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PubMed2003

A novel polyamine analog inhibits growth and induces apoptosis in human breast cancer cells.

Polyamine analogs have demonstrated considerable activity against many important solid tumor models including breast cancer. However, the precise mechanisms of antitumor activities of polyamine analogs are not entirely understood. The cytotoxicity of a newly developed polyamine analog compound, SL11144, against human breast cancer was assessed. Treatment of human breast cancer cell lines in culture with SL11144 decreased cell proliferation and induced programmed cell death in a time- and dose-dependent manner. SL11144 also profoundly inhibited the growth of MDA-MB-231 xenografts in host nude mice without overt toxic effects. Treatment of MDA-MB-435 cells with SL11144 led to the release of cytochrome c from mitochondria into cytosol, activation of caspase-3, and poly(ADP-ribose) polymerase cleavage. SL11144 decreased Bcl-2 and increased Bax protein levels in MDA-MB-231 cells. Furthermore, activator protein 1 transcriptional factor family member c-Jun was up-regulated by SL11144 in MDA-MB-435 and MDA-MB-231 cells, but not in MCF7 cells. In addition, significant inhibition of ornithine decarboxylase activity and a decrease in polyamine pools were demonstrated. These results demonstrate that the novel polyamine analog SL11144 has effective antineoplastic action against human breast cancer cells in vitro and in vivo and that multiple apoptotic mechanisms are associated with its cytotoxic effect in specific human breast cancer cell lines.

Animals↗

PubMed1997

Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line.

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4-24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (-25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

Adenosine Triphosphate↗

PubMedDate not supplied

Technetium-99m glucarate uptake in a swine model of limited flow plus increased demand.

BACKGROUND: Glucarate is a 6-carbon dicarboxylic acid shown to be taken up by necrotic myocytes, binding to nuclear histones in animal models of coronary occlusion, resulting in infarction. This study investigated glucarate uptake in a model of severe ischemia. METHODS AND RESULTS: Thirty-five experiments were performed, in which a catheter-mounted stenosis (reducing lumen dimensions by 80%) was placed in the left anterior descending coronary artery (LAD) of an anesthetized, instrumented domestic swine and technetium-99m glucarate (GLU) was injected during the last minute of 5 minutes of pacing. Hemodynamic and blood flow measurements were performed at control, during pacing, and during recovery. The animals were killed; their hearts were stained with fluorescein dye and triphenyl tetrazolium chloride (TTC). Electron micography (EM; n = 6) and cell centrifugation (n = 7) were also performed. On the basis of net lactate production and severe blood flow reduction in the risk region (RR), ischemia with pacing developed in 25 animals. Fifteen of 25 animals showed tracer uptake in the RR on in vivo and ex vivo imaging (scan positive), and 10 were scan negative in the RR. Endocardial blood flow in the RR during pacing was 0.28+/-0.16 mL/g/min for scan-positive and 0.30+/-0.17 mL/g/min for scan-negative experiments (P = not significant [NS]). Transmyocardial net lactate extraction during pacing was -63%+/-44% for scan-positive and -53%+/-60% for scan-negative experiments (P = NS). Control and recovery heart rates were higher in scan-positive experiments (108+/-14 vs. 92+/-17, and 125+/-24 vs. 104+/-18, P<.02). Lactate extraction was lower during control and recovery in scan-positive animals (2+/-29 vs. 30+/-19, P = .03). Scan-positive animals had a more proximal stenosis position. Minimal necrosis was documented by means of TTC negative staining in 8 of 15 scan-positive experiments (comprising 10%+/-4.3% of RR area). EM or cell fractionation was performed in 5 of the 7 remaining scan-positive and TTC-positive hearts, and in those 5 experiments, necrosis was documented by means of EM in 2 and by means of cell fractionation in 3. CONCLUSIONS: Uptake of Tc-99m glucarate was seen in the RR in a swine model of ischemia severe enough to produce myocyte injury and early cell death.

Animals↗

PubMed2006

Inhibitory effect of triptolide on lymph node metastasis in patients with non-Hodgkin lymphoma by regulating SDF-1/CXCR4 axis in vitro.

