Colon Cancer Cell Line WiDr Research Articles

Hypoxia-Inducible Factors Activate CD133 Promoter through ETS Family Transcription Factors

CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1–P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

Expression of MMP2 and MMP9 (Gelatinases A and B) in Human Colon Cancer Cells

Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases, collectively capable of degrading essentially all matrix components. Elevated levels of distinct MMPs are detected in tumor tissue or serum of patients with advanced cancer, and they are the major prognostic indicators in cancer. Inhibition of MMPs has been explored as a therapeutic goal for almost two decades. Nitric oxide (NO), a free radical plays an important role in signaling pathways in regulation of MMP expression. In the present study, we demonstrated the role of exogenous NO levels in the regulation of MMP2 and MMP9 (gelatinases A and B) in colon cancer cell line WiDr and its inhibition with emodin (a naturally occurring anthraquinone).

Ethanolic Extract of Moringa oleifera L. Increases Sensitivity of WiDr Colon Cancer Cell Line Towards 5-Fluorouracil

For more than four decades, combination chemotherapy (co-chemotherapy) has been employed as a means to increase the effectiveness of chemotherapy regiments. The aim of our research is to investigate the activity of Moringa oleifera L. (tanaman kelor) ethanolic extract (MEE) as a co-chemotherapy agent with 5-fluorouracil (5-FU) on WiDr colon cancer cell line. Evaluation of MEE potency as a co-chemotherapy agent with 5-FU was based on cytotoxic activity based on percent cell viability via MTT assay, and based on apoptosis observation via the double staining method using acrydin orange – ethidium bromide (AE) as the staining reagent.Cytotoxicity evaluation of single treatment using concentrations of 5, 20, 50, 100,125, and 250 µg/ml of MEE reduced cell viability 24 hours post-treatment. 5, 50, and 250 µg/ml of MEE was chosen as the combination concentrations with 1000 µM 5-FU. MTT assay 24 hours and 48 hours post-combination treatment showed significant cell viability reduction in comparison to those of single treatments. Apoptosis observation using the double staining method shows the presence of apoptotic cells 48 hours post combination treatment. MEE is a potential co-chemotherapy agent by increasing the sensitivity of WiDr colon cancer cell line towards 5-FU.Keywords: co-chemotherapy, 5-fluorouracil, Moringa oleifera L., colon cancer

Comparative Quantitation of Aberrant Glycoforms by Lectin-Based Glycoprotein Enrichment Coupled with Multiple-Reaction Monitoring Mass Spectrometry

Lectin enrichment-coupled multiple-reaction monitoring (MRM) mass spectrometry was employed to quantitatively monitor the variation of aberrant glycoforms produced under pathological states. For this, aberrant glycoforms of the tissue inhibitor of metalloproteinase 1 (TIMP1) and protein tyrosine phosphatase kappa (PTPkappa), previously known target proteins for N-acetylglucosaminyltransferase-V (GnT-V), were enriched by phytohemagglutinin-L(4) (L-PHA) lectin and comparatively analyzed in the conditioned medium of the WiDr colon cancer cell line and its GnT-V-overexpressing transfectant cells. Enriched glycoforms were digested, and the resultant peptides were comparatively quantified by MRM analysis. MRM quantitation data for the L-PHA-enriched samples revealed that the abundance of aberrant glycoforms of TIMP1 and PTPkappa was greatly increased (11.7- and 16.5-fold, respectively) in GnT-V-treated cells compared to the control cells, although the abundance of total TIMP1 and PTPkappa in GnT-V-treated cells was slightly different (1.1- and 0.5-fold, respectively) for unenriched samples compared to that in control cells. The dramatic variation in abundance of the aberrant glycoforms due to overexpressed GnT-V was confirmed quantitatively by comparative MRM analysis of lectin-enriched samples. This method is capable of comparatively quantitating the abundance of a protein of interest and its aberrant glycoform and will be useful for studying pathological mechanisms of cancer or verifying biomarker candidates.

