Volume 5, Issue 3, September 2020, Page: 56-64
Novel Targets of Protoporphyrin-IX Determined By Gene Expression Analysis
Yiyang Dai, Department of Gastroenterology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine, Yi Wu, China
Wolfgang Kemmner, Research Group Translational Oncology, Experimental Clinical Research Center at the Max-Delbrueck-Center for Molecular Medicine, Charitè Campus Buch, Berlin, Germany
Received: Jul. 8, 2020;       Accepted: Jul. 27, 2020;       Published: Aug. 18, 2020
DOI: 10.11648/j.ijcocr.20200503.12      View  52      Downloads  45
Treatment with 5-Aminolevulinic acid-mediated photody¬namic therapy is a promising therapeutic option for various carcinomas. An appropriate photosensitizer for photody¬namic therapy is protoporphyrin-IX (PpIX), a light sensitive metabolite of heme synthesis. Incorporation of iron into PpIX leading to heme is carried out by Ferrochelatase (FECH). Earlier, we described a significant down regulation of FECH mRNA-expression and enzyme activity in carcinoma cells leading to an endogenous accumulation of PpIX. How PpIX affects the cell metabolism has not been examined so far. Thus, we tried to identify novel targets of PpIX with regard to cell proliferation, apoptosis and invasion. Endogenous generation of PpIX was induced by silencing of FECH in breast carcinoma MDA-MB-231 cells using a specific siRNA. Successful silencing of FECH was confirmed by RT-PCR and induction of PpIX was assessed by flow cytometry for each experiment. Subsequently, gene expression was determined using Affymetrix GeneChip® Human Gene 1.0 ST. Validation of microarray data was achieved by quantitative RT-PCR. Expression of one of the newly discovered target genes, BAMBI, was assessed by immunohistochemistry. In addition, the effect of silencing of FECH was examined by functional studies of cell apoptosis, invasion and wound healing. According to the gene expression analysis, an enhancement of PpIX suppressed Hedgehog as well as TGF-beta signaling. Expression of HHIP, a negative regulator of the hedgehog pathway, was found to be strongly increased after silencing of FECH. With regard to TGF-beta signaling, expression of the signaling inhibitor SMAD7 was strongly upregulated while the positive mediators SMAD2 and SMAD4 were less expressed after silencing FECH. Similarly, apoptosis of tumor cells was promoted, probably due to an increased expression of the pro-apoptotic gene APAF1 and a reduced expression of anti-apoptotic protein API5. Moreover, a significantly reduced invasion capability after treatment of cells with FECH siRNA was found. Here, we report that an accumulation of PpIX due to silencing of FECH affects various pathways and promotes apoptosis of tumor cells in different ways. Thus, silencing of FECH might have a tumor-suppressive effect. The search for substances which block FECH activity in a direct way therefore might be of high relevance for future cancer therapy approaches.
Photodynamic Therapy, Protoporphyrin-IX, Ferrochelatase, Silencing RNA, Microarray, Gene Expression, Apoptosis, Invasion
To cite this article
Yiyang Dai, Wolfgang Kemmner, Novel Targets of Protoporphyrin-IX Determined By Gene Expression Analysis, International Journal of Clinical Oncology and Cancer Research. Vol. 5, No. 3, 2020, pp. 56-64. doi: 10.11648/j.ijcocr.20200503.12
Copyright © 2020 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ishizuka M, Abe F, Sano Y, Takahashi K, Inoue K, Nakajima M, et al. Novel development of 5-aminolevurinic acid (ALA) in cancer diagnoses and therapy. Int Immunopharmacol. 2011; 11 (3): 358-65.
Moesta KT, Ebert B, Handke T, Nolte D, Nowak C, Haensch WE, et al. Protoporphyrin IX occurs naturally in colorectal cancers and their metastases. Cancer Res. 2001; 61 (3): 991-9.
Kemmner W, Wan K, Ruttinger S, Ebert B, Macdonald R, Klamm U, et al. Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer. FASEB J. 2008; 22 (2): 500-9.
Wan K, Ebert B, Voigt J, Wang Q, Dai Y, Haag R, et al. In vivo tumor imaging using a novel RNAi-based detection mechanism. Nanomedicine. 2012; 8 (4): 393-8.
Zawacka-Pankau J, Issaeva N, Hossain S, Pramanik A, Selivanova G, Podhajska AJ. Protoporphyrin IX interacts with wild-type p53 protein in vitro and induces cell death of human colon cancer cells in a p53-dependent and -independent manner. J Biol Chem. 2007; 282 (4): 2466-72.
