Cytotoxicity Of SN-38 Research Articles

Roles of breast cancer resistance protein and organic anion transporting polypeptide 2B1 in gastrointestinal toxicity induced by SN-38 under inflammatory conditions

Drug transporters are among the factors that determine the pharmacokinetic profiles after drug administration. In this study, we investigated the roles of drug transporters involved in transport of SN-38, which is an active metabolite of irinotecan, in the intestine under inflammatory conditions in vitro and determined their functional consequences. The expression alterations of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 2B1 were determined at the mRNA and protein levels, and the subsequent functional alterations were evaluated via an accumulation study with the representative transporter substrates [prazosin and dibromofluorescein (DBF)] and SN-38. We also determined the cytotoxicity of SN-38 under inflammatory conditions. Decreased BCRP expression and increased OATP2B1 expression were observed under inflammatory conditions in vitro, which led to altered accumulation profiles of prazosin, DBF, and SN-38, and the subsequent cytotoxic profiles of SN-38. Treatment with rifampin or novobiocin supported the significant roles of BCRP and OATP2B1 in the transport and cytotoxic profile of SN-38. Collectively, these results suggest that BCRP and OATP2B1 are involved in the increased cytotoxicity of SN-38 under inflammatory conditions in vitro. Further comprehensive research is warranted to completely understand SN-38-induced gastrointestinal cytotoxicity and aid in the successful treatment of cancer with irinotecan.

Downregulation of breast cancer resistance protein by long-term fractionated radiotherapy sensitizes lung adenocarcinoma to SN-38.

Chemotherapy is usually the subsequent treatment for non-small cell lung cancer patients with acquired radioresistance after long-term fractionated radiotherapy. However, few studies have focused on the selection of chemotherapeutic drugs to treat lung adenocarcinoma patients with radioresistance. Our study compared the sensitivity changes of lung adenocarcinoma cells to conventional chemotherapeutic drugs under radioresistant circumstances by using three lung adenocarcinoma cell models, which were irradiated with fractionated X-rays at a total dose of 60Gy. The results showed that the toxicities of paclitaxel, docetaxel and SN-38 were increased in radioresistant cells. The IC50 values of docetaxel and SN-38 decreased 0 ~ 3 times and 3 ~ 36 times in radioresistant cells, respectively. Notably, the A549 radioresistant cells were approximately 36 times more sensitive to SN-38 than the parental cells. Further results revealed that the downregulation of the efflux transporter BCRP by long-term fractionated irradiation was an important factor contributing to the increased cytotoxicity of SN-38. In addition, the reported miRNAs and transcriptional factors that regulate BCRP did not participate in the downregulation. In conclusion, these results presented important data on the sensitivity changes of lung adenocarcinoma cells to chemotherapeutic drugs after acquiring radioresistance and suggested that irinotecan (the prodrug of SN-38) might be a promising drug candidate for lung adenocarcinoma patients with acquired radioresistance.

MRP3-Mediated Chemoresistance in Cholangiocarcinoma: Target for Chemosensitization Through Restoring SOX17 Expression.

A limitation for the treatment of unresectable cholangiocarcinoma (CCA) is its poor response to chemotherapy, which is partly due to reduction of intracellular levels of anticancer drugs through ATP-binding cassette (ABC) pumps. Low expression of SOX17 (SRY-box containing gene 17), a transcription factor that promotes biliary differentiation and phenotype maintenance, has been associated with cholangiocyte malignant transformation. Whether SOX17 is also involved in CCA chemoresistance is investigated in this study. SOX17 expression in human CCA cells (EGI-1 and TFK-1) selectively potentiated cytotoxicity of SN-38, 5-fluorouracil and mitoxantrone, but not that of gemcitabine, capecitabine, cisplatin, or oxaliplatin. The analysis of the resistome by TaqMan low-density arrays revealed changes affecting primarily ABC pump expression. Single-gene quantitative real-time PCR, immunoblot, and immunofluorescence analyses confirmed that MRP3 (multidrug resistance associated protein 3), which was highly expressed in CCA human tumors, was down-regulated in SOX17-transduced CCA cells. The substrate specificity of this pump matched that of SOX17-induced in vitro selective chemosensitization. Functional studies showed lower ability of SOX17-expressing CCA cells to extrude specific MRP3 substrates. Reporter assay of MRP3 promoter (ABCC3pr) revealed that ABCC3pr activity was inhibited by SOX17 expression and SOX2/SOX9 silencing. The latter was highly expressed in CCA. Moreover, SOX2/9, but not SOX17, induced altered electrophoretic mobility of ABCC3pr, which was prevented by SOX17. The growth of CCA tumors subcutaneously implanted into immunodeficient mice was inhibited by 5-fluorouracil. This effect was enhanced by co-treatment with adenoviral vectors encoding SOX17. SOX9/2/17 are involved in MRP3-mediated CCA chemoresistance. Restored SOX17 expression, in addition to its tumor suppression effect, induces selective chemosensitization due to MRP3 down-regulation and subsequent intracellular drug accumulation.

