ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136.

ATP-binding cassette transporter P-glycoprotein (ABCB1) is responsible for the multidrug resistance (MDR1) phenotype observed in cancer cells. SJG-136, a new pyrrolobenzodiazepine dimer, is a sequence-dependent DNA crosslinking agent and substrate of ABCB1. We previously showed that colon cancer cell lines expressing high levels of ABCB1 showed a lower sensitivity to SJG-136. Here, we show that in 3T3 isogenic fibroblasts, ABCB1 genetic polymorphism differentially affects ABCB1 gene expression and transport function. However, this genotype-phenotype relationship was not observed in immortalized lymphocytes, which expressed 10- to 1000-fold less ABCB1 than colon cancer cell lines. Consistent with this, the cytotoxicity of SJG-136 in 3T3 fibroblasts was affected by ABCB1 genetic polymorphism but not in immortalized lymphocytes. ABCB1 genetic polymorphism is therefore likely to affect drug sensitivity in tissues expressing high levels of the transporter and in which significant variability is observed.

Short hairpin RNAs targeting Bcl-xL modulate senescence and apoptosis following SN-38 and irinotecan exposure in a colon cancer model.

Bcl-xL is an anti-apoptotic protein over-expressed in colorectal cancers acting on both the intrinsic and extrinsic pathways. We stably expressed four different short hairpin RNA (pSNG-xL1-4) targeting Bcl-xL in HCT 116 cells. HCT 116 pSNG-xL#1 produced a modest (30%) decrease in Bcl-xL expression whilst Bcl-2 levels were similar to the parental cell line, HCT 116 pSNG-xL#2 and 3 showed 50% decrease in Bcl-xL and stable Bcl-2. HCT 116 pSNG-xL#3 showed a concomitant decrease (50%) in Bcl-2. A decrease in Bcl-xL sensitised cells to the small molecule inhibitor of Bcl-xL, Antimycin A3 and the DNA topoisomerase I inhibitors, SN-38 and camptothecin, but not to doxorubicin. HCT 116 pSNG-xL#1 produced a moderate increase in both senescence and apoptosis and a limited increase in SN-38 induced cell death while HCT 116 pSNG-xL#2 produced an increase in apoptosis but reduced senescence. Finally, when both Bcl-xL and Bcl-2 were decreased to a similar degree (HCT 116 pSNG-xL#3), senescence was significantly increased but apoptosis was limited. This effect was confirmed in vivo after administration of irinotecan and was associated with greater anti-tumour effect. Optimal growth inhibitory effect was therefore observed when both Bcl-xL and Bcl-2 were decreased to a similar extent. Antimycin A3, in combination with SN-38 recapitulated this phenotype in HCT 116 cells, suggesting a potential role for small molecule inhibitors of Bcl-xL/Bcl-2 in the treatment of colorectal cancer, potentially in combination with irinotecan.

Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds.

Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium(II) organometallic arene complexes in human ovarian cancer. Br J Cancer 2002;86(10):1652-7]. [(eta6-C6H5C6H5)Ru(en)Cl]+ (as a PF6 salt, where en=ethylenediamine (RM175)) has been evaluated in a 13-cell line panel. Particular sensitivity (approximately 10-fold lower than mean IC50) was noted in breast cancer and non-small cell lung cancer cell lines. In addition, IC50 in the A549 was 2 microM and RM175 (25 mg kg-1, days 1 and 5, i.p.) caused a significant (p=0.004) growth delay in a xenograft model. HC11 [(eta6-tetrahydroanthracene)Ru(en)Cl]PF6 was more potent in the A549 cell line (IC50 0.5 microM). HC11 (25 mg kg-1, days 1, 8 and 15, i.p.) was also active in vivo. Following RM175 25 mg kg-1, days 1 and 5, and 15 mg kg-1, days 1-5, HC11 25 and 40 mg kg-1, day 1, elevated alanine transaminase levels were detected, suggesting hepatotoxicity. No changes were observed in kidney or haematological parameters. In liver sections, multi-focal hepatic necrosis was seen, becoming confluent at high doses of HC11. In vitro studies confirmed that HC11 was more toxic than RM175 to fresh human hepatocytes and equitoxic to mithramycin. Liver toxicity may be related to the arene ligand and modification may reduce the potential for hepatic toxicity, while maintaining the anti-tumour activity seen.

A virus-directed enzyme prodrug therapy approach to purging neuroblastoma cells from hematopoietic cells using adenovirus encoding rabbit carboxylesterase and CPT-11.

Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%.

Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136.

SJG-136 is a novel pyrrolobenzodiazepine dimer analogue that acts as a minor-groove interstrand DNA cross-linking agent. The present study investigated the impact of ABCB1 (mdr-1) expression on the activity of SJG-136 using both in vitro and in vivo systems. SJG-136 was highly potent in the colon cancer cell lines HCT-116, HT-29 and SW620 (IC50 0.1-0.3 nM). However, HCT-8 and HCT-15 cells expressing significant levels of mdr-1 were less sensitive (IC50 2.3 and 3.7 nM, respectively) using a SRB assay. The cytotoxicity was increased in HCT-15 and A2780(AD) in presence of 5 microg/ml verapamil. Mdr-1 mRNA expression was determined by qRT-PCR and correlated to SJG-136 IC50s (r2=0.86, P=0.0001). Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were less sensitive to SJG-136 than the parental 3T3 cells (IC50 208 and 6.3 nM, respectively). Finally, SJG-136 (120 microg/kg/d dx5) was highly active against A2780 xenografts (SGD=275) but not A2780(AD) xenografts (SGD=67).