AIM: To investigate the antiproliferative effect of triptolide on B-NHL cell line Raji cells, to study its effect on lymph node metastasis in patients with non-Hodgkin's lymphoma (NHL) in vitro, and to explore the underlying mechanism regulating SDF-1/CXCR4 axis. METHODS: The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effects of triptolide on SDF-1 mRNA expression in lymph node stromal cells from patients with NHL were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effects of triptolide on CXCR4 expression on lymphoma cells freshly isolated from the lymph nodes of these patients were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of primary lymphoma cells towards recombinant human SDF-1 alpha (rhSDF-1 alpha) or cultured lymph node stromal cells in vitro. RESULTS: Triptolide inhibited the proliferation of B-NHL cell line Raji cells in a dose- and time-dependent manner with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. The expression of SDF-1alpha mRNA in lymph node stromal cells obtained from patients with NHL was decreased after treatment by triptolide at concentrations of 25 and 50 nmol/L for 24 h. Flow cytometry analysis showed that the CXCR4 expression on primary lymphoma cells were downregulated gradually in a dose-dependent manner following triptolide treatment. Chemotaxis assays revealed that the migration of freshly isolated lymphoma cells towards either rhSDF-1 or cultured lymph node stromal cells was markedly inhibited by the addition of triptolide in vitro, and the inhibition was dose-dependent. CONCLUSION: Triptolide can inhibit the proliferation of B-NHL cell line Raji cells. Moreover, triptolide is able to inhibit the migration of lymphoma cells via lymph nodes in vitro. The potential antitumor mechanisms of triptolide are related to the antiproliferative effect and the blockage of SDF-1/CXCR4 axis.

Antineoplastic Agents, Alkylating↗

PubMed1986

Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation.

To explore the structural characteristics of various derivatives of the anticancer drugs, doxorubicin and daunorubicin, for exhibiting redox activities believed to be associated with toxic radical production, we tested over fifty derivatives in a rapid screening procedure for augmenting oxygen consumption by rat liver microsomes. Measurement of parent drug disappearance and of metabolite appearance for fourteen anthracyclines with a broad range of activities for augmenting oxygen consumption indicated that a single reaction, conversion to the 7-deoxyaglycone, occurred. Multiple tests of selected compounds showed that the liver microsome system exhibited saturation kinetics, and calculated values of Vmax/Km gave the same relative order of activities as did the screening test. The liver microsome system was not found to be stereoselective. Measurements of the abilities of a number of the anthracycline derivatives after chemical activation by reduction with sodium borohydride to convert oxygen to superoxide anion, or to the hydroxyl radical, were also made. The reactivities of the anthracyclines in these latter two assays were positively related to the activities obtained in the rat liver microsome screening test, suggesting that all three tests were measuring various steps in the sequence from anthracycline semiquinone radical formation through oxygen activation and radical formation. Superoxide anion generation from chemically reduced anthracyclines was inhibited by the addition of calf thymus DNA, and the extent of inhibition was positively correlated with the measured DNA association constants of the anthracyclines. However, the DNA association constants were unrelated to superoxide anion generation in the absence of DNA or to the augmentation of oxygen consumption in liver microsomes. Half-wave potentials were negatively correlated with both the results of the microsomal oxygen consumption test and the production of superoxide anion in the chemical test system. No relationships were discerned among the DNA association constants, half-wave potentials, or reoxidizabilities of the anthracyclines tested. Comparisons of the relatively low activities of certain of the anthracyclines in the biochemical and chemical tests for oxygen activation with their known high activities against murine tumors in vivo, but low cardiotoxicities in animal model systems, suggest that the separation of the cytotoxic antitumor and cardiotoxic actions of these derivatives may have been achieved.

Animals↗

PubMed1998

Generation and characterization of a mutant of influenza A virus selected with the neuraminidase inhibitor BCX-140.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

Animals↗

PubMed1996

Chromium- and nickel-induced cytotoxicity in normal and transformed human keratinocytes: an investigation of pharmacological approaches to the prevention of Cr(VI)-induced cytotoxicity.

