Inhibition of P-glycoprotein transport function and reversion of MDR1 multidrug resistance by cnidiadin

Purpose Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene is one of the major obstacles to successful cancer chemotherapy. The goal of this study was to evaluate if, among other natural coumarins, cnidiadin, a furanocoumarin present in traditional Chinese medications and in a spice commonly used in Greek food, inhibits Pgp transport activity and has the potential to reverse MDR1 multidrug resistance.Methods Using MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells as a model of cells expressing the human MDR1 phenotype, and verapamil or CsA or both as positive control, we tested the capacity of six natural coumarins (umbelliferone, esculin, esculetin, cnidiadin, angelicin and psoralen) to induce the accumulation of rhodamine-123 (R-123) and [³H]-vinblastine ([³H]-VBL) and to modulate the photolabeling of Pgp by SDZ 212-122, a diazirin cyclosporin A. The growth-inhibitory effect of cnidiadin and its capacity to enhance the cell toxicity of vinblastine (VBL) or vincristine (VCR) was then evaluated by the WST-1 assay in two cell lines overexpressing Pgp (MDCK-MDR1 and vincristine-resistant KB/VCR).Results Cnidiadin was the only tested coumarin capable of significantly accumulating R-123 and [³H]-VBL and inhibiting Pgp photolabeling in MDCK-MDR1 cells. The dose-dependent increase in [³H]-VBL uptake (IC₅₀ 26.5 M) induced by cnidiadin in the dose range 1–100 M correlated with inhibition of Pgp photolabeling. At 10 M cnidiadin inhibited photolabeling by 59% and sensitized both MDCK-MDR1 and KB/VCR cells to vinca alkaloids.Conclusion Cnidiadin is a cytotoxic agent capable in vitro of competitively inhibiting the binding and efflux of drug by Pgp and of enhancing the cell toxicity of vinca alkaloids in two cell lines (MDCK-MDR1 and mutant human carcinoma KB/VCR) overexpressing Pgp. This suggests that diet or traditional preparation containing cnidiadin may contribute to the reversal of MDR1 multidrug resistance and may affect the bioavailability of Pgp substrates orally administered. However, due to its cell toxicity, clinical interest in cnidiadin as a chemosensitizer appears to be limited.     

Effect of tamoxifen on mitoxantrone cytotoxicity in drug-sensitive and multidrug-resistant MCF-7 cells

The influence of the antiestrogen tamoxifen (TAM) on the activity of mitoxantrone (MXN), was evaluated against wild-type MCF-7/WT and their multidrug-resistant variant MCF-7/ADR cells. Multidrug resistance (MDR) in this cell line which was selected for resistance to Adriamycin (ADR), is associated with increased expression of P-glycoprotein (P-gp). In a clonogenic assay it was observed that TAM (1–10 M) significantly enhanced the activity of MXN in the MCF-7/ADR but not in the drug-sensitive cell line. Isobologram analysis indicated that the effect of the combination was additive in the parental MCF-7/WT cells and strongly synergistic in the MDR MCF-7/ADR cells. Also, TAM (10 M) caused a three-fold increase in the steady-state levels (C_ss) of MXN in MCF-7/ADR cells but did not modulate MXN levels in MCF-7/WT cells. The observed synergism in MCF-7/ADR cells was perhaps due to the increase in C_ss of MXN that may involve interaction of TAM with P-gp. The combination of MXN and TAM may be useful in the treatment of drug-sensitive and drug-resistant breast cancer.     

