[Show abstract][Hide abstract] ABSTRACT: Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype.
The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype.
These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology.
PLoS ONE 01/2013; 8(2):e57099. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new tetrazolium salt, XTT, has been synthesized. XTT is reduced by a considerable variety of cell lines to a water-soluble formazan. XTT appears to merit further investigation as a reagent for broader application to cell culture assay systems.
[Show abstract][Hide abstract] ABSTRACT: Twelve artemisinin acetal dimers were synthesized and tested for antitumor activity in the National Cancer Institute (NCI) in vitro human tumor 60 cell line assay, producing a mean GI(50) concentration between 8.7 (least active) and 0.019 microM (most active). The significant activity of the compounds in this preliminary screen led to additional in vitro antitumor and antiangiogenesis studies. Several active dimers were also evaluated in the in vivo NCI hollow fiber assay followed by a preliminary xenograft study. The title compounds were found to be active against solid tumor-derived cell lines and showed good correlation with other artemisinin-based molecules in the NCI database. The dimers were also evaluated for their antimalarial and antileishmanial activities. The antimalarial activity ranged from 0.3 to 32 nM (IC(50)), compared to 9.9 nM for artemisinin.
[Show abstract][Hide abstract] ABSTRACT: A major limitation of adenovirus type 5-mediated cancer gene therapy is the inefficient infection of many cancer cells. Previously, we showed that treatment with low doses of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) increased coxsackie adenovirus receptor (CAR) levels, histone H3 acetylation, and adenovirus infection efficiencies as measured by viral transgene expression in cancer cell lines but not in cultured normal cells. To evaluate FK228 in vivo, the effects of FK228 therapy in athymic mice bearing LOX IMVI or UACC-62 human melanoma xenografts were examined. Groups of mice were treated with FK228 using several dosing schedules and the differences between treated and control animals were determined. In mice with LOX IMVI xenografts (n = 6), maximum CAR induction was observed 24 h following a single FK228 dose of 3.6 mg/kg with a 13.6 +/- 4.3-fold (mean +/- SD) increase in human CAR mRNA as determined by semiquantitative reverse transcription-PCR analysis. By comparison, mouse CAR levels in liver, kidney, and lung from the same animals showed little to no change. Maximum CAR protein induction of 9.2 +/- 4.8-fold was achieved with these treatment conditions and was associated with increased histone H3 acetylation. Adenovirus carrying a green fluorescent protein (GFP) transgene (2 x 10(9) viral particles) was injected into the xenografts and GFP mRNA levels were determined. A 7.4 +/- 5.2-fold increase in GFP mRNA was found 24 h following adenovirus injection into optimally FK228-treated mice (n = 10). A 4-fold increase in GFP protein-positive cells was found following FK228 treatment. These studies suggest that FK228 treatment prior to adenovirus infection could increase the efficiency of adenovirus gene therapy in xenograft model systems.
Molecular Cancer Therapeutics 03/2007; 6(2):496-505. · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increased CAR RNA levels and was associated with a 5-10 fold increase in adenovirus transgene expression following adenovirus infection of FK228-treated cells. Since treatment of cultured cells with 1 ng/ml FK228 caused increased acetylated histone H3, the mechanism for the FK228 effect in vitro may be through protein acetylation. This potential mechanism was also suggested because treatment with the histone deacetylase inhibitors sodium butyrate or trichostatin A also caused increased levels of CAR. Increases in CAR levels, transgene expression, and histone H3 acetylation were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment in vivo was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI human melanoma xenografts. Treatment efficacy was determined by analyzing CAR RNA levels by semi- quantitative RT-PCR analysis or by analyzing CAR protein by western blot analysis. A maximum human CAR increase of 13.6- fold (±4.3) compared to untreated xenografts was observed 24 h following i.v. injection of mice with 3.6 mg/kg FK228. By comparison, murine CAR RNA levels in livers, kidneys and lungs from the same animals remained unchanged. Xenograft protein from mice treated with 3.6 mg/kg FK228 and analyzed at 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (±4.8) increase in CAR levels. Based on these results xenograft-bearing mice treated with 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h later. Analysis of RNA from the entire xenografts showed a 7.2-fold (±4.5) increase in expression from the adenovirus GFP transgene in the FK228-treated mice compared to untreated controls. In order to determine the mechanism for this effect, the levels of histone H3 acetylation in treated compared to control xenografts were analyzed. Western blot analysis of xenografts from mice showed that 6 h following treatment with 3.6 mg/kg FK228, there was an average 28.2-fold (±22.9) increase in histone H3 acetylation that remained at 20.4-fold (±12.6) 24 h following treatment. These studies suggest that the mechanism by which FK228 increases the efficiency of adenovirus gene therapy in vivo is through protein acetylation.