Chromium and nickel compounds cause irritancy but can also induce allergic contact dermatitis. The aims of this study were to characterize the direct cytotoxic effects of Cr(VI), Cr(III) and Ni(II) salts on keratinocytes, and to investigate pharmacological strategies to protect cells against Cr(VI)-induced cytotoxicity. Normal human keratinocytes and the HaCaT keratinocyte cell line were used. Cell viability was assessed by neutral red dye uptake, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) eluted stain assay and measurement of lactate dehydrogenase (LDH) activity in the medium. The assays varied slightly in their sensitivities (neutral red > MTT > LDH) although all three gave similar results. In both cell types, the relative order of cytotoxicity of the salts was Cr(VI) >> Ni(II) > Cr(III). There were no major differences between chromium salts of a common valency. Normal human keratinocytes showed a much greater variability in their response to Cr(VI) and Ni(II) salts than HaCaT cells and were generally more resistant to Cr(VI)- and Ni(II)-induced cytotoxicity. Several drugs were screened for their potential to protect both cell types against the cytotoxic effects of Cr(VI), specifically the reducing agents ascorbic acid, cysteine and glutathione, and the Cr(VI) cellular uptake inhibitors 4,4'-diisothiocyanato-2,2'-stilbenedisulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulphonic acid (SITS). All five drugs provided concentration-dependent protection against Cr(VI)-induced cytotoxicity but only ascorbic acid offered complete protection. Several of these pharmacological approaches to the prevention of Cr(VI) cytotoxicity confirm previous clinical studies on the inactivation of Cr(VI), while the clinical potential of others has yet to be investigated.

Ascorbic Acid↗

PubMed2002

Enhanced antitumor activities of TZT-1027 against TNF-alpha or IL-6 secreting Lewis lung carcinoma in vivo.

PURPOSE: TZT-1027, an antimicrotubule agent that inhibits the polymerization of tubulin, shows potent antitumor activity in various transplantable tumor models in vivo. The high antitumor activity of TZT-1027 prompted us to speculate that this compound may have a mode of action other than its antimicrotubule and antimitotic activities. To elucidate the interaction of antitumor cytokines with TZT-1027 in tumors in vivo, we examined the antitumor activity of this agent against various cytokine gene-transfected Lewis lung carcinoma (LLC) cells inoculated into C57BL/6 mice. METHODS: In vitro growth inhibition was evaluated using the MTT assay, and in vivo activity was evaluated in subcutaneous models in C57BL/6 mice. The status of the vasculature in tumor tissues was evaluated immunohistochemically using anti-CD31 antibody. We used a cDNA macroarray to examine the gene expression profiles in tumor tissues removed from mice. RESULTS: TZT-1027 at 3 mg/kg showed potent antitumor activity in Mock (LLC-Neo cells) inoculated mice with a T/C% value of 16%. TZT-1027 at 3 mg/kg showed more potent antitumor activity in LLC-TNF cells and LLC-IL6 cells with T/C% values of 4% and 3%, respectively. TZT-1027 treatment destroyed the tumor vasculature as well as tumor cells in LLC-TNF and LLC-IL6 tissues of mice treated with TZT-1027. The LLC-TNF and LLC-IL6 tissues of mice treated with TZT-1027 had in common the independent alteration of the non-histone chromosomal protein HMG-14 and transcription factor 1 for heat shock gene. Focusing on the gene regulation related to angiogenesis, the alteration in transcriptional factors such as ets family genes and homeobox family genes was remarkable. CONCLUSIONS: These factors are candidates as determinants of the enhanced TZT-1027 antitumor activity in relation to these cytokines.

Animals↗

PubMed2004

Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection.