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil

Summary Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 M LCV concurrently with or after 10 M FUra following MTX (1 M) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV schedulling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-³H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 M LCV with 10 M FUra after 1 M MTX resulted in significantly more ternary complex formation than did 1 M MTX before 10 M FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-³H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.Abbreviations MTX methotrexate - FUra 5-fluorouracil - LCV d, 1-N ⁵-formyltetrahydrofolic acid, calcium salt (folinic acid, leucovorin) - F dUMP 5-fluoro-2-deoxyuridylate - FUTP 5-fluorouidine-5-triphosphate - PRPP 5-phosphoribosyl-1-pyrophosphate - CH₂FAH₄ N ^5,10-methylenetetrahydrofolate - dUMP 2-deoxyuridylate - dTMP thymidylate - TS thymidylate synthetase - PBS phosphate-buffered saline - NaCl 8.0 g - KCl 0.2 g - Na₂HPO₄ 1.15 g - KH₂PO₄ 0.2 g in 1 1 H₂O, pH 7.4 - FdUrd 5-fluoro-2-deoxyuridine This work was supported in part by grant CH-145 from the American Cancer Society and by grants CA-27130 and CA-08341 from the National Cancer Institute. LLD was supported by Postdoctoral Training Grant S-T32-CA09085-08 from the National Institutes of Health and EC was the recipient of a Cancer Research Award from the American Cancer Society     

Differential effects of estrogen,tamoxifen and the pure antiestrogen ICI 182,780 in human drug-resistant leukemia cell lines

ICI 182,780, a potent, new steroidal antiestrogen without apparent agonist activity, appears to be a potent modulator of the classic multidrug resistance (MDR) phenotype in the CEM/A7, CEM/VLB₁₀₀ and K562/VIN100 MDR cell lines. This reagent had no effect on the respective parental CCRF-CEM and K562 cell lines. The use of 1.25 M ICI 182,780 resulted in a 6- to 7-fold decrease in doxorubicin resistance in the CEM/A7 and CEM/VLB₁₀₀ cell lines. A dose-response effect was observed at ICI 182,780 concentrations of up to 5 M. As compared with tamoxifen (TAM), ICI 182,780 was 2 and 4 times more effective in the K562/VIN100 and CEM/A7 cell lines, respectively. ICI 182,780 at 0.625 M increased [³H]-daunomycin uptake (P<0.0001) as effectively as 5 M TAM in the resistant CEM/A7 line. Drug-efflux studies showed that 5 M ICI 182,780 significantly decreased drug efflux as compared with 5 M TAM (P<0.0001). Estradiol (EST) at 10 M increased doxorubicin resistance by 1.2–1.3 times in the CEM/A7 and CEM/VLB₁₀₀ cell lines and significantly decreased drug accumulation (P=0.002) and retention (P<0.001) in the CEM/A7 cell line. However, the addition of 10 M EST to 1–2 M ICI 182,780 did not inhibit the ability of ICI 182,780 to modulate doxorubicin resistance in the two resistant cell lines. Using reverse-phase high-performance liquid chromatography (HPLC) to measure lipophilicity, we found no apparent association between the ability of ICI 182,780, TAM or EST to modulate resistance and their relative hydrophobicity.This work was supported in part by grants from the Department of Veterans Affairs, Canberra, ICI Pharmaceuticals and the AntiCancer Council of Victoria, Australia     

Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target

This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 M. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3,4,7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 M Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anticancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.This work was partially supported by grants from MURST (60% and 40%) and CNR (Special Projects: A.C.R.O. 94.01098.PF 39); R. DeVincenzo and G. Ferrandina are recipients of fellowships from the Italian Association for Cancer Research (AIRC); M. Cianfriglia was partly supported by the AIDS research project (contract 720/P)     

Tamoxifen-Induced Increases in Cytoplasmic Free Ca2+ Levels in Human Breast Cancer Cells

