Inhibition of EGF and CoCl2-Induced HIF-1α and VEGF Production in Triple Negative MDA-MB-468 Cells by Umbelliprenin: Unveiling the Role of PI3K/AKT/mTOR and MAPK Pathways

Document Type : Original paper

Authors

1 Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

3 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Abstract

Background and objectives: Triple-negative breast cancer is a significant global health challenge, and there's growing interest in targeting multiple pathways for treatment. Umbelliprenin, derived from herbal sources, has shown anti-tumor potential. This study aimed to assess umbelliprenin's impact on key genes related to proliferation, metastasis, and angiogenesis. Methods: Umbelliprenin, which was synthesized by the Pharmaceutical Research Center (PRC) at Mashhad University of Medical Sciences in Iran, was utilized in this study. The study aimed to investigate the impact of umbelliprenin on EGF and CoCl2-induced signaling in the PI3K/AKT/mTOR and MAPK pathways. Quantitative PCR was employed to assess the expression of EGFR, PI3K, AKT, mTOR, S6K, ERK1, ERK2, 4EBP1, HIF-1α, HIF-1β, VEGF, and VEGFR genes. Additionally, immunoblot assays were conducted to evaluate the levels of VEGF and HIF-1α in MDA-MB-468 cells. Results: The study found that umbelliprenin had cytotoxic effects, with an IC50 value of 152.5 µM. At concentrations of 10 µM and 20 µM, it upregulated genes like EGFR, VEGFR, HIF-1α, VEGF, PI3K, ERK2, and mTOR while downregulating 4EBP1. Umbelliprenin also increased VEGF protein levels. When used on EGF-stimulated cells, it enhanced VEGF and PI3K expression while inhibiting AKT, ERK2, mTOR, and antiproliferative 4EBP1 genes. Notably, VEGF and HIF-1α protein levels remained unchanged. Conversely, umbelliprenin downregulated EGFR, AKT, ERK1/2, HIF-1α, and VEGF in CoCl2-stimulated cells, while elevating 4EBP1 and reducing VEGF and HIF-1α protein levels. Conclusion: Umbelliprenin inhibited MDA-MB-468 cell growth and impacted gene expression related to metastasis and angiogenesis, particularly under conditions of EGFR activation and hypoxia.

