Michael C. Alley

Cucurbitacin E-induced disruption of the actin and vimentin cytoskeleton in prostate carcinoma cells

Cucurbitacin E has been identified by an empiric screening strategy as a sterol with potent growth inhibitory activity in vitro directed against prostate carcinoma explants (IC50 of 7-50 nM in 2- to 6-day exposures). The mechanism of cucurbitacin cytoxicity has not been elucidated previously. In the present study, we observed that cucurbitacin E caused marked disruption of the actin cytoskeleton, and in a series of cucurbitacin analogues, anti-proliferative activity correlated directly with the disruption of the F-actin cytoskeleton. The distribution of vimentin was also altered in cells exposed to cucurbitacin E, as vimentin associated with drug-induced membrane blebs. The appearance of microtubules was unaffected. Western blot analysis of intracellular actin in cells exposed to cucurbitacins and quantitation of rhodamine-phalloidin binding support the hypothesis that cucurbitacin treatment leads to an inappropriate increase in the filamentous or polymerized actin fraction in prostate carcinoma cells. We conclude that cucurbitacins are potent disruptors of cytoskeletal integrity. Prostate carcinoma cells appear notably sensitive to growth inhibition by cucurbitacin E.

SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 1: Cellular Pharmacology, In vitro and Initial In vivo Antitumor Activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.

SJG-136 (NSC 694501), A Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 2: Efficacy Evaluations

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.

FLUORINATED 2-(4-AMINO-3-METHYLPHENYL)BENZOTHIAZOLES INDUCE CYP1A1 EXPRESSION, BECOME METABOLIZED, AND BIND TO MACROMOLECULES IN SENSITIVE HUMAN CANCER CELLS

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In “sensitive” cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any “resistant” cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.

The histone deacetylase inhibitor FK228 given prior to adenovirus infection can boost infection in melanoma xenograft model systems

A major limitation of adenovirus type 5–mediated cancer gene therapy is the inefficient infection of many cancer cells. Previously, we showed that treatment with low doses of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) increased coxsackie adenovirus receptor (CAR) levels, histone H3 acetylation, and adenovirus infection efficiencies as measured by viral transgene expression in cancer cell lines but not in cultured normal cells. To evaluate FK228 in vivo, the effects of FK228 therapy in athymic mice bearing LOX IMVI or UACC-62 human melanoma xenografts were examined. Groups of mice were treated with FK228 using several dosing schedules and the differences between treated and control animals were determined. In mice with LOX IMVI xenografts (n = 6), maximum CAR induction was observed 24 h following a single FK228 dose of 3.6 mg/kg with a 13.6 ± 4.3-fold (mean ± SD) increase in human CAR mRNA as determined by semiquantitative reverse transcription-PCR analysis. By comparison, mouse CAR levels in liver, kidney, and lung from the same animals showed little to no change. Maximum CAR protein induction of 9.2 ± 4.8-fold was achieved with these treatment conditions and was associated with increased histone H3 acetylation. Adenovirus carrying a green fluorescent protein (GFP) transgene (2 × 109 viral particles) was injected into the xenografts and GFP mRNA levels were determined. A 7.4 ± 5.2-fold increase in GFP mRNA was found 24 h following adenovirus injection into optimally FK228-treated mice (n = 10). A 4-fold increase in GFP protein–positive cells was found following FK228 treatment. These studies suggest that FK228 treatment prior to adenovirus infection could increase the efficiency of adenovirus gene therapy in xenograft model systems. [Mol Cancer Ther 2007;6(2):496–505]

Fatty Acid Cyclooxygenase Metabolism of Arachidonic Acid in Human Tumor Cells

The prostaglandins, leukotrienes and related eicosanoids have been implicated as mediators in human malignant disease, particularly in cellular events related to tumor metastasis, cell proliferation, tumor promotion and host immunoregulation (1–23). There is substantial evidence that human tumor cells may synthesize significant quantities of prostaglandins. Elevated production of prostaglandin E2 (PGE2) has been demonstrated in lung cancer patients in vivo (24,25). Other studies have shown that prostanoid biosynthesis is elevated in human tumor tissues in comparison with production in normal human tissues (26–28) and that cultured human tumor cells synthesize significant quantities of prostanoids (29–32). In the present studies, the profiles of prostanoid biosynthesis from endogenous arachidonic acid in 55 established cell lines derived from human tumors of the colon, lung, prostate, ovary, kidney, and the central nervous system were determined. The objective of these studies was the determination of PGH synthase activity in diverse histological classes of human tumor cells in order to discern whether fatty acid cyclooxygenase metabolism of arachidonic acid may be uniquely characteristic of certain histological classes of human tumors.