Tamoxifen has been shown to increase cytoplasmic free Ca²⁺ levels [Ca²⁺]_i in renal tubular cells and bladder cancer cells, and to alter Ca²⁺ signaling in MCF-7 breast cancer cells. The present study examined the effect of tamoxifen on [Ca²⁺]_i in ZR-75-1 human breast cancer cells using fura-2 as an indicator. Tamoxifen increased [Ca²⁺]_i at a concentration above 2M with an EC₅₀ of 5M. Removing extracellular Ca²⁺ reduced the response by 48±2%. In Ca²⁺-free medium, after tamoxifen-induced [Ca²⁺]_i increased had returned to baseline, adding 3mM Ca²⁺ increased [Ca²⁺]_i in a concentration-dependent manner. Further, pretreatment with 10M tamoxifen abolished the [Ca²⁺]_i increase induced by 1M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor); and conversely, pretreatment with thapsigargin prevented tamoxifen from releasing more Ca²⁺. Tamoxifen (10M)-induced Ca²⁺ release was not changed by inhibiting phospholipase C activity with 2M U73122. Trypan blue exclusion assay revealed that tamoxifen (1–10M) did not alter viability after 1min of incubation, but killed 10% of cells after 3–10min of incubation. Together, this study shows that tamoxifen (>2M) induced a significant, immediate increase in [Ca²⁺]_i in ZR-75-1 breast cancer cells. Tamoxifen acted by releasing Ca²⁺ from the endoplasmic reticulum Ca²⁺ stores in a manner independent of phospholipase C activity, and by inducing Ca²⁺ entry from extracellular medium. Tamoxifen may be of mild cytotoxicity after acute exposure.     

Bullatacin — in vivo and in vitro experience in an ovarian cancer model

The cytotoxicity and antitumor effects of the acetogenin Bullatacin were evaluated in vitro in multiple ovarian cancer cell lines and in vivo in a murine ovarian teratocarcinoma (MOT) model in C3HeB/FeJ mice. The in vitro cytotoxicity of Bullatacin against four human ovarian epithelial tumor cell lines (OC-194, OC-222, OVCAR-3, and A-2780) was assessed in 48- and 72-h tetrazolium-dye (MTT) cytotoxicity assays. The percentage of cytotoxicity was determined on the basis of the mean optical density of the respective untreated cells and the dose effective against 50% of the cells (ED₅₀) was calculated for each cell line. In vivo experiments were performed on adult female C3HeB/FeJ mice, which were injected i.p. with 10⁵ MOT cells and varying amounts of Bullatacin given either in a single dose or in 5 subsequent doses over 72 h. All mice were observed for survival relative to that of the control groups, which were injected either with 10⁵ MOT cells with or without serial injections of vehicle or with vehicle only. All four epithelial ovarian cancer cell lines displayed sensitivity to Bullatacin. The relative cytotoxic effects were very heterogeneous, with the ED₅₀ value ranging between 10^–7 g/ml for OC-194 and 4 g/ml for the cisplatin-resistant cell line OVCAR-3 in a 72-h MTT cytotoxicity assay. All mice that had been injected i.p. with 10⁵ MOT cells and 1.4 mg/kg or more of Bullatacin died within the first 24 h after injection, whereas all mice that had received 600 g/kg of Bullatacin or less survived equally as long as the controls that had been injected with MOT only (21.1±0.9 days). Mice that had received Bullatacin at a dose ranging from 600 g/kg to 1.4 mg/kg either died during the 1st day postinjection or survived, but not longer than the MOT control group. Serial i.p. injections of Bullatacin again either led to death of the mice within 24–48 h of the last dose of Bullatacin or did not have any effect on the survival of the mice as compared with the respective control groups, which had been injected with the tumor and serial injections of vehicle (22.5±2.2 days). In summary, Bullatacin showed no effect on MOT-caused animal death in C3HeB/FeJ mice at nonlethal dose ranges, whether it was given as a single i.p. dose or serially over 72 h. In vitro, however, it proved to be a very potent cytotoxic agent in a variety of ovarian cancer cell lines. As compared with other chemotherapeutic agents, which we accept as having clinically important antitumor efficacy against ovarian cancer, such as cisplatin or carboplatin, Bullatacin demonstrated a very favorable ED_{50 in vitro}/LD_{50 in vivo} (the dose lethal to 50% of the mice) ratio.This work was supported by the Bertha Warshaver Rubin Research Fund and was presented at the 21st annual meeting of the Western Association of Gynecologic Oncologists, May 19–23, 1993, Santa Monica, California     

