Cytotoxicity Of CPT-11 Research Articles

Binding of combined irinotecan and epicatechin to a pH-responsive DNA tetrahedron for controlled release and enhanced cytotoxicity

pH-Responsive aptamer-linked DNA nanostructures can actively target cancer cells to release drugs in the cells’ acidic environment. Tea polyphenols can be used as cancer-preventive agents to enhance the efficacy of chemotherapeutic drugs. In this study, we constructed a pH-responsive DNA tetrahedron with MUC1 aptamer (MUC1-TD) to co-load irinotecan hydrochloride (CPT-11) and (-)-epicatechin (EC). The thermodynamic parameters for the interaction of CPT-11 and/or EC with MUC1-TD were investigated using fluorescence spectroscopy and calorimetry. The binding of CPT-11 to MUC1-TD was found to be stronger than that of EC. Differential scanning calorimetry and fluorescence quenching experiments demonstrated the intercalative binding between CPT-11/EC and MUC1-TD. The in vitro release rate of CPT-11 was faster at pH 5.0 than at pH 7.4, confirming the pH-responsive of MUC1-TD. In vitro cytotoxicity results indicated that CPT-11 loaded by MUC1-TD was more cytotoxic than free CPT-11. Further, we found that the cytotoxicity of CPT-11 was further enhanced in the presence of EC due to a synergy between them. Additionally, cell uptake experiments showed that CPT-11 and EC loaded by MUC1-TD were mainly distributed in the nucleus of human breast cancer cells (MCF-7). These findings indicate that pH-responsive DNA nanostructures are a promising delivery system for co-loading chemotherapeutic drugs and natural polyphenols.

Drug Resistance of CPT-11 in Human DLD-1 Colorectal Cancer Cells through MutS Homolog 2 Upregulation.

Colorectal cancers (CRCs) is the most commonly diagnosed and deadly cancer types in the world. Despite advances in chemotherapy for CRCs, drug resistance remains a major challenge to high incurable and eventually deadly rates for patients. CPT-11 is one of the current chemotherapy agents for CRC patients and the CPT-11 resistance development of CRCs is also inevitable. Recently, accumulating data has suggested that DNA repair system might be an inducer of chemotherapy resistance in cancer cells. Thus, this study was aimed to examine whether MutS homolog (MSH) 2, one member of DNA repair system, plays a role to affect the cytotoxicity of CPT-11 to CRCs. Human DLD-1 CRC cells were used in this study. It was shown that MSH2 gene and protein expression could be upregulated in DLD-1 cells under CPT-11 treatment and this upregulation subsequently attenuates the sensitivity of DLD-1 cells to CPT-11. Moreover, ERK1/2 and Akt signaling and AP-1 transcription factor have been found to modulate these effects. These results elucidate the drug resistance role of MSH2 upregulation in the CPT-11-treated DLD-1 CRC cells. Our findings may provide a useful thought for new adjuvant drug development by controlling the DNA repair system.

Thermosensitive magnetic liposomes for alternating magnetic field-inducible drug delivery in dual targeted brain tumor chemotherapy

