Biochem. Pharmacol. 82(4), 418–425 (2011).
Medline CASGoogle Scholar
8. Li WH, Qiu Y, Zhang HQ et al. P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells. Br. J. Cancer 109(6), 1666–1675 (2013).
Medline CASGoogle Scholar
9. Shabbir M, Ryten M, Thompson C, Mikhailidis D, Burnstock G. Characterization of calcium-independent purinergic receptor-mediated apoptosis in hormone-refractory prostate cancer. BJU Int. 101(3), 352–359 (2008).
Medline CASGoogle Scholar
10. Le HTT, Rimpilainen T, Konda Mani S et al. Synthesis and preclinical validation of novel P2Y1 receptor ligands as a potent anti-prostate cancer agent. Sci. Rep. 9(1), 18938 (2019).
MedlineGoogle Scholar
11. Buvinic S, Bravo-Zehnder M, Boyer JL, Huidobro-Toro JP, González A. Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells. J. Cell Sci. 120(Pt 24), 4289–4301 (2007).
Medline CASGoogle Scholar
12. Woehrle T, Ledderose C, Rink J, Slubowski C, Junger WG. Autocrine stimulation of P2Y1 receptors is part of the purinergic signaling mechanism that regulates T cell activation. Purinergic Signal. 15(2), 127–137 (2019).
Medline CASGoogle Scholar
13. Niimi K, Ueda M, Fukumoto M et al. Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells. Biochem. Biophys. Res. Commun. 489(4), 413–419 (2017).
Medline CASGoogle Scholar
14. Mamedova LK, Gao ZG, Jacobson KA. Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors. Biochem. Pharmacol. 72(8), 1031–1041 (2006).
Medline CASGoogle Scholar
15. Harden TK, Waldo GL, Hicks SN, Sondek J. Mechanism of activation and inactivation of Gq/phospholipase C-β signaling nodes. Chem. Rev. 111(10), 6120–6129 (2011).
Medline CASGoogle Scholar
16. Mizuno N, Itoh H. Functions and regulatory mechanisms of Gq-signaling pathways. NeuroSignals 17(1), 42–54 (2009).
Medline CASGoogle Scholar
17. Xu KP, Dartt DA, Yu FSX. EGF-induced ERK phosphorylation independent of PKC isozymes in human corneal epithelial cells. Investig. Ophthalmol. Vis. Sci. 43(12), 3673–3679 (2002).
MedlineGoogle Scholar
18. Yin J, Yu FSX. ERK1/2 mediate wounding- and G-protein-coupled receptor ligands-induced EGFR activation via regulating ADAM17 and HB-EGF shedding. Investig. Ophthalmol. Vis. Sci. 50(1), 132–139 (2009).
MedlineGoogle Scholar
19. Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J. Recept. Signal Transduct. 35(6), 600–604 (2015).
Medline CASGoogle Scholar
20. Muscella A, Cossa LG, Vetrugno C, Antonaci G, Marsigliante S. Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway. J. Cell. Physiol. 233(3), 2526–2536 (2018).
Medline CASGoogle Scholar
21. Wong PC, Watson C, Crain EJ. The P2Y1 receptor antagonist MRS2500 prevents carotid artery thrombosis in cynomolgus monkeys. J. Thromb. Thrombolysis 41(3), 514–521 (2016).
Medline CASGoogle Scholar
22. Li Y, Yin C, Liu P, Li D, Lin J. Identification of a different agonist-binding site and activation mechanism of the human P2Y1 receptor. Sci. Rep. 7(1), 13764 (2017).
MedlineGoogle Scholar
23. Shen J, DiCorleto PE. ADP stimulates human endothelial cell migration via P2Y1 nucleotide receptor-mediated mitogen-activated protein kinase pathways. Circ. Res. 102(4), 448–456 (2008).
Medline CASGoogle Scholar
24. Borowicz S, Van Scoyk M, Avasarala S et al. The soft agar colony formation assay. J. Vis. Exp. (92), e51998 (2014).
MedlineGoogle Scholar
25. Pehkonen H, Lento M, Von Nandelstadh P et al. Liprin-α1 modulates cancer cell signaling by transmembrane protein CD82 in adhesive membrane domains linked to cytoskeleton. Cell Commun. Signal. 16(1), 41 (2018).