[Show abstract][Hide abstract] ABSTRACT: To describe the preclinical basis for further development of 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG, NSC 707545).
In vitro proliferation assays, and in vivo model studies in metastatic pancreatic carcinoma and subcutaneous xenograft melanoma and small-cell lung carcinoma models.
17-DMAG emerged from screening studies as a potent geldanamycin analog, with the average concentration inhibiting the growth of the NCI anticancer cell line drug screen by 50% being 0.053 microM. "Head to head" comparison with 17-allylamino-17-demethoxygeldanamycin (17-AAG, NSC 330507) revealed 17-DMAG to possess potent activity against certain cell types, e.g., MDA-MB-231 breast carcinoma and HL60-TB leukemia which were relatively insensitive to 17-AAG. Evidence of oral bioavailability of 17-DMAG in a saline-based formulation prompted more detailed examination of its antitumor efficacy in vivo. 17-DMAG inhibited the growth of the AsPC-1 pancreatic carcinoma xenografts growing as intrahepatic metastases at doses of 6.7-10 mg/kg twice daily for 5 days administered orally under conditions where 17-AAG was without activity. 17-DMAG in an aqueous vehicle at 7.5-15 mg/kg per day for 3 days on days 1-3, 8-10 and 13-17, or 1-5 and 8-12 showed evidence of antitumor activity by the parenteral and oral routes in the MEXF 276 and MEXF 989 melanomas and by the parenteral route in the LXFA 629 and LXFS 650 adenocarcinoma and small-cell carcinoma models. The latter activity was comparable to the historical activity of 17-AAG.
Taken together, the in vivo activity of 17-DMAG supports the further development of this water-soluble and potentially orally administrable geldanamycin congener.
Cancer Chemotherapy and Pharmacology 09/2005; 56(2):115-25. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S175|[ndash]|S176; doi: 10.1016/j.ymthe.2005.06.461
453. The Histone Deacetylase Inhibitor FK228 Can Increase Adenovirus Transgene Protein Expression in Human LOX IMVI Melanoma Xenografts
Merrill E. Goldsmith1, Alian Aguila1, Michael C. Alley2, William R. Waud3, Susan Bates1 and Tito Fojo11National Cancer Institute, National Institutes of Health, Bethesda, MD2National Cancer Institute, National Institutes of Health, Frederick, MD3Southern Research Institute, Birmingham, AL
[Show abstract][Hide abstract] ABSTRACT: Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any "resistant" cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.
Drug Metabolism and Disposition 01/2005; 32(12):1392-401. · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.
Cancer Research 10/2004; 64(18):6693-9. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250-400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd x 5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd x 5 schedule, the maximum-tolerated dose was approximately 120 microg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was approximately 16 microg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.
Cancer Research 10/2004; 64(18):6700-6. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antineoplastic agents often achieve antitumor activity at the expense of close to unacceptable toxicity. One potential avenue to improve therapeutic index might combine agents targeting distinct components of the same growth regulatory pathway. This might lead to more complete modulation of the target pathway at concentrations lower than those associated with limiting adventitious toxicities from either agent alone. The protein kinase antagonist UCN-01 is currently used in Phase I/II trials and has recently been demonstrated to inhibit potently PDK1. We have recently documented that the alkylphospholipid perifosine potently also inhibits Akt kinase (PKB) activation by interfering with membrane localization of Akt. This leads to the hypothesis that these two agents might act synergistically through distinct mechanisms in the PI3K/Akt proliferation and survival-related signaling pathway.
The synergistic effects of UCN-01 and perifosine, on two cell lines (A-549 and PC-3), were examined using various long-term in vitro assays for cell growth, cell cycle distribution, clonogenicity, survival morphology, and apoptosis. Along with Western blotting experiments were performed to determine whether this synergistic combination of two drugs has significant effect on their downstream targets and on biochemical markers of apoptosis.
After 72 h, perifosine at concentrations of 1.5 and 10 microM UCN-01 at 40 and 250 nM did not significantly affect the growth of PC-3 and A459 cells, respectively. However, in combination at the same respective individual concentrations (1.5 microM and 40 nM of perifosine and UCN-01, respectively, in PC-3 cells and 10 microM perifosine and 0.25 microM UCN-01 in the somewhat more resistant A549 cells), virtually complete growth inhibition of both the cell lines resulted. Supra-additive inhibition of growth was also demonstrated in independent clonogenic assays. Mechanistic studies in cell culture models suggest enhanced depletion of the S-phase population in cells treated by the combination. This correlated with enhanced inactivation of Akt along with activation of caspases 3 and 9 and poly(ADP-ribose) polymerase cleavage. Evidence of synergy was formally demonstrated and occurred across a wide range of drug concentrations and was largely independent of the order or sequence of drug addition.
As the concentrations of UCN-01 and perifosine causing synergistic inhibition of cell growth are clinically achievable without prominent toxicity, these data support the development of clinical studies with this combination.
Clinical Cancer Research 09/2004; 10(15):5242-52. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AH(R100), with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)-controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AH(R100)) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.
Molecular Cancer Therapeutics 07/2004; 3(6):715-25. · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Efficient adenovirus infection requires the presence of coxsackie-adenovirus receptor (CAR) and αv integrin on the surface of cells. Previously, we showed that treatment of several cancer cell lines with a low concentration of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) (1 ng/ml) caused an increase in the RNA levels of CAR and αv-integrin. FK228 pre-treatment was associated with a 5–10 fold increase in adenoviral transgene expression following adenovirus infection. The levels of CAR and αv integrin RNA were not increased in cultured normal cells from breast, liver or kidney following similar FK228 treatment. These results suggest that FK228, a drug currently in phase II clinical trials for the treatment of patients with peripheral or cutaneous T-cell lymphoma, may result in preferential enhancement of adenoviral transgene expression in cancer cells. To further evaluate this differential sensitivity between normal and cancer cells, we examined the effect of FK228 in athymic mice bearing advanced-stage subcutaneous LOX IMVI and UACC-62 human melanoma xenografts and MDA-MB-231 human breast cancer xenografts. Mice with melanoma xenografts were treated with FK228 iv q4d × 3 (days 1, 5, and 9) with one of three doses- 0.7, 1.6, or 3.6 mg/kg/dose. The highest dose was effective in causing tumor regression. Animals were sacrificed 6 h, 24 h, or 48 h following the last dose of FK228 and tissues were harvested. The levels of CAR and αv integrin RNA were monitored in mouse liver, kidney and lung and in the human xenografts using semi-quantitative RT-PCR analysis. The levels of CAR RNA were increased in the xenografts. The time of maximum increase in the levels of CAR RNA in the xenografts was 6 h after the administration of 3.6 mg/kg/dose FK228. The levels of αv integrin RNA in the xenografts were unchanged as were the levels of both CAR and αv integrin, in the livers, kidneys and lungs from the same animals. In order to define an optimal dose and schedule of FK228 administration for CAR induction, mice with LOX IMVI xenografts were treated with FK228 one, two, or three times. The tissues were then harvested either 6 or 24 hours following each dose and the levels of CAR and αv integrin RNA were determined. The results showed that the optimal dose for CAR induction was 24 hours after a single treatment with 3.6 mg/kg FK228 where CAR RNA levels were increased by 14-fold. Accordingly, CAR protein levels increased by 3–8 fold. In mice with MDA xenografts treated with a single dose of 1.6, 3.6 or 5.4 mg/kg FK228, CAR protein levels increased after 48 hours by 4-fold following the 3.6 mg/kg dose, whereas αv integrin levels remained unchanged. Because we have previously shown a correlation between the level of CAR and the extent of adenovirus infection in vitro, these results suggest that FK228 may preferentially increase the levels of adenovirus infection in cancer cells in vivo as it did in vitro. Experiments are in progress to determine if FK228 pre-treatment can increase the efficiency and selectivity of adenovirus gene therapy in vivo.