Abstract 3925: RNF138, a novel ubiquitin E3 ligase is overexpressed in colon cancer cell lines and interacts with RAD51D

Abstract The RAD51 protein family provides core components of the Homologous Recombination (HR) complex, which is critical for repairing DNA double-strand breaks and maintaining genome stability. The purpose of this study was to identify proteins that regulate HR processes through post-translational modification of the RAD51 paralogs. A yeast two-hybrid screen was performed using the RAD51 paralogs RAD51C, RAD51D, and XRCC2 as bait against a normalized universal mouse cDNA library. Fifty-eight primary candidates were isolated, and five are predicted to function in the ubiquitination pathway. Within this group, Ring Finger Protein 138 (RNF138) is of particular interest since RNF138 is a novel E3-ubiquitin ligase having both an N-terminal RING finger domain and a putative C-terminal ubiquitin interaction motif. Presence of both domains indicates RNF138 induces the transfer of ubiquitin to target substrates and also recognizes ubiquitin moieties on other proteins. Expression of RNF138 is increased by 12-fold in the HCC38 breast cancer cell line and 4-fold in SW-48, RKO, and Wi-DR colon cancer cell lines compared to non-tumorigenic controls. Yeast two-hybrid results indicate that RNF138 interaction is unique to RAD51D, as RNF138 failed to directly interact with RAD51C and XRCC2. Further analysis using N- and C-terminal truncated RAD51D constructs revealed that the RAD51D core domain rather than the N-terminal linker region that governs the interaction between RAD51D and XRCC2 mediates the association with RNF138. Moreover, K113A and K113R mutations of the highly conserved RAD51D Walker A ATP binding motif abolished RAD51D-RNF138 association, suggesting that ATP binding and hydrolysis may regulate the interaction. As further demonstration of this association, Myc-RNF138 co-immunoprecipitated with HA-RAD51D following transient transfections. Future studies will determine whether RNF138 is directly required for HR repair and if the mechanism specifically involves RAD51D ubiquitination. Understanding the regulation of HR proteins by ubiquitination processes will provide important insights into cancer progression and guide the identification of new therapeutic strategies. 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 3925.

Cell cycle effects of fatty acid derivatives of cytarabine, CP-4055, and of gemcitabine, CP-4126, as basis for the interaction with oxaliplatin and docetaxel

To bypass resistance due to limited entry into the cell derivatives of cytarabine (CP-4055, elacytarabine) and gemcitabine (CP-4126) containing a fatty acid chain at the 5' position of the nucleoside were developed. CP-4055 showed an increased retention of the active metabolite, the triphosphate. This characteristic was supposed to favor combinations, such as with the tubulin antagonist docetaxel, the platinum oxaliplatin and the antifolate pemetrexed. The role of the cell cycle effects of CP-4055 and CP-4126 on the efficacy of the combination with docetaxel or pemetrexed was determined. The combination of CP-4055 with oxaliplatin and docetaxel was also evaluated in a mouse xenograft model. CP-4055 induced a G2/M and S phase accumulation and CP-4126 an S phase accumulation. Both analogs induced a dose-dependent cell kill (apoptosis and necrosis). None of the docetaxel combinations induced a synergistic effect. The combination of docetaxel with CP-4055 or CP-4126 induced a G2/M accumulation in the A549 (lung cancer) cell line, but a G0/G1 accumulation in the WiDR (colon cancer) cell line. Preincubation with docetaxel induced an increased cell kill in both cell lines. The combination with oxaliplatin showed a synergistic effect in both cell lines. Combinations with pemetrexed were antagonistic in both cell lines. In the A549 cell line pemetrexed with CP-4055 led to an increase of the G0/G1 phase and the S phase. In WiDR the combination of pemetrexed with CP-4055 increased the G0/G1 phase and increased the cell kill. Pemetrexed with CP-4126 induced an increase in the G0/G1 phase and the S phase in the A549 cell line. In the xenograft study, on a colon cancer and a lung metastasis model, the combination of CP-4055 with docetaxel showed the best results. Treatment with CP-4055 followed by docetaxel after 4 h resulted in a reduction in metastasis in a lung metastasis model, and a favorable toxicity profile was observed. In conclusion, the combinations with oxaliplatin showed a synergistic effect in the combination studies. Although the combinations with docetaxel did not show an enhanced effect in the in vitro studies, this combination revealed an increased effect in the xenograft model.