BACKGROUND: Transient (low-conductance) opening of the mitochondrial permeability transition pore (mPTP) may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. We hypothesize that transient mPTP opening and ROS mediate the protection associated with myocardial preconditioning and mitochondrial uncoupling. METHODS AND RESULTS: Isolated perfused rat hearts were subjected to 35 minutes of ischemia/120 minutes of reperfusion, and the infarct-risk-volume ratio was determined by tetrazolium staining. Inhibiting mPTP opening during the preconditioning phase with cyclosporine-A (CsA, 0.2 micromol/L) or sanglifehrin-A (SfA, 1.0 micromol/L) abolished the protection associated with ischemic preconditioning (IPC) (20.2+/-3.6% versus 45.9+/-2.5% with CsA, 49.0+/-7.1% with SfA; P<0.001); and pharmacological preconditioning with diazoxide (Dzx, 30 micromol/L) (22.1+/-2.7% versus 46.3+/-3.0% with CsA, 48.4+/-5.5% with SfA; P<0.001), CCPA (the adenosine A1-receptor agonist, 200 nmol/L) (24.9+/-4.5% versus 54.4+/-6.6% with CsA, 42.6+/-9.0% with SfA; P<0.001), or 2,4-dinitrophenol (DNP, the mitochondrial uncoupler, 50 micromol/L) (15.7+/-2.7% versus 40.8+/-5.5% with CsA, 34.3+/-3.1% with SfA; P<0.001), suggesting that mPTP opening during the preconditioning phase is required to mediate protection in these settings. Inhibiting ROS during the preconditioning protocols with N-mercaptopropionylglycine (MPG, 1 mmol/L) also abolished the protection associated with IPC (20.2+/-3.6% versus 47.1+/-3.8% with MPG; P<0.001), diazoxide (22.1+/-2.7% versus 56.3+/-3.8% with MPG; P<0.001), and DNP (15.7+/-2.7% versus 50.7+/-6.6% with MPG; P<0.001) but not CCPA (24.9+/-4.5% versus 26.5+/-8.4% with MPG; P=NS). Further experiments in adult rat myocytes demonstrated that diazoxide induced CsA-sensitive, low-conductance transient mPTP opening (represented by a 28+/-3% reduction in mitochondrial calcein fluorescence compared with control; P<0.01). CONCLUSIONS: We report that the protection associated with IPC, diazoxide, and mitochondrial uncoupling requires transient mPTP opening and ROS.

2,4-Dinitrophenol↗

PubMed2004

Inhibition of tumor-associated fatty acid synthase activity enhances vinorelbine (Navelbine)-induced cytotoxicity and apoptotic cell death in human breast cancer cells.

The lipogenic enzyme fatty acid synthase (FAS) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for anti-neoplastic therapy development. Current trends in the treatment of human breast cancer are with drug combinations that result in improved responses as well as the ability to use less toxic concentrations of the drugs. Here, we envisioned that combinations of conventional chemotherapeutic agents with novel compounds directed against breast cancer-associated FAS hyperactivity may provide increased efficacy over existing therapy for human breast cancer. Specifically, we examined the ability of the mycotoxin cerulenin, a potent and non-competitive inhibitor of FAS activity, to enhance the cytotoxic effects of vinorelbine (Navelbine), a derivative of vinca alkaloid that interferes with tubulin assembly and exhibits activity against metastatic breast cancer. SK-Br3, MCF-7 and MDA-MB-231 human breast cancer cell lines were employed as models of high, moderate and low levels of FAS ('cerulenin-target'), respectively. Combinations of cerulenin with vinorelbine were tested for synergism, additivity or antagonism using the isobologram and the median-effect plot (Chou-Talalay) analyses. Breast cancer cells were either simultaneously exposed to cerulenin and vinorelbine for 24 h or sequentially to cerulenin for 24 h followed by vinorelbine for 24 h. Concurrent exposure to cerulenin and vinorelbine resulted in synergistic interactions in MCF-7 and MDA-MB-231 cell lines, while additivity was found in SK-Br3 cells. Sequencing cerulenin followed by vinorelbine resulted in synergism for SK-Br3 and MDA-MB-231 cells, whereas it showed additive effects in MCF-7 cells. FAS activity blockade was found to synergistically enhance apoptosis-inducing activity of vinorelbine, as determined by an enzyme-linked immunosorbent assay for histone-associated DNA fragments. To the best of our knowledge this is the first study demonstrating that breast cancer-associated FAS is playing an active role in human breast cancer chemosensitivity. We suggest that pharmacological inhibition of FAS activity is a novel molecular approach to enhance the cytotoxic effects of existing chemotherapeutic agents in human breast cancer.

