The Korean Journal of Medicine

The Korean Association of Internal Medicine (대한내과학회)
권호 표지

Peroxisome proliferator-activated receptor $\gamma$ ligands exert antineoplastic effects in hepatocellular carcinoma cells

Peroxisome proliferator-activated receptor $\gamma$ 배위자의 간암 세포에 대한 항암 효과

Chae, Myung-Jong;Shim, Jae-Jun;Kim, Byung-Ho;Hwangbo, Young;Lee, Young-Ju;Ha, Seung-Hyung;Jang, Jae-Young;Dong, Seok-Ho;Kim, Hyo-Jong;Chang, Young-Woo;Chang, Rin
채명종;심재준;김병호;황보영;이영주;하승형;장재영;동석호;김효종;장영운;장린

  • Published : 2008.09.01
⟨ Previous Next ⟩

Abstract

Background/Aims: Thiazolidinediones, which are synthetic insulin sensitizers, are known activators of peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ ligands, including endogenous 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), are thought to elicit antineoplastic effects in various cancer cells. In this study, the antineoplastic effects of PPARγ ligands against hepatocellular carcinoma (HCC) cells were investigated. Methods: HepG2, Hep3B, and PLC/PRF5 cells were cultured with troglitazone (TGZ), pioglitazone (PGZ), rosiglitazone (RGZ), or 15d-PGJ2 at concentrations of 20-100 μM.. Cell viability, cell cycle arrest, apoptosis, and caspase activity were measured using the MTT assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and colorimetric assays, respectively. The effects of various caspase inhibitors were also measured using a cell death detection ELISA. Results: All three cell lines expressed the PPARγ gene. TGZ and 15d-PGJ2 strongly inhibited growth in HepG2, Hep3B, and PLC/PRF5 cells. In contrast, PGZ and RGZ showed a much weaker effect in all cell lines. In terms of cell cycle arrest and apoptosis, TGZ induced G0/G1 arrest in HepG2 cells and increased the apoptotic fraction in Hep3B and PLC/PRF5 cells. In contrast, 15d-PGJ2 induced apoptosis only in HepG2 and Hep3B cells. TGZ and 15d-PGJ2 increased caspase-3 activity significantly and increased caspase-9 activity slightly. TGZ- and 15d-PGJ2-induced apoptoses were inhibited by a pancaspase inhibitor (Z-VAD-FMK) and a caspase-3 specific inhibitor (Z-DEVD-FMK) in a dose- dependent manner. Conclusions: TGZ and 15d-PGJ2 elicit antineoplastic effects in various HCC cells via caspase-dependent apoptotic induction. Their differential effects on similar cell types suggest that another antineoplastic mechanism, most likely a PPARγ-independent pathway, is involved.

목적: PPARγ는 핵 수용체의 일종으로 혈당 조절과 지방 세포의 분화에 작용하는 것뿐만 아니라 각종 암세포에 대한 항암 작용과도 연관되어 있다고 알려져 있다. 대표적인 합성 PPARγ배위자인 thiazolidinediones계 혈당 강하제와 내인성 배위자인 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2)가 간세포암(이하 간암)에도 항암 효과가 있는지 조사하였다. 방법: 간암 세포주로는 HepG2, Hep3B, PLC/PRF5를 사용하였는데, 이들 세포주에서 PPARγ유전자 발현은 역전사-중합효소연쇄반응으로 확인하였다. Thiazolidinediones계 약물인 troglitazone (TGZ), pioglitazone (PGZ) 그리고 rosiglitazone (RGZ)을 각각 25～100 μM의 농도로 각 세포주에 투여하였고, 15d-PGJ2는 20～80 μM의 농도로 투여하였다. MTT assay를 이용하여 세포 생존률을 72시간까지 대조군과 비교하였다. 세포 주기 분석은 flow cytometry를 이용하였고, 세포자멸사는 ELISA kit를 이용하였다. Caspase-3와 caspase-8 그리고 caspase-9의 활성도를 caspase colorimetric assay를 이용하여 분석하였다. Caspase 억제제를 투여하여 PPARγ배위자에 의한 세포자멸사가 억제되는지 조사하였다. 결과: 사용된 PPARγ배위자 중 TGZ과 15d-PGJ2가 HepG2, Hep3B 그리고 PLC/PRF/5에 뚜렷한 성장 억제를 나타냈다. 반면에 PGZ과 RGZ은 TGZ에 비해 거의 효과가 없었다. 세포 주기분석에서도 TGZ과 15d-PGJ2가 G0/G1 arrest와 세포자멸사를 일으킨 반면 PGZ과 RGZ은 효과가 없었다. 세포 주기 및 세포자멸사 분석에서 TGZ과 15d-PGJ2의 항암 효과는 세포주에 따라 서로 다르게 나타났다. TGZ은 HepG2에서 G0/G1 arrest를 일으켰고 PLC/PRF5의 세포자멸사를 유도한 반면 15d-PGJ2는 HepG2에서 세포자멸사를 일으키고 PLC/ PRF5에서는 뚜렷한 세포자멸사를 일으키지 못하였다. TGZ과 15d-PGJ2은 caspase-3 활성도를 크게 증가시켰으며(p<0.01) caspase-9의 활성도도 증가시켰다. TGZ과 15d-PGJ2 에 의해 유도된 세포자멸사는 pancaspase 억제제와 caspase-3 특이 억제제에 용량 의존적으로 억제되었다. 결론: Troglitazone과 15d-PGJ2는 간암 세포에서 세포자멸사를 유도하여 항암 작용을 나타내는데, PPARγ이외의 다른 기전이 작용하는 것으로 보이며 항암 기전도 서로 다른 것으로 생각한다.