MedlineGoogle Scholar
26. Hubbard T, Barker D, Birney E et al. The Ensembl genome database project. Nucleic Acids Res. 30(1), 38–41 (2002).
Medline CASGoogle Scholar
27. Babraham Institute. FASTQC: a quality control tool for high throughput sequence data. www.bioinformatics.babraham.ac.uk/projects/fastqc/
Google Scholar
28. Dobin A, Davis CA, Schlesinger F et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1), 15–21 (2013).
Medline CASGoogle Scholar
29. Li H, Handsaker B, Wysoker A et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16), 2078–2079 (2009).
MedlineGoogle Scholar
30. Anders S, Pyl PT, Huber W. HTSeq – a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2), 166–169 (2015).
Medline CASGoogle Scholar
31. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15(12), 550 (2014).
MedlineGoogle Scholar
32. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57(1), 289–300 (1995).
Google Scholar
33. Ashburner M, Ball CA, Blake JA et al. Gene ontology: tool for the unification of biology. Nat. Genet. 25(1), 25–29 (2000).
Medline CASGoogle Scholar
34. Yu G, Wang LG, Han Y, He QY. ClusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16(5), 284–287 (2012).
Medline CASGoogle Scholar
35. Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28(1), 27–30 (2000).
Medline CASGoogle Scholar
36. Wang HY, Kim NH. CDK2 is required for the DNA damage response during porcine early embryonic development. Biol. Reprod. 95(2), 31 (2016).
MedlineGoogle Scholar
37. Gao SY, Li J, Qu XY, Zhu N, Ji YB. Downregulation of Cdk1 and CyclinB1 expression contributes to oridonin-induced cell cycle arrest at G2/M phase and growth inhibition in SGC-7901 gastric cancer cells. Asian Pacific J. Cancer Prev. 15(15), 6437–6441 (2014).
MedlineGoogle Scholar
38. Hur W, Rhim H, Jung CK et al. SOX4 overexpression regulates the p53-mediated apoptosis in hepatocellular carcinoma: clinical implication and functional analysis in vitro. Carcinogenesis 31(7), 1298–1307 (2010).
Medline CASGoogle Scholar
39. Urist M, Tanaka T, Poyurovsky MV, Prives C. p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2. Genes Dev. 18(24), 3041–3054 (2004).
Medline CASGoogle Scholar
40. Mancini F, Pieroni L, Monteleone V et al. MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response. Oncogene 35(2), 228–240 (2016).
Medline CASGoogle Scholar
41. Toledo F, Wahl GM. MDM2 and MDM4: p53 regulators as targets in anticancer therapy. Int. J. Biochem. Cell Biol. 39(7–8), 1476–1482 (2007).
Medline CASGoogle Scholar
42. Hussain T, Saha D, Purohit G et al. Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex. Sci. Rep. 7(1), 11541 (2017).
MedlineGoogle Scholar
43. Nag S, Qin J, Srivenugopal KS, Wang M, Zhang R. The MDM2–p53 pathway revisited. J. Biomed. Res. 27(4), 254–271 (2013).
Medline CASGoogle Scholar
44. Siednienko J, Gajanayake T, Fitzgerald KA, Moynagh P, Miggin SM. Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-β and RANTES production. J. Immunol. 186(4), 2514–2522 (2011).
Medline CASGoogle Scholar
45. Matt S, Hofmann TG. The DNA damage-induced cell death response: a roadmap to kill cancer cells. Cell. Mol. Life Sci. 73(15), 2829–2850 (2016).
Medline CASGoogle Scholar
46. Das SP, Borrman T, Liu VWT, Yang SCH, Bechhoefer J, Rhind N. Replication timing is regulated by the number of MCMs loaded at origins. Genome Res. 25(12), 1886–1892 (2015).
