15d-PGJ2 Induces Apoptosis of MCF-7 and MDA-MB-231 Cells via Increased Intracellular Calcium and Activation of Caspases, Independent of ERα and ERβ

  • Muhammad, Siti Nur Hasyila (Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus) ;
  • Mokhtar, Noor Fatmawati (nstitute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Health Campus) ;
  • Yaacob, Nik Soriani (Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus)
  • Published : 2016.07.01


Reports indicate that 15-deoxy-delta-12,14-prostaglandin-J2 (15d-PGJ2) has anticancer activities, but its mechanisms of action have yet to be fully elucidated. We therefore investigated the effects of 15d-PGJ2 on the human breast cancer cell lines, MCF-7 (estrogen receptor $ER{\alpha}+/ER{\beta}+$) and MDA-MB-231 ($ER{\alpha}-/ER{\beta}+$). Cellular proliferation and cytotoxicity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays while apoptosis was determined by fluorescence microscopy and flow cytometry using annexin V-propidium iodide (PI) staining. ER expression was determined by Western blotting. Intracellular calcium was stained with Fluo-4 AM while intracellular caspase activities were detected with Caspase-$FLICA(R)$ and measured by flow cytometry. We showed that 15d-PGJ2 caused a significant increase in apoptosis in MCF-7 and MDA-MB-231 cells. $ER{\alpha}$ protein expression was reduced in treated MCF-7 cells but pre-incubation with the $ER{\alpha}$ inhibitor' ICI 182 780' did not affect the percentage of apoptotic cells. The expression of $ER{\beta}$ was unchanged in both cell lines. In addition, 15d-PGJ2 increased intracellular calcium ($Ca^{2+}$) staining and caspase 8, 9 and 3/7 activities. We therefore conclude that 15d-PGJ2 induces caspase-dependent apoptosis that is associated with an influx of intracellular $Ca^{2+}$ with no involvement of ER signaling.


