Histone Deacetylase Inhibitor FR901228 Research Articles

Apoptosis Induced by the Histone Deacetylase Inhibitor FR901228 in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

Apoptosis Induced by the Histone Deacetylase Inhibitor FR901228 in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction

Reveal mechanisms for the novel proapoptotic ability of oncogenic H-Ras to increase cell susceptibility to a histone deacetylase inhibitor (HDACI) FR901228 for inducing caspase activation and selective apoptosis. Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal differential induction of intracellular reactive oxygen species (ROS), caspase activation, and apoptosis by HDACI FR901228. ROS levels and caspase-8, -9, and -3/7 activities were measured by flow cytometry and luminescence assays, respectively. Specific inhibitors were used to suppress caspases and ROS. Western blot analysis determined modulators of caspase pathways. ROS, caspase activity, and cell death was differentially increased by FR901228 in oncogenic H-Ras-expressing J82 versus parental cells. Blocking ROS resulted in reduced FR901228-induced cell death and caspase activation. Suppression of caspase-8 resulted in reduced FR901228-activated caspase-9 and -3/7. Suppression of caspase-9 resulted in reduced FR901228-activated caspase-3/7. Although FR901228 induced an ROS-dependent increase of FasL, FasL failed to induce caspase activation and cell death. Increased ROS played an important role in the activation of the extrinsic and intrinsic caspase pathways to cooperatively induce executioner caspase-3/7 through a novel FasL-independent pathway in FR901228-induced selective apoptosis of oncogenic H-Ras-expressing J82 versus parental cells.

Enhanced transgene expression in urothelial cancer gene therapy with histone deacetylase inhibitor: Okegawa T, Nutahara K, Pong RC, Higashihara E, Hsieh JT. Department of Urology, University of Kyorin, Tokyo, Japan

Efficient adenoviral infection requires the presence of the coxsackievirus and adenovirus receptor (CAR). We determined whether the histone deacetylase inhibitor FR901228 (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) increases the efficiency of adenoviral gene therapy in bladder cancer in vivo and in vitro. Cytotoxicity studies were performed to determine a minimally cytotoxic FR901228 concentration for bladder cancer cells. The level of CAR expression was determined by fluorescence activated cell scanning and/or reverse transcriptase-polymerase chain reaction analysis in FR901228 treated bladder cell lines. The in vivo effect on adenoviral gene expression was investigated in athymic mice. The concentration of FR901228 showing no or minimal cytotoxicity that was selected for these studies was 0.5 ng/ml for bladder cancer cells. Treatment of cancer cells with 0.5 ng/ml histone deacetylase inhibitor increased CAR RNA levels and acetylated histone H3. This increase was associated with a 5 to 10-fold increase in adenoviral infection, as evidenced by increased transgene expression from a beta-galactosidase containing adenoviral vector. Intravenous administration of FR901228 enhanced CAR expression in athymic mice. The combination of p53 adenovirus and histone deacetylase inhibitor resulted in significant tumor inhibition in vitro and in vivo. Nontoxic doses of the histone deacetylase inhibitor FR901228 increased CAR RNA levels and resulted in the marked enhancement of transgene expression after adenoviral infections. FR901228 pretreatment may increase the sensitivity of tumor cells to adenoviral gene therapy vectors.

A Histone Deacetylase Inhibitor Enhances Recombinant Adeno-associated Virus-Mediated Gene Expression in Tumor Cells

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.

Histone deacetylase inhibitor FR901228 enhances the antitumor effect of telomerase-specific replication-selective adenoviral agent OBP-301 in human lung cancer cells

Replication-competent oncolytic viruses are being developed for human cancer therapy. We previously reported that an attenuated adenovirus OBP-301 (Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, could replicate in and causes selective lysis of human cancer cells. Infection efficiency in target cancer cells is the most important factor that predicts the antitumor effects of OBP-301. The objectives of this study are to examine the effects of the histone deacetylase inhibitor FR901228 on the level of coxsackie and adenovirus receptor (CAR) expression and OBP-301-mediated oncolysis in human non-small cell lung cancer cell lines. Flow cytometric analysis revealed up-regulated CAR expression in A549 and H460 cells following treatment with 1 ng/ml of FR901228, which was associated with increased infection efficiency as confirmed by replication-deficient beta-galactosidase-expressing adenovirus vector. In contrast, neither CAR expression nor infection efficiency was affected by FR901228 in H1299 cells. To visualize and quantify viral replication in the presence of FR901228, we used OBP-401 (Telomelysin-GFP) that expresses the green fluorescent protein (GFP) reporter gene under the control of the cytomegalovirus promoter in the E3 region. Fluorescence microscopy and flow cytometry showed that FR901228 increased GFP expression in A549 and H460 cells following OBP-401 infection in a dose-dependent manner, but this effect did not occur in H1299 cells. In addition, OBP-301 and FR901228 demonstrated a synergistic antitumor effect in A549 cells in vitro, as confirmed by isobologram analysis. Our data indicate that FR901228 preferentially increases adenovirus infectivity via up-regulation of CAR expression, leading to a profound oncolytic effect, which may have a significant impact on the outcome of adenovirus-based oncolytic virotherapy.