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Main Subjects

References

  • Arnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M, Vignat J, Gralow JR, Cardoso F, Siesling S, Soerjomataram I. Current and future burden of breast cancer: Global statistics for 2020 and 2040. Breast. 2022; 66: 15–23.
  • Khaghanzadeh N, Mojtahedi Z, Ramezani M, Erfani N, Ghaderi A. Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells. Daru. 2012; 20 (1): 1–6.
  • Nakai K, Hung MC, Yamaguchi H. A perspective on anti-EGFR therapies targeting triple-negative breast cancer. Am J Cancer Res. 2016; 6(8): 1609–1623.
  • Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. 2010; 363(20): 1938–1948.
  • Thomas ES. Ixabepilone plus capecitabine for metastatic breast cancer progressing after anthracycline and taxane treatment. J Clin Oncol. 2008; Article ID 2223.
  • Khan MA, Jain VK, Rizwanullah M, Ahmad J, Jain K. PI3K/AKT/mTOR pathway inhibitors in triple-negative breast cancer: a review on drug discovery and future challenges. Drug Discov Today. 2019; 24(11): 2181–2191.
  • Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol. 2009; 27(13): 2278–2287.
  • Palumbo C, Benvenuto M, Focaccetti C, Albonici L, Cifaldi L, Rufini A, Nardozi D, Angiolini V, Bei A, Masuelli L, Bei R. Recent findings on the impact of ErbB receptors status on prognosis and therapy of head and neck squamous cell carcinoma. Front Med (Lausanne). 2023; 10: 1–16.
  • Park JH, Yoon J, Park B. Pomolic acid suppresses HIF1alpha/VEGF-mediated angiogenesis by targeting p38-MAPK and mTOR signaling cascades. Phytomedicine. 2016; 23(14): 1716–1726.
  • Saryeddine L, Zibara K, Kassem N, Badran B, El-Zein N. EGF-induced VEGF exerts a pi3k-dependent positive feedback on ERK and AKT through VEGFR2 in hematological in vitro models. PLoS One. 2016; 11(11): 1–16.
  • Nielsen DL, Andersson M, Andersen JL, Kamby C. Antiangiogenic therapy for breast cancer. Breast Cancer Res. 2010; 12(5): 1–16.
  • Baselga J, Gomez P, Greil R, Braga S, Climent MA, Wardley AM, Kaufman B, Stemmer SM, Pego A, Chan A, Goeminne JC, Graas MP, Kennedy MJ, Ciruelos Gil EM, Schneeweiss A, Zubel A, Groos J, Melezinkova H, Awada A. Randomized phase II study of the anti-epidermal growth factor receptor monoclonal antibody cetuximab with cisplatin versus cisplatin alone in patients with metastatic triple-negative breast cancer. J Clin Oncol. 2013; 31(20): 2586–2592.
  • Carey LA, Rugo HS, Marcom PK, Mayer EL, Esteva FJ, Ma CX, Liu MC, Storniolo AM, Rimawi MF, Forero-Torres A, Wolff AC, Hobday TJ, Ivanova A, Chiu WK, Ferraro M, Burrows E, Bernard PS, Hoadley KA, Perou CM, Winer EP. TBCRC 001: randomized phase II study of cetuximab in combination with carboplatin in stage IV triple-negative breast cancer. J Clin Oncol. 2012; 30(21): 2615–2623.
  • Bell R, Brown J, Parmar M, Toi M, Suter T, Steger GG, Pivot X, Mackey J, Jackisch C, Dent R, Hall P, Xu N, Morales L, Provencher L, Hegg R, Vanlemmens L, Kirsch A, Schneeweiss A, Masuda N, Overkamp F, Cameron D. Final efficacy and updated safety results of the randomized phase III BEATRICE trial evaluating adjuvant bevacizumab-containing therapy in triple-negative early breast cancer. Ann Oncol. 2017; 28(4): 754–760.
  • Miles DW, Chan A, Dirix LY, Cortes J, Pivot X, Tomczak P, Delozier T, Sohn JH, Provencher L, Puglisi F, Harbeck N, Steger GG, Schneeweiss A, Wardley AM, Chlistalla A, Romieu G. Phase III study of bevacizumab plus docetaxel compared with placebo plus docetaxel for the first-line treatment of human epidermal growth factor receptor 2-negative metastatic breast cancer. J Clin Oncol. 2010; 28(20): 3239–3247.
  • Miles DW, Dieras V, Cortes J, Duenne AA, Yi J, O'Shaughnessy J. First-line bevacizumab in combination with chemotherapy for HER2-negative metastatic breast cancer: pooled and subgroup analyses of data from 2447 patients. Ann Oncol. 2013; 24(11): 2773–2780.
  • Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA, Shenkier T, Cella D, Davidson NE. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007; 357(26): 2666–2676.
  • Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001; 294(5545): 1337–1340.