The mechanism of action of Kahalalide F: variable cell permeability in human hepatoma cell lines.

Kahalalide F (KF) is a small natural peptide that showed activity in vitro and in vivo. The dose-limiting toxicity in clinical trials was transaminitis. We investigated the cytotoxicity of KF in cell lines from breast, ovary, prostate and colon cancers, but focused on hepatoma cell lines, performing mechanistic studies in HepG2 (IC50 = 0.3 microM) and PLC/PRF/5C (IC50 = 5 microM). Following KF exposure, HepG2 cells demonstrated profound ATP depletion, associated with cell swelling and cell blebbing, and increased permeability to propidium iodide (PI), annexin V (AV) and release of lactate dehydrogenase (LDH). PLC/PRF/5C cells retained their cell structure, but were permeable to PI and, following exposure to high concentrations of KF, to AV. The pattern of cell permeability is similar to maitotoxin, another small cytotoxic peptide, but the differential effects on the cell membrane induced by KF in HepG2 and PLC/PRF/5C suggest specific interactions with membranes or proteins. This could lead to better drug design aimed at exploiting the potential for cell selectivity.

Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase.

The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.

Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated.

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low. Data presented here demonstrate that nuclear p53 protein is expressed in a panel of neuroblastoma cell lines, and after exposure to DNA damage, transcriptionally active p53 expression can be induced. After exposure to both equitoxic IC80 and 10-Gy doses of ionizing radiation, both p53 and p21 were induced, but G1 cell cycle arrest was attenuated. To investigate whether the DNA damage signaling pathway was incapable of inducing sufficient p53 in these cells, we expressed additional wild-type p53 after adenoviral vector transduction. This exogenous p53 expression also resulted in p21 induction but was unable to enhance the G1 arrest, suggesting that the pathway downstream from p53 is nonfunctional. Although p53-mediated G1 arrest is attenuated in neuroblastoma cells, the ability of p53 to induce apoptosis appears functional, consistent with its chemosensitive phenotype. This work demonstrates that p53 is expressed in the nucleus of neuroblastoma cells and can mediate induction of p21. However, this cell type appears to have an attenuated ability to mediate a DNA damage-induced G1 cell cycle arrest.

Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice.

PURPOSE: To develop a highly reproducible model of disseminated childhood neuroblastoma in mice to allow secondary evaluation of therapeutics against microscopic disseminated disease. METHODS: CB17/Icr SCID were injected i.v. with 10(3) to 5 x 10(6) human NB-1691 neuroblastoma cells. NB-1691 cells were detected by PCR for synaptophysin and tyrosine hydroxylase in peripheral blood, and bone marrow. Therapeutic studies evaluated topotecan and vincristine as single agents or in combination. Topotecan was administered i.v. daily for 5 days on two consecutive weeks. Courses were repeated every 21 days for three cycles. Vincristine (1 mg/kg) was administered i.v. every 7 days for nine consecutive weeks. Treatment started 11-21 days after tumor cell inoculation. RESULTS: Following injection of > or = 1 x 10(5) cells 100% of mice developed disease. Mice inoculated with 10(7) cells survived a median of 42 days. Survival time was a linear function of the cell inoculum. At autopsy, gross tumor was routinely detected in many organs in particular liver, ovaries, kidneys and adrenals. NB-1691 cells were detected by PCR in peripheral blood, and bone marrow. Immunohistochemical staining showed that lesions were strongly positive for synaptophysin, chromogranin A and negative for leukocyte common antigen. Topotecan (0.6 mg/kg) alone extended median survival from 44 days (controls) to 95 days. When treatment was started 21 days after inoculation of NB-1691 cells, topotecan extended median survival from 39 days (controls) to 91 and 99 days at dose levels of 0.3 and 0.6 mg/kg, respectively. Vincristine (1 mg/kg) extended survival by a median of 9.5 days. In combination with vincristine (1 mg/kg), median survival was increased to 141 days (topotecan 0.6 mg/kg) and 159 days (topotecan 1.0 mg/kg). CONCLUSION: This model of disseminated neuroblastoma is highly reproducible. As this model may more closely simulate childhood disease it may be a valuable adjunct in developing new approaches to advanced stage, poor prognosis neuroblastoma.

Topoisomerase enzymes as drug targets.

DNA topoisomerases catalyze changes in the topology of DNA. Recently, other functions have also been reported for these enzymes. For example, topoisomerase I participates in transcription by RNA polymerases I, II, and III, and also has a kinase activity. Topoisomerase I binds directly to at least two helicases, nucleolin and SV40 T antigen, and mechanistic studies show that T antigen alters the function of topoisomerase I. Additional protein and nucleotide interactions for both topoisomerases I and II suggest that each protein is multifunctional. It may be that the multifunctional nature of these enzymes is the basis for the antitumor activity seen with inhibitors of these enzymes. Clinical trials with combinations of CPT-11 and 5-fluorouracil for the treatment of colon cancer, and preclinical studies with CPT-11 and vincristine are particularly encouraging. Protracted schedules of administration of topoisomerase inhibitors will likely have greater antitumor effect than more concentrated, higher dose exposures, but a systematic determination of optimal schedules of administration is needed.

Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells to CPT-11.

Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) and rhabdomyosarcoma cell lines (JR1neo20, JR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 microM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 microM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications.