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

Abstract

Purpose

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).

Methods

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.

Results

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).

Conclusion

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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Abbreviations

AEA:

Anandamide

AM-251:

N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide

AM-630:

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

capsazepine:

(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

COX:

Cyclooxygenase

L-PGDS:

Lipocalin-type prostaglandin D synthase

MA:

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

NS-398:

N-[2-(Cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide

PG:

Prostaglandin

PPARγ:

Peroxisome proliferator-activated receptor γ

RT-PCR:

Reverse transcriptase-polymerase chain reaction

siRNA:

Small-interfering RNA

TRPV1:

Transient receptor potential vanilloid-type 1

2-AG:

2-Arachidonylglycerol

15d-PGJ2 :

15-Deoxy-Δ12,14-PGJ2

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Acknowledgment

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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KEY WORDS

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