R(+)-Methanandamide-Induced Apoptosis of Human Cervical Carcinoma Cells Involves A Cyclooxygenase-2-Dependent Pathway



Cannabinoids have received renewed interest due to their antitumorigenic effects. Using human cervical carcinoma cells (HeLa), this study investigates the role of cyclooxygenase-2 (COX-2) in apoptosis elicited by the endocannabinoid analog R(+)-methanandamide (MA).


COX-2 expression was assessed by RT-PCR and Western blotting. PGE2/PGD2 levels in cell culture supernatants and DNA fragmentation were measured by ELISA.


MA led to an induction of COX-2 expression, PGD2 and PGE2 synthesis. Cells were significantly less sensitive to MA-induced apoptosis when COX-2 was suppressed by siRNA or the selective COX-2 inhibitor NS-398. COX-2 expression and apoptosis by MA was also prevented by the ceramide synthase inhibitor fumonisin B1, but not by antagonists to cannabinoid receptors and TRPV1. In line with the established role of peroxisome proliferator-activated receptor γ (PPARγ) in the proapoptotic action of PGs of the D and J series, inhibition of MA-induced apoptosis was also achieved by siRNA targeting lipocalin-type PGD synthase (L-PGDS) or PPARγ. A role of COX-2 and PPARγ in MA-induced apoptosis was confirmed in another human cervical cancer cell line (C33A) and in human lung carcinoma cells (A549).


This study demonstrates COX-2 induction and synthesis of L-PGDS-derived, PPARγ-activating PGs as a possible mechanism of apoptosis by MA.

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Fig. 7







(6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl) methanone


(N-[2-(4-Chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2- benzazepine-2-carbothioamide

CB1 :

Cannabinoid receptor 1

CB2 :

Cannabinoid receptor 2




Lipocalin-type prostaglandin D synthase


R(+)-methanandamide (R-(+)-arachidonyl-1′-hydroxy-2′-propylamide)






Peroxisome proliferator-activated receptor γ


Reverse transcriptase-polymerase chain reaction


Small-interfering RNA


Transient receptor potential vanilloid-type 1



15d-PGJ2 :



  1. 1.

    M. Guzman. Cannabinoids: potential anticancer agents. Nat. Rev. Cancer. 3:745–755 (2003) doi:10.1038/nrc1188.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    M. Bifulco, C. Laezza, S. Pisanti, and P. Gazzerro. Cannabinoids and cancer: pros and cons of an antitumour strategy. Br. J. Pharmacol. 148:123–135 (2006) doi:10.1038/sj.bjp.0706632.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    L. Matsuda, S. Lolait, M. Brownstein, A. Young, and T. Bonner. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 346:561–564 (1990) doi:10.1038/346561a0.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    S. Munro, K. Thomas, and M. Abu-Shaar. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 365:61–65 (1993) doi:10.1038/365061a0.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    S. Galiegue, S. Mary, J. Marchand, D. Dussossoy, D. Carriere, P. Carayon, M. Bouaboula, D. Shire, G. L. Fur, and P. Casellas. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur. J. Biochem. 232:54–61 (1995) doi:10.1111/j.1432-1033.1995.tb20780.x.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    W. A. Devane, L. Hanus, A. Breuer, R. G. Pertwee, L. A. Sevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, and R. Mechoulam. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science. 258:1946–1949 (1992) doi:10.1126/science.1470919.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    D. Smart, M. Gunthorpe, J. Jerman, S. Nasir, J. Gray, A. Muir, J. Chambers, A. Randall, and J. Davis. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br. J. Pharmacol. 129:227–230 (2000) doi:10.1038/sj.bjp.0703050.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    R. Mechoulam, S. Ben-Shabat, L. Hanus, M. Ligunsky, N. E. Kaninski, A. R. Schatz, A. Gopher, S. Almog, B. R. Martin, D. R. Compton, R. G. Pertwee, G. Griffin, M. Bayewitch, J. Barg, and Z. Vogel. Identification of an endogenous 2-monoglyceride, present in canine-gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50:83–90 (1995) doi:10.1016/0006-2952(95)00109-D.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    L. D. De Petrocellis, D. Melck, A. Palmisano, T. Bisogno, C. Laezza, M. Bifulco, and V. D. Marzo. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation. Proc. Natl. Acad. Sci. USA. 95:8375–8380 (1998) doi:10.1073/pnas.95.14.8375.

    PubMed  Article  Google Scholar 

  10. 10.