[Show abstract][Hide abstract] ABSTRACT: Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide prodrug (2b), sufficient to elicit cytocidal activity against ZR-75-1 and T47D human mammary carcinoma cell lines persist > 6 h. The growth of breast (MCF-7) and ovarian (IGROV-1) xenograft tumors is significantly retarded by 2b. Manageable toxic side effects are reported from preclinically efficacious doses of 2b. Cytochrome P 450 1A1 protein expression, selectively induced in sensitive carcinoma cells, was detected in MCF-7 and IGROV-1 tumors 24 h after treatment of mice with 2b (20 mg/kg). The lysyl amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is potentially suitable for clinical evaluation.
Molecular Cancer Therapeutics 02/2002; 1(4):239-46. · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). Both in vitro and in vivo studies demonstrated that SarCNU was more effective than BCNU against human gliomas. To clarify whether EMT expression correlates with antitumor efficacy of SarCNU, we determined human EMT (EMTh) and O(6)-methylguanine-DNA methyltransferase (MGMT) expression in nine human xenograft models using semiquantitative reverse-transcription polymerase chain reaction. These results were compared with the antitumor effects of SarCNU and the standard chloroethylnitrosourea antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There was no significant correlation between EMTh expression and antitumor efficacy of SarCNU or BCNU. Also, there was no significant correlation between MGMT expression and SarCNU efficacy. However, a significant correlation was found between MGMT expression and BCNU antitumor efficacy. Interestingly, multiple regression analysis demonstrated a significant correlation between SarCNU efficacy and EMTh plus MGMT expression, whereas there was no correlation between BCNU efficacy and MGMT plus EMTh expression. Thus, the absence of a linear correlation between SarCNU efficacy and EMTh expression appears to be due, at least in part, to the presence of DNA repair, specifically, MGMT, in these xenograft models. These studies suggest that MGMT expression alone correlates with BCNU activity, whereas both EMTh and MGMT expression are important determinants of SarCNU activity against human tumor xenograft models. SarCNU is in clinical trials and these results may have important clinical implications.
Journal of Pharmacology and Experimental Therapeutics 04/2001; 296(3):712-5. · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We were interested in identifying novel agents for renal cell carcinoma (RCC) by screening for activities that model renal tumor biology. Searching for relative renal cell sensitivity and leukemia insensitivity among cytotoxicity profiles in the NCI Drug Screen database, we identified 16 potential agents with renal selectivity. We evaluated the agents in 10 RCC cell lines (of primary and metastatic origin) isolated from 5 patients. The 50% inhibitory concentrations (IC50) in these cell lines ranged from 0.019 +/- 0.013 to 11.4 +/- 0.55 microM and were comparable with values obtained with renal cell lines in the NCI Drug Screen panel. Because RCC are slowly growing tumors, we evaluated the compounds on rapidly (27% S phase) or slowly (6% S phase) growing cells. In contrast to doxorubicin, where cytotoxicity was restricted to rapidly proliferating cells, three compounds (NSC 280074, 281613, and 281817) were more cytotoxic in slowly proliferating cells. NSC 72151 and 268965 were equitoxic for both populations. NSC 94889, 638850, and 630938 were more cytotoxic in rapidly growing cells. In in vitro time exposure studies, four compounds, NSC 268965, 280074, 281613, and 281817, were maximally cytotoxic with as little as 3 h exposure time. From an analysis comparing the p53 genotype of the 60 cell lines of the National Cancer Institute (NCI) Drug Screen with the cytotoxicity profiles for the 16 putative renal compounds, 13 compounds were classified as likely to be indifferent to p53 status. We also developed a panel specificity detection method for the NCI Drug Screen database to evaluate the prevalence of renal sensitive compounds. Of the 16 studied compounds, 14 were among those identified as renal sensitive by the statistical analysis. Lastly, we found reduced tumor growth in mice with established renal human tumor xenografts after treatment with two of the renal active compounds. These studies describe compounds with potential renal activity that are candidates for preclinical development for renal cell carcinoma.