Tin(IV) Cyanoximates: Synthesis, Characterization, and Cytotoxicity

In recent years, numerous organotin(IV) derivatives have exhibited remarkable cytotoxicity against several types of cancer. However, the properties of the cyanoxime-containing organotin(IV) complexes are unknown. Previously, it has been shown that cyanoximes displayed an interesting spectrum of biological activity ranging from growth-regulation to antimicrobial and pesticide detoxification actions. The work presented here attempts to combine the useful properties of both groups of compounds and investigate the likely antiproliferating activity of the new substances. A series of 19 organotin(IV) complexes, with nine different cyanoxime ligands, were anaerobically prepared by means of the heterogeneous metathesis reaction between the respective organotin(IV) halides (Cl, Br) and ML (M=Ag, Tl; L=cyanoximate anion), using an ultrasound in the CH3CN at room temperature. The compounds were characterized using spectroscopic methods (UV-visible, IR, 1H,13C NMR, 119Sn Mössbauer) and X-ray analysis. The crystal structures of the complexes revealed the formation of two types of tin(IV) cyanoximates: mononuclear five-coordinated compounds of R4-xSnLx composition (R=Me, Et, n-Bu, Ph; x=1, 2; L=cyanoximate anion), and the tetranuclear R8Sn4(OH)2O2L2 species (R=n-Bu, Ph). The latter complex contains a planar [Sn4(OH)2O2]2- core, consisting of three adjacent rhombs with bridging oxo and hydroxo groups. The tin(IV) atoms are five-coordinated and have distorted trigonal-pyramidal surrounding. This is the first instance when the organic anions were found to act as monodentate O-bound planar oxime ligands. All of the compounds were studied in vitro for antiproliferating activity, using human cervical cancer HeLa and WiDR colon cancer cell lines; cisplatin was used as a positive control substance. The two dibutyltin(IV) cyanoximates showed cytotoxicity similar and greater to that of cisplatin.

Simultaneous alteration of de novo and salvage pathway to the deoxynucleoside triphosphate pool by (E)-2′-Deoxy-(fluoromethylene)cytidine (FMdC) and zidovudine (AZT) results in increased radiosensitivity in vitro

To test whether a thymidine analog zidovudine (=AZT), is able to modify the radiosensitizing effects of (E)-2′-Deoxy-(fluoromethylene)cytidine (FMdC). A human colon cancer cell line Widr was exposed for 48 hours prior to irradiation to FMdC. Zidovudine was added at various concentrations immediately before irradiation. We measured cell survival and the effect of FMdC, AZT and FMdC + AZT on deoxynucleotide triphosphate pool. FMdC results in a significant increase of radiosensitivity. The enhancement ratios (ER =surviving fraction irradiated cells/surviving fraction drug treated and irradiated cells), obtained by FMdC or AZT alone are significantly increased by the combination of both compounds. Adding FMdC to AZT yields enhancement ratios ranging from 1.25 to 2.26. FMdC reduces dATP significantly, with a corresponding increase of TTP, dCTP and dGTP. This increase of TTP, dCTP and dGTP is abolished with the addition of AZT. Adding AZT to FMdC results in a significant increase of the radiosensitizing effect of FMdC. This combination appears to reduce the reactive enhancement of TTP, dCTP and dGTP induced by FMdC while it does not affect the inhibitory effect on dATP.

SMS 201-995 enhances S-phase block induced by 5-fluorouracil in a human colorectal cancer cell line

The action of the somatostatin analog SMS-201.995 (SMS) was tested in monotherapy and in combined therapy with the cytotoxic agent 5-fluorouracil (5-FU) on cell cycle kinetics of the human colon cancer cell line WiDr, expressing a mutant p53 (mp53). The data, obtained by flow cytometric DNA analysis, showed that SMS at 0.2 microg/ml increased apoptosis, augmenting the proportion of cells with subdiploid DNA content by 65 and 48% after 3 and 6 h, respectively. In cultures lasting 24 and 36 h, it also decreased the percentages of cells in G0/G1 phase by 22.9 and 14.3%; whereas at a dose of 0.1 microg/ml, SMS decreased the percentage of cells in G2/M by 14.3%. In contrast to SMS, 5-FU (0.1 microg/ml) augmented the apoptosis at 12 h, and markedly increased the fraction of cells in S phase, increasing its value from 24 and 72 h by 108 and 234%, respectively, in comparison to the control. The most evident finding after the combination of SMS (0.2 microg/ml) and 5-FU (0.1 microg/ml) was a potentiation of 5-FU-induced S-phase block by a further 7.9, 12.9 and 42.1% at 24, 36 and 72 h, respectively. Treatment with 5-FU also upregulated HLA class I expression of the cancer cells. In this sense, SMS was less effective and when given in combination with 5-FU did not change the effects induced by 5-FU. The data emphasize that SMS exhibits pro-apoptotic and anti-proliferative effects, which in proper dose combinations might enhance the effects of 5-FU on human colorectal cancer cells expressing mp53.