453. The Histone Deacetylase Inhibitor FK228 Can Increase Adenovirus Transgene Protein Expression in Human LOX IMVI Melanoma Xenografts

Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increases in CAR and |[alpha]|v-integrin RNA levels. Treatment of cells with 1 ng/ml FK228 prior to adenovirus infection was associated with a 5-10 fold increase in adenovirus transgene expression. Increases in transgene expression were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI or UACC-62 human melanoma xenografts. The efficacy of treatment was evaluated by analyzing CAR and |[alpha]|v-integrin RNA and protein. Both types of xenografts (n=3) had increased CAR RNA levels as determined by semi-quantitative RT-PCR analysis 6 h following the last of three treatments with 3.6 mg/kg FK228. The LOX IMVI xenografts showed an 8.0-fold (|[plusmn]|0.9) and the UACC-62 xenografts showed a 5.1-fold (|[plusmn]|0.4) increase over untreated mice. The LOX IMVI system was chosen for further study because of greater response to FK228. Mice with LOX IMVI xenografts (n=6) treated with single 3.6 mg/kg FK228 dose and analyzed at 6 h following drug administration showed a 10.7-fold (|[plusmn]|4.9) increase in CAR levels while at 24 h there was a 13.6-fold (|[plusmn]|4.3) increase. By comparison, CAR levels in the livers, kidneys and lungs from the same animals remained unchanged. Xenografts from mice (n=6) treated with 3.6 mg/kg and analyzed by western blot analysis 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR protein. Little change in |[alpha]|v-integrin RNA or protein was observed under any conditions. Based on these results xenograft-bearing mice (n=10) treated with a single dose of 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h following virus injection. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228 treated mice. Immunohistochemistry of formalin-fixed paraffin-embedded xenografts using an antibody to GFP showed higher levels of GFP protein expression diffusely spread throughout the xenografts in FK228 treated mice 18 h and 24 h following adenovirus injection. Thus, as previously observed in vitro, the FK228 induced increase in adenovirus transgene expression also occurs in vivo. These studies suggest that FK228 treatment can increase the efficiency of adenovirus gene therapy in vivo.

618. Coxsackie-Adenovirus Receptor (CAR) Levels Can Be Increased in Human Xenografts Followed by Treatment with FK228, a Histone Deacetylase Inhibitor

Efficient adenovirus infection requires the presence of coxsackie-adenovirus receptor (CAR) and αv integrin on the surface of cells. Previously, we showed that treatment of several cancer cell lines with a low concentration of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) (1 ng/ml) caused an increase in the RNA levels of CAR and αv-integrin. FK228 pre-treatment was associated with a 5–10 fold increase in adenoviral transgene expression following adenovirus infection. The levels of CAR and αv integrin RNA were not increased in cultured normal cells from breast, liver or kidney following similar FK228 treatment. These results suggest that FK228, a drug currently in phase II clinical trials for the treatment of patients with peripheral or cutaneous T-cell lymphoma, may result in preferential enhancement of adenoviral transgene expression in cancer cells. To further evaluate this differential sensitivity between normal and cancer cells, we examined the effect of FK228 in athymic mice bearing advanced-stage subcutaneous LOX IMVI and UACC-62 human melanoma xenografts and MDA-MB-231 human breast cancer xenografts. Mice with melanoma xenografts were treated with FK228 iv q4d × 3 (days 1, 5, and 9) with one of three doses- 0.7, 1.6, or 3.6 mg/kg/dose. The highest dose was effective in causing tumor regression. Animals were sacrificed 6 h, 24 h, or 48 h following the last dose of FK228 and tissues were harvested. The levels of CAR and αv integrin RNA were monitored in mouse liver, kidney and lung and in the human xenografts using semi-quantitative RT-PCR analysis. The levels of CAR RNA were increased in the xenografts. The time of maximum increase in the levels of CAR RNA in the xenografts was 6 h after the administration of 3.6 mg/kg/dose FK228. The levels of αv integrin RNA in the xenografts were unchanged as were the levels of both CAR and αv integrin, in the livers, kidneys and lungs from the same animals. In order to define an optimal dose and schedule of FK228 administration for CAR induction, mice with LOX IMVI xenografts were treated with FK228 one, two, or three times. The tissues were then harvested either 6 or 24 hours following each dose and the levels of CAR and αv integrin RNA were determined. The results showed that the optimal dose for CAR induction was 24 hours after a single treatment with 3.6 mg/kg FK228 where CAR RNA levels were increased by 14-fold. Accordingly, CAR protein levels increased by 3–8 fold. In mice with MDA xenografts treated with a single dose of 1.6, 3.6 or 5.4 mg/kg FK228, CAR protein levels increased after 48 hours by 4-fold following the 3.6 mg/kg dose, whereas αv integrin levels remained unchanged. Because we have previously shown a correlation between the level of CAR and the extent of adenovirus infection in vitro, these results suggest that FK228 may preferentially increase the levels of adenovirus infection in cancer cells in vivo as it did in vitro. Experiments are in progress to determine if FK228 pre-treatment can increase the efficiency and selectivity of adenovirus gene therapy in vivo.