ENHANCED TRANSGENE EXPRESSION IN UROTHELIAL CANCER GENE THERAPY WITH HISTONE DEACETYLASE INHIBITOR

Efficient adenoviral infection requires the presence of the coxsackievirus and adenovirus receptor (CAR). We determined whether the histone deacetylase inhibitor FR901228 (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) increases the efficiency of adenoviral gene therapy in bladder cancer in vivo and in vitro. Cytotoxicity studies were performed to determine a minimally cytotoxic FR901228 concentration for bladder cancer cells. The level of CAR expression was determined by fluorescence activated cell scanning and/or reverse transcriptase-polymerase chain reaction analysis in FR901228 treated bladder cell lines. The in vivo effect on adenoviral gene expression was investigated in athymic mice. The concentration of FR901228 showing no or minimal cytotoxicity that was selected for these studies was 0.5 ng/ml for bladder cancer cells. Treatment of cancer cells with 0.5 ng/ml histone deacetylase inhibitor increased CAR RNA levels and acetylated histone H3. This increase was associated with a 5 to 10-fold increase in adenoviral infection, as evidenced by increased transgene expression from a beta-galactosidase containing adenoviral vector. Intravenous administration of FR901228 enhanced CAR expression in athymic mice. The combination of p53 adenovirus and histone deacetylase inhibitor resulted in significant tumor inhibition in vitro and in vivo. Nontoxic doses of the histone deacetylase inhibitor FR901228 increased CAR RNA levels and resulted in the marked enhancement of transgene expression after adenoviral infections. FR901228 pretreatment may increase the sensitivity of tumor cells to adenoviral gene therapy vectors.

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer

We speculated whether or not the expression level of telomerase reverse transcriptase (hTERT) would be modulated by agents targeting epigenetics in oral cancer cell lines. Although hTERT is known to be targeted by epigenetic changes, it remains unclear how chemoagents targeting epigenetics work on hTERT transcription. In the present study, the epigenetic effects of the histone deacetylase (HDAC) inhibitor FR901228 on hTERT transcription in oral cancer cell lines were analyzed by RT-PCR. The mRNA expression of hTERT was upregulated after exposure to FR901228 in hTERT-negative Hep2 cells, and even SAS and KB cells expressed high levels of hTERT. Moreover, cotreatment of protein synthesis inhibitor cycloheximide (CHX) resulted in the induction of hTERT transcription by FR901228. This suggests that the induction of hTERT by FR901228 requires de novo protein synthesis to some extent and is more likely a direct than an indirect effect on epigenetic changes such as histone acetylation/deacetylation. We further examined the effect of FR901228 on c-myc protein, which is one of the main hTERT transcription activators. FR901228 repressed c-myc protein only in the absence of CHX, and depended on the enhancement of de novo protein synthesis. Our results indicate that c-myc protein is repressed indirectly by FR901228 but may not contribute to FR901228-induced hTERT transcription. The present study showed that the HDAC inhibitor FR901228 induced the hTERT gene by a complex mechanism that involved transcription factors other than c-myc, in addition to inhibition of histone deacetylation.

