Tetrazolium Dye Assay Research Articles

Synergistic Growth Inhibition with a PI3 Kinase/mTOR Inhibitor Plus Lapatinib.

Abstract The mammalian target of rapamycin, mTOR, is a member of the phosphatidylinositol 3-kinase (PI3-K) /Akt family and plays a key role in cell growth and survival. The PI3-K/mTOR pathway is activated by many growth factor receptors in human tumor cells. In addition, the PI3-K pathway is constitutively activated by mutation in one-third of breast cancers. Therefore, blockade of the PI3-K/mTOR pathway may sensitize breast cancers to HER-2/neu inhibition. BEZ235 (Novartis Pharma AG) is a potent, oral, dual PI3-K/mTOR inhibitor currently in Phase I clinical trials. The purpose of this study was to assess the in vitro effects of combining a dual PI3-K/mTOR inhibitor (BEZ235, Novartis Pharma AG) and a lapatinib-like dual inhibitor of EGFR and HER-2/neu (GW2974) on the growth of HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to the lapatinib-like compound [GW2974 (0.05-5.0µM)] or the dual PI3-K/mTOR inhibitor [BEZ235 (0.5-3nM)] alone or in combination for 3 days (12 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. The Chou-Talalay method of isobologram analysis was performed to determine the inhibitory effect. Dose-dependent growth inhibition was observed in HER-2/neu-transfected MCF-7 cells after 3 days exposure to GW2974 (IC50: 5.0 mM) or BEZ235 (IC50: 2.2 nM). Exposure to the combination of GW2974 (0.05-0.50 mM) and BEZ235 (0.50-1.0 nM) resulted in synergistic growth inhibition in HER-2/neu-transfected MCF-7 human breast cancer cells. In summary, the combination of lapatinib plus PI3-K/mTOR warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6114.

Growth Inhibition of Human Breast Cancer Cells with an Iron Chelator (Deferasirox) and Docetaxel.

Abstract Treatment of cells with an iron chelator results in G1/S arrest and apoptosis. Tumor cells appear to be more sensitive to iron chelation probably because they express higher levels of the iron containing enzyme, ribonucleotide reductase. Previously, our laboratory has shown that an iron chelator (deferoxamine) inhibited the growth of a variety of cancer cell lines and also prolonged survival in tumor bearing mice. Docetaxel is an antineoplastic agent belonging to the taxane family. Docetaxel induces bcl-2 phosphorylation and subsequent apoptosis and is effective in the treatment of patients with breast cancer. The purpose of this study was to assess the in vitro effects of combining an iron chelator, deferasirox (Exjade: Novartis Pharma AG), and docetaxel on the growth of MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to deferasirox or docetaxel alone or in combination for 3 days (9 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. Dose-dependent growth inhibition was observed in both the MCF-7 and the HER-2/neu-tranfected MCF-7 cells after 3 days exposure to deferasirox (IC50: 1.5 and 2.3 μM, respectively) or docetaxel (IC50: 0.9 nM and 2.0 nM, respectively). Exposure to the combination of deferasirox (0.25 – 1.5 μM) and docetaxel (0.1 – 0.5 nM) resulted in synergistic growth inhibition in both the MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Sequencing experiments with the HER-2/neu-transfected MCF-7 cells showed that simultaneous exposure of the cells to deferasirox and docetaxel was superior to pretreating the cells with either of the agents 6 or 24 hours prior to addition of the second agent. These results suggest this combination warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5075.

1251 Evaluation of pharmacokinetics and safety profiles between S-1 granule and S-1 capsule in patients with solid tumors