The nonspecific distribution and non-targeted heating of chemotherapeutic agents in the human body commonly produce adverse side effects during brain cancer management. Even though an external magnetic field can partially gather extracellular magnetic drug carriers near brain tumor, magnetic guidance alone still cannot precisely identify and target either tumors or healthy tissues due to a lack of selectivity. In this study, we successfully developed thermal and magnetic dual-responsive thermosensitive magnetic liposomes (TML), which co-encapsulates Camptosar (CPT-11) and citric acid-coated magnetic Fe3O4 nanoparticles within the aqueous core and surface-conjugated with Cetuximab (CET) for recognizing over-expressed epidermal growth factor receptors on cancer cell surface. This drug carrier system can control the release of encapsulated drug when exposed to a high-frequency alternating magnetic field (AMF) that elevates the temperature of the liposomal membrane and triggers drug release from TML after their selective endocytosis by cancer cells. By detailed characterizing the physico-chemical and biological properties of liposomes, we demonstrated the liposomal formulation is with high biocompatibility and showed no hemolysis in vitro. Enhanced intracellular uptake of TML-CPT-11-CET by human primary glioblastoma cells (U87) also supported targeted delivery through CET-mediated endocytosis. The treatment of TML-CPT-11-CET solutions by AMF in vitro showed rise in solution temperature and enhanced drug release, which enhanced cytotoxicity of CPT-11 toward U87 though cell apoptosis as revealed from flow cytometry analysis of apoptotic cells and Western blot studies of marker proteins. Finally, the in vivo therapeutic efficacy was demonstrated in mice orthotopic xenograft brain tumor model from IVIS and PET/MRI studies.

Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity.

CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.

Influence of UGT1A1 gene methylation level in colorectal cancer cells on the sensitivity of the chemotherapy drug CPT-11

ObjectiveTo study the influence of the methylation level of UGT1A1 gene related to CPT-11 metabolic enzymes in colorectal cancer cells on the sensitivity of chemotherapy drugs. MethodsTest the changes in sensitivity of seven colorectal cancer cell strains that have been/not been subject to DAC treatment to CPT-11, analyze its correlation with CES2, UGT1A1 and GUSB mRNA expression according to IC50; screen the effective interference sequence of UGT1A1 siRNA, test the changes in cytotoxicity of CPT-11 after UGT1A1 siRNA is transfected, select RK0 cells and make them transfected with the chemosynthetic UGT1A1 siRNA after their UGT1A1 expression is restored with or without demethylation treatment. ResultsThe sensitivity of different colorectal cancer cell strains to CPT-11 showed difference (P<0.05), UGT1A1 expression in colorectal cell lines had a negative correlation with the IC50 (r=0.790648, P<0.05), the interference efficiency of the screened UGT1A1 siRNA was up to 78%. The IC50 value of siRNA decreased by nearly one time after transfected with HT-29 (P<0.01); which of methylated RK0 cells of UGT1A1 gene increased instead after the demethylation treatment. However, the IC50 value of the demethylation treatment group increased compared with the non-demethylation treatment group after UGT1A1 siRNA was transfected. ConclusionsThe cytotoxicity of CPT-11 to colorectal cancer cells has a negative correlation with UGT1A1 expression, and positive correlation with CES2 and GUSB. The specific silencing UGT1A1 gene of siRNA could significantly increase the sensitivity of CPT-11 to the chemotherapy of colorectal cancer cells. UGT1A1 methylation was an important factor affecting the chemosensitivity of CPT-11.

Neural Stem Cell-Mediated Delivery of Irinotecan-Activating Carboxylesterases to Glioma: Implications for Clinical Use

CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediated CE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor-tropic properties, and mediated an increase in the cytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demonstrate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.

Hepatocyte growth factor suppresses the anticancer effect of irinotecan by decreasing the level of active metabolite in HepG2 cells