The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma.

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.

Layered superhydrophobic meshes for controlled drug release

Layered superhydrophobic electrospun meshes composed of poly(ε-caprolactone) (PCL) and poly(glycerol monostearate-co-ε-caprolactone) (PGC-C18) are described as a local source of chemotherapeutic delivery. Specifically, the chemotherapeutic agent SN-38 is incorporated into a central ‘core’ layer, between two ‘shield’ layers of mesh without drug. This mesh is resistant to wetting of the surface and throughout the bulk due to the pronounced hydrophobicity imparted by the high roughness of a hydrophobic polymer, PGC-C18. In serum solution, these meshes exhibit slow initial drug release over 10days corresponding to media infiltrating the shield layer, followed by steady release over >30days, as the drug-loaded core layer is wetted. This sequence of events is supported by X-ray computed tomography imaging of a contrast agent solution infiltrating the mesh. In vitro cytotoxicity data collected with Lewis Lung Carcinoma (LLC) cells are consistent with this release profile, remaining cytotoxic for over 20days, longer than the unlayered version. Finally, after subcutaneous implantation in rats, histology of meshes with and without drug demonstrated good integration and lack of adverse reaction over 28days. The drug release rates, robust superhydrophobicity, in vitro cytotoxicity of SN-38 loaded meshes, and compatibility provide key design parameters for the development of an implantable chemotherapeutic-loaded device for the prevention of local lung cancer recurrence following surgical resection.

Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewing's family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression.

To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI). Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting. Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines. In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.

Abstract 2274: RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines

Abstract BACKGROUND: TNF is a dual role cytokine involved both in cell death and survival that is expressed in the tumor microenvironment. However, little is known about the molecular events that mark the biological outcomes of such opposite biological events. Recently, the protein kinase RIP1 has been identified as a new intermediate involved in the responses activated after DNA damage induction. PURPOSE: In this study we investigated how TNF modulates the sensitivity to SN38 in colon cancer cell lines. We focused further on HT29- and HCT116-derived cell lines in which RIP1 or RIP3 proteins expression were stably downregulated. RESULTS: We found that TNF increases cellular sensitivity to different chemotherapeutic drugs under both normoxic and hypoxic conditions over a panel of different colon adenocarcinoma cell lines. Combinations with SN38 were highly synergistic. In HT29 and HCT116 cell lines (TNF resistant and TNF sensitive respectively), we analyzed this interaction by studying P-H2-AX induction, cell proliferation (clonogenic assays and BrdU incorporation) and cell cycle distribution. To study the role of RIP1 and RIP3 on the biological responses triggered by SN38, HT29- and HCT116-derived cell lines expressing low levels of RIP1 and RIP3 were analyzed. While knockdown of RIP3 had no effect on SN38 cytotoxicity, RIP1 down regulation produced a 2.5-fold increased resistance to SN38 under hypoxic conditions. This change in SN38 sensitivity also correlated with a reduction in the levels of P-H2-AX in the presence of TNF, suggesting dependence of DNA damage induction by SN38 on RIP1. CONCLUSIONS: These data suggest that RIP1 may be a key intermediate in determining cell death following SN38 treatment. Additional studies in tumor samples are in progress. Citation Format: Lucia Cabal-Hierro, Peter J. O'Dwyer. RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2274. doi:10.1158/1538-7445.AM2014-2274

Abstract 3972: Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewings family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression

Abstract Introduction: Pharmacokinetic properties of irinotecan limit tumor exposure to the active metabolite SN-38. MM-398, a nanoliposomal irinotecan (nal-IRI) developed to attempt to enhance delivery of irinotecan and SN-38 to tumor cells is being tested in clinical trials. We assessed the pharmacokinetics and anti-tumor activity of nal-IRI in comparison to the current clinical formulation of irinotecan (free irinotecan) in preclinical models of pediatric cancer in relationship to expression of the putative DNA/RNA helicase SLFN11. Experimental Design: Plasma and tissue samples were analyzed by liquid chromatography-tandem mass spectrometry for irinotecan and SN-38. In vivo activity of free irinotecan and nal-IRI was compared in subcutaneous xenograft models (3 each) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 mRNA expression (real-time RT-PCR) was correlated to in vitro cytotoxicity of SN-38 (by DIMSCAN) in 20 EWS, 13 RMS, and 20 NB cell lines. SLFN11 mRNA expression in primary tumors (10 EFT, 10 NB, and 39 RMS) was assessed by Affymetrix HuEx microarrays. Results: Plasma and tumor concentrations of irinotecan and SN-38 were ∼ 10-fold higher for nal-IRI than for free irinotecan at 4h, 6h, and 24h post-intravenous injection (p<0.01). In 7 of 9 xenograft models, nal-IRI significantly extended event-free survival (EFS) compared to controls, and for 6 of 9 models compared to irinotecan. Combining the 3 EFT models tested, 23 of 25 nal-IRI treated mice achieved complete responses for > 100 days (88% EFS on day 120). EFS for mice with RMS (median 39 days) and NB (median 81 days) was significantly shorter (P<0.001) than for EFT. SN-38 exposure in tumors from nal-IRI may be due to tumor-associated macrophage accumulation, but neither tumor infiltration of macrophages nor tumor SN-38 levels showed significant differences between EFT and RMS or NB xenografts. SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents and the median SLFN11 mRNA expression was > 100-fold higher in EFT cell lines and > 10-fold in EFT primary tumors compared with NB or RMS cell lines or primary tumors (P<0.0001). Relative cytotoxic/inhibitory concentrations (rIC50) of SN-38 showed a significant inverse correlation with SLFN11 expression in 20 EWS cell lines (r=-0.63, P=0.004). Conclusions: Nal-IRI demonstrated higher anti-tumor activity than free irinotecan in pediatric solid tumor xenografts, presumably due to increased systemic and tumor exposures to the SN-38 active metabolite. The high SLFN11 expression reported in EFT cell lines and the strong nal-IRI activity we have observed in EFT xenografts warrants testing in correlative clinical studies to determine if SLFN11 can reliably be used as a biomarker to predict nal-IRI activity in patients. Citation Format: Min H. Kang, Michael M. Song, Monish Makena, Joo-Sang Lee, Connor P. Hall, Ashly Hindle, Winford Ko, Nancy Paz, Jonathan Fitzgerald, Daryl C. Drummond, Timothy J. Triche, C. Patrick Reynolds. Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewings family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2014-3972

The Use of Olaparib (AZD2281) Potentiates SN-38 Cytotoxicity in Colon Cancer Cells by Indirect Inhibition of Rad51-Mediated Repair of DNA Double-Strand Breaks

Potent application of topoisomerase I inhibitor plus PARP inhibitor has been suggested to be an effective strategy for cancer therapy. Reportedly, mismatch repair (MMR)-deficient colon cancer cells are sensitive to topoisomerase I inhibitor, presumably due to microsatellite instability (MSI) of the MRE11 locus. We examined the synergy of SN-38, an active metabolite of irinotecan, in combination with the PARP inhibitor olaparib in colon cancer cells showing different MMR status, such as MSI or microsatellite stable (MSS) phenotype. Treatment with SN-38 and olaparib in combination almost halved the IC50 of SN-38 for a broad spectrum of colon cancer cells independent of the MMR status. Furthermore, olaparib potentiated S-phase-specific double-strand DNA breaks (DSB) induced by SN-38, which is followed by Rad51 recruitment. siRNA-mediated knockdown of Rad51, but not Mre11 or Rad50, increased the sensitivity to olaparib and/or SN-38 treatment in colon cancer cells. In vivo study using mouse xenograft demonstrated that olaparib was effective to potentiate the antitumor effect of irinotecan. In conclusion, olaparib shows a synergistic effect in colon cancer cells in combination with SN-38 or irinotecan, potentiated by the Rad51-mediated HR pathway, irrespective of the Mre11-mediated failure of the MRN complex. These results may contribute to future clinical trials using PARP inhibitor plus topoisomerase I inhibitor in combination. Furthermore, the synergistic effect comprising topoisomerase I-mediated DNA breakage-reunion reaction, PARP and Rad51-mediated HR pathway suggests the triple synthetic lethal pathways contribute to this event and are applicable as a potential target for future chemotherapy.

SN-38-Cyclodextrin Complexation and Its Influence on the Solubility, Stability, and In Vitro Anticancer Activity Against Ovarian Cancer

SN-38, an active metabolite of irinotecan, is up to 1,000-fold more potent than irinotecan. But the clinical use of SN-38 is limited by its extreme hydrophobicity and instability at physiological pH. To enhance solubility and stability, SN-38 was complexed with different cyclodextrins (CDs), namely, sodium sulfobutylether β-cyclodextrin (SBEβCD), hydroxypropyl β-cyclodextrin, randomly methylated β-cyclodextrin, and methyl β-cyclodextrin, and their influence on SN-38 solubility, stability, and in vitro cytotoxicity was studied against ovarian cancer cell lines (A2780 and 2008). Phase solubility studies were conducted to understand the pattern of SN-38 solubilization. SN-38-βCD complexes were characterized by differential scanning calorimetry (DSC), X-ray powder diffraction analysis (XRPD), and Fourier transform infrared (FTIR). Stability of SN-38-SBEβCD complex in pH 7.4 phosphate-buffered saline was evaluated and compared against free SN-38. Phase solubility studies revealed that SN-38 solubility increased linearly as a function of CD concentration and the linearity was characteristic of an AP-type system. Aqueous solubility of SN-38 was enhanced by about 30-1,400 times by CD complexation. DSC, XRPD, and FTIR studies confirmed the formation of inclusion complexes, and stability studies revealed that cyclodextrin complexation significantly increased the hydrolytic stability of SN-38 at physiological pH 7.4. Cytotoxicity of SN-38-SBEβCD complex was significantly higher than SN-38 and irinotecan in both A2780 and 2008 cell lines. Results suggest that SBEβCD encapsulated SN-38 deep into the cavity forming stable inclusion complex and as a result increased the solubility, stability, and cytotoxicity of SN-38. It may be concluded that preparation of inclusion complexes with SBEβCD is a suitable approach to overcome the solubility and stability problems of SN-38 for future clinical applications.

Abstract 3353: Effect of erlotinib on the pharmacokinetics of irinotecan and its metabolites: A drug-drug interaction study.

Abstract Purpose: Since irinotecan and erlotinib are used in treatment for non-small cell lung cancer (NSCLC), co-administration of these drugs might be an effective treatment for NSCLC. However, drug-drug interaction should be concerned because these drugs are metabolized by CYP3A4 and excreted into bile by ABC transporters. To clarify the drug-drug interaction when these drugs are co-administered, we examined the effect of this co-administration on the pharmacokinetics of irinotecan, its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), SN-38 glucuronide (SN-38G) in animal model. Methods: Cytotoxicity of irinotecan and SN-38 with erlotinib against NSCLC cell line (A549) was assessed. To elucidate the effect of drug-drug interaction between irinotecan and erlotinib, male Sprague-Dawley rats were treated with 10 mg/kg irinotecan and 25 mg/kg erlotinib. The concentrations of irinotecan, SN-38, SN-38G and erlotinib in plasma, bile, and tissues were analyzed by high-performance liquid chromatography. Pharmacokinetic parameters were calculated by non-compartmental model methods. Results: Co-exposured erlotinib enhanced the cytotoxicity of SN-38 in a dose-dependent manner, however it did not significantly affect the cytotoxicity of irinotecan against A549 that is resistant to erlotinib. Additionally, co-administration of erlotinib resulted in a significant increase of plasma concentration of irinotecan, SN-38 and SN-38G compared to irinotecan alone, but decrease in the biliary excretion. Erlotinib significantly increased the concentration of irinotecan and SN-38G in kidney and that of SN-38 in lung and jejunum. On the other hand, co-administration of irinotecan intended to decrease the plasma concentration of erlotinib but significant differences of pharmacokinetic parameters were not observed. Also, levels of erlotinib in tissues were not affected by co-administration of irinotecan. Conclusions: We proposed the possibility of combination therapy of irinotecan with erlotinib because the plasma concentration of irinotecan and its active metabolite SN-38 significantly increased in vivo and the cytotoxicity of SN-38 against NSCLC cell line enhanced. New effective salvage chemotherapy regimens are needed in patients with NSCLC. We conducted a phase I/II clinical study of combination chemotherapy with irinotecan and erlotinib for recurrent NSCLC because our data have shown synergistic/additive effects between irinotecan and epidermal growth factor receptor-tyrosine kinase inhibitor. Citation Format: Emiko Sanematsu, Issei Toyooka, Yuki Takashima, Tamio Okimoto, Yukari Tsubata, Hideyuki Saito, Takeshi Isobe, Akinobu Hamada. Effect of erlotinib on the pharmacokinetics of irinotecan and its metabolites: A drug-drug interaction study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3353. doi:10.1158/1538-7445.AM2013-3353

