Isabelle Hennebelle

Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines

Oxaliplatin (L-OHP) is a new platinum analogue that has shown antitumour activity against colon cancer both in vitro and in vivo and is now used in the chemotherapeutic treatment of metastatic colon and rectal cancer. L-OHP like cisplatin (CDDP), is detoxified by glutathione (GSH)-related enzymes and forms platinum (Pt)-DNA adducts lesions that are repaired by the nucleotide excision repair system (NER). We investigated the cytotoxicity and the pharmacology of L-OHP and CDDP on a panel of six colon cell lines in vitro. We showed that GSH and glutathione S-transferase (GST) activity were not correlated to oxaliplatin cytotoxicity. Pt-DNA adducts formation and repair were correlated with CDDP, but not with L-OHP cytotoxicity. The determination of ERCC1 and XPA expression, two enzymes of the NER pathway, by reverse transcriptase-polymerase chain reaction (RT-PCR), demonstrated that ERCC1 expression was predictive of L-OHP sensitivity (r(2)=0.67, P=0.02) and XPA level after oxaliplatin exposure was also correlated to L-OHP IC(50) (r(2)=0.5; P=0.04). The knowledge of such correlations could help predict the sensitivity of patients with colon cancer to L-OHP.

Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line☆

CPT-11 (irinotecan) is a DNA topoisomerase I inhibitor active against metastatic colorectal carcinoma. We investigated, in a human colon carcinoma cell line, HT-29, the effects of CPT-11 and 5-fluorouracil (5FU) combinations. A strong synergism between CPT-11 and 5FU was observed after sequential exposure and only additivity or antagonism after simultaneous exposure. When cells were first exposed to 5FU, the product of cellular CPT-11 concentrations versus time (CxT) was 6895 +/- 1020 pmol x hr/10(6) cells, while it was 3875 +/- 121 pmol x hr/10(6) cells with CPT-11 alone (p < 0.01). The same phenomenon was observed with SN-38: 148.2 +/- 49.5 versus 83.4 +/- 23.6 pmol x hr/10(6) cells (p < 0.05). Consequently, the formation of protein-DNA complexes was 1.4 times greater with 5FU pretreatment than with CPT-11 alone (p = 0.03). Moreover, the incorporation of 5FU derivatives into DNA was multiplied by a factor of 1.5 24 hr after CPT-11 exposure. When cells were first incubated with CPT-11, the decrease in thymidylate synthase (TS) activity was identical to that obtained after 5FU exposure (1.09 to 0.023 pmol/min/mg protein), but this decrease persisted for 24 hr (0.014 pmol/min/mg protein) (p = 0.035). At the same time, a 1.8-fold increase in the incorporation of 5FU derivatives into DNA and a 2-fold increase in DNA-protein complex formation were evidenced. With the two sequential associations, we observed a persistent S-phase arrest, as compared with CPT-11 alone. These results suggest that CPT-11 and 5FU combinations are of clinical interest, and mechanisms of interaction between the two drugs seem to be multifactorial.

Hospicells (ascites-derived stromal cells) promote tumorigenicity and angiogenesis

The microenvironment is known to play a dominant role in cancer progression. Cells closely associated with tumoral cells, named hospicells, have been recently isolated from the ascites of ovarian cancer patients. Whilst these cells present no specific markers from known cell lineages, they do share some homology with bone marrow‐derived or adipose tissue‐derived human mesenchymal stem cells (CD9, CD10, CD29, CD146, CD166, HLA‐1). We studied the role of hospicells in ovarian carcinoma progression. In vitro, these cells had no effect on the growth of human ovarian carcinoma cell lines OVCAR‐3, SKOV‐1 and IGROV‐1. In vivo, their co‐injection with adenocarcinoma cells enhanced tumor growth whatever the tumor model used (subcutaneous and intraperitoneally established xenografts in athymic mice). In addition, their injection increased the development of ascites in tumor‐bearing mice. Fluorescent macroscopy revealed an association between hospicells and ovarian adenocarcinoma cells within the tumor mass. Tumors obtained by coinjection of hospicells and human ovarian adenocarcinoma cells presented an increased microvascularization indicating that the hospicells could promote tumorigenicity of ovarian tumor cells in vivovia their action on angiogenesis. This effect on angiogenesis could be attributed to the increased HIF1α and VEGF expression associated with the presence of the hospicells. Collectively, these data indicate a role for these ascite‐derived stromal cells in promoting tumor growth by increasing angiogenesis.

Combination of oxaliplatin and irinotecan on human colon cancer cell lines: activity in vitro and in vivo.

