Antineoplastic Effects of Various Peroxisome Proliferator Activated Receptor(PPAR)-  Agonists against Hepatitis B virus (HBV)-associated Hepatocellular.

Presentation on theme: "Antineoplastic Effects of Various Peroxisome Proliferator Activated Receptor(PPAR)-  Agonists against Hepatitis B virus (HBV)-associated Hepatocellular."— Presentation transcript:

1 Antineoplastic Effects of Various Peroxisome Proliferator Activated Receptor(PPAR)-  Agonists against Hepatitis B virus (HBV)-associated Hepatocellular carcinoma (HCC) cells Jaejun Shim, Byung-Ho Kim, Young Whangbo, Sung Hoon Jung, Chang Kyun Lee, Jae-Young Jang, Seok Ho Dong, Hyo Jong Kim, Young Woon Chang and Rin Chang Department of Internal Medicine, Kyung Hee University School of Medicine

2 Background PPAR Antineoplastic effects of the PPAR  ligands
Glucose and insulin homeostasis lipid metabolism, adipocyte differentiation Antineoplastic effects of the PPAR  ligands Prostate, colon, pancreas, breast, lung cancer Hepatocellular carcinoma Through G1 arrest and induction of apoptosis PPAR gamma는 steroid 호르몬과 같은 핵수용체의 일종으로 여러 ligand와 결합하여 세포 핵내에서 특정 유전자의 발현을 조절하여 혈당과 인슐린의 조절, 지질 대사, 지방 세포의 분화에 영향을 미치는 것으로 알려져 있습니다. 최근 PPAR gamma ligand의 일종인 글리타존계 약물들이 항암 효과가 있는 것으로 보고되고 있는데 대표적으로 전립선, 대장, 췌장, 유방, 폐암 등이 있으며 HepG2를 포함하여 여러 간암 세포에서도 유사한 보고들이 나오고 있으며 그 기전은 주로 G1 arrest와 apoptosis의 유도에 의한 것으로 생각되고 있으나 자세한 것은 아직 밝혀져 있지 않은 상태입니다.

3 Background Hepatocelluar carcinoma cell lines Thiazolidinediones
HepG2, HuH-7, HLF, KYN-1, KYN-2, HAK(-1A,-1B,-5 ) : HBV(-) Thiazolidinediones Troglitazone (TGZ) : used in the most studies Pioglitazone (PGZ) : more potent affinity to PPAR  Rosiglitazone (RGZ) 지금까지 보고된 PPAR gamma ligand가 효과를 보였던 간암세포주들을 살펴보면 HepG2와 HuH-7가 대표적인데 대부분 HBV와 관련이 없는 간암 세포들이어서 우리나라에서 흔하게 발생하는 HBV와 연관된 간암을 충분히 반영해 주지 못한다는 단점이 있습니다. 또한 연구에 사용된 PPAR ligand도 대부분 idiosyncratic hepatitis와 같은 간독성 때문에 사용이 중지된 TGZ을 이용하였다는 한계가 있었습니다. 최근에 TGZ의 부작용을 줄이고 PPAR gamma에 더 효과적으로 작용하는 PGZ 과 RGZ이 환자들에게 사용되고 있습니다. 따라서 이들 약제가 PPAR gamma를 통해 간암 세포에 대해 뚜렷한 항암 효과가 있다는 것이 밝혀 진다면 간암 환자나 간암 발생의 고위험환자에게 보다 쉽게 예방적인 항암 약제로 사용될 가능성도 생각해 볼 수 있겠습니다.

4 Aim To evaluate the antineoplastic effect of synthetic PPAR ligands with less liver toxicity on various HCC cell lines that were established from Korean HCC patients with chronic HBV infection 이에 저자들은 만성 HBV 감염과 관련된 간암 세포주들을 대상으로 최근 새롭게 사용되고 있는 PPAR gamma ligand들의 항암 효과를 알아보고자 이 연구를 계획하였습니다.

5 Materials and Methods HBV-related HCC cell lines PPAR ligands
SNU-739, SNU-761, SNU-878, SNU-886 PPAR ligands Synthetic ligands TGZ, PGZ, RGZ Intrinsic ligand 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) 실험에 사용된 세포 SNU-739부터 886은 모두 만성 B형 간염과 간경변이 동반된 50대 우리나라 남성 및 여성에서 얻은 것으로 모두 HBV DNA의 integration이 있으며 HBs Ag을 분비하는 세포들입니다. 사용된 PPAR ligand는 다음과 같으며 최근 내인성 PPAR gamma ligand 중 가장 강력한 것으로 알려진 cyclopentenone PG인 15deoxy-PGJ2도 사용하였습니다.