Low-dose SN-38 with paclitaxel induces lethality in human uterine cervical adenocarcinoma cells by increasing caspase activity

Combination of anticancer drugs may provide a rational molecular basis for novel chemotherapeutic strategies. Paclitaxel and SN-38 (an active metabolite of CPT-11) are effective for many kinds of cancer. Therefore, we investigated the possibility that combination of these drugs could be effective against cervical adenocarcinoma cells. In this study, we examined cell growth inhibition after 96h using the MTT assay and examined the release of fragmented DNA into the cytoplasm during apoptotic cell death by PI staining. Single and combined use of paclitaxel and SN-38 produced significant cytolethality against the cervical adenocarcinoma cell line CAC-1. Addition of a low concentration of SN-38 reduced the IC50 value of paclitaxel compared to that without SN-38, although the low concentration of paclitaxel did not enhance the cytotoxicity of SN-38. FACS scan analysis suggested that these drugs induced apoptosis and cell cycle arrest, and that caspase-3 and -7 were activated in the process. MTT assay and the IC50 demonstrated that paclitaxel had strong cytotoxicity against CAC-1 as well as other cancer cells. In this study, though only a single cell line was used for the experiment and the data are limited, our results suggest that paclitaxel together with low-dose CPT-11 is a promising basis for a new combination cancer chemotherapy.

Bicarbonate Attenuates Irinotecan-Induced Cytotoxicity through Regulation of Both Extracellular and Intracellular <i>pHs</i> in Intestine Cell Line

The anti-cancer therapy of irinotecan (CPT-11) is often limited due to severe late-onset diarrhea. Because the higher toxic form of CPT-11/its active metabolite (SN-38) is produced at acidification, the usefulness of oral sodium bicarbonate treatment against the CPT-11/SN-38-induced intestinal injuries and diarrhea has been confirmed. However, the roles of bicarbonate have been suggested to affect not only intestinal pH environment but also intracellular pH and CPT-11/SN-38 dynamics. The present study proposed to clarify the hypothesis in CPT-11/SN-38-exposed colon cell line in various pH conditions adjusted by bicarbonate. HT29 cell pre-exposed to ~1.0 μM SN-38 lactone or carboxylate forms was incubated at different pH adjusted by either bicarbonate or HCl/NaOH. The degrees of SN-38-induced cell injury depended on the higher proportion of the toxic form (lactone) of SN-38 rather than mere pH condition of medium. Apoptosis and cell injury induced by SN-38 were significantly inhibited by bicarbonate in a dose-dependent manner. Intercellular pH acidification induced by SN-38 was significantly prevented by 30 mM bicarbonate. Cell cytotoxicity of SN-38 depended on not only extracellular but also intracellular pH that converts the SN-38 form, while the intracellular acidification was prevented by bicarbonate. The multiple regulations of bicarbonate on both exracellular and intracellular pH would be essential mechanism against intestinal cell injury by CPT-11/SN-38.