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the pathway of uracil and thymine catabolism. DPD is also the principal enzyme involved in the degradation of 5-fluorouracil (5-FU), an anticancer chemotherapeutic agent that is used clinically to treat renal cell carcinoma (RCC). Little is known about the significance of DPD activity in human cancers. We investigated the activity of DPD in 68 RCC and the relationship between DPD activity and the sensitivity to 5-FU. The levels of DPD activity in RCC and normal kidney samples were determined by the 5-FU degradation assay. The sensitivity to 5-FU was assessed by the microculture tetrazolium dye assay. The activity of DPD was approximately 2-fold higher in normal kidney compared with RCC. DPD activity in Stage I/II RCC was approximately 2-fold higher than that in Stage III/IV RCC. The levels of DPD activity in Grade 1 and Grade 2 RCC were 3 and 2-fold higher, respectively, than that in the Grade 3 cancers. There was an inverse correlation between DPD activity in RCC cells and their sensitivity to 5-FU. Furthermore, 5-chloro-2,4-dihydroxypyridine (CDHP), a potent DPD inhibitor, enhanced the sensitivity to 5-FU. The current study is the first to demonstrate that the level of DPD activity was inversely correlated with both the progression of the disease and increased grade of RCC, and that DPD activity was inversely associated with the sensitivity of RCC to 5-FU, which was enhanced by a DPD inhibitor. These results suggest that a low DPD activity may be associated with the malignant potential of RCC. In addition, it may be possible to overcome 5-FU resistance by using DPD inhibitors in the treatment protocols of 5-FU-based chemotherapy for RCC.

1,3-Dinitrobenzene induces apoptosis in TM4 mouse Sertoli cells: Involvement of the c-Jun N-terminal kinase (JNK) MAPK pathway

Several studies have shown that 1,3-dinitrobenzene (1,3-DNB) causes injury to Sertoli cells and induces apoptosis in the surrounding germinal cells in male laboratory rats; however, the mechanism by which 1,3-DNB functions is not well understood. In this study, we investigated whether 1,3-DNB induces apoptosis and which pathways are undertaken in TM4 cells. When cells were treated with 1,3-DNB, a dose-dependent reduction in cell viability was observed by tetrazolium dye assay and LDH assay. The reduced cell viability by 1,3-DNB treatment appeared to involve necrosis as well as apoptosis, based on staining with annexin V-FITC and propidium iodide (PI) staining and Western blotting for PARP protein. 1,3-DNB treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and increased expression levels of the pro-apoptotic protein Bax. In addition, using FACS analysis we detected G2/M phase cell cycle arrest by 1,3-DNB, concurrent with a remarkable increase in p21 expression and decrease in cdc2 expression. Interestingly, we found that the phosphorylation of c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) was promoted by 1,3-DNB, furthermore, 1,3-DNB-induced cell death was significantly inhibited by the JNK inhibitor, but not by ERK inhibitor or the p38 inhibitor. Together, our results suggest that 1,3-DNB induces apoptotic cell death and G2/M phase cell cycle arrest, at least in part, via JNK signaling in TM4 mouse Sertoli cells.

Synergistic interactions between the synthetic retinoid tamibarotene and glucocorticoids in human myeloma cells

Tamibarotene (TM411) is a synthetic retinoic acid receptor-alpha/-beta selective retinoid that is chemically more stable than all-trans retinoic acid. This study was designed to evaluate the activity of TM411 in multiple myeloma (MM) and the effects of TM411 combined with a glucocorticoid (GC). In vitro, five human myeloma cells were treated with TM411 alone, GC alone, or TM411 + GC. Cell survival was analyzed by the tetrazolium dye assay and the Hoechst 33342/propidium iodide double-staining method. The effect of TM411 + GC was assessed by the isobologram method. In vivo, the growth-inhibitory effects of the drugs on RPMI-8226 cell xenografts established in SCID mice were examined. The effects of the agents on IL-6-mediated signaling pathways were also analyzed by Western blotting. TM411 was 2- to 10-fold more potent, in terms of its growth-inhibitory effect, than all-trans retinoic acid. The combination of TM411 and GC was found to show a markedly synergistic interaction. While increased expressions of the IL-6 receptor, phosphorylated MAPK, and Akt were observed after exposure to GC, TM411 attenuated this increase in the expressions, suggesting that such modification of the effect of GC by TM411 might be the possible mechanism underlying the synergistic interaction. Furthermore, TM411 + GC showed a supra-additive inhibitory effect in a xenograft model as compared with TM411 or GC alone. These results imply that the combination of TM411 + GC might be highly effective against MM, and suggest the need for clinical evaluation of TM411 + GC for the treatment of MM.