Keywords

References

  1. Korea National Statistical Office. The cause of the death. Avail from: http://www.nso.go.kr
  2. El-Serag HB, Mason AC, Key C. Trends in survival of patients with hepatocellular carcinoma between 1977 and 1996 in the United States. Hepatology 33:62-65, 2001 https://doi.org/10.1053/jhep.2001.21041
  3. Grommes C, Landreth GE, Heneka MT. Antineoplastic effects of peroxisome proliferator-activated receptor $\gamma$ agonists. Lancet Oncol 5:419-429, 2004 https://doi.org/10.1016/S1470-2045(04)01509-8
  4. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR$\gamma$, a lipid-activated transcription factor. Cell 79:1147-1156, 1994 https://doi.org/10.1016/0092-8674(94)90006-X
  5. Frohlich E, Machicao F, Wahl R. Action of thiazolidinediones on differentiation, proliferation and apoptosis of normal and transformed thyrocytes in culture. Endocr Relat Cancer 12:291-303, 2005 https://doi.org/10.1677/erc.1.00973
  6. Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, Koeffler HP. Ligand for peroxisome proliferator-activated receptor $\gamma$ (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 58:3344-3352, 1998
  7. Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, Holden SA, Chen LB, Singer S, Fletcher C, Spiegelman BM. Differentiation and reversal of malignant changes in colon cancer through PPAR$\gamma$. Nat Med 4:1046- 1052, 1998 https://doi.org/10.1038/2030
  8. Han S, Roman J. Peroxisome proliferator-activated receptor : a novel target for cancer therapeutics? Anticancer Drugs 18:237-244, 2007 https://doi.org/10.1097/CAD.0b013e328011e67d
  9. Koga H, Sakisaka S, Harada M, Takagi T, Hanada S, Taniguchi E, Kawaguchi T, Sasatomi K, Kimura R, Hashimoto O, Ueno T, Yano H, Kojiro M, Sata M. Involvement of p21 (WAF1/Cip1), p27 (Kip1), and p18 (INK4c) in troglitazone-induced cell-cycle arrest in human hepatoma cell lines. Hepatology 33:1087-1097, 2001 https://doi.org/10.1053/jhep.2001.24024
  10. McIntyre TM, Pontsler AV, Silva AR, St Hilaire A, Xu Y, Hinshaw JC, Zimmerman GA, Hama K, Aoki J, Arai H, Prestwich GD. Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARgamma agonist. Proc Natl Acad Sci U S A 100:131- 136, 2003
  11. Schopfer FJ, Lin Y, Baker PR, Cui T, Garcia-Barrio M, Zhang J, Chen K, Chen YE, Freeman BA. Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor gamma ligand. Proc Natl Acad Sci U S A 102:2340-2345, 2005
  12. Clay CE, Namen AM, Atsumi G, Willingham MC, High KP, Kute TE, Trimboli AJ, Fonteh AN, Dawson PA, Chilton FH. Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells. Carcinogenesis 20:1905- 1911, 1999 https://doi.org/10.1093/carcin/20.10.1905
  13. Shimada T, Kojima K, Yoshiura K, Hiraishi H, Terano A. Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells. Gut 50:658-664, 2002 https://doi.org/10.1136/gut.50.5.658
  14. Kojima K, Shimada T, Mitobe Y, Yoshiura K, Hiraishi H, Terano A. Effect of PPARgamma ligands on the viability of gastric epithelial cells. Aliment Pharmacol Ther 16(Suppl 2):67-73, 2002 https://doi.org/10.1046/j.1365-2036.16.s2.16.x
  15. Ray DM, Bernstein SH, Phipps RP. Human multiple myeloma cells express peroxisome proliferator-activated receptor gamma and undergo apoptosis upon exposure to PPARgamma ligands. Clin Immunol 113:203-213, 2004 https://doi.org/10.1016/j.clim.2004.06.011