In the liver, carboxylesterase (CES) converts irinotecan (CPT-11) to its active metabolite SN-38, which exerts anticancer effects. SN-38 is metabolized to an inactive metabolite SN-38 glucuronide by uridine 5′-diphospho-glucuronosyltransferase 1A1 (UGT1A1). Therefore, single nucleotide polymorphisms (SNPs) of the UGT1A1 gene are responsible for the severe adverse effects associated with the disruption of SN-38 metabolism. However, despite having SNPs of the UGT1A1 gene, many patients metabolize SN-38 sufficiently to avoid severe adverse effects. Among these patients, we found individuals with elevated serum concentrations of hepatocyte growth factor (HGF). The aim of this study was to evaluate whether HGF alters the metabolism of CPT-11, resulting in a reduction in the anticancer effect of CPT11. The cytotoxicity of CPT-11 and SN-38 was evaluated in HepG2 cells pretreated with HGF. Furthermore, we explored the level of expression and mechanisms of activity of CES and UGT1A1. HGF suppressed the cytotoxicity of CPT-11 by decreasing intracellular SN-38 levels that resulted from a decrease in CES2 and an increase in UGT1A1. Furthermore, this HGF-induced suppression was improved by pretreatment with an inhibitor of HGF receptor c-Met, and the improvement was synergistically potentiated by epidermal growth factor receptor (EGFR) inhibitors. Moreover, HGF induced phosphorylation of signal transducer and activator of transcription 3 and transactivated EGFR. These results suggest that HGF is a possible causative agent of acquired clinical resistance in chemotherapy with CPT-11 and could be useful as a predictor of clinical resistance. Additional treatment using c-Met and/or EGFR inhibitors could be a novel strategy to overcome resistance.

Role of Glucocorticoid Receptor in the Regulation of Cellular Sensitivity to Irinotecan Hydrochloride

In clinical practice, glucocorticoids are often used with the aim of modulating the efficacy and toxicity of chemotherapeutic agents. However, how glucocorticoids modulate the pharmacological action of chemotherapeutic agents remains to be clarified. In this study, we generated glucocorticoid receptor (GR)-deficient rat-1 cells to investigate the role of GR in the regulation of cellular sensitivity to irinotecan hydrochloride (CPT-11). Treatment of wild-type rat-1 cells with dexamethasone (DEX) significantly enhanced the cytotoxic effect of CPT-11, whereas the treatment had little effect on the cytotoxicity of CPT-11 in GR-deficient cells. Topoisomerase-I activity in wild-type cells after concomitant treatment with DEX and CPT-11 was significantly lower than that after treatment with CPT-11 alone. DEX treatment also enhanced the inhibitory action of CPT-11 on the phosphatidylinositol 3-kinase–Akt signaling pathway in wild-type cells, accompanied by facilitating caspase-3 activity. These modulatory effects of DEX on the CPT-11–induced cytotoxicity were not observed in GR-deficient cells. Our present findings reveal the underlying mechanism by which GCs enhance the chemotherapeutic effect of CPT-11 and indicate the possibility that the dosage of CPT-11 could be reduced by the combination treatment with GCs, which may attenuate the adverse effect without decreasing anti-tumor activity.[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.08219FP]

A phase II randomized multicenter trial of gefitinib plus FOLFIRI and FOLFIRI alone in patients with metastatic colorectal cancer

BackgroundGefitinib inhibits the epidermal growth factor receptor tyrosine kinase and preclinical studies indicate that it may enhance CPT-11 cytotoxicity. This randomized phase II trial investigates the feasibility and efficacy of gefitinib and 5-fluorouracil, folinic acid, irinotecan (FOLFIRI) in patients with metastatic colorectal cancer. Patients and methodsPatients were randomized to FOLFIRI ± gefitinib 250 mg daily p.o. Patients randomized to FOLFIRI + gefitinib without disease progression after 6 months continued to receive gefitinib alone until disease progression. ResultsFrom October 2002 to September 2004, 100 patients were enrolled. Twenty-three patients (47.9%) in the FOLFIRI arm and 23 (45.1%) in the FOLFIRI + gefitinib arm experienced an objective response. The median progression-free survival and overall survival were 8.3 and 18.6 months in the FOLFIRI arm, and 8.3 and 17.1 months in the FOLFIRI + gefitinib arm, respectively. In the combination arm, grades 3–4 adverse events were experienced by 35 (67.3%) patients versus 25 patients (52.1%) in the FOLFIRI arm; 12 patients (23.1%) withdrew for an adverse event in the FOLFIRI + gefitinib arm and 5 (10.4%) in the FOLFIRI arm. ConclusionsThese data show that adding gefitinib to FOLFIRI does not improve the efficacy of FOLFIRI regimen. These disappointing results could be related to the high toxicity observed that led to significant dose reductions and delays.