Antitumor activity of pyrvinium pamoate, 6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-quinolinium pamoate salt, showing preferential cytotoxicity during glucose starvation.

An anthelminthic, pyrvinium pamoate (PP), 6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-quinolinium pamoate salt, has been found to be extremely toxic to PANC-1 cells in glucose-free medium, but not to be toxic to the same cells cultured in ordinary medium, Dulbecco's modified Eagle's medium (DMEM). It showed the same preferential toxicity for various cancer cell lines during glucose starvation. When 0.1 microg/ml PP was added to the medium, spheroid growth of human colon cancer cell line WiDr was strongly inhibited to a diameter of 750 microm, and this finding is consistent with the concept of anti-austerity. PP was also found to exert antitumor activity against human pancreatic cancer cell line PANC-1 in nude mice and SCID mice when it was administered subcutaneously or orally. Regarding the mechanism of PP action, inhibition of Akt phosphorylation, which has been found to be essential for the austerity mechanism, was observed in vitro and in vivo. These findings indicate that PP may be useful for anticancer therapy and that anti-austerity therapy could be a novel strategy for anticancer therapy.

Combined administration of imatinib mesylate and ionizing radiation leads to increased radiosensitivity in the human glioblastoma cell line RuSi RS1

1564 Background: The selective tyrosine kinase inhibitor imatinib mesylate (Gleevec) effectively inhibits the activity of the abl, PDGF receptor (PDGFR) and c-kit tyrosine kinases. Because of the specific target profile of this orally available and generally well tolerated drug, imatinib is under intensive investigation for the potential treatment of other diseases. Malignant gliomas frequently show a high expression of PDGF receptor which is involved in tumorigenesis by formation of an autocrine loop. A role in DNA repair carried out through c-abl and c-kit was previously described both for c-abl and c-kit. In this study we address the potential use of imatinib mesylate and concomitant ionizing radiation (IR) to improve the radiosensitivity of the glioblastoma cell line RuSi RS1. Methods: Cells of the primary glioblastoma cell line RuSi RS1, as well as of the colon cancer cell line WiDr and of BT20, a human breast cancer cell line, were incubated with different concentrations of imatinib mesylate 30 minutes prior to irradiation (doses between 0 and 16 Gy). Western blots were done comparing the different expression of c-abl, PDGFR alpha and beta, and c-kit in the different cell lines. Immunoprecipitation was performed to assess inhibition of the different kinases by imatinib mesylate. Results: Under combined treatment with imatinib and IR a significant shift of the IC50 (dose needed to reduce the survival fraction by 50%) was detected in RuSi RS1 compared to IR alone. This effect was not seen in WiDr or BT20. RuSi RS1 showed an overexpression of PDGFR beta. Inhibition of tyrosine kinase phosphorylation by imatinib could be shown for PDGFR beta and c-abl (the IP failed for c-kit). Conclusion: Concomitant administration of imatinib mesylate and ionizing radiation increases the radiosensitivity of the glioblastoma cell line RuSi RS1. This effect might partly be due to disruption of an autocrine loop of the PDGF/PDGFR system. Inhibition of DNA repair mechanisms in which the tyrosine kinase activity of c-abl and c-kit is involved might be important causes of this observed phenomenon as well. No significant financial relationships to disclose.