Cytotoxicity of antitumor platinum complexes withl-buthionine-(R,S)-sulfoximine and/or etanidazole in human carcinoma cell lines sensitive and resistant to cisplatin

Human 2008 ovarian carcinoma cells and the C13 CDDP-resistant subline and human MCF-7 breast carcinoma cells and the MCF-7/CDDP CDDP-resistant subline were exposed tol-buthionine-(S, R)-sulfoximine (50 M) for 48 h prior to and during exposure for 1 h to the antitumor platinum complexes,cis-diamminedichloroplatinum(II), carboplatin or D,L-tetraplatin and/or to etanidazole (1 mM) for 2 h prior to and during exposure for 1 to the antitumor platinum complexes. These modulators alone did not significantly alter the cytotoxicity of CDDP toward either parental line. A twofold enhancement in cytotoxicity was observed with carboplatin in the 2008 cells and with D,L-tetraplatin in both parental lines with the single modulators. The modulator combination (buthionine sulfoximine/etanidazole) was very effective along with D,L-tetraplatin in both the MCF-7 parent and MCF-7/CDDP cell lines where at the higher platinum complex concentrations there was 1.5 to 3 logs increased killing of cells by the drug plus the modulators compared with the drug alone. Similarly, when C13 cells were exposed to CDDP (100 M) or D,L-tetraplatin (100 M) along with buthionine sulfoximine and etanidazole there was a 2-log increase in cell killing compared with exposure to the platinum complex alone. Treatment of each of the four cell lines with buthionine sulfoximine decreased both the non-protein and total sulfhydryl content of the cells. Treatment with the combination of modulators did not produce a further decrease in cellular sulfhydryl content compared with buthionine sulfoximine alone. The total sulfhydryl content in MCF-7 cells and 2008 cells exposed to buthionine sulfoximine and etanidazole was 58% and 31% of normal and the total sulfhydryl content of MCF-7/CDDP cells and C13 cells treated the same way was 54% and 23% of normal, respectively. DNA alkaline elution was used to assess the impact of exposure to the modulators, buthionine sulfoximine and etanidazole, alone and in combination on the cross linking of DNA by the antitumor platinum complexes in the MCF-7 and MCF-7/CDDP cell lines. Overall, the increases in DNA cross linking factors were greater in the MCF-7 cells than in the MCF-7/CDDP cells. These results indicate a possible clinical potential for this modulator combination.This work supported by NIH grant P01-CA 38493.     

Assessment of antineoplastic agents by MTT assay: partial underestimation of antiproliferative properties

