Dvorit Samid

A phase II study of 5-aza-2'deoxycytidine (decitabine) in hormone independent metastatic (D2) prostate cancer.

Aims and Background Decitabine (5-aza-2′-deoxycytidine) is an S-phase-specific pyrimidine analog with hypomethylation properties. In laboratory models of prostate cancer (PC-3 and DU-145), decitabine induces cellular differentiation and enhanced expression of genes involved in tumor suppression, immunogenicity, and programmed cell death. Methods We conducted a phase II study of decitabine in 14 men with progressive, metastatic prostate cancer recurrent after total androgen blockade and flutamide withdrawal. Decitabine was administered at a dose of 75 mg/m2/dose IV as a 1 hour infusion every 8 hours for three doses. Cycles of therapy were repeated every 5 to 8 weeks to allow for resolution of toxicity. Results Two of 12 patients evaluable for response had stable disease with a time to progression of more than 10 weeks. This activity was seen in 2 of 3 African-American patients. Toxicity was similar to previously reported experience. No significant changes in urinary concentrations of the angiogenic factor bFGF, a potential biomarker of tumor activity, were identified over time in 7 unselected patients with progressive disease. Conclusions We conclude that decitabine is a well tolerated regimen with modest clinical activity against hormone-independent prostate cancer. Further investigations in patients of African-American origin may be warranted.

Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs

The aromatic fatty acid phenylacetate and its analogs induce tumor cytostasis and differentiation in experimental models. Although the underlying mechanisms of action are not clear, effects on lipid metabolism are evident. We have now examined whether these compounds, structurally similar to the peroxisome proliferator clofibrate, affect the human peroxisome proliferator-activated receptor (hPPAR), a homolog of the rodent PPAR alpha, a transcriptional factor regulating lipid metabolism and cell growth. Gene transfer experiments showed activation of hPPAR, evident by the increased expression of the reporter gene chloramphenicol acetyltransferase linked to PPAR-response element from either the rat acyl-CoA oxidase or rabbit CYP4A6 genes. The relative potency of tested drugs in the co-transfection assay was: 4-iodophenylbutyrate > 4-chlorophenylbutyrate > clofibrate > phenylbutyrate > naphthylacetate > 2,4-D > 4-chlorophenylacetate > phenylacetate >> indoleacetate. Phenylacetylglutamine, in which the carboxylic acid is blocked, was inactive. The ability of the aromatic fatty acids to activate PPAR was confirmed in vivo, as CYP4A mRNA levels increased in hepatocytes of treated rats. Further studies using human prostate carcinoma, melanoma, and glioblastoma cell lines showed a tight correlation between drug-induced cytostasis, increased expression of the endogenous hPPAR, and receptor activation documented in the gene-transfer model. These results identify phenylacetate and its analogs as a new class of aromatic fatty acids capable of activating hPPAR, and suggest that this nuclear receptor may mediate tumor cytostasis induced by these drugs.

Lipid Metabolism as a Target for Brain Cancer Therapy: Synergistic Activity of Lovastatin and Sodium Phenylacetate Against Human Glioma Cells

Abstract: Malignant gliomas, the most common form of primary brain tumors, are highly dependent on the mevalonate (MVA) pathway for the synthesis of lipid moieties critical to cell replication. Human glioblastoma cells were found to be uniquely vulnerable to growth arrest by lovastatin, a competitive inhibitor of the enzyme regulating MVA synthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. The sodium salt of phenylacetic acid (NaPA), an inhibitor of MVA‐pyrophosphate decarboxylase, the enzyme that controls MVA use, acted synergistically with lovastatin to suppress malignant growth. When used at pharmacologically attainable concentrations, the two compounds induced profound cytostasis and loss of malignant properties such as invasiveness and expression of the transforming growth factor‐β2 gene, coding for a potent immunosuppressive cytokine. Supplementation with exogenous ubiquinone, an end product of the MVA pathway, failed to rescue the cells, suggesting that decreased synthesis of intermediary products are responsible for the antitumor effects observed. In addition to blocking the MVA pathway, lovastatin alone and in combination with NaPA increased the expression of the peroxisome proliferator‐activated receptor, a transcription factor implicated in the control of lipid metabolism, cell growth, and differentiation. Our results indicate that targeting lipid metabolism with lovastatin, used alone or in combination with the aromatic fatty acid NaPA, may offer a novel approach to the treatment of malignant gliomas.

