Inhibition of the membrane translocation and activation of protein kinase C, and potentiation of doxorubicin-induced apoptosis of hepatocellular carcinoma cells by tamoxifen.

Hepatocellular carcinoma (HCC) is characterized by high drug resistance to currently available chemotherapeutic agents. In a prospective clinical study, we have demonstrated that high-dose tamoxifen significantly enhanced the therapeutic efficacy of doxorubicin in patients with far-advanced HCC. In a search for a possible mechanism, we found that tamoxifen at a clinically achievable concentration (2.5 microM) significantly enhanced doxorubicin-induced cytotoxicity and apoptosis of Hep-3B cells, a multidrug resistance (MDR)-1 expressing HCC cell line. This synergistic cytotoxic effect of tamoxifen, at this concentration, however, was not mediated by MDR inhibition. Instead, as evidenced by both western blot and immunofluorescence studies, tamoxifen inhibited the cytoplasmic-membrane translocation of protein kinase C (PKC)-alpha. 12-O-Tetradecanoylphorbol-13-acetate (TPA) restored the membrane translocation of PKC-alpha and abrogated the synergistic cytotoxicity of tamoxifen. We also showed that tamoxifen, at this concentration, did not directly affect the enzyme activity of PKC. Further, membrane translocation of other membrane-bound proteins, such as Ras protein, was similarly inhibited by tamoxifen, but could not be restored by the addition of TPA. Together, these data suggested that tamoxifen may act on the cytoplasmic membrane, and thereby inhibit PKC-alpha translocation to the membrane where it is activated. We hypothesize that high-dose tamoxifen may be an effective modulator of doxorubicin in the treatment of HCC, and suggest that biochemical modulation of PKC as a measure to improve systemic chemotherapy for HCC deserves further investigation.

Upregulation of tumor necrosis factor-alpha gene by Epstein-Barr virus and activation of macrophages in Epstein-Barr virus-infected T cells in the pathogenesis of hemophagocytic syndrome.

A potentially fatal hemophagocytic syndrome has been noted in patients with malignant lymphomas, particularly in EBV-infected T cell lymphoma. Cytokines, such as interferon-gamma (IFN-gamma), TNF-alpha, and IL-1alpha, are elevated in patients' sera. To verify whether infection of T cells by EBV will upregulate specific cytokine genes and subsequently activate macrophages leading to hemophagocytic syndrome, we studied the transcripts of TNF-alpha, IFN-gamma, and IL-1alpha in EBV-infected and EBV-negative lymphoma tissues. By reverse transcription PCR analysis, transcripts of TNF-alpha were detected in 8 (57%) of 14 EBV-infected T cell lymphomas, higher than that detected in EBV-negative T cell lymphoma (one of six, 17%), EBV-positive B cell lymphoma (two of five, 40%) and EBV-negative B cell lymphomas (one of seven, 14%). Transcripts of IFN-gamma were consistently detected in T cell lymphoma and occasionally in B cell lymphoma, but were independent of EBV status. IL-1alpha expression was not detectable in any category. Consistent with these in vivo observations, in vitro EBV infection of T cell lymphoma lines caused upregulation of TNF-alpha gene, and increased secretion of TNF-alpha, but not IFN-gamma or IL-1alpha. Expression of TNF-alpha, IFN-gamma, and IL-1alpha was not changed by EBV infection of B cell lymphoma lines. To identify the specific cytokine(s) responsible for macrophage activation, culture supernatants from EBV-infected T cells were cocultured with a monocytic cell line U937 for 24 h. Enhanced phagocytosis and secretion of TNF-alpha, IFN-gamma, and IL-1alpha by U937 cells were observed, and could be inhibited to a large extent by anti-TNF-alpha (70%), less effectively by anti-IFN-gamma (31%), but almost completely by the combination of anti-TNF-alpha and anti-IFN-gamma (85%). Taken together, the in vivo and in vitro observations suggest that infection of T cells by EBV selectively upregulates the TNF-alpha expression which, in combination with IFN-gamma and probably other cytokines, can activate macrophages. This study not only highlights a probable pathogenesis for virus-associated hemophagocytic syndrome, but also suggests that anti-TNF-alpha will have therapeutic potential in the context of their fatal syndrome.

Genomic analysis of Epstein-Barr virus in nasal and peripheral T-cell lymphoma: a comparison with nasopharyngeal carcinoma in an endemic area.