Hypoxia‐targeted over‐expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT‐11)

The induced expression of carboxylesterase (CE) enzymes, which convert the prodrug irinotecan (CPT-11) into its active cytotoxic metabolite SN-38, constitutes a promising strategy for cancer gene therapy. By incorporating hypoxia-responsive elements (HREs) in conjunction with the transgene, expression can be targeted specifically to hypoxic tissues (such as solid tumours), expressing the hypoxia-inducible factor 1 (HIF-1). We have constructed a recombinant adenoviral vector, AdHRE-rCE, encoding the cDNA for the highly efficient rabbit liver CE (rCE), under the control of a HRE derived from the human phosphoglycerate kinase 1 (PGK-1) gene in conjunction with a minimal SV40 promoter. In vitro, HT1080 fibrosarcoma and SW480 colon carcinoma cells demonstrated an approximately 10-fold hypoxia-dependent induction in CE expression following pre-infection with AdHRE-rCE, which led to a15-30-fold increased sensitivity to CPT-11. Furthermore, in vivo, SW480 tumour xenografts infected with AdHRE-rCE demonstrated a 2-fold decrease in tumour doubling time, when combined with 7 days of CPT-11 treatment, in comparison to mock-infected controls, with rCE expression shown to be limited to hypoxic regions only. As the cytotoxicity of CPT-11 is reduced under hypoxic conditions, over-expression of a highly efficient CE such as rCE under hypoxia control within these hypoxic cells could reverse this effect and, therefore, form the basis for future clinical treatment strategies.

Schedule-dependent cytotoxicity of 5-fluorouracil and irinotecan in a colon cancer cell line

Our aim was to clarify the significance of widely accepted irinotecan (CPT-11)/5-fluorouracil (5-FU) combinations in colon cancer by investigating their sequential effect. The sequential effect of CPT-11/5-FU in two colon cancer cell lines, LoVo and SW480, was evaluated by WST-8 colorimetric assay. The cell cycle distributions of each drug were analyzed by flow cytometry, and then the chemoresistant mechanisms and expression of a drug transporter (MDR1), the bcl-2 apoptotic pathway, metabolizing enzymes [carboxylesterase (CE), dihydropyrimidine dehydrogenase], and target enzymes (topoisomerase I, thymidine synthase) associated with sequence-dependent cytotoxicity were examined. The cytotoxicity of 5-FU (10, 100, 1000 microM) followed by CPT-11 (1 microM) was significantly greater than that of CPT-11 (1 microM) followed by 5-FU (10, 100, 1000 microM) (P < 0.05). Reverse transcription-polymerase chain reaction analysis revealed that exposure to 5-FU downregulated both MDR1 and bcl-2 mRNA and simultaneously upregulated CE2 mRNA expression, suggesting enhancement of subsequent CPT-11 cytotoxicity. The cytotoxic effects of the CPT-11/5-FU combinations were shown to be schedule-dependent in human colon cancer cells. The findings suggest that 5-FU followed by CPT-11 administration might be the optimal sequence for CPT-11/5-FU treatment of advanced colon cancer.

Human carboxylesterase 1A2 expressed from carboxylesterase 1A1 and 1A2 genes is a potent predictor of CPT-11 cytotoxicity in vitro