Summary In the present study a slightly modified MTT assay was used in conjunction with cell counting to determine the antiproliferative efficacy ofN-(2-chloroethyl)-N-nitroso-N-2-hydroxyethylurea (HECNU), vinblastine, and hexadecylphosphocholine (HPC) in a panel of six tumor cell lines. This panel consisted of two human (MDA-MB231, MCF-7) and two rodent (1/C2, 1/C32) mammary-carcinoma cell lines as well as of two tumor cell lines of gastrointestinal origin (HT-29, KB). It was shown that the use of acid isopropanol as a solvent of the formazan crystals produced correlations between cell number and absorption that were as good as, if not better than, those seen after dimethylsulfoxide (DMSO) application. The optimal period of incubation with the MTT dye was 2 h. A comparison of the antiproliferative activity of HECNU revealed that the HT-29 cell line was most resistant [50% inhibition concentration (IC₅₀), 138.7 mol/l], followed by MCF-7 cells (IC₅₀, 127.7 mol/l), whereas MDA-MB231 cells showed the highest sensitivity (IC₅₀, 6 mol/l). Vinblastine induced the highest (MCF-7 cells; IC₅₀, 0.68 nmol/l) and the lowest (1/C2 cells; IC₅₀, 7.69 nmol/l) degrees of growth inhibition in cell lines derived from mammary carcinoma. This contrasted with the activity of HPC, which was considerably less effective in the four mammary-carcinoma cell lines (IC₅₀ from 29.4 to 69.9 mol/l) than in the two cell lines of gastrointestinal origin (IC₅₀, 1.9 and 3.1 mol/l). Interestingly, treatment with HPC stimulated the growth of 1/C32 cells in the lower dose range. After treatment with HECNU, the average IC₅₀ value determined in the MTT assay was 2.4-fold that disclosed by cell counting, whereas the average values found for HPC and vinblastine by both methods corresponded fairly well, with the respective values obtained using the MTT assay being only 26% and 14% higher than those measured by cell counting. A dose-dependent increase in the mean size of MCF-7 cells was observed after exposure to HECNU, which — if taken into account — considerably reduced underestimation of this parameter by the MTT assay. No variation in cell size was noted following treatment with HPC and vinblastine. Thus, depending on the antitumor agent used, the MTT assay can result in slight or even considerable understimation of the antitumor efficacy of certain compounds and may need correction by consideration of the effect of the drugs on cell size.Abbreviations MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - HECNU N-(2-ehloroethyl)-N-nitroso-N-2-hydroxyethylurea - HPC hexadecylphosphocholine - MNU methylnitrosourea - DMSO dimethylsulfoxide - IC₅₀ 50% inhibitory concentration - APC alkylphosphocholines - PBS phosphoate-buffered saline - fl femto litre     

Cytotoxicity of ketoconazole in malignant cell lines

Summary The cytotoxic effects of ketoconazole, an antifungal agent known to have some activity against human prostate cancer, adrenal cancer, and male metastatic breast cancer, were evaluated using colony-growth and clonogenic assays in eight malignant cell lines. The cytotoxicity of ketoconazole showed a dose-and time-dependent pattern, with the following concentrations, inhibiting 90% of the growing colonies (IC₉₀): MCF 7 (human breast cancer) 7.25 g/ml, T 47 D (human breast cancer) 9.0 g/ml, MiaPaCa (human pancreatic carcinoma) 10.0 g/ml, COLO 357 (human pancreatic carcinoma) 9.5 g/ml, HCT 8 (human colonic adenocarcinoma) 27.1 g/ml, DU 145 (human prostatic cancer) 40.0 g/ml, AR 42 J (rat pancreatic carcinoma) 9.0 g/ml, and L1210 (murine leukemia) 8.6 g/ml. Since a concentration of 10 g/ml can be achieved in humans, the use of ketoconazole in human malignancies might be worthy of clinical evaluation.This investigation was supported in part by a grant from the German Volkswagen Foundation, Hannover, Federal Republic of Germany and by a gift from Dr Virgil Gianelli, Stockton, California     

In vitro evaluation of a novel chemotherapeutic agent,adozelesin, in gynecologic-cancer cell lines

Summary Adozelesin is a derivative of an extremely cytotoxic compound, CC1065. This entirely new class of drug binds preferentially to DNA and facilitates alkylation reaction. In the present study, we used the adenosine triphosphate (ATP) chemosensitivity assay to compare the cytotoxic potency of Adozelesin with that of common chemotherapeutic agents in ten gynecologic-cancer cell lines. Flow cytometry was also used to study its effects on cell-cycle kinetics. The mean drug concentrations required to produce a 50% reduction in ATP levels as compared with controls [IC₅₀] were: Adriamycin, 0.17±0.06 m; 4OH-Cytoxan, 18±3 m; cisplatin, 17±7 m; 5-fluorouracil, 183±116 m; and Adozelesin, 11.0±5.4pm. Thus, Adozelesin was 10⁴–10⁷ times more potent than Adriamycin, cisplatin, 5-fluorouracil, and Cytoxan. Cell kinetics studies revealed significant S and G2 blocks such as those previously reported for other alkylating agents.Supported in part by an American Cancer Society Clinical Oncology Fellowship and an American Cancer Society/Florida Division Startup Grant (both awarded to H. N. N.)     