The in vitro and in vivo combination of oxaliplatin and irinotecan was investigated in a panel of four human colon cancer cell lines and their counterpart xenografts. In vitro and in vivo experiments demonstrated a synergistic or additive interaction in three cell lines (HCT-116, HCT-8 and HT-29) and an antagonism in SW-620 cells. Since there were clearly opposite interactions depending on the cell line, we further investigated cellular determinants possibly involved in the interaction between the two drugs in HCT-8 and SW-620 cells. Irinotecan slowed down the early platinum-DNA adducts repair (1 h after oxaliplatin exposure) in the presence of irinotecan only in HCT-8 cells (p=0.03, n=3). Moreover, a decrease of the expression of two proteins of the nucleotide excision repair (NER) system, ERCC1 and XPA, was observed. None of these effects was seen in SW-620 cells. Irinotecan induced apoptosis with an increase of poly(ADP-ribose) polymerase (PARP) cleavage in SW-620 cells (60 versus 7% basal level). Pretreatment of these cells with oxaliplatin abolished the increase in PARP cleavage induced by irinotecan (29%). In HCT-8 cells, a very little PARP cleavage was observed whatever the drug treatment. The persistence of platinum-DNA adducts in the presence of irinotecan could be due to a direct impact of irinotecan on NER gene expression or to an indirect effect on topoisomerase I activity. Complementary studies are required to determine if the cellular parameters identified in this study could be translated at the clinical level to predict clinical response after combined treatment with oxaliplatin and irinotecan in humans.

Population Analysis of Erlotinib in Adults and Children Reveals Pharmacokinetic Characteristics as the Main Factor Explaining Tolerance Particularities in Children

Purpose: The aim of this pharmacokinetic–pharmacodynamic (PK–PD) analysis was to evaluate the pharmacologic characteristics of erlotinib and its main metabolite (OSI-420) in pediatric patients compared with those in adult patients. Experimental Design: Plasma concentrations of erlotinib and OSI-420 of 46 children with malignant brain tumors included in a phase I study and 42 adults with head and neck carcinoma were analyzed by a population-pharmacokinetic method (NONMEM). The effect of several covariates and single nucleotide polymorphisms (SNP) in ABCB1, ABCG2, and CYP3A5 on pharmacokinetic parameters was evaluated. PK/PD relationships between plasma drug exposure Area Under the Curve (AUC) at day 1 and skin toxicity were studied in children and compared with the relationship observed in adults. Results: A significant difference in erlotinib clearance (P = 0.0001), when expressed in L·h−1·kg−1, was observed between children and adults with mean values of 0.146 and 0.095, respectively (mean difference = 0.051 L·h−1·kg−1, SD = 0.0594). However, a common covariate model was obtained describing erlotinib clearance according to body weight, alanine aminotransferase, ABCB1, and CYP3A5 polymorphisms (2677G > T/A and 6986G > A) for both children and adult patients. The PK–PD relationship was very consistent between the children and adult groups with risk of skin toxicity rising with increasing erlotinib AUC. Conclusions: The nonlinear population approach applied to pharmacokinetic data combined with a pharmacokinetic–pharmacodynamic analysis revealed that the higher recommended dose in children (125 mg/m2/day) compared with adults (90 mg/m2/day) is mainly due to pharmacokinetic rather than pharmacodynamic particularities. Clin Cancer Res; 17(14); 4862–71. ©2011 AACR.

Contribution of apoptosis in the cytotoxicity of the oxaliplatin–irinotecan combination in the HT29 human colon adenocarcinoma cell line

Interactions between the topoisomerase I inhibitor irinotecan (CPT-11) and the platinum derivative oxaliplatin (L-OHP) were investigated in HT29 colon cancer cell line. Synergism was observed when cells were simultaneously exposed to drugs or when cells were first exposed to CPT-11. Flow cytometric studies showed a G(2)/M accumulation when cells were exposed to the simultaneous and CPT-11-->L-OHP combinations whereas a persistent S phase delay was observed when cells were first exposed to L-OHP. We characterised the cytotoxic effect by assessing the induction of apoptosis. Irinotecan induced substantial DEVDase activity and poly(ADP-ribose) polymerase cleavage while this activity was moderate and delayed after exposure to L-OHP. Combination experiments showed a sequence-dependent onset of apoptosis, the CPT-11-->L-OHP schedule being the earliest and the most effective; on the other hand the apoptotic signaling generated by CPT-11 was partly inhibited in the simultaneous combination and in the L-OHP-->CPT-11 sequence. Cell death studies using a dual staining technique showed a shift from apoptosis to necrosis when combining these drugs at high concentrations. Synergistic interactions observed using CPT-11 before L-OHP may be linked to an early apoptotic signaling while the L-OHP-induced S phase block could account for the observed additive effect in the reverse sequence. An additional phenomenon might work towards synergism for the simultaneous combination.