The conversion of CPT-11 to its active form, SN-38, by carboxylesterases (CESs) is a critical event in CPT-11-induced cytotoxicity. Among the CESs, CES1 and CES2 probably play a major role in the metabolism, but the functional significance and molecular basis of CES1 on CPT-11 response remain unclear. We investigated CES1A1 (AB119997) and CES1A2 (AB187225), whose coding sequences were recently registered in GenBank, for CPT-11-induced cytotoxicity, anticipating novel biomarkers of CPT-11 response. Their coding sequences showed high homology, with only four amino acid differences in the N-terminal region, but our sequencing study of the 5' regions revealed that CES1A1 and CES1A2 had distinctive consensus sequences for transcription factors in the regions, implying differences in transcriptional regulation of the genes. We also identified three isoforms of CES1A1 gene--CES1A1a, CES1A1b and CES1A1c--and developed a detection method for CES1A1 and CES1A2 types of mRNA expression. Interestingly, CES1A2 type of mRNA was found to be expressed from both CES1A1b and CES1A1c isoforms and CES1A2, the promoter activity of the former was higher than that of the original CES1A2 gene. Finally, CES1A2 type of mRNA expression correlated with CPT-11 sensitivities of cancer cells. We demonstrated novel sequence structures and a functional role of CES1A genes in CPT-11 responses. We believe that our novel findings will be of key importance in developing a really useful prediction method for CPT-11 chemosensitivity.

Intracellular inhibition of carboxylesterases by benzil: modulation of CPT-11 cytotoxicity

Carboxylesterases are ubiquitous proteins responsible for the detoxification of xenobiotics. However, these enzymes also activate prodrugs, such as the anticancer agents capecitabine and CPT-11. As a consequence, overexpression of carboxylesterases within tumor cells sensitizes these cells to CPT-11. We have recently identified two classes of carboxylesterase inhibitors based on either a benzil (diphenylethane-1,2-dione) or a benzene sulfonamide scaffold and showed that these compounds inhibit carboxylesterases with Kis in the low nanomolar range. Because both classes of inhibitors show reversible enzyme inhibition, conventional in vitro biochemical assays would not accurately reflect the in situ levels of carboxylesterase activity or inhibition. Therefore, we have developed a novel assay for the determination of intracellular carboxylesterase activity using 4-methylumbelliferone as a substrate. These studies show that benzil and a dimethylbenzil analogue efficiently enter cells and inhibit human intestinal carboxylesterase and rabbit liver carboxylesterase intracellularly. This inhibition results in reduced cytotoxicity to CPT-11 due to the lack of carboxylesterase-mediated conversion of the prodrug to SN-38. These results suggest that intracellular modulation of carboxylesterase activity with benzil or its analogues may be applied to minimize the toxicity of normal cells to CPT-11.

In vitro synergistic antitumor activity of a combination of 5-fluorouracil and irinotecan in human colon cancer

The combination of irinotecan and a fluoro-pyrimidine is widely accepted as a treatment for advanced colorectal cancer. However, evaluable data on the feasibility of these combinations has not been presented, and an optimal sequence for administration has not been experimentally and clinically determined. The sequential effect of a combination of 5-FU and CPT-11 in the human colon cancer cell line LoVo was evaluated by WST-8 colorimetric assay. The cytotoxicity and cell cycle distributions of each drug were analyzed by apoptosis assay and flow cytometry. Further, the potential mechanisms of the sequence-dependent effects were investigated by a microarray technique, and confirmed by Western blot analysis. The cytotoxicity of 5-FU (10, 100, 1000 microM) followed by CPT-11 (1 microM) was significantly greater than that of CPT-11 (1 microM) followed by 5-FU (10, 100, 1000 microM) (p<0.05). In cell cycle distribution, 5-FU exposure for 24 h increased the S phase fraction in a dose-dependent manner; though there was no significant difference in cell cycle distribution in 24 h CPT-11 (0.01-1 microM) exposure. Microarray analysis revealed that expressions of some apoptosis related genes such as Bcl-2 changed, and were correlated with sequence-dependent cytotoxicity of the 5-FU --> CPT-11 sequence. Western blot analysis confirmed that the Bcl-2/Bax ratio was lower after 5-FU --> CPT-11 sequence than before. The sequence-dependent cytotoxic effect may depend on the sensitizing effect of 5-FU pretreatment on CPT-11 cytotoxicity. 5-FU followed by CPT-11 administration may be an optimal sequence for IFL treatment of advanced colon cancer.