Combined administration of imatinib mesylate and ionizing radiation leads to increased radiosensitivity in the human glioblastoma cell line RuSi RS1

1564 Background: The selective tyrosine kinase inhibitor imatinib mesylate (Gleevec) effectively inhibits the activity of the abl, PDGF receptor (PDGFR) and c-kit tyrosine kinases. Because of the specific target profile of this orally available and generally well tolerated drug, imatinib is under intensive investigation for the potential treatment of other diseases. Malignant gliomas frequently show a high expression of PDGF receptor which is involved in tumorigenesis by formation of an autocrine loop. A role in DNA repair carried out through c-abl and c-kit was previously described both for c-abl and c-kit. In this study we address the potential use of imatinib mesylate and concomitant ionizing radiation (IR) to improve the radiosensitivity of the glioblastoma cell line RuSi RS1. Methods: Cells of the primary glioblastoma cell line RuSi RS1, as well as of the colon cancer cell line WiDr and of BT20, a human breast cancer cell line, were incubated with different concentrations of imatinib mesylate 30 minutes prior to irradiation (doses between 0 and 16 Gy). Western blots were done comparing the different expression of c-abl, PDGFR alpha and beta, and c-kit in the different cell lines. Immunoprecipitation was performed to assess inhibition of the different kinases by imatinib mesylate. Results: Under combined treatment with imatinib and IR a significant shift of the IC50 (dose needed to reduce the survival fraction by 50%) was detected in RuSi RS1 compared to IR alone. This effect was not seen in WiDr or BT20. RuSi RS1 showed an overexpression of PDGFR beta. Inhibition of tyrosine kinase phosphorylation by imatinib could be shown for PDGFR beta and c-abl (the IP failed for c-kit). Conclusion: Concomitant administration of imatinib mesylate and ionizing radiation increases the radiosensitivity of the glioblastoma cell line RuSi RS1. This effect might partly be due to disruption of an autocrine loop of the PDGF/PDGFR system. Inhibition of DNA repair mechanisms in which the tyrosine kinase activity of c-abl and c-kit is involved might be important causes of this observed phenomenon as well. No significant financial relationships to disclose.

Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 μM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 μM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:β actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold ( P=0.01) and 18-fold ( P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 μM dUMP increased from 30 pmol/hr/10 6 cells in WiDr cells to 2201 and 7663 pmol/hr/10 6 cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.

Impact of the oxaliplatin-5 fluorouracil-folinic acid combination on respective intracellular determinants of drug activity

The combination of 5-fluorouracil-folinic acid and oxaliplatin has led to a significant improvement of chemotherapy efficacy in advanced pretreated colorectal cancer. The objective of the present study was, considering the oxaplatin-5-fluorouracil-folinic acid combination, to examine the impact of one given drug on the cellular determinants of cytotoxic activity of the other drug. These cellular factors were analysed on the human colon cancer cell line WiDr in clinically relevant conditions of drug exposure (‘De Gramont’ schedule) with oxaliplatin-folinic acid during 2 h followed by 5-fluorouracil 48 h. The DNA binding of oxaliplatin was significantly reduced by the presence of 5-fluorouracil but this effect was time-dependent and after 50 h the platinum incorporated into DNA was identical in controls and in the drug combination. In the presence of oxaliplatin, there was less formation of FUH2 which is the first catabolite produced in the cascade of 5-fluorouracil metabolic degradation. The effects of drugs on cell cycle were quite different from one drug to the other with oxaliplatin inducing a shift towards G2 accumulation and 5-fluorouracil-folinic acid to a greater proportion of cells in G1–S. When oxaliplatin and 5-fluorouracil-folinic acid were combined the cell cycle effects were very similar to that of the 5-fluorouracil-folinic acid sequence alone. Oxaliplatin was able to reduce thymidylate synthase activity with a marked impact 28 h after the beginning of cell exposure to the drug. The 5-fluorouracil-folinic acid drug sequence led to a profound reduction in thymidylate synthase activity and this decrease was not markedly enhanced by the presence of oxaliplatin. Regarding apoptosis, changes in mitochondrial membrane permeability were observed in the presence of the tested drugs and the impact of 5-fluorouracil-folinic acid was greater than that of oxaliplatin. The addition of oxaliplatin did not amplify the action of 5-fluorouracil-folinic acid upon mitochondrial membrane permeability change. The presence of oxaliplatin itself did not modify the intracellular concentration of total reduced folates. The fact that oxaliplatin may reduce 5-fluorouracil catabolism could be central in explaining the supra-additive interaction between these drugs.