Jiang L, Malik N, Acedo P, Zawacka-Pankau J. Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukemia cells. Cell Death Discov. 2019; 5: 77.
Zawacka-Pankau J, Kowalska A, Issaeva N, Burcza A, Kwiek P, Bednarz N, et al. Tumor suppressor Fhit protein interacts with protoporphyrin IX in vitro and enhances the response of HeLa cells to photodynamic therapy. J Photochem Photobiol B. 2007; 86 (1): 35-42.
Lee JM, Heo MJ, Lee CG, Yang YM, Kim SG. Increase of miR-199a-5p by protoporphyrin IX, a photocatalyzer, directly inhibits E2F3, sensitizing mesenchymal tumor cells to anti-cancer agents. Oncotarget. 2015; 6 (6): 3918-31.
Carvalho BS, Irizarry RA. A framework for oligonucleotide microarray preprocessing. Bioinformatics. 2010; 26 (19): 2363-7.
Fischer M. Census and evaluation of p53 target genes. Oncogene. 2017; 36 (28): 3943-56.
Matozaki T, Sakamoto C, Suzuki T, Matsuda K, Uchida T, Nakano O, et al. p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. Cancer Res. 1992; 52 (16): 4335-41.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012; 149 (5): 1060-72.
Rubie C, Kempf K, Hans J, Su T, Tilton B, Georg T, et al. Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues. Molecular and cellular probes. 2005; 19 (2): 101-9.
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.
May BK, Dogra SC, Sadlon TJ, Bhasker CR, Cox TC, Bottomley SS. Molecular regulation of heme biosynthesis in higher vertebrates. Prog Nucleic Acid Res Mol Biol. 1995; 51: 1-51.
Elbirt KK, Bonkovsky HL. Heme oxygenase: recent advances in understanding its regulation and role. Proc Assoc Am Physicians. 1999; 111 (5): 438-47.
Shakeri R, Kheirollahi A, Davoodi J. Apaf-1: Regulation and function in cell death. Biochimie. 2017; 135: 111-25.
Leu JI, Dumont P, Hafey M, Murphy ME, George DL. Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol. 2004; 6 (5): 443-50.
Cho H, Chung JY, Song KH, Noh KH, Kim BW, Chung EJ, et al. Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer. BMC Cancer. 2014; 14: 545.
Massague J. TGFbeta signalling in context. Nat Rev Mol Cell Biol. 2012; 13 (10): 616-30.
Miyazono K. Transforming growth factor-beta signaling in epithelial-mesenchymal transition and progression of cancer. Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85 (8): 314-23.
Zeng H, Tang L. CRIM1, the antagonist of BMPs, is a potential risk factor of cancer. Curr Cancer Drug Targets. 2014; 14 (7): 652-8.
Yuan CL, Liang R, Liu ZH, Li YQ, Luo XL, Ye JZ, et al. Bone morphogenetic protein and activin membrane-bound inhibitor overexpression inhibits gastric tumor cell invasion via the transforming growth factor-beta/epithelial-mesenchymal transition signaling pathway. Exp Ther Med. 2018; 15 (6): 5422-30.
Briscoe J, Therond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol. 2013; 14 (7): 416-29.
25. Sun H, Ni SJ, Ye M, Weng W, Zhang Q, Zhang M, et al. Hedgehog Interacting Protein 1 is a Prognostic Marker and Suppresses Cell Metastasis in Gastric Cancer. J Cancer. 2018; 9 (24): 4642-9.
Zhan Y, Zheng N, Teng F, Bao L, Liu F, Zhang M, et al. MiR-199a/b-5p inhibits hepatocellular carcinoma progression by post-transcriptionally suppressing ROCK1. Oncotarget. 2017; 8 (40): 67169-80.
Kim BK, Yoo HI, Kim I, Park J, Kim Yoon S. FZD6 expression is negatively regulated by miR-199a-5p in human colorectal cancer. BMB Rep. 2015; 48 (6): 360-6.
Song H, Hogdall E, Ramus SJ, Dicioccio RA, Hogdall C, Quaye L, et al. Effects of common germ-line genetic variation in cell cycle genes on ovarian cancer survival. Clin Cancer Res. 2008; 14 (4): 1090-5.
Yokota I, Sasaki Y, Kashima L, Idogawa M, Tokino T. Identification and characterization of early growth response 2, a zinc-finger transcription factor, as a p53-regulated proapoptotic gene. Int J Oncol. 2010; 37 (6): 1407-16.
Browse journals by subject