    S. Jacobsson, T. Wallin, and C. Fowler. Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors. J. Pharmacol. Exp. Ther. 299:951–959 (2001).

    PubMed  CAS  Google Scholar 

  11. 11.

    E. Contassot, M. Tenan, V. Schnuriger, M. F. Pelte, and P. Y. Dietrich. Arachidonyl ethanolamide induces apoptosis of uterine cervix cancer cells via aberrantly expressed vanilloid receptor-1. Gynecol. Oncol. 93:182–188 (2004) doi:10.1016/j.ygyno.2003.12.040.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    A. Ligresti, T. Bisogno, I. Matias, L. D. Petrocellis, M. Cascio, V. Cosenza, G. D’argenio, G. Scaglione, M. Bifulco, I. Sorrentini, and V. D. Marzo. Possible endocannabinoid control of colorectal cancer growth. Gastroenterology. 125:677–687 (2003) doi:10.1016/S0016-5085(03)00881-3.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    M. Bifulco, C. Laezza, M. Valenti, A. Ligresti, G. Portella, and V. DiMarzo. A new strategy to block tumor growth by inhibiting endocannabinoid inactivation. FASEB J. 18:1606–1608 (2004).

    PubMed  CAS  Google Scholar 

  14. 14.

    V. DiMarzo, T. Bisogno, L. De Petrocellis, D. Melck, and B. R. Martin. Cannabimimetic fatty acid derivatives: the anandamide family and other endocannabinoids. Curr. Med. Chem. 6:721–744 (1999).

    CAS  Google Scholar 

  15. 15.

    V. DiMarzo, C. S. Breivogel, Q. Tao, D. T. Bridgen, R. K. Razdan, A. M. Zimmer, A. Zimmer, and B. R. Martin. Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain. J. Neurochem. 75:2434–2444 (2000) doi:10.1046/j.1471-4159.2000.0752434.x.

    Article  CAS  Google Scholar 

  16. 16.

    E. Berdyshev, P. Schmid, R. Krebsbach, C. Hillard, C. Huang, N. Chen, Z. Dong, and H. Schmid. Cannabinoid-receptor-independent cell signalling by N-acylethanolamines. Biochem. J. 360:67–75 (2001) doi:10.1042/0264-6021:3600067.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    K. Sarker, and I. Maruyama. Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts. Cell. Mol. Life Sci. 60:1200–1208 (2003).

    PubMed  CAS  Google Scholar 

  18. 18.

    E. Ellis, S. Moore, and K. Willoughby. Anandamide and Δ 9-THC dilation of cerebral arterioles is blocked by indomethacin. Am. J. Physiol. 269:H1859–1864 (1995).

    PubMed  CAS  Google Scholar 

  19. 19.

    S. Burstein, K. Hull, S. Hunter, and J. Shilstone. Immunization against prostaglandins reduces Δ1-tetrahydrocannabinol-induced catalepsy in mice. Mol. Pharmacol. 35:6–9 (1989).

    PubMed  CAS  Google Scholar 

  20. 20.

    D. Pate, K. Jarvinen, A. Urtti, P. Jarho, M. Fich, V. Mahadevan, and T. Jarvinen. Effects of topical anandamides on intraocular pressure in normotensive rabbits. Life Sci. 58:1849–1860 (1996) doi:10.1016/0024-3205(96)00169-5.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    K. Green, E. Kearse, and O. McIntyre. Interaction between Δ9-tetrahydrocannabinol and indomethacin. Ophthalmic Res. 33:217–220 (2001) doi:10.1159/000055673.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    G. Chan, T. Hinds, S. Impey, and D. Storm. Hippocampal neurotoxicity of Δ9- tetrahydrocannabinol. J. Neurosci. 18:5322–5332 (1998).

    PubMed  CAS  Google Scholar 

  23. 23.

    B. Hinz, R. Ramer, K. Eichele, U. Weinzierl, and K. Brune. Upregulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells. Mol. Pharmacol. 66:1643–1651 (2004) doi:10.1124/mol.104.002618.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    K. Eichele, U. Weinzierl, R. Ramer, K. Brune, and B. Hinz. R(+)-methanandamide elicits a cyclooxygenase-2-dependent mitochondrial apoptosis signaling pathway in human neuroglioma cells. Pharm. Res. 23:90–94 (2006) doi:10.1007/s11095-005-8815-2.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Y. C. Chen, S. C. Shen, and S. H. Tsai. Prostaglandin D2 and J2 induce apoptosis in human leukemia cells via activation of the caspase 3 cascade and production of reactive oxygen species. Biochim. Biophys. Acta. 1743:291–304 (2005) doi:10.1016/j.bbamcr.2004.10.016.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    M. Maccarrone, R. Pauselli, M. DiRienzo, and A. Finazzi-Agro. Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells. Biochem. J. 366:137–144 (2002).