Conjugation with α-linolenic acid improves cancer cell uptake and cytotoxicity of doxorubicin

The synthetic DOX–LNA conjugate was characterized by proton nuclear magnetic resonance and mass spectrometry. In addition, the purity of the conjugate was analyzed by reverse-phase high-performance liquid chromatography. The cellular uptake, intracellular distribution, and cytotoxicity of DOX–LNA were assessed by flow cytometry, fluorescence microscopy, liquid chromatography/electrospray ionization tandem mass spectrometry, and the tetrazolium dye assay using the in vitro cell models. The DOX–LNA conjugate showed substantially higher tumor-specific cytotoxicity compared with DOX.

Low osmolar contrast medium induces cellular injury and disruption of calcium homeostasis in rat glomerular endothelial cells in vitro

The mechanisms of contrast medium (CM)-induced renal impairment at cellular level are not fully understood. The purpose of this study was to investigate the toxic effects of non-ionic low osmolar contrast medium (LOCM) on glomerular endothelial cells (GECs). Ioversol, the most representative LOCM used in clinic, was chosen to act on primary cultured rat GECs. When rat GECs were treated with various amount of ioversol, a dose-dependent reduction in cell viability was observed by tetrazolium dye (MTT) assay. After exposure to ioversol at dose of 100 μl/ml for 24 h, nuclear condensation, nuclear fragmentation, cell apoptosis and necrosis were found in GECs. The intracellular free Ca 2+ concentrations ([Ca 2+]i) detected by confocal laser scanning were markedly elevated in GECs treated with ioversol. The [Ca 2+]i increase, LDH release and expression changes of apoptotic associated proteins in GECs induced by ioversol could be reversed by pretreatment with intracellular Ca 2+ chelator, 1,2-bis ( o-aminophenoxy) ethane- N, N, N′, N′-tetra-acetic acid (BAPTA/AM). These results suggest that LOCM can decrease cell viability and cause cell injury of GECs. The elevation of [Ca 2+]i released from intracellular calcium stores likely contribute to the LOCM-induced cell injury of GECs.

Developmental neurotoxicity role of cyclophosphamide on post-neural tube closure of rodents in vitro and in vivo

To evaluate the possible developmental role of cyclophosphamide on post-neural tube closure of rodents, the 46 pregnant rats of 2.5–3 months old were randomly divided into five groups. On the 13th day of gestation, the rats in control and experimental groups were treated with physiological saline and cyclophosphamide (7.5, 10.5, 12.5 and 15.0mg/kgbw), respectively. On the 14th day of gestation, three rats each selected randomly from control and experimental (12.5mg/kgbw) group, respectively, were executed and the embryo brains were checked using both light microscope and transmission electron microscope. Neurocytotoxic effects of cyclophosphamide were determined using tetrazolium dye (MTT) assay, single cell gel electrophoresis, flow cytometry and scanning electron microscope. Compared with control group, the malformation incidence of the experimental groups (except the group 7.5mg/kgbw) was significantly higher, especially in 12.5mg/kgbw group (malformation incidence 98%, lethality 0). Neural tube serial section of fetus showed the apoptotic morphological features after 24h cyclophosphamide administrated. Cyclophosphamide (8μg/ml) decreased the growth and viability of neurons, damaged nuclear DNA and induced early apoptotic morphological changes. Our study shows that the teratogenesis of cyclophosphamide after neural tube closure are involved in cyclophosphamide-induced neuronal apoptosis and DNA damage.

Involvement of microtubules in the tolerance of cardiomyocytes to cold ischemia-reperfusion

Before transplantation, the heart graft is preserved by the use of cold storage in order to limit ischemia-reperfusion stress. However, sustained exposure to low temperature may induce myocardial ultrastructural damage, particularly microtubules (MT) disruption. Previous data suggested that tubulin-binding agents are able to attenuate cold-induced cytoskeleton alterations. Thus, the aim of the present work was to study the influence of docetaxel (DX, a tubulin-binding taxane) on the effects of deep hypothermia (4 degrees C) and of simulated cold ischemia-reperfusion on the MT network and oxidative stress of cardiomyocyte (CM) in monolayer cultures prepared from newborn rat ventricles. The MT network was explored by immunocytochemistry and Western-blotting, the cell stress by tetrazolium dye assay (MTT) and lactate dehydrogenase (LDH) release, and the superoxide production by the dihydroethidium probe (DHE). The MT assembly remained stable after 4 and 8 h of hypothermia. Tubulin acetylation was promoted in CM subjected to 4-h hypothermia. Low temperature reduced the mitochondrial function and increased the basal LDH release. The cold ischemia during 4 and 8 h preserved MT network. Docetaxel promoted MT polymerization and tubulin acetylation in basal and in cold conditions. This drug decreased the release of LDH induced by cold ischemia. Moreover, hypothermia (4 h) significantly raised the anion superoxide production. Docetaxel decreased this oxidative stress in the control CM and in CM submitted to 4 h of hypothermia. These data demonstrated that stabilizing MT with DX exerted a protective effect on CM subjected to hypothermia and to cold ischemia-reperfusion. Tubulin-ligands should be thus considered to improve the tolerance of the heart graft toward stressing conservative conditions.