Clinical Cancer Research 04/2001; 7(3):620-33. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To clarify whether 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) has an anti-tumor effect in DNA repair gene expressing tumors.
Human non-small cell lung cancer cell line,NCI-H522,was implanted into 25 athymic mice and 6 were treated with SarCNU 120mg/kg once a day for 5 times intraperitoneally (ip).The left ones were given normal saline.The extraneuronal monoamine transporter (EMT) expression,DNA repair gene O⁶-methylguanine-DNA methyltransferase (MGMT) and excision repair cross-complementing rodent repair deficiency gene (ERCC1-6) expressions were detected in the tumor specimens by using reverse-transcription polymerase chain reaction (RT-PCR).Comparison of tumor size change between two groups was illustrated with T/C%.
All the tumors were reduced in size through the treatment of SarCNU with the optimal T/C% of 23 at day 28.The tumor growth delay was 55 days,but no tumor free animals were observed.Positive EMT and DNA repair gene expression were observed in all tumor samples.
The results suggest that anti-tumor effect of SarCNU in EMT positive tumor is satisfactory even though the tumor exhibits DNA repair gene expression,specifically MGMT and ERCC1-6.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 10/2000; 3(5):359-62.
[Show abstract][Hide abstract] ABSTRACT: Altered cell cycle control has emerged as a recurring theme in neoplasia. Strategies that would return toward normal the altered cell cycle control present in tumor cells have appeal as novel approaches to cancer treatment. Cyclin-dependent kinases (CDKs) control the progression through the cell cycle, operating at the transition from the G2 to M and G1 to S phases, and progression through S. CDKs are regulated by a complex set of mechanisms, including the presence of activating cyclins, regulatory phosphorylations, and endogenous CDK inhibitors at "checkpoints." This overview focuses on progress in defining compounds that can antagonize directly the action of CDKs. These have emerged as various types of ATP site-directed inhibitors, including flavopiridol, N-substituted adenine derivatives, the natural product butyrolactone, staurosporine derivatives, and, more recently, the synthetic paullones. Paullones appear to be of interest in that one of the most active members of the class, 9-nitropaullone (alsterpaullone), requires relatively brief periods of exposure to living cells in order to effect lasting effects on cellular and proliferative potential. Two of these compounds, flavopiridol and UCN-01 (7-hydroxy-staurosporine), have entered early clinical trials and achieved concentrations that might potentially modulate CDK function. In the case of UCN-01, unexpected human plasma protein binding might prevent direct inhibition of CDKs but allow drug concentrations to be achieved that indirectly affect CDKs by checkpoint abrogation. Further studies with CDK inhibitors should define the expected end point of CDK inhibition more clearly in preclinical models and clinical systems, including cytostasis, apoptosis, or differentiation.
Annals of the New York Academy of Sciences 07/2000; 910:207-21; discussion 221-2. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.
[Show abstract][Hide abstract] ABSTRACT: The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1–2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80–90 μM and was cleared rapidly, with T1/2α and T1/2β values of 7.4 and 31.3 min, respectively. Bioavailability was 80–100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.