On the role of agonist-evoked Ca2+ mobilization in sustaining the ongoing phosphoinositide hydrolysis

Stimulation of SK-N-BE(2) cells with 1 mM carbachol (Cch) elicited phosphoinositide (PPI) hydrolysis and a rapid elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) from 115 nM to about 500 nM, followed by a plateau around 200 nM. In myo [³H]inositol-labelled cells, Cch-evoked accumulation of [³H]inositol phosphates (IPs) was not affected when [Ca²⁺]_i was clamped at resting by cell loading with 10 M BAPTA/AM; under these conditions, maximal 1,4,5-inositol trisphosphate accumulation was not reduced either. When [Ca²⁺]_i was clamped around 700 nM by cell treatment with 600 nM ionomycin, Cch-evoked [³H]IPs accumulation was enhanced by less than 20%, but it was impaired by a 30% and a 55% after [Ca²⁺]_i reduction to about 70 nM and 35—50 nM, by cell loading with 20 M or 40 M BAPTA/AM, respectively. These results show that, in SK-N-BE(2) cells, Cch-activated PPI-specific phospholipase C is sensitive to [Ca²⁺]_i but it already operates under suboptimal conditions at resting [Ca²⁺]_i.     

Taxol and taxotere in bladder cancer: in vitro activity and urine stability

In this study the antimicrotubular agents taxol, taxotere, and vinblastine were compared for their ability to inhibit the clonal growth of human bladder tumor cell lines using a soft-agar clonogenic assay. The stability of taxol and taxotere was evaluated by high-performance liquid chromatography over a range of pH in human urine. Both taxol and taxotere were shown to maximally inhibit the clonal growth of human bladder cell lines within 1 h of drug incubation. The most active agent in the panel of tumor lines was taxotere, with 6 of 12 lines being sensitive to the agent at 0.01 M and all cell lines being sensitive at 0.1 M. Taxol was active in 1 of 12 lines at 0.01 M and in 11 of 12 at 0.1 M. Only 2 of 12 cell lines were sensitive to vinblastine over the 0.01- to 0.1-M dose range. Taxol and taxotere were found to be stable in human urine for 4 h over a pH range of 5–7. At least 85% of both drugs were present during this period of drug incubation. Our findings suggest that both taxol and taxotere may be clinically useful agents for systemic and intravesical use in bladder cancer.This work was supported in part by the Veterans Administration Research Service     

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis

N-{4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl}-l-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED₅₀ values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNP-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (K _m) of 1.45 M and a maximum of velocity (V_max) of 1,925 pmol h^–1 mg^–1. Inhibitory activities of TNP-351-G_n (n=1–6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (K _i): G₁, 52 M; G₆, 0.07 M]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.     

H1, a novel derivative of tetrandrine reverse P-glycoprotein-mediated multidrug resistance by inhibiting transport function and expression of P-glycoprotein

Purpose

H1 is a novel derivative of tetrandrine (Tet). Here we investigate the ability of H1 to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) and its mechanisms.

Methods

KBv200, MCF-7/adr and their parental sensitive cell lines KB, MCF-7 were used for reversal study. The intracellular accumulation and efflux studies with Pgp substrates of doxorubicin and rhodamine 123 were determined by flow cytometry. The expression of Pgp was investigated by Western blot and RT-PCR analysis. ATPase activity of Pgp was performed by Pgp-Glo^? assay systems. The ubiquitination level of Pgp was determined by immunoprecipitation analysis. The effect of ERK1/2 on Pgp expression in KBv200 cells were investigated by RNA interference.