Ovarian ascites-derived Hospicells promote angiogenesis via activation of macrophages

Within the microenvironment, Carcinoma-associated mesenchymal stem cells (Hospicells) are able to influence ovarian tumor development via, among others, the facilitation of angiogenesis in the tumor site allowing an accelerated tumor growth. We demonstrate the presence of a chemotactism between endothelial cells and Hospicells, and a cell line specific increased secretion of pro-angiogenic cytokines such as IL-6, IL-8 and VEGF from ovarian adenocarcinoma cells. Hospicells are also able to attract and activate macrophages to a M2 phenotype and allow them to secrete a huge quantity of pro-angiogenic cytokines, favorable to tumor progression of all the associated ovarian adenocarcinoma cells tested.

Characterization of CPT-11 converting carboxylesterase activity in colon tumor and normal tissues: comparison with p-nitro-phenylacetate converting carboxylesterase activity.

Irinotecan (CPT-11) is a topoisomerase I inhibitor commonly used in the treatment of colorectal tumors. It is a prodrug, converted to an active metabolite, SN-38, by carboxylesterases (CEs). CEs are ubiquitary enzymes that react with numerous substrates. A specific CPT-11 converting enzyme was isolated from rat serum, with different kinetic properties than other CEs. We determined kinetic properties of specific CPT-11 CE activity (CPT-CE) in human normal liver and colon tumors. Km were very similar (3.4 μM in liver and 3.8 μM in colon tumors), but Vmax was higher in liver (2.7 pmol/min/mg protein) than in colon tumor (1.7 pmol/min/mg protein). CPT-CE and total CE (using p-nitro-phenylacetate as substrate) were weakly correlated in colon tumors. The large interpatient variability observed in liver CPT-CE activity could play a potential role in the pharmacokinetic variability observed with irinotecan.

Unexpected High Levels of Vorinostat when Combined with Vinorelbine in Patients with Advanced Cancer

BACKGROUND This study was a multi-centre, dose-escalation trial in patients with advanced cancers. Primary objective was to determine maximum tolerated dose (MTD) of vorinostat, a competitive inhibitor of histone deacetylase (HDAC), in combination with vinorelbine. Secondary aims were to determine (1) corresponding pharmacokinetics, (2) safety of this regimen, and (3) impact of UGT1A1 and 2B17 polymorphisms on vorinostat pharmacokinetics. METHODS Starting dose of once daily oral vorinostat was 200 mg for 7 days every 21 days in combination with a 20-min intraveinous weekly infusion of vinorelbine 25 mg/m2, starting 4 hours after the first vorinostat dose. During cycle 1, blood samples were collected at day 1 for vorinostat and at days 1 and 8 for vinorelbine for pharmacokinetic evaluation. RESULTS Seven patients were included. Most of adverse events observed were mild (grades 0-2) and reversible after treatment discontinuation (hemotological toxicity, asthenia, diarrhea, dyspnea, fever, hyperglycemia and nausea). Two patients had a dose limiting toxicity at the first dose level that consisted of grade 3 hyperglycemia and vinorelbine administration was delayed. The first dose-level was considered as the MDT and therefore dose escalation was stopped. Mean vorinostat plasma AUC was higher than reported previously at a similar dose when used as single agent or in combination with other cytotoxics. There was no obvious vinorelbine-vorinostat interaction nor any correlation with UGT1A1 or 2B17 polymorphisms. CONCLUSION MDT of the combination was 200 mg oral vorinostat for 7 days in combination with 25 mg/m2 weekly vinorelbine. Severity of hyperglycemia was most likely related to unexpected high vorinostat exposures.

Irreversible hepatotoxicity after administration of trabectedin to a pleiomorphic sarcoma patient with a rare ABCC2 polymorphism: a case report

We describe here the case of a 60-year old male patient treated for an extensive local progression of a pleiomorphic sarcoma on the right tibial crest with second-line trabectedin. Two cycles were administrated before a major liver toxicity was retrieved, with both cytolytic and cholestatic hepatitis quickly associated with irreversible jaundice. The radiological, histological, chemistry and pharmacogenetic investigations led us to diagnose chronic hepatobiliary toxicity with portal fibrosis, cholangiolitis damages and chronic hepatopathy. The patient had a deficient variant genotype of ABCC2 (c.-24TT, c.4488CT and c.4544GA), which has been suggested to play a role in excretion of toxic metabolites of trabectedin. This case report is, to our knowledge, the first description of trabectedins irreversible liver toxicity in a human patient. Supported by a thorough review of the literature, this hepatitis is thought to have resulted from a multihit process involving genetic variants of ABC proteins and comedication.