Medline CASGoogle Scholar
47. Gordon E, Ravicz J, Liu S, Chawla S, Hall F. Cell cycle checkpoint control: the cyclin G1/Mdm2/p53 axis emerges as a strategic target for broad-spectrum cancer gene therapy – a review of molecular mechanisms for oncologists. Mol. Clin. Oncol. 9(2), 115–134 (2018).
Medline CASGoogle Scholar
48. Neganova I, Vilella F, Atkinson SP et al. An important role for CDK2 in G1 to S checkpoint activation and DNA damage response in human embryonic stem cells. Stem Cells 29(4), 651–659 (2011).
Medline CASGoogle Scholar
49. Peyressatre M, Prével C, Pellerano M, Morris MC. Targeting cyclin-dependent kinases in human cancers: from small molecules to peptide inhibitors. Cancers (Basel) 7(1), 179–237 (2015).
Medline CASGoogle Scholar
50. Doan P, Nguyen T, Yli-Harja O et al. Effect of alkylaminophenols on growth inhibition and apoptosis of bone cancer cells. Eur. J. Pharm. Sci. 107, 208–216 (2017).
Medline CASGoogle Scholar
51. Haque S, Morris JC. Transforming growth factor-β: a therapeutic target for cancer. Hum. Vaccines Immunother. 13(8), 1741–1750 (2017).
MedlineGoogle Scholar
52. Xu J, Lamouille S, Derynck R. TGF-β-induced epithelial to mesenchymal transition. Cell Res. 19(2), 156–172 (2009).
Medline CASGoogle Scholar
53. Ampuja M, Jokimäki R, Juuti-Uusitalo K, Rodriguez-Martinez A, Alarmo EL, Kallioniemi A. BMP4 inhibits the proliferation of breast cancer cells and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells in 3D environment. BMC Cancer 13, 429 (2013).
MedlineGoogle Scholar
54. Du R, Liu B, Zhou L et al. Downregulation of annexin A3 inhibits tumor metastasis and decreases drug resistance in breast cancer. Cell Death Dis. 9(2), 126 (2018).
MedlineGoogle Scholar
55. Peitsch WK, Doerflinger Y, Fischer-Colbrie R et al. Desmoglein 2 depletion leads to increased migration and upregulation of the chemoattractant secretoneurin in melanoma cells. PLoS ONE 9(2), e89491 (2014).
MedlineGoogle Scholar
56. Shen H, Xu L, You C et al. miR-665 is downregulated in glioma and inhibits tumor cell proliferation, migration and invasion by targeting high mobility group box 1. Oncol. Lett. 21(2), 156 (2021).
Medline CASGoogle Scholar
57. Woodfield SE, Shi Y, Patel RH et al. MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma. Sci. Rep. 11(1), 2967 (2021).
Medline CASGoogle Scholar
58. Fukasawa K, Vande Woude GF. Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability. Mol. Cell. Biol. 17(1), 506–518 (1997).
Medline CASGoogle Scholar
59. Mor O, Yaron P, Huszar M et al. Absence of p53 mutations in malignant mesotheliomas. Am. J. Respir. Cell Mol. Biol. 16(1), 9–13 (1997).
Medline CASGoogle Scholar
60. Okuda T, Otsuka J, Sekizawa A et al. p53 mutations and overexpression affect prognosis of ovarian endometrioid cancer but not clear cell cancer. Gynecol. Oncol. 88(3), 318–325 (2003).
Medline CASGoogle Scholar
61. Chen L, Wang S, Zhou Y et al. Identification of early growth response protein 1 (EGR-1) as a novel target for JUN-induced apoptosis in multiple myeloma. Blood 115(1), 61–70 (2010).
Medline CASGoogle Scholar
62. Fan M, Goodwin ME, Birrer MJ, Chambers TC. The c-Jun NH2-terminal protein kinase/AP-1 pathway is required for efficient apoptosis induced by vinblastine. Cancer Res. 61(11), 4450–4458 (2001).
Medline CASGoogle Scholar
63. Saha MN, Jiang H, Yang Y et al. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma. PLoS ONE 7(1), e30215 (2012).
Medline CASGoogle Scholar