Results

H1 significantly potentiated the sensitivity of Pgp substrates in KBv200 and MCF-7/adr cells, but not in parental cells KB and MCF-7. H1 inhibited Pgp expression in KBv200 cells in a dose-dependent manner, but had no effect on MDR1 expression. Further studies showed that H1 prompted the degradation of Pgp and decreased Pgp protein half-life by enhancing the ubiquitination of Pgp, which may be related to downregulated MEK-ERK signal pathway. We also found H1 inhibited ATPase activity of Pgp in a dose-dependent manner.

Conclusions

H1 is an effectively and potential agent in reversing Pgp-mediated MDR by inhibiting the transport function and expression of Pgp.     

Phosphodiesterase specific inhibitors control cell growth of a human neuroepithelioma cell line

The effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cell proliferation of SK-N-MC human neuroepithelioma cell line was studied. Clonal density experiments in the presence of 100 M rolipram and zaprinast showed respectively 27% and 91% inhibition. The effects of PDE inhibitors were then investigated on crude cell extracts; the calculated IC₅₀ were 32 M for zaprinast and 16 nM for DC-TA-46; the latter inhibitor was used instead of rolipram for itshigher efficacy. Dose-response experiments in clonal density conditions showed IC₅₀ of 5 M and 1.8 M in the presence respectively of zaprinast and rolipram. These data show that both inhibitors are effective in reducing cell growth, although the response was quantitatively different.     

In vitro enhancement of fluoropyrimidine-induced cytotoxicity by leucovorin in colorectal and gastric carcinoma cell lines but not in non-small-cell lung carcinoma cell lines

Summary Leucovorin (LV) increases the cytotoxic effect of fluorouracil (FUra) and 5-fluoro-2-deoxyuridine (FdUrd) by enhancing the formation of the fluorodeoxyuridine monophosphate (FdUMP) thymidylate synthase (TS) 5,10-methylenetetrahydrofolate (mTHF) ternary complex. To study the difference in the efficacy of this combination against different tumors, we compared the effect of LV (20 m) on the cytotoxicity of FUra, FdUrd, and 5-fluorouridine (FUrd) in vitro against cell lines of five colorectal carcinomas (CC), five gastric carcinomas (GC), and four non-small-cell lung carcinomas (NSCLC) using the colony-forming assay. At the concentration used in the experiments, LV alone failed to inhibit colony formation in any of the cell lines tested. The NSCLC cell lines were more resistant to FdUrd than were the CC and GC lines. LV modulated the cytotoxicity of FdUrd in all five CC lines and in three of the five GC lines but failed to do so in any of the NSCLC lines. In addition, following 20 h treatment with 1 m [³H]-FdUrd, formation of the FdUMP/TS/mTHF ternary complex was enhanced by LV in the LV-sensitized CC and GC cell lines but not in the LV-refractory NSCLC lines. These in vitro data corresponded well to the results of clinical trials. Therefore, the colony-forming assay may be useful for the identification of the sensitivity of tumors according to phenotype.Abbreviations FUra 5-fluorouracil - FdUrd 5-fluoro-2-deoxyuridine - FUrd 5-fluorouridine - FdUMP 5-fluoro-2-deoxyuridine monophosphate - FUTP fluorouridine triphosphate - FBS fetal bovine serum - LV leuvovorin - mTHF 5, 10-methylenetetrahydrofolate - NSCLC non-small-cell lung carcinoma - SDS sodium dodecyl sulfate - TS thymidylate synthase - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - IC₅₀ 50% inhibitory concentration - TCA trichloroacetic acid This research was supported in part by Grants-In-Aid from the Ministry of Health and Welfare for Comprehensive 10-Year Strategy for Cancer Control and from the Ministry of Education, Science, and Culture, Japan     

Effects of N-trifluoroacetyladriamycin-14-valerate (AD-32) on human bladder tumor cell lines