Supported by : Ministry of Science Technology and Innovation


  1. Zingarelli B, Sheehan M, Hake PW, et al (2003). Peroxisome proliferator activator receptor-gamma ligands, 15-deoxy-Delta (12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsis by modulation of signal transduction pathways. J Immunol, 171, 6827-37.
  2. Zoli W, Ulivi P, Tesei A. et al (2005). Addition of 5-fluorouracil to doxorubicin- paclitaxel sequence increases caspasedependent apoptosis in breast cancer cell lines. Breast Cancer Res, 7, 681-9.
  3. Mughal A, Kumar D, Vikram A (2015). Effects of Thiazolidinediones on metabolism and cancer: Relative influence of $PPAR{\gamma}$ and IGF-1 signaling. Eur J Pharmacol, 768, 217-25.
  4. Napimoga MH, Demasib APD, Bossonarob JP et al (2013). Low doses of 15d-PGJ2 induce osteoblast activity in a PPAR-gamma independent manner. International Immunopharmacol, 16, 131-8.
  5. Orrenius S, Zhivotovsky B, Nicotera P (2003). Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol, 4, 552-65.
  6. Pacher P, Hajnoczky G (2001). Propagation of the apoptotic signal by mitochondrial waves. EMBO J, 20, 4107-21.
  7. Parrish AB, Freel CD and Kornbluth S (2013). Cellular Mechanisms Controlling Caspase Activation and Function Cold Spring Harb Perspect Biol, 5, 8672.
  8. Popolo A, Piccinelli AL, Morello S, et al (2011). Cytotoxic activity of nemorosone in human MCF-7 breast cancer cells. Can J Physiol Pharmacol, 89, 50-7.
  9. Qin C, Burghardt R, Smith R, et al (2003). Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells. Cancer Res, 63, 958-64.
  10. Rao GK, Zhang W, Kaminski NE (2004). Cannabinoid receptormediated regulation of intracellular calcium by delta(9)-tetrahydrocannabinol in resting T cells. J Leukoc Biol, 75, 884-92.
  11. Rizzuto R, Pinton P, Ferrari D, et al (2003). Calcium and apoptosis: facts and hypotheses. Oncogene, 22, 8619-27.
  12. Shin SW, Seo CY, Han H, et al (2009). 15d-PGJ2 induces apoptosis by reactive oxygen species-mediated inactivation of Akt in leukemia and colorectal cancer cells and shows in vivo antitumor activity. Clin Cancer Res, 15, 5414-25.
  13. Twiddy D, Cohen GM, Macfarlane M, et al (2006). Caspase-7 is directly activated by the approximately 700-kDa apoptosome complex and is released as a stable XIAPcaspase-7 approximately 200-kDa complex. J Biol Chem, 281, 3876-88.
  14. Wen J, Huang YC, Xiu HH, et al (2016). Altered expression of stromal interaction molecule (STIM)-calcium releaseactivated calcium channel protein (ORAI) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in cancer: will they become a new battlefield for oncotherapy? Chin J Cancer, 35, 32.
  15. Yaacob NS, Nasir R, Norazmi MN (2013). Influence of 17betaestradiol on 15- deoxy-delta12,14 prostaglandin J2 -induced apoptosis in MCF-7 and MDA-MB-231 cells. Asian Pac J Cancer Prev, 14, 6761-7.
  16. Yen CC, Hsiao CD, Chen WM, et al. (2014). Cytotoxic effects of 15d-PGJ2 against osteosarcoma through ROS-mediated AKT and cell cycle inhibition. Oncotarget, 5, 716-25.
  17. Yu K, He Y, Yeung LW, et al (2008). DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondriacaspase protease pathway in human neuroblastoma cells in vitro. Toxicol Sci, 104, 341-51.
  18. Chaffer CL, Thomas DM, Thompson EW, et al (2006). PPARgamma-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma. BMC Cancer, 6, 53.
  19. Chen Y, Li Z, He Y, et al (2014). Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell-matrigel adhesion of MCF-7 breast cancer cells through a 14 novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis. Toxicol Appl Pharmacol, 275, 176-81.
  20. Cho WH, Choi CH, Park JY, et al (2006). 15-deoxy-(Delta12,14)-prostaglandin J2 (15d-PGJ2) induces cell death through caspase-independent mechanism in A172 human glioma cells. Neurochem Res, 31, 1247-54.
  21. Ciucci A, Gianferretti P, Piva R, et al (2006). Induction of apoptosis in estrogen receptor-negative breast cancer cells by natural and synthetic cyclopentenones: role of the IkappaB kinase/nuclear factor-kappaB pathway. Mol Pharmacol, 70, 1812-21.
  22. Croasdell A, Duffney PF, Kim N, et al (2015). $PPAR{\gamma}$ and the innate immune system mediate the resolution of inflammation. PPAR Res, 2015, 549691.
  23. Decuypere J-P, Monaco G, Bultynck G, et al (2011). The IP3 receptor-mitochondria connection in apoptosis and autophagy. Biochimica et Biophysica Acta, 1813, 1003-13.
  24. Dejeana LM, Martinez-Caballeroa S, Manonb S, et al (2006). Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins. Biochimica et Biophysica Acta, 1762, 191-201.
  25. Dionne S, Levy E, Levesque D, et al (2010). PPARgamma ligand 15-deoxy-delta 12,14-prostaglandin J2 sensitizes human colon carcinoma cells to TWEAK-induced apoptosis. Anticancer Res, 30, 157-66
  26. Egger J, Fischer S, Bretscher P, et al (2015). Total synthesis of prostaglandin 15d- PGJ2 and investigation of its effect on the secretion of IL-6 and IL-12. Org. Lett, 17, 4340-3.
  27. Fujita M, Tohji C, Honda Y, et al (2012). Cytotoxicity of 15-deoxy-Delta(12,14)- prostaglandin J(2) through PPARgamma-independent pathway and the involvement of the JNK and Akt pathway in renal cell carcinoma. Int J Med Sci, 9, 555-66.
  28. Jo DG, Jun JI, Chang JW, et al (2004). Calcium binding of ARC mediates regulation of caspase 8 and cell death. Mol Cell Biol, 24, 9763-70.
  29. Kim EJ, Park KS, Chung SY, et al (2003). Peroxisome proliferator-activated receptor-gamma activator 15-deoxy-Delta12,14-prostaglandin J2 inhibits neuroblastoma cell growth through induction of apoptosis: association with extracellular signal-regulated kinase signal pathway. J Pharmacol Exp Ther, 307, 505-17.
  30. Kim HJ, Kim JY, Meng Z, et al (2007). 15-deoxy-Delta12,14-prostaglandin J2 inhibits transcriptional activity of estrogen receptor-alpha via covalent modification of DNA-binding domain. Cancer Res, 67, 2595-602.
  31. Kim JH, Song J, Park KW (2015). The multifaceted factor peroxisome proliferator- activated receptor ${\gamma}$ ($PPAR{\gamma}$) in metabolism, immunity, and cancer. Arch Pharm Res, 38, 302-12.
  32. Kocanova S, Mazaheri M, Caze-Subra S, et al (2010). Ligands specify estrogen receptor alpha nuclear localization and degradation. BMC Cell Biol, 11, 98.
  33. Landar A, Shiva S, Levonen AL, et al (2006). Induction of the permeability transition and cytochrome c release by 15-deoxy-Delta12,14-prostaglandin J2 in mitochondria. Biochem J, 394, 185-95.
  34. Lecomte J, Flament S, Salamone S, et al (2008). Disruption of ERalpha signalling pathway by PPARgamma agonists: evidences of PPARgamma-independent events in two hormone-dependent breast cancer cell lines. Breast Cancer Res Treat, 112, 437-51.
  35. Li H, Pauza CD (2009). Effects of 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and rosiglitazone on human gammadelta2 T cells. PLoS One, 4, 7726.
  36. McKeage K, Curran MP, Plosker GL (2004). Fulvestrant: a review of its use in hormone receptor-positive metastatic breast cancer in postmenopausal women with disease progression following antiestrogen therapy. Drugs, 64, 633-8.
  37. Monteith GR, Mcandrew D, Faddy HM, et al (2007). Calcium and cancer: targeting $Ca^{2+}$ transport. Nat Rev Cancer, 7, 519-30.