Impact of the oxaliplatin-5 fluorouracil-folinic acid combination on respective intracellular determinants of drug activity

The combination of 5-fluorouracil-folinic acid and oxaliplatin has led to a significant improvement of chemotherapy efficacy in advanced pretreated colorectal cancer. The objective of the present study was, considering the oxaplatin-5-fluorouracil-folinic acid combination, to examine the impact of one given drug on the cellular determinants of cytotoxic activity of the other drug. These cellular factors were analysed on the human colon cancer cell line WiDr in clinically relevant conditions of drug exposure (‘De Gramont’ schedule) with oxaliplatin-folinic acid during 2 h followed by 5-fluorouracil 48 h. The DNA binding of oxaliplatin was significantly reduced by the presence of 5-fluorouracil but this effect was time-dependent and after 50 h the platinum incorporated into DNA was identical in controls and in the drug combination. In the presence of oxaliplatin, there was less formation of FUH2 which is the first catabolite produced in the cascade of 5-fluorouracil metabolic degradation. The effects of drugs on cell cycle were quite different from one drug to the other with oxaliplatin inducing a shift towards G2 accumulation and 5-fluorouracil-folinic acid to a greater proportion of cells in G1–S. When oxaliplatin and 5-fluorouracil-folinic acid were combined the cell cycle effects were very similar to that of the 5-fluorouracil-folinic acid sequence alone. Oxaliplatin was able to reduce thymidylate synthase activity with a marked impact 28 h after the beginning of cell exposure to the drug. The 5-fluorouracil-folinic acid drug sequence led to a profound reduction in thymidylate synthase activity and this decrease was not markedly enhanced by the presence of oxaliplatin. Regarding apoptosis, changes in mitochondrial membrane permeability were observed in the presence of the tested drugs and the impact of 5-fluorouracil-folinic acid was greater than that of oxaliplatin. The addition of oxaliplatin did not amplify the action of 5-fluorouracil-folinic acid upon mitochondrial membrane permeability change. The presence of oxaliplatin itself did not modify the intracellular concentration of total reduced folates. The fact that oxaliplatin may reduce 5-fluorouracil catabolism could be central in explaining the supra-additive interaction between these drugs.British Journal of Cancer (2002) 86, 1162–1168. DOI: 10.1038/sj/bjc/6600185 www.bjcancer.com© 2002 Cancer Research UK

Potentiation of cytotoxicity and radiosensitization of (E)-2-deoxy-2'-(fluoromethylene) cytidine by pentoxifylline in vitro.

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide-diphosphate reductase, has been shown to have anti-tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNA-damaging agents through abrogation of DNA-damage-dependent G2 checkpoint. We investigated the cytotoxic, radiosensitizing and cell-cycle effects of FMdC and PTX in a human colon-cancer cell line WiDr. PTX at 0.25-1.0 mM enhanced the cytotoxicity of FMdC and lowered the IC50 of FMdC from 79 +/- 0.1 to 31.2 +/- 2.1 nM, as determined by MTT assay. Using clonogenic assay, pre-irradiation exposure of exponentially growing WiDr cells to 30 nM FMdC for 48 hr or post-irradiation to 0.5 to 1.0 mM PTX alone resulted in an increase in radiation-induced cytotoxicity. Moreover, there was a significant change of the radiosensitization if both drugs were combined as compared with the effect of either drug alone. Cell-cycle analysis showed that treatment with nanomolar FMdC resulted in S-phase accumulation and that such an S-phase arrest can be abrogated by PTX. Treatment with FMdC prior to radiation increased post-irradiation-induced G2 arrest, and such G2 accumulation was also abrogated by PTX. These results suggest that pharmacological abrogation of S and G2 checkpoints by PTX may provide an effective strategy for enhancing the cytotoxic and radiosensitizing effects of FMdC.

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines.

Paclitaxel and irinotecan are important new anticancer agents. The combination of these two agents has been considered for use against a variety of advanced solid tumors. Since the schedule-dependent effects of this combination may be crucial to its use, we studied the interaction of paclitaxel and SN-38 (the active metabolite of irinotecan) in various schedules in four human cancer cell lines in culture. Cell growth inhibition after 5 days was determined using an MTT assay. The effects of drug combinations at the IC80 level were analyzed by the isobologram method. Simultaneous exposure to paclitaxel and SN-38 for 24 h produced antagonistic (subadditive and protective) effects in the human lung cancer cell line A549, the breast cancer cell line MCF7, and the colon cancer cell line WiDr, and produced additive effects in the ovarian cancer cell line PA1. Sequential exposure to paclitaxel for 24 h followed by SN-38 for 24 h, and the reverse sequence, produced additive effects in all four cell lines. These findings suggest that sequential administration, not simultaneous administration, may be the appropriate schedule for the therapeutic combination of paclitaxel and irinotecan. Continued preclinical and clinical studies should provide further insights and assist in determining the optimal schedule for this combination in clinical use.