  • Khong TL, Thairu N, Larsen H, Dawson PM, Kiriakidis S, Paleolog EM. Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer. BMC Cancer. 2013; 13: 1–17.
  • Semenza GL. Life with oxygen. Science. 2007; 318(5847): 62–64.
  • Soggia A, Ramond C, Akiyama H, Scharfmann R, Duvillie B. von Hippel-Lindau gene disruption in mouse pancreatic progenitors and its consequences on endocrine differentiation in vivo: importance of HIF1-alpha and VEGF-A upregulation. Diabetologia. 2014; 57(11): 2348–2356.
  • Piret JP, Mottet D, Raes M, Michiels C. CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci. 2002; 973(1): 443–447.
  • Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA. 1998; 95(14): 7987–7992.
  • Dengler VL, Galbraith M, Espinosa JM. Transcriptional regulation by hypoxia inducible factors. Crit Rev Biochem Mol Biol. 2014; 49(1): 1–15.
  • Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999; 15(1): 551–578.
  • Yang Y, Cong H, Han C, Yue L, Dong H, Liu J. 12-Deoxyphorbol 13-palmitate inhibits the expression of VEGF and HIF-1alpha in MCF-7 cells by blocking the PI3K/Akt/mTOR signaling pathway. Oncol Rep. 2015; 34(4): 1755–1760.
  • Dai ZJ, Gao J, Ma XB, Yan K, Liu XX, Kang HF, Ji ZZ, Guan HT, Wang XJ. Up-regulation of hypoxia inducible factor-1alpha by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells. J Exp Clin Cancer Res. 2012; 31(1): 1–7.
  • Ward C, Langdon SP, Mullen P, Harris AL, Harrison DJ, Supuran CT, Kunkler IH. New strategies for targeting the hypoxic tumour microenvironment in breast cancer. Cancer Treat Rev. 2013; 39(2): 171–179.
  • Rashidi M, Ziai SA, Moini Zanjani T, Khalilnezhad A, Jamshidi H, Amani D. Umbelliprenin is potentially toxic against the HT29, CT26, MCF-7, 4T1, A172, and GL26 cell lines, potentially harmful against bone marrow-derived stem cells, and non-toxic against peripheral blood mononuclear cells. Iran Red Crescent Med J. 2016; 18(7): 1–7.
  • Gholami O, Jeddi-Tehrani M, Iranshahi M, Zarnani AH, Ziai SA. Umbelliprenin from Ferula szowitsiana activates both Intrinsic and extrinsic pathways of apoptosis in jurkat T-CLL cell line. Iran J Pharm Res. 2013; 12(3): 371–376.
  • Iranshahi M, Sahebkar A, Takasaki M, Konoshima T, Tokuda H. Cancer chemopreventive activity of the prenylated coumarin, umbelliprenin, in vivo. Eur J Cancer Prev. 2009; 18(5): 412–415.
  • Zamani Taghizadeh Rabe S, Iranshahi M, Mahmoudi M. In vitro anti-inflammatory and immunomodulatory properties of umbelliprenin and methyl galbanate. J Immunotoxicol. 2016; 13(2): 209–216.
  • John A, Tuszynski G. The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol Oncol Res. 2001; 7(1): 14–23.
  • Shahverdi AR, Saadat F, Khorramizadeh MR, Iranshahi M, Khoshayand MR. Two matrix metalloproteinases inhibitors from Ferula persica persica. Phytomedicine. 2006; 13(9-10): 712–717.
  • Barthomeuf C, Lim S, Iranshahi M, Chollet P. Umbelliprenin from Ferula szowitsiana inhibits the growth of human M4Beu metastatic pigmented malignant melanoma cells through cell-cycle arrest in G1 and induction of caspase-dependent apoptosis. Phytomedicine. 2008; 15(1-2): 103–111.
  • Iranshahi M, Arfa P, Ramezani M, Jaafari MR, Sadeghian H, Bassarello C, Piacente S, Pizza C. Sesquiterpene coumarins from Ferula szowitsiana and in vitro antileishmanial activity of 7-prenyloxycoumarins against promastigotes. Phytochemistry. 2007; 68(4): 554–561.
  • Safdari H, Neshani A, Sadeghian A, Ebrahimi M, Iranshahi M, Sadeghian H. Potent and selective inhibitors of class A beta-lactamase: 7-prenyloxy coumarins. J Antibiot (Tokyo). 2014; 67(5): 373–377.
  • Rashidi M, Khalilnezhad A, Amani D, Jamshidi H, Muhammadnejad A, Bazi A, Ziai SA. Umbelliprenin shows antitumor, antiangiogenesis, antimetastatic, anti-inflammatory, and immunostimulatory activities in 4T1 tumor-bearing Balb/c mice. J Cell Physiol. 2018; 233(11): 8908–8918.
  • Atabakhshian R, Salami S, Mirfakhraie R, Mahmoodi Khatonabadi S, Sirati-Sabet M, Gholamali Yaghmaei BG, Ghafghazi S, Dowlati Beirami A, Sadat Rezaei M. Umbelliprenin suppresses angiogenesis signaling in SKBR-3 cell line by downregulation of EGF/CoCl2-mediated PI3K/AKT/MAPK. Res J Pharmacogn. 2021; 8(1): 7–18.