The Epstein-Barr virus (EBV) is prevalent in nasal and peripheral T-cell lymphoma (NPTL) in Taiwan, where nasopharyngeal carcinoma (NPC) is endemic. In order to understand the pathogenesis of these two malignancies in this endemic area, genomic analysis of EBV in NPTL with comparison to NPC is important. We investigated the EBV subtype (types A and B), BamH-I "f" variant, and the Xho-I site mutant of the latent membrane protein-1 (LMP-1) gene in 19 cases of EBV-associated NPTL and in 30 cases of NPC. EBV DNA from three patients with infectious mononucleosis (IM) was simultaneously studied as representative of normal healthy carriers. Similar to NPC and IM, the EBV in NPTL was found to belong to the type A strain in the majority (18 of 19) of cases by analyzing the 3' divergence of EBNA-2 genes. The extra restriction enzyme site in the BamHI-F region ("f" variant) of EBV DNA was frequently (15 of 30) demonstrated in NPC, but only rarely (1 of 19) was it detected in NPTL and IM (0 of 3). The Xho-I site mutant of the LMP-1 gene previously characterized in Chinese NPC also prevailed in NPTL and IM with an identical nucleotide sequence. No correlation exists between the EBV subtype and its variants. In conclusion, type A EBV is prevalent in Taiwanese NPTL, a finding much distinct from the dominance of type B virus in nonendemic European patients. The EBV genomes in NPTL are closely similar to those in IM or normal healthy carriers, but are distinct from NPC for the infrequency of the "f" variant. The prevalence of the LMP-1 mutant in this endemic region suggests that this EBV strain may confer a growth advantage role in the pathogenesis of these EBV-associated diseases. The rarity of the "f" variant in NPTL and its high frequency in NPC may explain the differential tumorigenesis of different EBV strains.

Use of retinoic acids in the treatment of peripheral T-cell lymphoma: a pilot study.

PURPOSE: We have systemically analyzed, both in vitro and in vivo, the effect of 13-cis-retinoic acids (RA) on non-Hodgkin's lymphoma (NHL). METHODS: The in vitro growth-inhibitory effect of 13-cis-RA was examined in 11 (T cell, five; B cell, six) lymphoma cell lines by a tetrazolium colorimetric assay. A pilot clinical trial with oral 13-cis-RA 1 mg/kg/d was conducted in a selected group of 18 lymphoma patients, of whom 16 had failed to respond to at least one regimen of intensive chemotherapy. The in vitro and in vivo effects of 13-cis-RA were correlated with immunophenotypes, RA-induced changes of morphology, and patterns of DNA fragmentation of the lymphoma cells. RESULTS: Four of five T-lymphoma cell lines and none of six B-lymphoma cell lines were sensitive (concentration of 50% growth inhibition [IC50] < 1.5 microns) to 13-cis-RA (P = .015). In the clinical trial, five (two Ki-1, one angioinvasive type, one diffuse mixed cell, and one diffuse large cell) complete remissions and one (Ki1) partial remission were observed in 12 patients with peripheral T-cell lymphoma (PTCL), while none of six patients with B-cell lymphoma responded to 13-cis-RA. 13-cis-RA-induced cellular differentiation and apoptosis, as evidenced by the more mature morphology, characteristic nuclear condensation, and DNA ladder pattern signifying internucleosomal fragmentation, were demonstrated in the sensitive cell lines, as well as in the remitting lymphoma tissues. CONCLUSION: The 13-cis-RA appears to be active on lymphomas of T-lineage and their therapeutic indication may be extended to include some subtypes of PTCL. The mechanisms of action are related to differentiation and apoptosis of lymphoma cells. There appears to be no cross-resistance between 13-cis-RA and conventional chemotherapy.

Retinoic acid-induced apoptosis and regression of a refractory Epstein-Barr virus-containing T cell lymphoma expressing multidrug-resistance phenotypes.

The virus-associated T cell leukaemias/lymphomas are characterized by a poor prognosis primarily because of the rapid emergence of drug resistance which may lead to failure of subsequent chemotherapy. We report here a case of Epstein-Barr virus-associated T cell lymphoma which relapsed soon after chemotherapy and radiotherapy. The neoplastic cells of the relapsed tumour expressed high levels of multi-drug resistance gene (mdr1)-related P-glycoprotein and glutathione-S-transferase-pi, both of which were absent in the pre-chemotherapy tumour tissues. Empirical treatment with oral 13-cis-retinoic acid (RA) was then given with subsequent complete disappearance of the tumour. The therapeutic effect of RA appears to act through an apoptotic process. In accordance with our previous report of a successful salvage of a refractory Ki-1 large cell lymphoma. RA appears to be a potentially useful drug for some specific type T-cell lymphomas.