Therapeutic potential and molecular mechanism of a novel sulfonamide anticancer drug, indisulam (E7070) in combination with CPT-11 for cancer treatment

Indisulam (N-(-3-chloro-7-indolyl)-1,4-benzenedisulfonamide; E7070) is an experimental anticancer agent. Microarray analysis indicates that indisulam downregulates several genes involved in drug resistance, and this finding led us to test the effect of combining indisulam with other anticancer drugs. We investigated the antitumor effect and mechanism of synergism when indisulam was administered in combination with CPT-11. In vitro cytotoxic activity was examined using a cell counter kit, and the combination effect was determined by isobologram analysis. The level of topoisomerase IIα was measured by Western blotting. The in vivo antitumor effect was assessed in mice inoculated with human colorectal cancer SW620 cells. Isobologram analysis indicated that a 24-h exposure to indisulam and SN-38, an active metabolite of CPT-11, had a synergistic effect in HCT116 and SW620 cells and an additive effect in HCT15 and WiDr cells. Prolongation of exposure to 48 h resulted in a synergistic effect in HCT15 and WiDr cells. Treatment with SN-38 alone increased the amount of intracellular topoisomerase IIα in all cell lines tested. Co-treatment with indisulam suppressed the SN-38-induced upregulation of topoisomerase IIα after 24 h of exposure in HCT116 and SW620 cells and after 48 h of exposure in HCT15 and WiDr cells. This apparent association between a synergistic effect and suppression of SN-38-mediated upregulation of topoisomerase IIα suggests that indisulam enhances SN-38 cytotoxicity by suppressing topoisomerase IIα upregulation to compensate for topoisomerase I inhibition by SN-38. Synergy was also observed in xenografted tumors and was accompanied by complete suppression of topoisomerase IIα upregulation induced by CPT-11 treatment. These observations prompted the clinical evaluation of indisulam and CPT-11 combination therapy.

Novel Acrylonitrile Derivatives, YHO-13177 and YHO-13351, Reverse BCRP/ABCG2-Mediated Drug ResistanceIn VitroandIn Vivo

Breast cancer resistance protein (BCRP/ABCG2) confers resistance to anticancer drugs such as 7-ethyl-10-hydroxycamptothecin (SN-38, an active metabolite of irinotecan), mitoxantrone, and topotecan. In this study, we examined the reversing effects of YHO-13177, a novel acrylonitrile derivative, and its water-soluble diethylaminoacetate prodrug YHO-13351 on the BCRP-mediated drug resistance. YHO-13177 potentiated the cytotoxicity of SN-38, mitoxantrone, and topotecan in both BCRP-transduced human colon cancer HCT116 (HCT116/BCRP) cells and SN-38-resistant human lung cancer A549 (A549/SN4) cells that express BCRP, but had little effect in the parental cells. In addition, YHO-13177 potentiated the cytotoxicity of SN-38 in human lung cancer NCI-H460 and NCI-H23, myeloma RPMI-8226, and pancreatic cancer AsPC-1 cells that intrinsically expressed BCRP. In contrast, it had no effect on P-glycoprotein-mediated paclitaxel resistance in MDR1-transduced human leukemia K562 cells and multidrug resistance-related protein 1-mediated doxorubicin resistance in MRP1-transfected human epidermoid cancer KB-3-1 cells. YHO-13177 increased the intracellular accumulation of Hoechst 33342, a substrate of BCRP, at 30 minutes and partially suppressed the expression of BCRP protein at more than 24 hours after its treatment in both HCT116/BCRP and A549/SN4 cells. In mice, YHO-13351 was rapidly converted into YHO-13177 after its oral or intravenous administration. Coadministration of irinotecan with YHO-13351 significantly increased the survival time of mice inoculated with BCRP-transduced murine leukemia P388 cells and suppressed the tumor growth in an HCT116/BCRP xenograft model, whereas irinotecan alone had little effect in these tumor models. These findings suggest that YHO-13351, a prodrug of YHO-13177, could be clinically useful for reversing BCRP-mediated drug resistance in cancer chemotherapy.

