1246 Induction of apoptosis in human hepatocellular carcinoma by photodynamic therapy is associated with the activation of caspase 2 and deamidation of BCL-XL

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

progression of HCC and the interaction between ephrin-Bs and RhoA. Methods: Ephrin-Bs transcripts in 26 HCC and their corresponding non-tumor liver tissues were analyzed by the quantitative reverse transcription-polymerase chain reaction (RT-PCR). We established ephirn-B1 overexpressing cells in a h u m a n HCC cell line, PLC/PRF/5 cell, and their in vivo growth monitored after subcutaneous injection into nude mice. Neovascularization in the inoculated tumors was evaluated by the immunohistochemical staining of CD31. A cell proliferation and migration assay was performed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and modified Boyden chamber. To analyze the effect of ephrin-B1 on the proliferation and migration of endothelial cells, h u m a n umbilical vein endothelial cells (HUVECs) was examined in response to soluble ephrin-B1-Fc. The activity of the RhoA protein in the ephrin-B1 overexpressing cells was analyzed by immunoblot analysis. Results: The ephrin-B1 transcript was detected in all HCC tissues, and the level was significantly higher than in non-tumor tissues (p<0.05). The ephrin-B2 transcript was detected in all tissues investigated, but the level was the same as that from non-tumor tissues. The ephrin-B3 transcript was detected in 7 of 26 non-tumor tissues and only 2 of 26 HCC tissues. No significant difference in cell growth was found among the ephrin-B1 overexpressing cells, the parental cells and mock cells in vitro. Moreover, soluble form of EphB1, EphB2 and EphB3, which are known to bind ephrin-B1 ligand, had no influence on the proliferation of ephrin-B1 overexpressing cells in vitro. However, the ephrin-B1 overexpressing cells showed a rapid growth in vivo compared with the parental and mock cells (p<0.05). The tumor vessel number significantly increased in the ephrin-B1 overexpressing tumors (p<0.0001). In vitro studies also revealed that ephrin-B1-Fc specifically induced migration and proliferation of HUVECs that expressed EphB1, EphB2 and EphB3 (p<0.05). Additionaly, peritoneal and/or abdominal cavity invasion was found in 3 of 16 ephrin-B1 overexpressing tumors that were tightly adhering to the peritoneum and bone. Histological examination also revealed that cells had invaded into the peritoneal tissue. On the other h a n g no such invasion was found in the parental and mock cells, which were easily separated from stromal tissue. The m e m b r a n e b o u n d RhoA (active form) was elevated in the ephrin-B1 overexpressing cells, compared with the parental and mock cells. Conclusions: Ephrin-B1 may be involved in the progression of HCC in vivo by promoting neovascularization and invasion. Disclosures: Koji Fukui - No relationships to disclose Kazuho Imanaka - No relationships to disclose Nobuyuki Ito - No relationships to disclose Shinichi Kiso - No relationships to disclose Yuji Matsuzawa - No relationships to disclose Ayuko Saeki - No relationships to disclose Shigeru Sakuda - No relationships to disclose Yoshiyuki Sawai - No relationships to disclose Shinji Tamura - No relationships to disclose

1246

INDUCTION OF APOPTOSIS IN HUMAN HEPATOCELLULAR CARCINOMA BY PHOTODYNAMIC THERAPY IS ASSOCIATED WITH THE ACTIVATION OF CASPASE 2 AND DEAMIDATION OF BCL-XL. Tetsuya

Yoshikawa, Koichi Nariai, Hideko Uga, Yoko Yumoto, Masataka Date, Hiroshi Takahashi, Instffute of Clinical Medicine and Research, Jikei University School of Medicine, Kashiwa, Japan Bcl-xL is an anti-apoptotic m e m b e r of the Bcl-2 family, which inhibits apoptosis initiated by various cellular stresses, and has a

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pivotal role in the survival of tumor cells. We have demonstrated that the rate of Bcl-xL deamidation, which imports a complete "loss of function" of this anti-apoptotic molecule, is significantly suppressed in hepatocellular carcinomas (HCCs) than in normal or adjacent non-tumor liver tissues. Photodynamic therapy (PDT) is an effective local cancer treatment using tissue-penetrating visible laser light after the administration of a tumor-localizing photosensitizer. We examined the effect of PDT on h u m a n HCC cells using mono-L-aspartyl chlorin e6 (NPe6), which is a second generation photosensitizer, and found that PDT induced activation of endogenous caspases and apoptosis in HCC cells as well as inactivation of antiapoptotic molecules including Bcl-xL and survivin. METHODS: Expression of Bcl-xL and survivin was examined by Western blot analysis. H u m a n HCC cell line with p53 mutation, Huh-7, was incubated with NPe6 for 2 hours in culture dish, and irradiated with diode laser (665 nm, 10 J/cm2). Cell viabilities were examined by MTF assay. Apoptosis was assessed by the detection of histon-associated DNA fragments using ELISA (Cell Death Detection ELISAPLUs, Roche Diagnostics GmbH). The activities of caspase-2, -3, -8, or -9-1ike proteases were assessed by fluorometric assay using VDVAD-AFC, DEVD-AFC, IETD-AFC or LEHDAFC as substrates, respectively. RESULTS: Laser irradiation killed more than 95% of Huh-7 cells in the presence of NPe6. Cytotoxicity was d e p e n d e n t on the concentration of NPe6 and dose of laser irradiation. On the other h a n g laser irradiation alone or NPe6 alone had no cytotoxicity to Huh-7 cells. Histon-associated DNA fragments were produced in Huh-7 cells by PDT treatment. Caspase-3, and -9, but not caspase-8 were activated. Interestingly, caspase-2 was significantly activated. Inhibition of caspase-2 by specific pentapeptide inhibitor completely inhibited the activation of caspase-3 in Huh-7 cells, and the level of inhibition was 100-fold stronger than that of caspase-9 inhibitor. Of interest, deamidation of Bcl-xL was accelerated in Huh-7 cells after PDT treatment, and the levels of survivin in PDT-treated HCCs significantly decreased as well. Furthermore, PDT using NPe6 significantly inhibited the growth of Huh-7 tumor xenograft in nude mice without severe side effects. CONCLUSION: PDT employing NPe6 induced cell death of HCC cells by apoptosis. We found that cell death was mediated by the activation of caspase-2, -3 and -9 without the activation of caspase-8. Interestingly, apoptosis was associated with deamidation of Bcl-xL as well as the down-regulation of survivin. We also found that the activation of caspase-2, which is thought to be required for permeabilization of mitochondria played a critical role in the induction of apoptosis by PDT. Thus, present study supports an idea that PDT using NPe6 may serve as a potent treatment of h u m a n HCCs and the activation of caspase-2 and the inactivation of anti-apoptotic molecules are important for the induction of apoptosis. Disclosures: Masataka Date - No relationships to disclose Koichi Nariai - No relationships to disclose Hiroshi Takahashi - No relationships to disclose Hideko Uga - No relationships to disclose Tetsuya Yoshikawa - No relationships to disclose Yoko Yumoto - No relationships to disclose