The expressions of p21 and pRB may be good indicators for the sensitivity of esophageal squamous cell cancers to CPT-11: Cell proliferation activity correlates with the effect of CPT-11.

Previously, we demonstrated that CPT-11 is an effective agent against esophageal squamous cell cancers (ESCC), and that the protein level of DNA topoisomerase I can be a predictor for sensitivity to CPT-11 (Jpn J Cancer Res 2001; 92: 1335-41). Here, we describe our search for additional predictors of sensitivity to CPT-11, mainly among cell cycle-regulating proteins, because the cytotoxicity of CPT-11 is significantly correlated with the percentage of ESCC cells in S-phase. To this end, we selected and examined the expressions of 5 proteins involved in G1-S transition, i.e., p53, cyclin D1, p21, p27, and pRB, in 14 ESCC cell lines by western blot analysis. Among these proteins, the expression levels of p21 and pRB showed significant differences that were associated with the IC50 values for CPT-11 (P = 0.0339 and P = 0.0109, respectively). Namely, the expression of p21 or pRB independently could be a good indicator of CPT-11 efficacy in ESCC. In addition, the cell proliferation activities examined by enzyme-linked immunosorbent assay (ELISA) using 5-bromo-2'-deoxyuridine (BrdU) showed a significant correlation with the percentage of total S-phase cells (correlation coefficient = 0.568, P = 0.0324), and an inverse correlation with the IC50 values for CPT-11 (correlation coefficient =-0.601, P = 0.0213). Because, as in the case of DNA topoisomerase I, the cell proliferation activity determined using BrdU shows a close relationship with the MIB-1 labeling index, immunohistochemical studies of p21, pRB, and MIB-1 in resected ESCC specimens and/or biopsy samples could make it possible to predict more precisely the sensitivity of ESCC patients to CPT-11 prior to treatment.

Thrombospondin-1 plus irinotecan: a novel antiangiogenic-chemotherapeutic combination that inhibits the growth of advanced human colon tumor xenografts in mice.

Chemotherapy for the treatment of advanced or metastatic colon cancer, utilizing agents such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), produce a 5-year survival of about 10%. Thus, the identification of new, effective, therapeutic regimens to treat this disease remains critically important. To this end, selected antiangiogenic agents, compounds that inhibit neovascularization, have been shown to produce a modest tumor growth-inhibitory effect with little systemic toxicity. Thus these agents are attractive candidates for use with conventional chemotherapeutic agents to treat this disease. To evaluate this approach, experiments were undertaken to assess the cytotoxic and antineoplastic activity of CPT-11 and the antiangiogenic agent thrombospondin-1 (TSP-1) in the HT-29 model of human colon cancer. These agents were chosen since CPT-11 is a camptothecin analogue efficacious in the treatment of colon cancer and TSP-1 is a human glycoprotein that possess antiangiogenic activity. As expected, in vitro studies revealed that a 5-day exposure to TSP-1 at concentrations up to 130 microg/ml was not cytotoxic alone and did not affect the cytotoxicity of CPT-11, or of its active metabolite SN38, in HT-29 cells. Similarly, in human umbilical vein endothelial cells, TSP-1 alone induced only a slight cell growth-inhibitory effect and did not significantly increase the cytotoxicity of either CPT-11 or SN38. The antineoplastic activities of TSP-1 and CPT-11 were assessed in athymic (nude) female mice bearing advanced subcutaneous xenografts of HT-29 cells. Mice received TSP-1 alone (5-40 mg/kg per day) intraperitoneally (i.p.), CPT-11 alone (100-300 mg/kg, i.p.), TSP-1 (10 mg/kg per day) plus CPT-11 (125 mg/kg), or TSP-1 (20 mg/kg per day) plus CPT-11 (150 mg/kg). TSP-1 was injected daily (Monday through Friday) for 4 weeks (20 injections in total) whereas CPT-11 was administered once weekly on days 0, 7, 14 and 21. By day 28, treatment with TSP-1 alone (5, 10 or 20 mg/kg per day) induced a dose-dependent inhibition of xenograft growth. Further, treatment with 10 or 20 mg/kg per day resulted in an average treated tumor size/control tumor size (T/C) on day 28 of 0.68 (range 0.64-0.71) or 0.58 (range 0.54-0.60), respectively. CPT-11 at all doses significantly inhibited tumor growth with an average T/C value of 0.21 (range 0.15-0.27). However, the 250 and 300 mg/kg regimens induced significant toxicity and mortality. When TSP-1 was combined with CPT-11, a significant ( P< or = 0.05) inhibition of tumor growth also was observed (T/C < or = 0.17, range 0.11-0.20). Importantly, this enhanced tumor growth inhibition was obtained without significant toxicity. The therapeutic implications of these findings are discussed.