  16. Kim JR, Kim JW, Lee HS, Yoon YB, Song IS. Natural history and survival rate of chronic liver diseases in Korea: 20 years prospective analysis. Korean J Med 46:168-180, 1994
  17. Nolte W, Hartmann H, Ramadori G. Glucose metabolism and liver cirrhosis. Exp Clin Endocrinol Diabetes 103:63-74, 1995 https://doi.org/10.1055/s-0029-1211331
  18. Yoshizawa K, Cioca DP, Kawa S, Tanaka E, Kiyosawa K. Peroxisome proliferator-activated receptor $\gamma$ ligand troglitazone induces cell cycle arrest and apoptosis of hepatocellular carcinoma cell lines. Cancer 95:2243-2251, 2002 https://doi.org/10.1002/cncr.10906
  19. Toyoda M, Takagi H, Horiguchi N, Kakizaki S, Sato K, Takayama H, Mori M. A ligand for peroxisome proliferator activated receptor $\gamma$ inhibits cell growth and induces apoptosis in human liver cancer cells. Gut 50:563-567, 2002 https://doi.org/10.1136/gut.50.4.563
  20. Motomura W, Takahashi N, Nagamine M, Sawamukai M, Tanno S, Kohgo Y, Okumura T. Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells. Int J Cancer 108:41-46, 2004 https://doi.org/10.1002/ijc.11561
  21. Weng JR, Chen CY, Pinzone JJ, Ringel MD, Chen CS. Beyond peroxisome proliferator-activated receptor $\gamma$ signaling: the multi-facets of the antitumor effect of thiazolidinediones. Endocr Relat Cancer 13:401-413, 2006 https://doi.org/10.1677/erc.1.01182
  22. Bae MA, Rhee H, Song BJ. Troglitazone but not rosiglitazone induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines. Toxicol Lett 139:67-75, 2003 https://doi.org/10.1016/S0378-4274(02)00468-X
  23. Fulzele SV, Chatterjee A, Shaik MS, Jackson T, Ichite N, Singh M. 15-Deoxy-Delta12,14-prostaglandin J2 enhances docetaxel anti-tumor activity against A549 and H460 non-small-cell lung cancer cell lines and xenograft tumors. Anticancer Drugs 18:65-78, 2007 https://doi.org/10.1097/CAD.0b013e3280101006
  24. Shiau CW, Yang CC, Kulp SK, Chen KF, Chen CS, Huang JW, Chen CS. Thiazolidenediones mediate apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/ Bcl-2 functions independently of PPARgamma. Cancer Res 65:1561-1569, 2005 https://doi.org/10.1158/0008-5472.CAN-04-1677
  25. Rumi MA, Sato H, Ishihara S, Kawashima K, Hamamoto S, Kazumori H, Okuyama T, Fukuda R, Nagasue N, Kinoshita Y. Peroxisome proliferator-activated receptor $\gamma$ ligand- induced growth inhibition of human hepatocellular carcinoma. Br J Cancer 84:1640-1647, 2001 https://doi.org/10.1054/bjoc.2001.1821
  26. Nakata S, Yoshida T, Shiraishi T, Horinaka M, Kouhara J, Wakada M, Sakai T. 15-Deoxy-Delta12,14-prostaglandin J(2) induces death receptor 5 expression through mRNA stabilization independently of PPAR$\gamma$ and potentiates TRAIL-induced apoptosis. Mol Cancer Ther 5:1827-1835, 2006 https://doi.org/10.1158/1535-7163.MCT-06-0023
  27. Ray DM, Akbiyik F, Phipps RP. The peroxisome proliferator- activated receptor gamma (PPARgamma) ligands 15-deoxy- Delta12,14 -prostaglandin J2 and ciglitazone induce human B lymphocyte and B cell lymphoma apoptosis by PPARgamma- independent mechanisms. J Immunol 177: 5068-5076, 2006 https://doi.org/10.4049/jimmunol.177.8.5068