The histone deacetylase inhibitor FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway in small cell lung cancer cells

Histone deacetylase inhibitors modulate the transcription of target genes and represent a new class of anticancer agents. The histone deacetylase inhibitor FR901228 has been reported to show antiproliferative and apoptotic effects in various malignancies including small cell lung cancer (SCLC) in vitro; however, the underlying mechanism is not fully understood. BCL-2 and BCL-XL are antiapoptotic proteins, of which overexpression has been reported to confer resistance to anticancer agents. High levels of BCL-2 and BCL-XL are frequently expressed in SCLC tumors. The present study was designed to clarify the apoptotic pathway of FR901228 in SCLC cells in vitro. FR901228 induced apoptosis in three SCLC cell lines after 24 hours of treatment. FR901228 activated caspase-9 and caspase-3 but not caspase-8, and the caspase-3 inhibitor Z-DEVD-fmk blocked the cytotoxicity of FR901228. FR901228 down-regulated the expression of bcl-2 and bcl-xL mRNA through de novo protein synthesis and suppressed the expression of BCL-2 and BCL-XL proteins. In addition, the combination of bcl-2 antisense oligonucleotides with FR901228 enhanced FR901228-induced caspase-3 activity and cytotoxicity. These findings suggest that FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway rather than the death receptor pathway. Considering the possible contributions of BCL-2 and BCL-XL to multidrug resistance, FR901228 is a promising agent in the treatment of refractory as well as primary SCLC tumors.

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.

Effects of demethylating agent 5-aza-2 ′-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines

Maspin, which belongs to the serine protease inhibitor (serpin) superfamily, has been proposed as a potent tumor suppressor that inhibits cell motility, invasion, angiogenesis, and metastasis. In the present study, we examined the effects of 5-aza-2 ′-deoxycytidine (5-aza-dC), a demethylating agent, and FR901228, a histone deacetylase (HDAC) inhibitor, on maspin expression in oral cancer cell lines. The expression levels of maspin mRNA were divided into two groups, which was the maspin low-expressed and high-expressed cell lines in the 12 oral cancer cell lines. The maspin promoter contained only a few methylated CpG sites in the maspin low-expressed cell lines. Moreover, the methylation status was not altered after 5-aza-dC treatment. However, the transcription of the maspin gene was clearly increased following 5-aza-dC treatment in a number of oral cancer cell lines. These results imply that an action of 5-aza-dC is separate from induction of promoter demethylation. Treatment with FR901228 resulted in a time-dependent stimulation of the re-expression of maspin mRNA as early as 4 h after treatment in the maspin downregulated cells. The re-expression of the maspin gene may contribute to the recuperation of biological functions linked to FR901228 such as an inhibitory effect on tumor angiogenesis and cell invasion. These results indicate that maspin and its target genes may be excellent leads for future studies on the potential benefits of FR901228, a HDAC inhibitor, in cancer therapy.

Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines.

FR901228 is a novel histone deacetylase (HDAC) inhibitor, and its antiproliferative effects on non-small cell lung cancer cells have been shown in vitro. However, there have been no reports concerning the effects on small-cell lung cancer (SCLC). We have recently demonstrated that the HDAC inhibitors trichostatin A and sodium butyrate inhibit expression of the catalytic subunit of telomerase (hTERT) mRNA and telomerase activity in prostate cancer cells. The present study was designed to evaluate the effects of FR901228 on proliferation and telomerase activity in SCLC cells in vitro. FR901228 at 5 to 10 nM increased the fraction of cells in the G(2)/M and sub-G(1) phases of the cell-cycle, and inhibited the growth of H69, H526 and H82 cell lines. The expression of hTERT mRNA was inhibited 6 hr after treatment, prior to obvious inhibition of cell growth or cell-cycle distribution shifts. The inhibition of hTERT mRNA expression and telomerase activity was not a consequence of cell-growth arrest or apoptosis. Cycloheximide blocked the suppression of hTERT mRNA induced by FR901228, and the inhibition of hTERT mRNA by FR901228 required newly synthesized proteins. FR901228 also effectively inhibited growth of etoposide-resistant UMCC-1/VP-16, irinotecan-resistant PC-6/SN2-5H and cisplatin-resistant H526/CDDP cells having decreased expression of hTERT mRNA and telomerase activity, as well as their parental cells. This implies that SCLC resistant to these key drugs are not cross-resistant to FR901228. The present study suggests that FR901228 may be a promising drug for chemotherapy of cancers including SCLC, even for refractory or relapsing tumors after conventional chemotherapy.

488. Adenovirus Transgene Expression in Human Umbilical Vein Endothelila Cells (HUVEC) Is Enhanced by Treatment with the Histone Deacetylase Inhibitor FR901228

488. Adenovirus Transgene Expression in Human Umbilical Vein Endothelila Cells (HUVEC) Is Enhanced by Treatment with the Histone Deacetylase Inhibitor FR901228

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228.

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21WAF1/CIP1in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity. © 2000 Cancer Research Campaign