576 Phase I dose-finding study of the combination of alimta (pemetrexed) and paclitaxel in patients with solid tumors

Objective.Irinotecan (CPT-11) is a DNA topoisomerase I inhibitor which has shown activityin vitroagainst several cancer cell lines and some promising clinical responses in phase I and II trials in various solid tumors. The cytotoxicity of CPT-11 on human ovarian epithelial malignancies was testedin vitroutilizing the ATP chemosensitivity assay. Flow cytometry was also performed on the fresh carcinoma specimens.Methods.Fresh tumor samples were obtained at laparotomy from 20 patients with primary adenocarcinoma of the ovary and 1 patient with heavily pretreated recurrent ovarian carcinoma. Tumors were plated in anin vitrosystem and treated with varying doses of both CPT-11 and its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin), in addition to a panel of standard chemotherapeutic agents used in treating ovarian cancer. On the sixth day after incubation, ATP levels in remaining cells were measured using a luminometer as an indication of the amount of cell kill compared to untreated controls.Results.Twelve of the 21 tumors were evaluable for a response to CPT-11. Of the 12 tumors tested, 7 were found to have median effect doses of CPT-11 significantly under the clinically achievable peak plasma concentration (range 1.00 × 10−4to 44.67 μM) and 11 of the 12 tumors showed sensitivity to the active metabolite SN38 with median effect doses ranging from 2.00 × 10−4to 0.147 μM. Some of these tumors showed resistance to the standard agents, such as platinum, based on our assay. The heavily pretreated recurrent tumor tested was very sensitive to both CPT-11 and SN38 by our assay. The results of flow cytometry showed that the tumors were composed of heterogeneous populations of cells with mean S phase fractions varying from 7.0 to 17.3%. The only tumor not sensitive to SN38 showed the highest mean S phase activity of 17.3%.Conclusions.Overall, these results suggest that CPT-11 deserves further investigation as a clinically useful chemotherapeutic agent for ovarian cancer.

Carboxylesterase-Mediated Sensitization of Human Tumor Cells to CPT-11 Cannot Override ABCG2-Mediated Drug Resistance

The recently introduced camptothecin-derived chemotherapeutic agents have demonstrated remarkable promise in cancer therapy and as such have been approved for use in humans for the treatment of ovarian, lung, and colon cancer. CPT-11 is a prodrug that is activated by esterases to yield the potent topoisomerase I inhibitor, SN-38. Considerable success has been achieved in the treatment of both naïve and drug-resistant colon cancer with CPT-11. However, mechanisms of resistance to this agent have not been explored in detail. The role of the ATP-dependent drug transporter ABCG2 in CPT-11 cytotoxicity is unclear because some ABCG2 mutants confer camptothecin resistance, whereas others do not. Because CPT-11 is activated by carboxylesterases (CEs), we assessed the relative contribution of each protein in mediating CPT-11 toxicity by both drug accumulation and cell growth-inhibition assays. Our results indicate that the expression of ABCG2 protects cells from CPT-11 toxicity, even in the presence of high levels of a rabbit liver carboxylesterase (rCE), which can efficiently activate the drug. However, this can be partially overcome by the ABCG2 inhibitor reserpine. These studies indicate that overexpression of ABCG2 in vivo would probably overcome any increased drug activation that might be achieved by gene delivery or antibody-directed enzyme prodrug therapy methods using rCE.