6 Materials and Methods Cell proliferation assay Cell cycle analysis
Determined by a colorimetric assay (MTT assay) Cell cycle analysis Flow cytometric analysis of DNA content with a FACScalibar Apoptosis and caspase inhibitor assay The cell death detection ELISAplus kit Measurement of caspases activity Caspase colorimetric protease assay sampler kit Cell proliferation assay 10,25,50,100uM, 24,48,72h까지 증식시킨 후 MTT assay Cell cycle 50uM씩 처리후 24시간후 Apoptosis 0-50uM씩 48시간 배양 후 DNA fragments를 측정, capase inhibitor 48시간후 Caspase 활성도 50uM씩 처리후 12시간 후 배양세포내에서 capase의 활성을 각각의 caspase 특이 substrate(기질)을 처리후 특정 파장에서 색변화를 비교하여 활성도를 비교함

7 Materials and Methods mRNA expression of PPAR , Bcl-2, Bax, β- actin
RT-PCR (reverse transcription-polymerase chain reaction) Gene Sequences Product size (bp) PPARγ F:5’ TCTCTCCGTAATGGAAGACC-3’ 474 R:5’-GCATTATGAGACATCCCCAC-3’ Bcl-2 F:5-CTTTGAGTTCGGTGGGGTCATGTG-3’ 275 R:5’-TGACTTCACTTGTGGCCCAGATAGG-3’ Bax F:5’-GCATCGGGGACGAACTGG-3’ 306 R:5’-GTCCCAAAGTAGGAGAGGA-3’ β-actin F:5’-CTTCTACAATGAGCTGCGTG-3’ 305 R:5’-TCATGAGGTAGTCAGTCAGG-3’ 각세포주마다 PPAR gamma의 expression 여부를 확인해 보고자 PPAR mRNA에 대한 RT-PCR을 시행하였고 Apoptosis의 기전을 알아보고자 내인성 apoptosis의 initiator인 Bcl-2와 Bax의 mRNA expression도 RT-PCR을 이용하여 분석하였습니다.

8 Results PPAR expression in HBV-related HCC cell lines Marker HepG2
SNU-739 SNU-761 SNU-878 SNU-886 474 bp PPAR  305 bp β-actin

9 Effects of PPARγ ligands on cell proliferation of SNU-739, -761
TGZ PGZ RGZ 15d-PGJ2 SNU-739 TGZ PGZ RGZ 15d-PGJ2 SNU-761

10 Effects of PPARγ ligands on cell proliferation of SNU-878, -886
TGZ PGZ RGZ 15d-PGJ2 SNU-878 TGZ PGZ RGZ 15d-PGJ2 SNU-886

11 Effects of PPARγ ligands on cell cycle progression
Control Troglitazone Pioglitazone Rosioglitazone 15d-PGJ2 G0/G1 48.3% G0/G1 54.3% G0/G1 35% G0/G1 53.8% G0/G1 13.8% SubG1 30.7% SubG1 28.8% SubG1 48.6% SubG1 26.5% SubG1 71.6% SNU-739 G0/G1 34.6% G0/G1 36.5% G0/G1 34.3% G0/G1 44.8% G0/G1 18.9% SubG1 28.6% SubG1 18.7% SubG1 30.4% SubG1 15.9% SubG1 41.4% Control Troglitazone Pioglitazone Rosioglitazone 15d-PGJ2 SNU-761

12 Effects of PPARγ ligands on cell cycle progression
G0/G1 34.9% G0/G1 36.6% G0/G1 28.1% G0/G1 37.1% G0/G1 20.1% SubG1 35.5% SubG1 29.5% SubG1 54.5% SubG1 29.9% SubG1 55% Control Troglitazone Pioglitazone Rosioglitazone 15d-PGJ2 SNU-878 G0/G1 51.9% G0/G1 53.1% G0/G1 41.4% G0/G1 57.4% G0/G1 16.1% SubG1 24.9% SubG1 16.4% SubG1 35.4% SubG1 20.3% SubG1 66.2% Control Troglitazone Pioglitazone Rosioglitazone 15d-PGJ2 SNU-886

13 Effects of PPARγ ligands on apoptosis
SNU SNU SNU SNU-886

14 Caspase activation induced by pioglitazone
Control PGZ Caspase-8 Caspase-9 SNU-739 Caspase-3 Control PGZ Caspase-8 Caspase-9 SNU-761 Caspase-3 Control PGZ Caspase-8 Caspase-9 SNU-878 Caspase-3 Control PGZ Caspase-8 Caspase-9 SNU-886

15 Effects of caspase inhibitors on the PPARγ ligands induced apoptosis of SNU-886
Control +Z-VAD (µM) +Z-DEVD (µM) 15d-PGJ2 Control +Z-VAD (µM) +Z-DEVD (µM) PGZ General caspase inhibitor Caspase-3 inhibitor General caspase inhibitor Caspase-3 inhibitor

16 Effects of PPARγ-ligands on Bcl-2 and Bax mRNA expression
h 24h 48h h 24h 48h Bcl-2 275 bp Bax 306 bp β-actin 305 bp Pioglitazone 15d-PGJ2 SNU-878

17 Summary All HCC cells (SNU-739,-761,-878,-886)
were expressing PPAR mRNA Decreased cell proliferation by various PPAR ligands dose- and time-dependently The effect was prominent in the PGZ and 15d-PGJ2 → about 50% cell decrease in 50μM/24h G1 arrest and apoptosis were induced by PGZ and 15d-PGJ2, but not by TGZ and RGZ Decreased Bcl-2 expression and increased caspase-3 and-9 activities were observed → intrinsic pathway of apoptosis by mitochondria

18 Conclusion In the various PPAR  ligands, PGZ showed more prominent antineoplastic effect on HCC cell lines related to chronic HBV infection PGZ can be a candidate for chemopreventive or therapeutic medicine to HCC patients with chronic HBV infection, but further investigations are needed