Antifungal Agents↗

PubMed2005

Comparative analysis of mutagenic potency of 1-nitro-acridine derivatives.

The anticancer effect of 1-nitro-9-hydroxyethylamino acridine (C-857), a compound belonging to the 1-nitroacridine class, has been well documented. Despite its therapeutic efficacy, the clinical development of C-857 has been impeded partly due to its high systemic toxicity. In an effort to enhance antitumor efficacy and lower toxicity, derivatives of C-857 have been synthesized with substitutions made at position C-4 and/or an esterified hydroxyl group in side chain at the C-9 position. The introduction of a methyl group at C-4 resulted in C-1748, which has a significantly higher therapeutic efficacy and is being clinically developed as an anticancer agent for solid tumors. The present study was undertaken to correlate the mutagenicity of C-857, C-1748, C-1790, C-1872 and C-1873 with their cytotoxicity and their anti-tumor efficacy. The mutagenicity of these drugs was determined using three Ames Salmonella typhimurium strains TA1537, TA98 and TA102. The bacteria were treated with different molar concentrations, ranging from 10(-3) to 10(-12) M, of the drugs and drug-induced histidine revertants were then counted after a 48 h incubation. C-1748 did not induce any revertants in both TA1537 and TA98 at a dose of 10(-6) M, whereas, C-857 at the same dose induced approximately 842 and approximately 1034 revertants respectively. In TA102, mutagenicity was lower than observed with TA98 and TA1537 with highest revertants observed at 10(-5) M with C-857 (approximately 606) and C-1748 (approximately 108). Higher mutagenicity was observed in the derivatives C-1790, C-1872 and C-1873 compared to C-1748, but lower than C-857. These studies demonstrate that C-1748 has the least mutagenic potential, with a much higher antitumor effect in prostate cancer and is a promising chemotherapeutic agent for clinical development.

Aminoacridines↗

PubMed2006

Induction of apoptosis by disturbing mitochondrial-membrane potential and cleaving PARP in Jurkat T cells through treatment with acetoxyscirpenol mycotoxins.

Paecilomyces tenuipes is a famous Chinese medicinal entomopathogenic fungus that grows within the larvae of silkworms. 4beta-acetoxyscirpendiol (4-MAS), a cytotoxic compound belonging to the scirpenol subfamily of trichothecene mycotoxin, was isolated from Paecilomyces tenuipes. To further elucidate the cytotoxic mechanism of 4-MAS, evidences of its induction of apoptosis, together with the structurally related acetoxyscirpenol moiety mycotoxins (ASMs) such as, 15-acetoxyscirpenol (15-MAS), 4,15-diacetoxyscirpenol (4,15-DAS), and 3alpha-acetyldiacetoxyscirpenol (TAS), in the human Jurkat T cell line were reported herein. In the MTT reduction and time-course cytotoxicity assays for monitoring cell viability, all the four ASMs that were tested exhibited cytotoxicity; single acetoxylation at C-4 of the scirpenol family resulted in relatively weak cytotoxicity, while acetoxylation at C-15 resulted in strong cytotoxicity regardless of the other acetoxylations at the C-3 and/or C-4 positions. Phosphatidylserine externalization was induced by all the ASMs that were treated at an early phase in a time-dependent manner, showing a typical apoptotic phenomenon, not a necrotic one. The ASMs also reduced the mitochondria's inner-membrane potential (deltaPsim) through flow cytometry analysis after staining these with DiOC6, a mitochondria-specific and voltage-dependent dye. Acetoxylation of ASM at C-15 increased deltaPsim disruption, but that at C-3 reduced the deltaPsim. The ASMs that were tested also cleaved 113 kDa PARP to an 89-kDa fragment through Western blot assay, suggesting the activation of caspase-3 and/or caspase-7 in the Jurkat T cell. DNA fragmentation was also observed to have been increased in a time-dependent manner by the ASMs that were tested in Jurkat T cells, resulting in the DNA fragmentation intensity order of 4,15-DAS>15-MAS>TAS>4-MAS. These data indicate that the Jurkat T cells that were treated with ASMs underwent typical cascades of apoptotic cell death.