  • Mousavi SH, Davari AS, Iranshahi M, Sabouri-Rad S, Tayarani Najaran Z. Comparative analysis of the cytotoxic effect of 7-prenyloxycoumarin compounds and herniarin on MCF-7 cell line. Avicenna J Phytomed. 2015; 5(6): 520–530.
  • Burness ML, Grushko TA, Olopade OI. Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker? Cancer J. 2010; 16(1): 23–32.
  • Siziopikou KP, Ariga R, Proussaloglou KE, Gattuso P, Cobleigh M. The challenging estrogen receptor-negative/ progesterone receptor-negative/HER-2-negative patient: a promising candidate for epidermal growth factor receptor-targeted therapy? Breast J. 2006; 12(4): 360–362.
  • Paplomata E, O'Regan R. The PI3K/AKT/mTOR pathway in breast cancer: targets, trials and biomarkers. Ther Adv Med Oncol. 2014; 6(4): 154–166.
  • Lee SH, Jee JG, Bae JS, Liu KH, Lee YM. A group of novel HIF-1alpha inhibitors, glyceollins, blocks HIF-1alpha synthesis and decreases its stability via inhibition of the PI3K/AKT/mTOR pathway and Hsp90 binding. J Cell Physiol. 2015; 230(4): 853–862.
  • Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci. 2011; 36(6): 320–328.
  • Wu N, Zhang J, Zhao J, Mu K, Zhang J, Jin Z, Yu J, Liu J. Precision medicine based on tumorigenic signaling pathways for triple-negative breast cancer. Oncol Lett. 2018; 16(4): 4984–4996.
  • Baselga J, Campone M, Piccart M, Burris HA, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI, Lebrun F, Beck JT, Ito Y, Yardley D, Deleu I, Perez A, Bachelot T, Vittori L, Xu Z, Mukhopadhyay P, Lebwohl D, Hortobagyi GN. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012; 366(6): 520–529.
  • Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418): 61–70
  • Schmid P, Abraham J, Chan S, Wheatley D, Brunt AM, Nemsadze G, Baird RD, Park YH, Hall PS, Perren T, Stein RC, Mangel L, Ferrero JM, Phillips M, Conibear J, Cortes J, Foxley A, de Bruin EC, McEwen R, Stetson D, Dougherty B, Sarker SJ, Prendergast A, McLaughlin-Callan M, Burgess M, Lawrence C, Cartwright H, Mousa K, Turner NC. Capivasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer: the PAKT trial. J Clin Oncol. 2020; 38(5): 423–433.
  • O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006; 66(3): 1500–1508.
  • Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbovic-Huezo O, Serra V, Majumder PK, Baselga J, Rosen N. AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity. Cancer Cell. 2011; 19(1): 58–71.
  • Serra V, Scaltriti M, Prudkin L, Eichhorn PJ, Ibrahim YH, Chandarlapaty S, Markman B, Rodriguez O, Guzman M, Rodriguez S, Gili M, Russillo M, Parra JL, Singh S, Arribas J, Rosen N, Baselga J. PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer. Oncogene. 2011; 30(22): 2547–2557.
  • Rojo F, Najera L, Lirola J, Jiménez J, Guzmán M, Sabadell MD, Baselga J, Cajal SRy. 4E-binding protein 1, a cell signaling hallmark in breast cancer that correlates with pathologic grade and prognosis. Clin Cancer Res. 2007; 13(1): 81–89.
  • Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010; 40(2): 310–322.
  • Avdulov S, Li S, Michalek V, Burrichter D, Peterson M, Perlman DM, Manivel JC, Sonenberg N, Yee D, Bitterman PB. Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells. Cancer cell. 2004; 5(6): 553–563.
  • Giltnane JM, Balko JM. Rationale for targeting the Ras/MAPK pathway in triple-negative breast cancer. Discov Med. 2014; 17(95): 275–283.
  • Bartholomeusz C, Gonzalez-Angulo AM, Liu P, Hayashi N, Lluch A, Ferrer-Lozano J, Hortobagyi GN. High ERK protein expression levels correlate with shorter survival in triple-negative breast cancer patients. Oncologist. 2012; 17(6): 766–774.
  • McKay MM, Morrison DK. Integrating signals from RTKs to ERK/MAPK. Oncogene. 2007; 26(22): 3113–3121.
  • Rozengurt E. Mitogenic signaling pathways induced by G protein-coupled receptors. J Cell Physiol. 2007; 213(3): 589–602.
  • Adam A, Kenny LM. Interventional oncology in multidisciplinary cancer treatment in the 21st Nat Rev Clin Oncol. 2015; 12(2): 105–113.
Research Journal of Pharmacognosy
Volume 11, Issue 1
January 2024
Pages 19-31

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