Enhanced orotate phosphoribosyltransferase activity in renal cell carcinoma and its prognostic significance

15590 Background: 5-Fluorouracil ( 5-FU ) is an anticancer agent clinically used against various cancers including renal cell carcinoma ( RCC ). 5-FU is a prodrug and orotate phosphoribosyltransferase ( OPRT ) is the principal enzyme which directly converts 5-FU to an active anticancer metabolite, 5-fluoro-2’-deoxyuridine 5’-monophosphate. Furthermore, OPRT is the key enzyme in the de novo DNA and RNA synthetic process, which converts orotic acid to orotidine 5’-phosphate. Little is known about the significance of OPRT in a variety of cancers including RCC. We investigated OPRT activity in 83 RCC and evaluated the association between OPRT activity and the stage/grade of RCC. The relationship between OPRT activity in RCC cells and their sensitivity to 5-FU was also examined. Methods: OPRT activity in non-fixed fresh frozen RCC and normal kidney were determined enzymatically by the 5-FU phosphorylation assay. The sensitivity of RCC cells to 5-FU was assessed by the microculture tetrazolium dye assay. Results: OPRT activity was approximately 8.5-fold higher in RCC compared to normal kidney. OPRT activity in T3/4 RCC was 3-fold higher than that in T1/2 RCC. OPRT activity in M1 RCC was 2.5-fold higher than that in M0 RCC. In addition, OPRT activity in Stage III/IV RCC was 3-fold higher than that in Stage I/II RCC. The level of OPRT activity in Grade 3 RCC was 3-fold higher than that in Grade 1/2 cancer. Patients with RCC with low OPRT activity had a longer postoperative disease-specific survival than those with high activity in the 5-year follow-up. OPRT activity in RCC cells positively correlated with their sensitivity to 5-FU. Conclusions: The present study has demonstrated that OPRT activity in RCC was higher than that in normal kidney, and that OPRT activity positively correlated with the stage/grade of RCC. Moreover, higher OPRT activity in RCC predicted worse prognosis and higher sensitivity to 5-FU. These results suggest that OPRT activity may be used as both a prognostic parameter and a predictive indicator for 5-FU efficacy in RCC. No significant financial relationships to disclose.

Optimization of UV cross‐linking density for durable and nontoxic collagen GAG dermal substitute

Artificial dermal constructs, based upon collagen-glycosaminoglycan matrices (CGMs), provide new options in treating skin defects. However, their clinical effectiveness may be limited by cytotoxicity related to residual aldehydes left over from the manufacturing process. Although both chemical and dehydrothermal (DHT) cross-linking are used to produce CGMs, we hypothesize that optimized nonchemical cross-linking, using ultra-violet (UV) and DHT treatment combinations, may limit cytotoxicity without sacrificing mechanical strength. Porous CGMs were physically cross-linked using a combination of DHT and varying intensities of UV light. These were compared to glutaraldehyde cross-linked controls. Human keratinocytes were seeded in each matrix, and cellular proliferation measured using a microculture tetrazolium dye assay. A scoring system (based on the in vitro contraction rate, stiffness, and cellular growth of a small cylindrical specimen) was developed to assess the best overall physical cross-linking method. More cellular growth was observed in the 90-120 min UV cross-linked group than in the glutaraldehyde-treated group (p < 0.05). Stiffness was maximized after 0-30 min of UV cross-linking. On the basis of our scoring system, DHT combined with 45 min of UV cross-linking produced the best overall matrix in terms of cellular growth and physical durability. UV cross-linked collagen-based biomaterials could be a viable alternative for use in biological applications to eliminate glutaraldehyde-associated cytotoxicity.