Animals↗

PubMed2004

RWJ-241947 (MCC-555), a unique peroxisome proliferator-activated receptor-gamma ligand with antitumor activity against human prostate cancer in vitro and in beige/nude/ X-linked immunodeficient mice and enhancement of apoptosis in myeloma cells induced by arsenic trioxide.

PURPOSE: RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-gamma ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds. EXPERIMENTAL DESIGN: Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined. RESULTS: In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21(WAF1), deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-gamma ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells. CONCLUSIONS: RWJ-241947 has surprisingly potent antiproliferative effects against prostate cancer cells in vivo, and it enhances the antitumor activity of As2O3 against myeloma cells. Small, well-defined clinical studies using RWJ-241947 are in order for these cancers.

Agar↗

PubMed2000

Cytotoxicity of PM(2.5) and PM(2.5--10) ambient air pollutants assessed by the MTT and the Comet assays.

Ambient air particulate matters are classified into two distinct modes in size distribution, namely the coarse and fine particles. Correlation between high particulate concentration and adverse effects on human populations has long been recognized, however, the toxicology of these adverse effects has not been clarified. In the current report, the cytotoxic effects of the solvent-extractable organic compounds (SEOC) from fine particles smaller than 2.5 microm (PM(2.5)) and from coarse particles between 2.5-10 microm (PM(2.5-10)) were studied. Nine 24h consecutive monthly samples were tested to determine the correlation between cytotoxicity and total SEOC in two size fractions of particulate air pollution. Cytotoxicity of SEOC was measured by two micro-scale mammalian cells-based bioassays: the MTT cell proliferation assay, and the Comet assay for the detection of DNA damage. A well-defined mammalian cell line - Rat 6 rodent fibroblast was employed in the study. The SEOC extracts of air particulate matters were sub divided into two equal parts. One part was dissolved in DMSO, the other in KOH/hexane and then conjugated with bovine serum albumin to produce a lipid-soluble fraction for testing. The DMSO fraction would contain mainly the polycyclic aromatic hydrocarbons (PAH), alkanes and alkanols, while the lipid-soluble fraction would be enriched with fatty acids. The results from MTT assay showed that cytotoxicity of the PM(2.5) was much more severe than the PM(2.5-10), suggesting that toxic SEOC were confined to the fine particles. By and large, the DMSO solubles were much more toxic than the lipid solubles. The degree of cytotoxicity of the DMSO soluble samples is positively correlated to the amount of particulates present in the ambient air. For the PM(2.5), the winter samples were significantly more toxic than the summer samples in terms of cell killing, which seemed to be a direct reflection of the total loading of organic matter in the samples. Results from Comet assays showed that SEOC samples of PM(2.5) derived from winter months induced DNA damage at dosages resulting in no obvious cell killing in the MTT assay. Thus, long-term exposure to non-killing dosage of air pollutants may lead to the accumulation of DNA lesions, which may be one of the mechanisms responsible for the chronic adverse health effects of particulate air pollution.

Aerosols↗

PubMed2005

Effect of NQO1 induction on the antitumor activity of RH1 in human tumors in vitro and in vivo.