Abstract 3660: Bicarbonate attenuates irinotecan-induced cytotoxicity by altering intracellular pH in HT29 cells

Abstract [Background/Aim] The therapeutic efficacy of irinotecan (CPT-11) is often limited by the induction of severe late-onset diarrhea. CPT-11 and its active metabolite (SN-38) have a labile α-hydroxy-lactone ring that undergoes pH-dependent reversible hydrolysis, and the toxicity is more potent in the lactone (LA) form at acidic pH compared to that in the carboxylate (CA) form in physiological and higher pH. We have previously demonstrated that sodium bicarbonate (Bicarb) reduced the intestinal toxicity of CPT-11/SN-38 in in vivo, in a mouse model, and in a cultured intestinal cancer (HT29) cell line. The hypothesis was that protective action of Bicarb might be due, at least in part, to reduced CPT-11/SN-38-induced intracellular (pHi) acidification. [Method] HT29 cells were exposed to ∼1µM SN-38 LA or CA form for 2h and further incubated in fresh medium at two different extracellular pH (6.2 and 7.4) adjusted by HCl with 44 mM Bicarb for an additional 96h. In addition, the HT29 cells exposed to SN-38 LA were also incubated at 5-set points of extracellular pH at 6.8, 7.0, 7.4, 7.8, 8.2 adjusted by either increasing concentrations of Bicarb (5, 10, 20, 44, 100mM, respectively) or HCl/NaOH in combination with Trice, Tris, and NaH2PO4 to maintain extracellualr pH during culture. The cell proliferation and apoptosis were examined by MTT assay and Flow Cytometry, respectively. The pHi following SN-38 exposure was also analyzed using Snarf-1 with two concentrations (10 and 30mM) of Bicarb. [Result] The cell proliferation was significantly decreased on exposure to SN-38 LA form compared to the CA form, while there was no difference in the lower cell growth on exposure to LA form between pH 6.2 and 7.4. In the SN-38 exposed cells, the cell proliferation and apoptosis were significantly higher and lower, respectively, dependent on the concentration of Bicarb. However, the decreased cell viabilities were not influenced by extracellular pH without Bicarb. The SN-38 LA exposure induced a significant decrease of pHi, and the incubation with 30mM Bicarb significantly prevented from the acidification of pHi compared to that in 10mM Bicarb. [Conclusion] The present study showed that cell cytotoxicity of SN-38 was independent of extracellular pH but dependent on pHi that converts the SN-38 form, while the acidification of pHi was reduced by Bicarb. The novel protective mechanism of Bicarb action against intestinal cell injury by CPT-11/SN-38 would be beneficial for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3660.

Vorinostat enhances the cytotoxic effects of the topoisomerase I inhibitor SN38 in glioblastoma cell lines

Histone deacetylase (HDAC) inhibitors represent a promising class of anti-cancer agents that are actively being evaluated in the context of clinical trials in solid tumors, including glioblastoma. What makes these agents particularly attractive is their capacity to enhance the activity of commonly used cytotoxics in cancer therapy, including both chemotherapy and ionizing radiation. As recent investigations suggest HDAC inhibitors may potentiate the cytotoxicity of topoisomerase inhibitors, which continue to be a commonly used class of agents in the treatment of glioblastoma, we performed preclinical studies to determine if this combination may be a promising strategy in glioblastoma. The effects of the HDAC inhibitor vorinostat and SN38, which is the active metabolite of the topoisomerase I inhibitor CPT-11, was evaluated using the clonogenic assay. Various treatment schedules were tested to determine optimum drug sequencing. Induction of DNA double strand breaks (DSBs) with the combination of vorinostat and SN38 was evaluated using the neutral comet assay, and their subsequent repair was evaluated by gammaH2AX foci kinetics using immunofluorescent cytochemistry. Vorinostat enhanced the cytotoxicity of SN38 in glioblastoma cell lines. Optimal treatment schedules involved maximal concurrent administration of agents. Pretreatment with either agent did not enhance cytotoxicity. Vorinostat potentiated SN38-induced DNA DSBs and attenuated their subsequent repair. These results indicate vorinostat enhances the cytotoxicity of SN38 in glioblastoma cell lines, suggesting this combination may be a worthwhile strategy to test in the context of a clinical trial.

Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1α

Hypoxic tumor cells overexpressing hypoxia-inducible factor 1alpha (HIF-1alpha) are generally resistant to chemo/radiotherapy. We have reported that Se-methylselenocysteine (MSC) therapeutically enhances the efficacy and selectivity of irinotecan against human tumor xenografts. The aim of this study was to delineate the mechanism responsible for the observed efficacy targeting on HIF-1alpha and its transcriptionally regulated genes VEGF and CAIX. We investigated the mechanism of HIF-1alpha inhibition by MSC and its critical role in the therapeutic outcome by generating HIF-1alpha stable knockdown (KD) human head and neck squamous cell carcinoma, FaDu by transfecting HIF-1alpha short hairpin RNA. While cytotoxic efficacy in combination with methylselenic acid (MSA) with SN-38 (active metabolites of MSC and irinotecan) could not be confirmed in vitro against normoxic tumor cells, the hypoxic tumor cells were more sensitive to the combination. Reduction in HIF-1alpha either by MSA or shRNA knockdown resulted in significant increase in cytotoxicity of SN38 in vitro against hypoxic, but not the normoxic tumor cells. Similarly, in vivo, either MSC in combination with irinotecan treatment of parental xenografts or HIF-1alpha KD tumors treated with irinotecan alone resulted in comparable therapeutic response and increase in the long-term survival of mice bearing FaDu xenografts. Our results show that HIF-1alpha is a critical target for MSC and its inhibition was associated with enhanced antitumor activity of irinotecan. Inhibition of HIF-1alpha appeared to be mediated through stabilization of PHD2, 3 and downregulation of ROS by MSC. Thus, our findings support the development of MSC as a HIF-1alpha inhibitor in combination chemotherapy.

Abstract C217: Genz-644282, a novel non-camptothecin topoisomerase I inhibitor for cancer treatment

Abstract Topoisomerase I (Top1), a well-established anticancer target, is a nuclear enzyme producing a single-strand DNA break to allow transcription. Camptothecin Top1 inhibitors are important drugs for the treatment of a variety of cancers including colon cancer, leukemias and ovarian cancer. Potent non-camptothecin Top1 inhibitors were identified. One of these, Genz-644282, binds tightly and selectively to the DNA:Top1 complex. The effects of Genz-644282, topotecan and irinotecan/SN38 in cell-based assays were compared. The colony formation assay is the gold-standard for determination of the cytotoxicity of compounds toward bone marrow and malignant cells as it measures the survival/killing of cells capable of self-renewal. Mouse and human bone marrow were subjected to colony forming unit -granulocyte/macrophage (CFU-GM) assays over a 5-log concentration range with continuous exposure to the compounds for 13–15 days. The IC90s for topotecan and SN38 for mouse CFU-GM were 519 nM and 331 nM, respectively. For human CFU-GM, the IC90s for topotecan and SN38 were 19 nM and 26 nM, resulting in mouse to human differentials of 28- and 13-fold. Genz-644282 was more potent, with IC90s of 8.4nM for mouse CFU-GM and 1.2 nM for human CFU-GM; thus only a 7-fold differential between species. The cytotoxicities of topotecan, SN38 and Genz-644282 were compared in the ability of seven human tumor cell lines of varied histologies to form colonies after a week of exposure to a 5-log concentration range of each compound. Human bone marrow CFU-GM was much more sensitive to topotecan than were RPMI-8226 multiple myeloma, KBV-1 cervical carcinoma or NCI-H460 non-small cell lung carcinoma cells. The IC90s for topotecan ranged between 11 and 257 nM. SN38 cytotoxicity to human bone marrow CFU-GM and to several human tumor cell lines was very similar with IC90s in the cancer lines ranging between 2 and 55 nM. HT29 human colon cancer cells, however, were markedly sensitive to SN38 (IC90 = 2 nM). Genz-644282 was a more potent cytotoxic agent than topotecan or SN38 with IC90 values ranging between 0.7 and 9.1 nM across the seven cell lines. MDA-MB-231 human breast carcinoma, HT29 colon carcinoma and RPMI-8226 cells were most sensitive to Genz-644282 with IC90s ranging between 0.7 and 7.0 nM. The growth inhibition produced by 72-hr exposure to Genz-644282 was examined in 26 established human tumor cell lines representing pancreatic carcinoma, melanoma, non-small cell lung cancer, breast carcinoma, ovarian carcinoma and renal cell carcinoma using a Cell TiterGlo ATP content endpoint. The IC90s for Genz-644282 ranged between 0.054 micromolar and 5 micromolar in this assay. The most sensitive tumor lines were 4 of 6 pancreatic cancer lines and the least sensitive lines included 3 of 4 melanoma lines. Colony formation data were analyzed by non-linear regression using SAS version 8.2. Based upon these findings and other data, Genz-644282 was selected as a development candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C217.

Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines

Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3sigma, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza-irinotecan interaction.