Activation of caspase-8 during N-(4-hydroxyphenyl)retinamide-induced apoptosis in Fas-defective hepatoma cells.

We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD-FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N-acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation.

Establishment and characterization of chromosomal aberrations in human cholangiocarcinoma cell lines by cross-species color banding.

Cholangiocarcinoma (CC), a malignant neoplasm of the biliary epithelium, is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. Furthermore, little is known about the genetics and biology of CC. Only a few reports concerning cytogenetic studies of CC have been published, and few cell lines have been established. We recently established four CC cell lines, designated as SCK, JCK, Cho-CK, and Choi-CK, and report the first application of cross-species color banding (RxFISH) and multiple chromosome painting for the characterization of the chromosomal rearrangements of these CC cell lines. Each cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. Chromosomes 3, 6, 7, 8, 12, 14, 17, and 18 were commonly involved in structural abnormalities. Homogeneously staining regions were determined in SCK and JCK, and double minute chromosomes were found in Cho-CK. The chromosomal aberrations of the four CC cell lines were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The nonrandom rearrangements suggest candidate regions for isolation of genes related to CC.

Down-regulation of insulin-like growth factor binding proteins and growth modulation in hepatoma cells by retinoic acid.

We observed that all-trans-retinoic acid (RA) down-regulated insulin-like growth factor binding proteins (IGFBPs) in cultured human hepatoma cells (Hep 3B, PLC/PRF/5, and Hep G2); therefore, we characterized the role of this down-regulation in cell growth. Treatment with 10 micromol/L RA revealed a rapid decrease in IGFBP-3 within 2 days, and continued treatment with RA for 6 days resulted in a time-dependent stimulation of Hep 3B cell growth. However, RA treatment decreased IGFBP-1 in PLC/PRF/5 cells and in Hep G2 cells, and the growth-stimulatory activity of RA was transient and less prominent, and was finally obliterated in both cell lines. The addition of 5 ng/mL or 50 ng/mL insulin-like growth factors (IGFs) did not change the growth effects elicited by RA. The addition of IGFBP-3 (1,000 ng/mL) inhibited the growth of Hep 3B cells and counteracted the growth-stimulatory activity of RA, but not completely, suggesting that RA has direct growth-stimulatory activity and that this is enhanced by autocrine down-regulation of IGFBP-3. IGFBP-3 also inhibited the growth of PLC/PRF/5 cells and of Hep G2 cells. Treatment with phosphorylated IGFBP-1 (1,000 ng/mL) alone or with RA did not affect the growth of PLC/PRF/5 cells or Hep G2 cells. However, addition of dephosphorylated IGFBP-1, derived from in vivo dephosphorylation of the phosphorylated form, stimulated the growth of both cell lines, independent of interaction with IGF-I. From these observations, we propose that RA down-regulates IGFBPs, which in turn causes autocrine modulation of cell growth independent of IGF in hepatoma cells in vitro or in vivo. In addition, RA regulates IGFBPs at the posttranscriptional (Hep 3B cells and Hep G2 cells) or transcriptional level (PLC/PRF/5 cells) in a cell-specific manner.

Lincomycin abrogates dexamethasone-enhanced melanogenesis in B16 melanoma cells.

The effects of lincosamide, and interference between the effects of glucocorticoid and lincosamide, on melanogenesis were determined in B16 melanoma cells. Cells were treated for 4 days with lincomycin (LM) and/or dexamethasone (DX) at equimolar concentrations ranging from 10(-9) M to 10(-5) M, or at various concentrations of DX with 10(-6) M LM. Effects on proliferation, tyrosinase activity, melanin biosynthesis, and levels of mRNA for tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) were examined. Treatment with LM or LM + DX stimulated proliferation of melanoma cells with minimal cytotoxicity, while DX did not influence cell proliferation either alone or in combination with LM. Treatment with LM alone increased tyrosinase activity slightly and reduced melanin content in a dose-dependent manner. However, LM counteracted the pronounced increase in tyrosinase elicited by DX and also abrogated the dose-dependent increase in melanin content elicited by DX. Treatment with LM alone did not affect mRNA levels for tyrosinase, TRP1, or TRP2. Furthermore, LM abrogated the DX-induced up-regulation of mRNAs for tyrosinase and the down-regulation of TRP1 mRNA. These results suggest that LM inhibits melanogenesis post-transcriptionally and abrogates glucocorticoid-induced melanogenesis at the transcriptional level in B16 melanoma cells.

Apoptosis induced by retinoic acid in Hep 3B cells in vitro.

Human hepatoma Hep 3B cells underwent apoptosis in response to 100 microM all-trans retinoic acid (RA) in full serum (10% fetal calf serum) condition in vitro. Cell death began approximately 24 h following treatment, with more than 80% of the cells dead after 60 h. The dead cells, mainly detached cells, exhibited condensed chromatin and DNA fragmentation, which are indicative of endonuclease activation and are the hallmarks of apoptosis in epithelial cells. Concurrent exposure to 1 microM cycloheximide (CX) prevented approximately 50% of cell death and DNA fragmentation induced by RA. Thus, other toxic injury to the cells as well as apoptosis might be involved in cell death. Sixty hours exposure of RA decreased the percentage of cells in G1 phase (16.3 +/- 0.4% versus 52.4 +/- 2.1%; P < or = 0.01) and in G2/M phase (13.4 +/- 1.2% versus 21.2 +/- 0.7%; P < or = 0.01), but did not change percent of cells in S phase (20.8 +/- 0.2% versus 20.7 +/- 0.5%) of the cell cycle compared with control. RA may have caused accumulation of Hep 3B cells before G1 phase, and that G0/G1 transition is a main check point in the active process of apoptosis. Electron micrographs of the cells treated with RA revealed typical morphologic changes of apoptosis, besides toxic injury to the cells. These data strongly indicate that RA is able to induce apoptosis and the induction of apoptosis may contribute to the antitumor activity of RA against hepatoma cells.