Cytostatic activity of phenylacetate and derivatives against tumor cells: Correlation with lipophilicity and inhibition of protein prenylation

The aromatic fatty acid phenylacetate, a common metabolite of phenylalanine, shows promise as a relatively non-toxic drug for cancer treatment. This slowly metabolized fatty acid alters tumor cell lipid metabolism causing, among other effects, inhibition of protein prenylation critical to malignant growth. In pursuit of more potent analogues, we have examined the activity of related compounds against tumor cell lines established from patients with advanced prostatic carcinoma, glioblastomas, and malignant melanoma. Like phenylacetate, derivatives containing alpha-carbon or ring substitutions induced cytostasis and phenotypic reversion at non-toxic concentrations. Potency was correlated with the degree of calculated lipophilicity of the aromatic fatty acid, and the extent of inhibition of protein prenylation. Remarkably, a parallel cytostatic activity was reported in embryonic plant cells, which respond to phenylacetate and its analogues in the same concentration range and the same rank order of lipophilicity. These data suggest that phenylacetate and its analogues may act through common mechanisms to inhibit the growth of vastly divergent, undifferentiated cell types, and provide a basis for the development of new agents for the treatment of human malignancies.

Transcriptional upregulation of γ-globin by phenylbutyrate and analogous aromatic fatty acids

Abstract Phenylbutyrate has been shown recently to induce fetal hemoglobin (HbF) production in patients with sickle cell anemia and β thalassemia. We have now examined related aromatic fatty acids in order to define the range of active structures and identify plausible mechanisms of action. Structure-function analysis revealed that for effective stimulation of HbF in erythroid precursors: (1) the ideal length for the aliphatic side chain is four carbons; (2) oxygen or sulfur substitutions in the carboxylic chain are allowed, as evidenced by the equal or increased activity of phenoxypropionate, benzylthioglycolate, and benzyloxyacetate compared with phenylbutyrate; and (3) blocking the carboxylate group by conversion to the amide form greatly reduces potency. Molecular analysis indicated that the prototype agent, phenylbutyrate, increases HbF production through transcriptional activation of the γ-globin gene. The latter contains a butyrate responsive promoter known to up-regulate transcription in the presence of short-chain fatty acids of three to five carbons. To determine whether stimulation of an element in this promoter by phenylbutyrate and its analogues might contribute to their mechanism of action, we used a transient expression system involving K562 erythroleukemia cells transfected with a luciferase reporter gene driven by the minimum γ-globin promoter. Transcriptional activation in this experimental system correlated well with the capacity of an aromatic fatty acid to increase HbF production in erythroid precursors ( r = 0.94). Our studies identify potent analogues of phenylbutyrate for the treatment of β-chain hemoglobinopathies, and suggest that stimulation of a butyrate responsive promoter may be responsible for their activity.

The differentiating agent phenylacetate increases prostate-specific antigen production by prostate cancer cells

The prostatic‐specific antigen (PSA) is the tumor marker most widely relied upon for the monitoring of patients with prostate cancer. Recently, declines in the serum concentrations of PSA have been advocated as a surrogate marker of tumor response in clinical trials of investigational antitumor agents. We examined the hypothesis that this postulate may not apply to the evaluation of drugs such as phenylacetate, a differentiating agent endowed with mechanisms of action different from those of classic cytotoxic chemotherapy. Using human prostatic carcinoma LNCaP cells as a model, we show that phenylacetate induces PSA production despite inhibition of tumor cell proliferation. Incubation of LNCaP cultures with cytostatic doses of phenylacetate (3–10 mM) resulted in a three‐ to fourfoLd increase in PSA secretion per cell. This appears to result from upregulation of PSA gene expression, as indicated by elevated PSA mRNA steady‐state levels in treated cells. The increase in PSA production per cell was confirmed in rats bearing subcutaneous LNCaP tumor implants that were treated systemically with phenylacetate. Further comparative studies indicate that upregulation of PSA is common to various differentiation inducers, including all, trans‐retinoic acid, 1,25‐dihydroxyvitamin D3, and butyrate but is not induced by other antitumor agents of clinical interest such as suramin. We conclude that declines in PSA may be treatment specific and that the exclusive use of this criterion as a marker of disease response may mislead the proper evaluation of differentiating agents in prostate cancer patients.