NQO1 is a reductive enzyme that is important for the activation of many bioreductive agents and is a target for an enzyme-directed approach to cancer therapy. It can be selectively induced in many tumor types by a number of compounds including dimethyl fumarate and sulforaphane. Mitomycin C is a bioreductive agent that is used clinically for treatment of solid tumors. RH1 (2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1,4-benzoquinone) is a new bioreductive agent currently in clinical trials. We have shown previously that induction of NQO1 can enhance the antitumor activity of mitomycin C in tumor cells in vitro and in vivo. As RH1 is activated selectively by NQO1 while mitomycin C is activated by many reductive enzymes, we investigated whether induction of NQO1 would produce a greater enhancement of the antitumor activity of RH1 compared with mitomycin C. HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with or without dimethyl fumarate or sulforaphane followed by mitomycin C or RH1 treatment, and cytotoxic activity was measured by a clonogenic (HCT116) or MTT assay (T47D). Dimethyl fumarate and sulforaphane treatment increased NQO1 activity by 1.4- to 2.8-fold and resulted in a significant enhancement of the antitumor activity of mitomycin C, but not of RH1. This appeared to be due to the presence of a sufficient constitutive level of NQO1 activity in the tumor cells to fully activate the RH1. Mice were implanted with HL60 human promyelocytic leukemia cells, which have low levels of NQO1 activity. The mice were fed control or dimethyl fumarate-containing diet and were treated with RH1. NQO1 activity in the tumors increased but RH1 produced no antitumor activity in mice fed control or dimethyl fumarate diet. This is consistent with a narrow window of NQO1 activity between no RH1 activation and maximum RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is not likely to be an effective strategy for enhancing the antitumor activity of RH1. In addition, we found that RH1 treatment produced significant leukopenia in mice that may be of concern in the clinic. These results suggest that the ease of reduction of RH1 by NQO1 makes it a poor candidate for an enzyme-directed approach to cancer therapy.

Animals↗

PubMedDate not supplied

Cutaneous inflammation and proliferation in vitro: differential effects and mode of action of topical glucocorticoids.

The nonhalogenated double ester of prednisolone, prednicarbate (PC), is the first topical glucocorticoid with an improved benefit/risk ratio verified clinically and in vitro. To evaluate if this is due to unique characteristics of this steroid, a new compound created according to an identical concept, prednisolone 17-ethylcarbonate, 21-phenylacetate (PEP), and the new halogenated monoester desoximetasone 21-cinnamate (DCE) were tested and compared to PC, desoximetasone (DM) and betamethasone 17-valerate (BMV). Isolated foreskin keratinocytes served for in vitro investigations of anti-inflammatory processes in the epidermis, fibroblasts of the same origin were used to investigate the atrophogenic potential. Inflammation was induced by TNFalpha, resulting in an increased interleukin 1alpha (Il-1alpha) synthesis. As quantified by ELISA, all drugs significantly reduced Il-1alpha production. But PC and BMV appeared particularly potent, followed by DM and the two new congeners, which revealed minor anti-inflammatory activity. Glucocorticoid esters including PEP are rapidly degraded in keratinocytes (85% within 12 h). Hence, a ribonuclease protection assay of Il-1alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation. This assay confirmed the anti-inflammatory potency of native PC and BMV. In contrary DCE and PEP did not reduce Il-1alpha mRNA to a significant extent. Therefore PEP acts as a prodrug only. In fibroblasts, Il-1alpha and Il-6 syntheses indicate proliferation and inflammation, respectively. Whereas PC and PEP inhibited Il-1alpha and Il-6 production in fibroblasts only to a minor extent, cytokine synthesis was strongly affected by the conventional glucocorticoids BMV and DM, but also by DCE. The minor unwanted effect of PC and PEP on fibroblasts was also reflected by their low influence on cell proliferation as derived from (3)H-thymidine incorporation. Again, more pronounced antiproliferative features were seen with the halogenated glucocorticoids. In the following, the correlation between antiphlogistic effects in keratinocytes (suppression of Il-1alpha) and antiproliferative effects in fibroblasts (suppression of Il-1alpha and Il-6; (3)H-thymidine incorporation) was analyzed. Here, PC is revealed as the only glucocorticoid with an improved benefit/risk ratio. Native PEP is shown to be almost ineffective and DCE presents exactly the opposite features of PC. It is tempting to speculate if this is due to different glucocorticoid receptor subtypes or different signaling pathways in keratinocytes and fibroblasts.

Administration, Topical↗

PubMed2004

Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C.

PURPOSE: To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD: Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS: Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS: Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.

Animals↗