Biomedical evaluation of polyvinyl alcohol–gelatin esterified hydrogel for wound dressing

The wound is a biosynthetic environment in which numerous cellular processes are interlinked in the process of repair. Modern dressings are designed to facilitate wound healing rather than just to cover it. Hydrogel dressing can protect injured skin and keep it appropriately moist to speed the healing process by absorbing exudates while maintaining the products of tissue repair, including growth factor and lysosomes, in contact with the wound. The design and development of novel membrane of hydrogels prepared by esterification of polyvinyl alcohol with gelatin were attempted. Contact angle of goat blood was determined. The hydrogel was characterized by hemolysis test and water vapor transmission rate. Diffusion coefficient of salicylic acid (SA) and gatifloxacin, a fourth generation fluoroquinolone, through the membrane was determined. Both the drugs were used as model drug. Methyl tetrazolium dye assay of the membrane was done using L929 fibroblast cell line and mice splenocytes to establish the biocompatibility of the membrane. The equilibrium goat blood-in-air contact angles of measured ester films ranged from 56 to 60 degrees . The hydrogel was found to be hemocompatible and moisture retentive indicating its possible use in moist wound care. The diffusion coefficient of SA and gatifloxacin through the membrane was found to be 1.49 x 10(-5) and 3.97 x 10(-6) cm(2)/s respectively. The membrane was found to be compatible with the L929 fibroblast cell line and mice splenocytes.

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.

Effect of root canal sealers on mouse peritoneal macrophage functions

Three root canal filling materials, viz. calcium hydroxide-based cement (Apexit, resin-based cement (AH-plus) and glass-ionomer based material (Ketac Endo) were tested for their influence on several functions of peritoneal macrophages from Balb/c mice. Macrophage functions were evaluated by the adherence, phagocytic, candidacidal and Nitro blue tetrazolium-dye assays. Ketac-Endo enhanced all macrophage functions in the first 2 d (p < or = 0.05), when compared to the positive control, but this effect had changed after 7 and 14 d, causing inhibition of these functions. Other materials suppressed substrate adherence capacity and phagocytosis, while significantly stimulating macrophage microbicidal activity (p < or = 0.05) in a time-dependent manner.

Proliferative Defect but Normal Transcriptional Response to Erythropoietin in Diamond Blackfan Anemia Erythroid Progenitor Cells in 2-Phase Erythroid Culture.

Diamond Blackfan anaemia (DBA) is a rare congenital red cell aplasia, characterised by a steroid responsive intrinsic erythropoietic failure. Mutations affecting RPS19 are found in up to 25% of DBA, but the pathophysiological link between ribosomal gene haploinsufficiency and the specific erythroid phenotype of DBA remains elusive. One potential mechanism is that ribosomal insufficiency might affect the level of expression of critical erythroid proteins, resulting in a block in terminal erythroid differentiation and expansion. The aim of this study was to elucidate the cellular and molecular events occurring in response to erythropoietin (EPO) in DBA. We applied a 2-phase erythroid culture system in which we have previously demonstrated a consistent and profound failure of erythroid expansion in DBA cultures, localised to early in the second EPO-dependent phase of culture. The timing of the onset of proliferation in response to EPO was studied by tetrazolium dye assay of enriched erythroid progenitor cells in phase II of culture. An increase in cell number was detectable following 72hrs of EPO exposure in DBA (n=5) as well as control cultures, but with a subsequently slower proliferation rate in DBA. The proliferation rate of both DBA and control cells was enhanced by the addition of dexamethasone. Transcriptional profiles of control and DBA erythroid progenitors were then compared by microarray analysis after 24hrs exposure to EPO. Cy3/Cy5 labelled aRNA was competitively hybridised on an in-house array containing 395 transcript exon identifiers in ENSEMBL. The results showed little difference in expression between control and DBA after 24hrs of EPO exposure, the only significant changes being in the expression of just 4 of the 251 genes expressed above background (CDKN2D, TNFRSF6, MAPKK and CXCR), which were upregulated in DBA (p<0.0001 on t-test analysis). The gene expression profile from 0–96 hrs following EPO exposure was then studied in control and DBA (n=3) erythroid progenitors by real time PCR. There was little change in the first 48hr of exposure to EPO. At 72hrs the expression of both EPOR and α-GLOBIN was increased 3–6 fold in both DBA and controls in comparison with pre-EPO, confirming the occurrence of an erythroid-specific transcriptional response to EPO in DBA erythroid progenitors. These results are consistent with a qualitatively normal onset of terminal erythroid maturation in DBA, rather than a block in differentiation, and tend to favour an alternative hypothesis that the erythroid failure in DBA is the result of ribosomal insufficiency at the simultaneous onset of both rapid cell proliferation and a high rate of globin synthesis. In keeping with this, the expression of RPS19 showed 1.5–2 fold increase at 72hrs after EPO exposure, with 3.5 fold increase in the presence of dexamethasone, with equivalent results in both DBA and controls. We now aim to study translational and cell cycle events during this important wave of erythroid proliferation to further elucidate the molecular basis for the specific erythroid defect in DBA, despite the ubiquitous cellular requirement for ribosomal biogenesis.