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4‐chlorophenylacetate (4‐CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4‐CPA were estrogen receptor (ER) positive (T47‐D, MCF‐7 and ZR‐75‐1) whereas those that were little affected by these compounds were ER‐negative (MDA‐MB‐157, MDA‐MB‐231 and SK‐Br‐3). Dose response studies indicated that 4‐CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3–4 times that of PA. These findings suggest that there is “cross‐talk” between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen‐dependent events. This hypothesis was directly tested in vitro using ER+ MCF‐7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF‐7‐D1). Our experiments with MCF‐7‐D1 cells indicated that PA and 4‐CPA inhibited basal and estrogen‐induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen‐dependent cyclin D1 gene activation. Using a reporter gene construct (EREV‐tk‐Luc) containing a canonical estrogen response element that was transiently transfected into MCF‐7 and MDA‐MB‐231 cells, we have also demonstrated inhibition of promoter activity by PA and 4‐CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.

Plasma protein binding of phenylacetate and phenylbutyrate, two novel antineoplastic agents.

Phenylacetate and phenylbutyrate, two novel inducers of tumor cytostasis and differentiation, are currently in clinical trials for the treatment of cancer in adults. The purpose of our study was to evaluate the plasma protein-binding characteristics of phenylacetate and phenylbutyrate in the plasma of normal volunteers and that of patients with cancer. Drug plasma protein-binding analysis was examined using three separate devices: a micropartition system and two equilibrium dialysis systems, all of which exhibited similar results. Phenylacetate and phenylbutyrate concentrations were determined by high-performance liquid chromatography. Both drugs exhibited concentration-dependent binding. Our results showed sodium phenylacetate to have a higher free fraction than sodium phenylbutyrate at corresponding concentrations (> 0.442 +/- 0.008 and > 0.188 +/- 0.001, respectively). Plasma pH did not greatly affect protein binding of either drug. As albumin concentration decreased, an increase in free fraction of both drugs was observed, however alpha 1-acid glyco-protein showed no change in free fraction as its concentration increased. Patients with cancer with lower levels of albumin showed an increase in free fraction with both phenylacetate and phenylbutyrate. When phenylacetate and phenylbutyrate were added together in plasma, the free fraction of phenylacetate increased, whereas the phenylbutyrate free fraction slightly decreased. We conclude that phenylacetate and phenylbutyrate have high free fractions that change with varying albumin levels and when both phenylacetate and phenylbutyrate are present together in plasma.

In vitro antitumor effect of hydroxyurea on hormone-refractory prostate cancer cells and its potentiation by phenylbutyrate.

Previous clinical trials have suggested that hydroxyurea may possess some activity against prostate cancer. The in vitro antiproliferative activity of hydroxyurea was evaluated in three hormone-refractory prostate cancer cell lines, PC-3, DU-145 and PC-3M. Fifty-percent inhibition of growth in all three cell lines required prolonged (120 h) exposure to hydroxyurea at a concentration of approximately 100 microM. Using pharmacokinetic data obtained during the course of a clinical trial of hydroxyurea, we simulated a dosing regimen that would sustain plasma drug concentrations above 100 microM for 120 h (1 g loading dose, followed by 500 mg every 6 h for 5 days in a 70 kg man). Since this dosing regimen is likely to generate an unacceptable degree of myelosuppression, in vitro combination studies were conducted with hydroxyurea and phenylbutyrate, a new differentiating agent with no myelosuppressive effects. These studies resulted in a reduction of the hydroxyurea concentration necessary for 50% growth inhibition (50 microM of hydroxyurea plus 0.5 mM of phenylbutyrate). A regimen designed to achieve that hydroxyurea concentration (400 mg loading dose, followed by 200 mg every 6 h for 5 days) should be clinically achievable. Based on these results, this combination deserves further evaluation in patients with stage D prostate cancer.