Novel combination therapy for human colon cancer with adenovirus-mediated wild-type p53 gene transfer and DNA-damaging chemotherapeutic agent.

Alteration of the wild-type (wt) p53 gene by mutation, deletion or re-arrangement is a major factor in the development of human colon cancer. Recent studies have demonstrated that p53 might be an essential component of the apoptotic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We examined the anti-tumor effects of adenovirus-mediated wt-p53 gene transfer in combination with a chemotherapeutic drug on the human colon cancer cell line WiDr, which is homozygous for a mutation in the p53 gene. Treatment with the chemotherapeutic drug cisplatin following infection with a replication-deficient, recombinant adenoviral vector expressing wt-p53 (termed AdCMVp53) significantly suppressed the growth of WiDr cells compared to single treatments alone. To evaluate the in vivo efficacy of AdCMVp53 and cisplatin given sequentially, WiDr cells were inoculated s.c. in nu/nu mice. After 3 days, AdCMVp53 was injected s.c. into the area where tumor cells were implanted, followed by i.p. administration of cisplatin. Analysis of initial growth inhibition at 21 days demonstrated a profound therapeutic cooperativity, though administration of either AdCMVp53 or cisplatin alone was followed only by a slowing of growth. Our results suggest that gene therapy using wt-p53-expressing adenovirus in combination with a chemotherapeutic DNA-damaging drug could be a useful strategy for treating human colon cancer.

Butyrate Activates the WAF1/Cip1 Gene Promoter through Sp1 Sites in a p53-negative Human Colon Cancer Cell Line

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. We also found that a TATA element at -46 and two overlapping consensus Sp1 sites at -60 and -55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.

Receptors for Interleukin (IL)-4 Do Not Associate with the Common γChain, and IL-4 Induces the Phosphorylation of JAK2 Tyrosine Kinase in Human Colon Carcinoma Cells

We have previously reported on the expression of interleukin-4 receptors (IL-4R) on many human epithelial cancer cells; however, the binding characteristics, structure, function, and signal transduction through the IL-4R in cancer cells is not known. IL-4 binding characteristics were determined in human colon carcinoma cell lines by a 125I-IL-4 binding assay, which demonstrated that the HT-29 and WiDr colon cancer cell lines expressed high affinity IL-4R (Kd = 200 pM). Cross-linking experiments revealed a major band of 140 kDa and a broad band at 70 kDa. While the common gamma chain of IL-2R is associated with IL-4R in immune cells and is similar in size to the 70-kDa protein, this chain was not expressed in these colon cancer cells. Interestingly, IL-13, which has many functions similar to IL-4, inhibited 125I-IL-4 binding to both the 140- and 70-kDa molecules. Next, we investigated the mechanism of IL-4-induced signal transduction in colon cancer cells. After stimulation with IL-4, a 170-kDa band was primarily phosphorylated within 1 min of exposure and was identified as insulin receptor substrate-1. In addition, by immunoprecipitation assay, three other phosphorylated bands were identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation; however, Tyk2 was constitutively phosphorylated, and IL-4 treatment further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were phosphorylated within minutes and that JAK1 and JAK2 were activated after IL-4 exposure. Contrary to observations in immune cells. JAK3 mRNA was neither detected in colon cancer cells nor did IL-4 treatment cause phosphorylation of JAK3. These data indicate that in colon carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are phosphorylated after IL-4 stimulation. In addition, as is the case in lymphoid cells, IL-4 activated and phosphorylated signal transducers and activators of transcription (IL-4-STAT or STAT-6) protein in both colon cancer cell lines. These results indicate that the IL-4R complex is composed of different subunits in different tissues and shares a component with the IL-13R complex. In addition, we demonstrate for the first time that like its family members (e.g. IL-3 and GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.