Synergistic inhibition of breast cancer cell lines with the combination of a dual inhibitor of EGFR/HER-2/neu and a Bcl-2 inhibitor

13100 Background: The epidermal growth factor receptor (EGFR; ErbB1) and HER-2/neu (ErbB2), members of the ErbB family of receptor tyrosine kinases, are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Use of inhibitors of these receptors as monotherapies, e.g., trastuzumab, Iressa, and erlotinib, has led to advances in treatment, but many patients do not respond or develop resistance. The anti-apoptotic protein, Bcl-2, is also overexpressed in a number of human tumors. Inhibitors of Bcl-2 induce apoptosis and sensitize cancer cells to other therapies. This study assesses the effects of a combination of a reversible inhibitor of both EGFR and HER-2/neu that is similar to lapatinib (GW2974) and a pan inhibitor of the Bcl-2 family (GX15–070: Gemin X Biotechnologies, Inc.) on the growth of human breast cancer cells. Methods: The MCF-7 human breast cancer cell line transfected with a control vector, MCF/neo, and the HER-2/neu transfected MCF-7 cell line, MCF/18, were treated with various concentrations of GW2974 (0.25–10 μM) and/or the GX15–070 pan Bcl-2 inhibitor (50–500 nM). After a 3 day exposure, cell number was determined using the colorimetric MTT tetrazolium dye assay. Percent of control was normalized to corresponding concentrations of the solvent for both agents (DMSO). Results: Treatment with the GW2974 dual inhibitor or the GX15–070 pan Bcl-2 inhibitor resulted in dose-dependent growth inhibition in both the control and HER-2/neu transfected MCF-7 cell lines. The combination of both agents produced synergistic growth inhibition in both cell lines as confirmed by isobologram analysis. Conclusions: This study has demonstrated synergy with the combination of a dual inhibitor of EGFR and HER-2/neu and an inhibitor of Bcl-2 in control and HER-2/neu overexpressing MCF-7 human breast cancer cells. This finding warrants an evaluation of this combination in clinical trials for the treatment of patients with metastatic breast cancer. [Table: see text]

The synergism between Belotecan and cisplatin in gastric cancer.

We wanted to demonstrate the anti-cancer effect and interaction between belotecan and cisplatin on gastric cancer cell line and we evaluated the mechanisms of this synergistic effect in vitro. The growth inhibitory effect of belotocan and cisplatin against several gastric cancer cell lines (SNU-5, SNU-16 and SNU-601) was estimated by tetrazolium dye assay. The effect of a combination treatment was evaluated by the isobologram method. The biochemical mechanisms for the interaction between the drugs were analyzed by measuring the formation of DNA interstrand cross-links (ICLs) and DNA topo-I activity. Belotecan showed synergism with cisplatin for growth inhibitory effect on the gastric cancer cell lines SNU-5, and SNU-16, but this was subadditive on the SNU-601 cell line. The formation of DNA ICLs in SNU-16 cells by cisplatin was increased by combination with belotecan, but this was not affected in SNU-601 cells. The topo-I inhibition by belotecan was enhanced at high concentrations of cisplatin in SNU-16, but not in SNU-601 cells. Belotecan and cisplatin show various combination effect against gastric cancer cells. The synergism between cisplatin and belotecan could be the result of one of the following mechanisms: the modulating effect of belotecan on the repair of cisplatin-induced DNA adducts and the enhancing effect of cisplatin on the belotecan-induced topo-I inhibitory effect.

