Olaparib in combination with irinotecan, cisplatin, and mitomycin C in patients with advanced pancreatic cancer.

BACKGROUND: Olaparib is an oral inhibitor of polyadenosine 5'-diphosphoribose polymerization (PARP) that has previously shown signs of activity in patients with BRCA mutations and pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: In this phase 1 dose-escalation trial in patients with unresectable PDAC, we determined the maximum tolerated dose (MTD) of olaparib (tablet formulation) in combination with irinotecan 70 mg/m2 on days 1 and 8 and cisplatin 25 mg/m2 on days 1 and 8 of a 28-day cycle (olaparib plus IC). We then studied the safety and tolerability of adding mitomycin C 5 mg/m2 on day 1 to this regimen (olaparib plus ICM). RESULTS: 18 patients with unresectable PDAC were enrolled. The MTD of olaparib plus IC was olaparib 100 mg twice-daily on days 1 and 8. The addition of mitomycin C to this dose level was not tolerated. Grade ≥3 drug-related adverse events (AEs) were encountered in 16 patients (89%). The most common grade ≥3 drug-related toxicities included neutropenia (89%), lymphopenia (72%), and anemia (22%). Two patients (11%), both of whom had remained on study for more than 12 cycles, developed drug-related myelodysplastic syndrome (MDS). The objective response rate (ORR) for all evaluable patients was 23%. One patient who carried a deleterious germline BRCA2 mutation had a durable clinical response lasting more than four years, but died from complications of treatment-related MDS. CONCLUSIONS: Olaparib had substantial toxicity when combined with IC or ICM in patients with PDAC, and this treatment combination did not have an acceptable risk/benefit profile for further study. However, durable clinical responses were observed in a subset of patients and further clinical investigation of PARP inhibitors in PDAC is warranted. TRIAL REGISTRATION: This clinical trial was registered on ClinicalTrials.gov as NCT01296763.

Sulforaphane retards the growth of human PC-3 xenografts and inhibits HDAC activity in human subjects.

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 mumol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.

Dietary agents as histone deacetylase inhibitors.

In cancer cells, an imbalance often exists between histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, and various drug companies are actively seeking competitive HDAC inhibitors for chemotherapeutic intervention. Cancer cells appear to be more sensitive than nontransformed cells to HDAC inhibitors, which disrupt the cell cycle and induce apoptosis via derepression of genes such as P21 and BAX. However, in the search for potent HDAC inhibitors with cancer therapeutic potential, a tendency exists to overlook or dismiss weak ligands that could prove effective in cancer prevention. Butyrate, diallyl disulfide (DADS), and sulforaphane (SFN) are three dietary agents that exhibit HDAC inhibitory activity in vitro and/or in vivo, and other such dietary agents probably will be discovered that affect HDAC activity. We make the distinction between 'pharmacologic' agents that potently derepress gene expression, during therapeutic intervention, and dietary HDAC inhibitors that, as weak ligands, might subtly regulate the expression of genes involved in cell growth and apoptosis. An important issue for future study is to determine the extent to which dietary HDAC inhibitors, by modulating genes such as p21 and Bax, enable normal, nontransformed cells to respond most effectively to external stimuli and toxic insults.

Histone deacetylases as targets for dietary cancer preventive agents: lessons learned with butyrate, diallyl disulfide, and sulforaphane.

Cancer is a multi-factorial process involving genetic and epigenetic events which result in neoplastic transformation. Reversal of aberrant epigenetic events, including those that modulate the transcriptional activity of genes associated with various signaling pathways, holds the prospect of influencing multiple stages of tumorigenesis. Perturbation of normal histone acetylation status can result in undesirable phenotypic changes, including developmental disorders and cancer. Indeed, aberrant histone acetylation may be an etiological factor in several, if not all, types of cancer. In general, histone acetylation leads to chromatin remodeling and a de-repression of transcription. Histone deacetylase (HDAC) inhibitors may be useful for cancer prevention and therapy by virtue of their ability to 'reactivate' the expression of epigenetically silenced genes, including those involved in differentiation, cell cycle regulation, apoptosis, angiogenesis, invasion, and metastasis. Several natural and synthetic HDAC inhibitors have been shown to affect the growth and survival of tumor cells in vitro and in vivo. Interestingly, three dietary chemopreventive agents, butyrate, diallyl disulfide, and sulforaphane, also have HDAC inhibitory activity. This review discusses the role of aberrant histone acetylation in tumorigenesis and describes the potential for cancer chemoprevention and therapy with a particular emphasis on dietary HDAC inhibitors.

Chemoprotection by sulforaphane: keep one eye beyond Keap1.

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables, with particularly high levels detected in broccoli and broccoli sprouts. Over a decade ago, this phytochemical was identified as a likely chemopreventive agent based on its ability to induce Phase 2 detoxification enzymes, as well as to inhibit Phase 1 enzymes involved in carcinogen activation. Considerable attention has focused on SFN as a 'blocking' agent, with the ability to modulate the Nrf2/Keap1 pathway, but recent evidence suggests that SFN acts by numerous other mechanisms. SFN induces cell cycle arrest and apoptosis in cancer cells, inhibits tubulin polymerization, activates checkpoint 2 kinase, and inhibits histone deacetylase activity. The latter findings suggest that SFN may be effective during the post-initiation stages of carcinogenesis, as a 'suppressing' agent. Moreover, pharmacological administration of SFN may be a promising therapeutic approach to the treatment of cancers, including those characterized by increased inflammation and involving viral or bacterial-related pathologies. The present review discusses the more widely established chemoprotective mechanisms of SFN, but makes the case for additional work on mechanisms that might be of importance during later stages of carcinogenesis, beyond Keap1.

Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apc-minus mice.

Sulforaphane (SFN) is an isothiocyanate from broccoli that induces phase 2 detoxification enzymes. We recently reported that SFN acts as a histone deacetylase (HDAC) inhibitor in human colon cancer cells in vitro, and the present study sought to extend these findings in vivo. In mice treated with a single oral dose of 10 mumol SFN, there was significant inhibition of HDAC activity in the colonic mucosa after 6 h, and immunoblots revealed a concomitant increase in acetylated histones H3 and H4, which returned to control levels by 48 h. Longer-term treatment with SFN in the diet resulted in levels of acetylated histones and p21(WAF1) in the ileum, colon, prostate, and peripheral blood mononuclear cells that were elevated compared with controls. Consistent with these findings, SFN suppressed tumor development in Apc(min) mice, and there was an increase in acetylated histones in the polyps, including acetylated histones specifically associated with the promoter region of the P21 and bax genes. These results provide the first evidence for HDAC inhibition by SFN in vivo and imply that such a mechanism might contribute to the cancer chemoprotective and therapeutic effects of SFN, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells.

Sulforaphane (SFN), an isothiocyanate first isolated from broccoli, exhibits chemopreventive properties in prostate cancer cells through mechanisms that are poorly understood. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells, namely the inhibition of histone deacetylase (HDAC). Here, we show that addition of 15 microM SFN also inhibited HDAC activity by 40, 30 and 40% in BPH-1, LnCaP and PC-3 prostate epithelial cells, respectively. The inhibition of HDAC was accompanied by a 50-100% increase in acetylated histones in all three prostate cell lines, and in BPH-1 cells treated with SFN there was enhanced interaction of acetylated histone H4 with the promoter region of the P21 gene and the bax gene. A corresponding 1.5- to 2-fold increase was seen for p21Cip1/Waf1 and Bax protein expression, consistent with previous studies using HDAC inhibitors, such as trichostatin A. The downstream events included cell cycle arrest and activation of apoptosis, as evidenced by changes in cell cycle kinetics and induction of multi-caspase activity. These findings provide new insight into the mechanisms of SFN action in benign prostate hyperplasia, androgen-dependent prostate cancer and androgen-independent prostate cancer cells, and they suggest a novel approach to chemoprotection and chemotherapy of prostate cancer through the inhibition of HDAC.

Dietary HDAC inhibitors: time to rethink weak ligands in cancer chemoprevention?

There is growing interest in the various mechanisms that regulate chromatin remodeling, including modulation of histone deacetylase (HDAC) activities. Competitive HDAC inhibitors disrupt the cell cycle and/or induce apoptosis via de-repression of genes such as P21 and BAX, and cancer cells appear to be more sensitive than non-transformed cells to trichostatin A and related HDAC inhibitory compounds. This apparent selectivity of action in cancer cells makes HDAC inhibitors an attractive avenue for drug development. However, in the search for potent HDAC inhibitors with cancer therapeutic potential there has been a tendency to overlook or dismiss weak ligands that could prove effective in cancer prevention, including agents present in the human diet. Recent reports have described butyrate, diallyl disulfide and sulforaphane as HDAC inhibitors, and many other dietary agents will be probably discovered to attenuate HDAC activity. Here we discuss 'pharmacologic' agents that potently de-repress gene expression (e.g. during therapeutic intervention) versus dietary HDAC inhibitors that, as weak ligands, might subtly regulate the expression of genes involved in cell growth and apoptosis. An important question is the extent to which dietary HDAC inhibitors, and other dietary agents that affect gene expression via chromatin remodeling, modulate the expression of genes such as P21 and BAX so that cells can respond most effectively to external stimuli and toxic insults.

A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase.

Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival and/or apoptosis in various cancer cells, through mechanisms that are poorly understood. We tested the hypothesis that SFN acts as an inhibitor of histone deacetylase (HDAC). In human embryonic kidney 293 cells, SFN dose-dependently increased the activity of a beta-catenin-responsive reporter (TOPflash), without altering beta-catenin or HDAC protein levels. Cytoplasmic and nuclear extracts from these cells had diminished HDAC activity, and both global and localized histone acetylation was increased, compared with untreated controls. Studies with SFN and with media from SFN-treated cells indicated that the parent compound was not responsible for the inhibition of HDAC, and this was confirmed using an inhibitor of glutathione S-transferase, which blocked the first step in the metabolism of SFN, via the mercapturic acid pathway. Whereas SFN and its glutathione conjugate (SFN-GSH) had little or no effect, the two major metabolites SFN-cysteine and SFN-N-acetylcysteine were effective HDAC inhibitors in vitro. Finally, several of these findings were recapitulated in HCT116 human colorectal cancer cells: SFN dose-dependently increased TOPflash reporter activity and inhibited HDAC activity, there was an increase in acetylated histones and in p21(Cip1/Waf1), and chromatin immunoprecipitation assays revealed an increase in acetylated histones bound to the P21 promoter. Collectively, these findings suggest that SFN may be effective as a tumor-suppressing agent and as a chemotherapeutic agent, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

Phosphorylation and ubiquitination of oncogenic mutants of beta-catenin containing substitutions at Asp32.

Beta-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3beta (GSK-3beta)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3beta region of beta-catenin stabilize the protein and activate beta-catenin/TCF/LEF target genes. This study examined three oncogenic beta-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3beta. Compared with wild-type beta-catenin (WT), the beta-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of beta-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the beta-catenin mutants. Specifically, antiphospho-beta-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated beta-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant beta-catenins, as well as on WT beta-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of beta-catenin.

Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid.

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.