64. Chen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harb. Perspect. Med. 6(3), a026104 (2016).
MedlineGoogle Scholar
65. Domercq M, Brambilla L, Pilati E, Marchaland J, Volterra A, Bezzi P. P2Y1 receptor-evoked glutamate exocytosis from astrocytes. J. Biol. Chem. 281(41), 30684–30696 (2006).
Medline CASGoogle Scholar
66. Shadfan M, Lopez-Pajares V, Yuan ZM. MDM2 and MDMX: alone and together in regulation of p53. Transl. Cancer Res. 1(2), 88–89 (2012).
MedlineGoogle Scholar
67. Pearson AS, Spitz FR, Swisher SG et al. Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells. Clin. Cancer Res. 6(3), 887–890 (2000).
Medline CASGoogle Scholar
68. Zhu J, Chen M, Chen N et al. Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway. Int. J. Oncol. 46(3), 981–988 (2015).
Medline CASGoogle Scholar
69. Higgins SP, Tang Y, Higgins CE et al. TGF-β1/p53 signaling in renal fibrogenesis. Cell. Signal. 43, 1–10 (2018).
Medline CASGoogle Scholar
70. Oh IR, Raymundo B, Kim MJ, Kim CW. Mesenchymal stem cells co-cultured with colorectal cancer cells showed increased invasive and proliferative abilities due to its altered p53/TGF-β1 levels. Biosci. Biotechnol. Biochem. 84(2), 256–267 (2020).
Medline CASGoogle Scholar
71. Gen SW. The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther. 3(2), 156–161 (2004).
MedlineGoogle Scholar
72. van der Vorst EPC, Theodorou K, Wu Y et al. High-density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-κB/STAT1-IRF1 signaling. Cell Metab. 25(1), 197–207 (2017).
MedlineGoogle Scholar
73. Chen L, He HY, Li HM et al. ERK1/2 and p38 pathways are required for P2Y receptor-mediated prostate cancer invasion. Cancer Lett. 215(2), 239–247 (2004).
Medline CASGoogle Scholar
74. Hafner A, Bulyk ML, Jambhekar A, Lahav G. The multiple mechanisms that regulate p53 activity and cell fate. Nat. Rev. Mol. Cell Biol. 20(4), 199–210 (2019).
Medline CASGoogle Scholar
75. Muscella A, Vetrugno C, Antonaci G, Cossa LG, Marsigliante S. PKC-δ/PKC-α activity balance regulates the lethal effects of cisplatin. Biochem. Pharmacol. 98(1), 29–40 (2015).
Medline CASGoogle Scholar
76. Basu A. Involvement of protein kinase C-δ in DNA damage-induced apoptosis. J. Cell. Mol. Med. 7(4), 341–350 (2003).
Medline CASGoogle Scholar
77. Chuderland D, Seger R. Calcium regulates ERK signaling by modulating its protein–protein interactions. Commun. Integr. Biol. 1(1), 4–5 (2008).
Medline CASGoogle Scholar
78. Liu Y, Song X, Shi Y et al. WNK1 activates large-conductance Ca²⁺-activated K⁺ channels through modulation of ERK1/2 signaling. J. Am. Soc. Nephrol. 26(4), 844–854 (2015).
Medline CASGoogle Scholar

Vol. 13, No. 21

Supplemental Materials

Metrics

Downloaded 65 times

History

Received 27 May 2021

Accepted 20 August 2021

Published online 10 September 2021

Published in print November 2021

Information

Keywords

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.4155/fmc-2021-0159

Acknowledgements

The authors thank A Musa, Tampere University for the technical support in gene expression analysis.

Financial & competing interests disclosure

H Le acknowledges TUT-RAE for the project grant support and Tamper University for Instrumental facility grant support. N Candeias acknowledges the Janne and Aatos Erkko Foundation, Academy of Finland (Decision 326487) and Fundação para a Ciência e Tecnologia (CEE-CINST/2018) for financial support. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

PDF download