Synergistic cytotoxicity and apoptosis of JTE-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer.

Cyclooxygenase-2 (COX-2) is a key inducible enzyme involved in the production of prostaglandins that has been shown to induce apoptosis in various cancer cells. Several anticancer agents also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. Since over expression of COX-2 has been demonstrated in bladder cancer, we reasoned that combination treatment with a COX-2 inhibitor and anticancer agents in bladder cancer cells may result in synergistic apoptosis. We examined whether the selective COX-2 inhibitor JTE-522 induces apoptosis in bladder cancer cells and whether JTE-522 may act synergistically with anticancer agents to achieve cytotoxicity and apoptosis in these cells. Cytotoxicity was determined by microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. COX-2 mRNA expression was observed in the HT1197 bladder cancer cell line. JTE-522 was cytotoxic in HT1197 cells. Treating HT1197 cells with JTE-522 combined with doxorubicin or mitomycin C did not show synergistic cytotoxicity. However, combination treatment of HT1197 cells with JTE-522 and 5-fluorouracil (5-FU) resulted in a synergistic cytotoxic effect. Synergy was also achieved in the T24 bladder cancer line. Synergistic cytotoxicity was noted irrespective of treatment sequence but the highest percent cytotoxicity was obtained when HT1197 cells were treated with JTE-522 and 5-FU simultaneously. The synergy achieved in cytotoxicity with JTE-522 and 5-FU was shown to be due to apoptosis. The mechanisms responsible for synergistic cytotoxicity and apoptosis was examined. Treating HT1197 cells with 5-FU enhanced expression of the pro-apoptotic molecule Bax, while JTE-522 treatment reduced expression of the anti-apoptotic molecule Bcl-XL, resulting in a significantly higher ratio of Bax-to-Bcl-XL. This study shows that combination treatment of bladder cancer cells with the selective COX-2 inhibitor JTE-522 and 5-FU results in synergistic cytotoxicity and apoptosis due to the enhanced Bax-to-Bcl-XL expression ratio. These findings support the in vivo potential application of a combination of JTE-522 and 5-FU for bladder cancer.

Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines

Cytotoxic and antiproliferative effects of halothane, isoflurane, and sevoflurane in anesthetic doses on human colon carcinoma (Caco-2), larynx carcinoma (HEp-2), pancreatic carcinoma cells (MIA PaCa-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), and normal fibroblasts were investigated. Cells were exposed to anesthetic gas mixture consisting of O 2: N2O (35:60 vol.%), halothane (1.5 vol.%) or isoflurane (2.0 vol.%) or sevoflurane (3.0 vol.%), and CO 2 (5 vol.%), for 2, 4, and 6 h. Cytotoxicity of anesthetics was analyzed by validated tetrazolium dye assay MTT test. All anesthetics expressed cytotoxic effects on treated tumor cells in time and cell line dependent manner. Growth suppression in cells exposed to halothane was enhanced in HEp-2 (to 67.7%), Caco-2 (to 76.3%), and SW620 cells (to 80.9%), and was minimal in normal fibroblasts (to 89.4%). Antiproliferative activity of halothane was measured via radioactive precursors incorporation assay. In Caco-2 cells treated by halothane, decrease in DNA synthesis (52.4%, p = 0.001), RNA synthesis (39.2%, p < 0.001), and protein synthesis (19.2%, p = 0.004) was observed. In HEp-2 cells, DNA and RNA syntheses were decreased to 72.5% and 79.9%, whereas protein synthesis was 14.0% of control ( p < 0.001). In SW620 cells, protein synthesis after 4 h was 24.4% ( p = 0.007). A DNA fragmentation was observed in Caco-2 and MIA PaCa-2 cells. Exposition of phosphatidylserine on outer lipid bilayer plasma membrane of tumor cell treated by halothane proved apoptosis as mode of cell death.

Gefitinib (“Iressa”, ZD1839), an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Reverses Breast Cancer Resistance Protein/ABCG2–Mediated Drug Resistance

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.