In VitroSensitivity of Fresh Ovarian Carcinoma Specimens to CPT-11 (Irinotecan)

Objective.Irinotecan (CPT-11) is a DNA topoisomerase I inhibitor which has shown activityin vitroagainst several cancer cell lines and some promising clinical responses in phase I and II trials in various solid tumors. The cytotoxicity of CPT-11 on human ovarian epithelial malignancies was testedin vitroutilizing the ATP chemosensitivity assay. Flow cytometry was also performed on the fresh carcinoma specimens.Methods.Fresh tumor samples were obtained at laparotomy from 20 patients with primary adenocarcinoma of the ovary and 1 patient with heavily pretreated recurrent ovarian carcinoma. Tumors were plated in anin vitrosystem and treated with varying doses of both CPT-11 and its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin), in addition to a panel of standard chemotherapeutic agents used in treating ovarian cancer. On the sixth day after incubation, ATP levels in remaining cells were measured using a luminometer as an indication of the amount of cell kill compared to untreated controls.Results.Twelve of the 21 tumors were evaluable for a response to CPT-11. Of the 12 tumors tested, 7 were found to have median effect doses of CPT-11 significantly under the clinically achievable peak plasma concentration (range 1.00 × 10−4to 44.67 μM) and 11 of the 12 tumors showed sensitivity to the active metabolite SN38 with median effect doses ranging from 2.00 × 10−4to 0.147 μM. Some of these tumors showed resistance to the standard agents, such as platinum, based on our assay. The heavily pretreated recurrent tumor tested was very sensitive to both CPT-11 and SN38 by our assay. The results of flow cytometry showed that the tumors were composed of heterogeneous populations of cells with mean S phase fractions varying from 7.0 to 17.3%. The only tumor not sensitive to SN38 showed the highest mean S phase activity of 17.3%.Conclusions.Overall, these results suggest that CPT-11 deserves further investigation as a clinically useful chemotherapeutic agent for ovarian cancer.

A lymphoma cell line resistant to 4-piperidinopiperidine was less sensitive to CPT-11.

CPT-11 is a promising new anticancer drug in which 4-piperidinopiperidine is a side-chain structure. In the present studies, we examined the role played by 4-piperidinopiperidine in the pharmacological activity of CPT-11. When T-cell lymphoma RVC cells were incubated with 4-piperidinopiperidine at concentrations higher than 50 micrograms/ml, the cells underwent apoptosis with a nucleosomal ladder of chromosomal DNA on agarose gels in a dose-dependent manner. We then established a cell line resistant to 4-piperidinopiperidine (4-pp-R), which was about 20-fold more resistant to 4-piperidinopiperidine than the parent RVC cells. Moreover, 4-pp-R cells showed coresistance to CPT-11. However, the growth rate and cell cycle population of 4-pp-R cells were not different from those of the parent RVC cells, and there were no differences between the two cells lines with regard to their drug transport system. CPT-11-metabolizing activity, their activity and amount of topoisomerase I, or their sensitivity to either SN-38 or etoposide, suggesting that the cytotoxicity of CPT-11 is not a consequence of the activity of its metabolite SN-38. The present studies suggested that resistance to CPT-11 is in part due to insensitivity to 4-piperidinopiperidine and its metabolites, since 4-piperidinopiperidine was cytotoxic and 4-pp-R cells were less sensitive to CPT-11.