    PubMed  CAS  Google Scholar 

  27. 27.

    Y. E. Dommels, M. M. Haring, N. G. Keestra, G. M. Alink, P. J. van Bladeren, and B. van Ommen. The role of cyclooxygenase in n-6 and n-3 polyunsaturated fatty acid mediated effects on cell proliferation, PGE2 synthesis and cytotoxicity in human colorectal carcinoma cell lines. Carcinogenesis. 24:385–392 (2003) doi:10.1093/carcin/24.3.385.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    H. K. Na, H. Inoue, and Y. J. Surh. ET-18-O-CH3-induced apoptosis is causally linked to COX-2 upregulation in H-ras transformed human breast epithelial cells. FEBS Lett. 579:6279–6287 (2005) doi:10.1016/j.febslet.2005.09.094.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    K. Eichele, R. Ramer, and B. Hinz. Decisive role of cyclooxygenase-2 and lipocalin-type prostaglandin D synthase in chemotherapeutics-induced apoptosis of human cervical carcinoma cells. Oncogene. 27:3032–3044 (2008) doi:10.1038/sj.onc.1210962.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    H. K. Na, and Y. J. Surh. Peroxisome proliferator-activated receptor γ (PPARγ) ligands as bifunctional regulators of cell proliferation. Biochem. Pharmacol. 66:1381–1391 (2003) doi:10.1016/S0006-2952(03)00488-X.

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    J. Kim, P. Yang, M. Suraokar, A. L. Sabichi, N. D. Llansa, G. Mendoza, V. Subbarayan, C. J. Logothetis, R. A. Newman, S. M. Lippman, and D. G. Menter. Suppression of prostate tumor cell growth by stromal cell prostaglandin D synthase-derived products. Cancer Res. 65:6189–6198 (2005) doi:10.1158/0008-5472.CAN-04-4439.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    B. Gardner, L. X. Zhu, S. Sharma, D. P. Tashkin, and S. M. Dubinett. Methanandamide increases COX-2 expression and tumor growth in murine lung cancer. FASEB J. 17:2157–2159 (2003).

    PubMed  CAS  Google Scholar 

  33. 33.

    L. Mestre, F. Correa, F. Docagne, D. Clemente, and C. Guaza. The synthetic cannabinoid WIN 55,212–2 increases COX-2 expression and PGE2 release in murine brain-derived endothelial cells following Theiler’s virus infection. Biochem. Pharmacol. 72:869–880 (2006) doi:10.1016/j.bcp.2006.06.037.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Y. Hannun, and L. Obeid. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 277:25847–25850 (2002) doi:10.1074/jbc.R200008200.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    K. Subbaramaiah, W. Chung, and A. Dannenberg. Ceramide regulates the transcription of cyclooxygenase-2. evidence for involvement of extracellular signal-regulated kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways. J. Biol. Chem. 273:32943–32949 (1998) doi:10.1074/jbc.273.49.32943.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    R. Ramer, K. Brune, A. Pahl, and B. Hinz. R(+)-methanandamide induces cyclooxygenase-2 expression in human neuroglioma cells via a non-cannabinoid receptor-mediated mechanism. Biochem. Biophys. Res. Commun. 286:1144–1152 (2001) doi:10.1006/bbrc.2001.5518.

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    G. Velasco, I. Galve-Roperh, C. Sanchez, C. Blazquez, A. Haro, and M. Guzman. Cannabinoids and ceramide: two lipids acting hand-by-hand. Life Sci. 77:1723–1731 (2005) doi:10.1016/j.lfs.2005.05.015.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    E. Wang, W. P. Norred, C. W. Bacon, R. T. Riley, and A. H. Jr Merrill. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J. Biol. Chem. 266:14486–14490 (1991).

    PubMed  CAS  Google Scholar 

  39. 39.

    R. Ramer, U. Weinzierl, B. Schwind, K. Brune, and B. Hinz. Ceramide is involved in R(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells. Mol. Pharmacol. 64:1189–1198 (2003) doi:10.1124/mol.64.5.1189.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    B. Hinz, K. Brune, and A. Pahl. Cyclooxygenase-2 expression in lipopolysaccharide-stimulated human monocytes is modulated by cyclic AMP, prostaglandin E2, and nonsteroidal anti-inflammatory drugs. Biochem. Biophys. Res. Commun. 278:790–6 (2000) doi:10.1006/bbrc.2000.3885.

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    S. Debey, J. Meyer-Kirchrath, and K. Schror. Regulation of cyclooxygenase-2 expression by iloprost in human vascular smooth muscle cells. Role of transcription factors CREB and ICER. Biochem. Pharmacol. 65:979–988 (2003) doi:10.1016/S0006-2952(02)01661-1.

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    S. Rosch, R. Ramer, K. Brune, and B. Hinz. Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases. Biochem. Biophys. Res. Commun. 338:1171–1178 (2005) doi:10.1016/j.bbrc.2005.10.051.

    PubMed  Article  Google Scholar 

  43. 43.

    L. Lalier, P. F. Cartron, F. Pedelaborde, C. Olivier, D. Loussouarn, S. A. Martin, K. Meflah, J. Menanteau, and F. M. Vallette. Increase in PGE2 biosynthesis induces a Bax dependent apoptosis correlated to patients’ survival in glioblastoma multiforme. Oncogene. 26:4999–5009 (2007) doi:10.1038/sj.onc.1210303.

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    J. L. Herlong, and T. R. Scott. Positioning prostanoids of the D and J series in the immunopathogenic scheme. Immunol. Lett. 102:121–131 (2006) doi:10.1016/j.imlet.2005.10.004.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    J. K. Maesaka, T. Palaia, L. Frese, S. Fishbane, and L. Ragolia. Prostaglandin D2 synthase induces apoptosis in pig kidney LLC-PK1 cells. Kidney Int. 60:1692–1698 (2001) doi:10.1046/j.1523-1755.2001.00989.x.

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    L. Ragolia, T. Palaia, L. Frese, S. Fishbane, and J. K. Maesaka. Prostaglandin D2 synthase induces apoptosis in PC12 neuronal cells. Neuroreport. 12:2623–2628 (2001) doi:10.1097/00001756-200108280-00008.

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    S. Han, and J. Roman. Peroxisome proliferator-activated receptor γ: a novel target for cancer therapeutics. Anticancer Drugs. 18:237–244 (2007) doi:10.1097/CAD.0b013e328011e67d.

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    M. Bouaboula, S. Hilairet, J. Marchand, L. Fajas, G. Le Fur, and P. Casellas. Anandamide induced PPARγ transcriptional activation and 3T3-L1 preadipocyte differentiation. Eur. J. Pharmacol. 517:174–181 (2005) doi:10.1016/j.ejphar.2005.05.032.

    PubMed  Article  CAS  Google Scholar 

  49. 49.

    S. Burstein. PPAR-γ: a nuclear receptor with affinity for cannabinoids. Life Sci. 77:1674–1684 (2005) doi:10.1016/j.lfs.2005.05.039.

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    S. E. O’Sullivan, E. J. Tarling, A. J. Bennett, D. A. Kendall, and M. D. Randall. Novel time-dependent vascular actions of Δ9-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma. Biochem. Biophys. Res. Commun. 337:824–831 (2005).

    PubMed  CAS  Google Scholar 

  51. 51.

    C. E. Rockwell, N. T. Snider, J. T. Thompson, J. P. Vanden Heuvel, and N. E. Kaminski. Interleukin-2 suppression by 2-arachidonyl glycerol is mediated through peroxisome proliferator-activated receptor γ independently of cannabinoid receptors 1 and 2. Mol. Pharmacol. 70:101–111 (2006).

    PubMed  CAS  Google Scholar 

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This study was supported by grants from the Deutsche Krebshilfe e.V. (Bonn, Germany), Deutsche Forschungsgemeinschaft (SFB 539 TP BI.6) and Johannes und Frieda Marohn Stiftung (Erlangen, Germany).

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Correspondence to Burkhard Hinz.

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Karin Eichele and Robert Ramer contributed equally to this work.

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Eichele, K., Ramer, R. & Hinz, B. R(+)-Methanandamide-Induced Apoptosis of Human Cervical Carcinoma Cells Involves A Cyclooxygenase-2-Dependent Pathway. Pharm Res 26, 346–355 (2009). https://doi.org/10.1007/s11095-008-9748-3

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  • Apoptosis
  • cyclooxygenase-2
  • lipocalin-type prostaglandin D synthase
  • peroxisome proliferator-activated receptor γ
  • R(+)-methanandamide