Summary We have compared the in vitro activity of N-trifluoroacetyladriamycin-14-valerate (AD-32) and doxorubicin hydrochloride (ADR) on the clonal growth of human bladder tumor cell lines (HBTCL). In order to determine the relative toxicity of ADR and AD-32 on hematopoietic stem cells, CFU-GM assays were set up using 10 normal human bone marrow samples. The mean lethal dose for 50% of the colonies (LD-₅₀) for ADR was 1.6±1.4 M and that for AD-32, 3.9±4.9 M (P<0.55),suggesting that these agents have similar bone marrow toxicity. Both drugs produced enhanced inhibition of clonal growth of HBTCL with increasing C × Ts. The spectrum of activity of the two drugs was similar against a panel of seven HBTCL. The activity of ADR was inhibited at 4 °C while the activity of AD-32 was unaffected by temperature. ADR was more effective against HBTCL in the log growth phase than the plateau phase while the reverse was found using AD-32. Verapamil was found to enhance the activity of both ADR and AD-32 against a HBTCL (T24), found to be resistant to both agents. The lipophilic properties of AD-32, along with its enhanced activity when used over prolonged periods of time and its activity against tumor cells in the plateau phase, suggest that AD-32 could be useful in the management of patients with superficial bladder cancer.Abbreviations used ADR doxorubicin hydrochloride - AD-32 N-trifluoroacetyladriamycin-14-valerate - HBTCL human bladder tumor cell lines - CFU-GM corony forming units-granulocyte, macrophage - CFU-T colony-forming units-tumor - C × T concentration × time of drug exposure - HBSS Hank's balanced salt solution - FCS fetal calf serum     

Effect of pharmacologic doses of zinc on the therapeutic index of brain tumor chemotherapy with carmustine

To evaluate the potential differential effect of pretreatment with pharmacologic doses of the trace element zinc on the chemosensitivity of glioma cells and bone marrow cells for carmustine (BCNU), we performed in vitro and in vivo studies of zinc toxicity as well as of the combined treatment with zinc and the anticancer drug. We studied the in vitro effects on established human and rat glioma cell lines using a microcolorimetric growth assay and on murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage. Zinc exposures of up to 100 M for 120 h did not influence the growth of six of seven human glioma cell lines. Only U87MG demonstrated statistically significant toxicity during high zinc exposure (100 M over 120 h). Dose-response growth curves generated for BCNU did not show protection against the anticancer agents by a 48-h pretreatment with different zinc concentrations. The clonogenic capacity of bone marrow cells was slightly reduced by in vitro culture for 24 and 48 h. Although this effect appeared to be more prominent in the presence of zinc supplementation, overall a statistically significant inhibition was seen only after exposure to a concentration of 100 M zinc over 48 h. As compared with chemotherapy alone, in vitro pretreatment with 50 M zinc over 48 h followed by chemotherapy resulted in an increased number of colony-forming unit-granulocyte monocyte (CFU-GM): CFU-GM increased by a factor of 2 for BCNU (60 M×2 h). This statistically significant in vitro chemoprotection would translate into a dose-protection factor of 1.5, i.e., for the same level of myelosuppression, zinc pretreatment would allow administration of a 50% increased dose of BCNU. The in vivo studies were performed in an s.c. xenograft model of the human glioma cell line U87MG in athymic mice. The maximal tolerable pretreatment with zinc was determined to be a 10-day course of daily i.p. injections of 10 mg/kg ZnCl₂. The subsequent i.p. administration of the dose lethal to 10% of the mice (LD₁₀) and of a 1.5×LD₁₀ dose of BCNU resulted in less bone marrow toxicity in pretreated animals than in non-zinc-pretreated mice as determined in a CFU-GM assay. Glioma colony-forming efficiency (CFE) assays, on the other hand, did not show any zinc-related difference in the BCNU sensitivity of U87MG. Our in vitro and in vivo results suggest the potential usefulness of high-zinc pretreatment for improving the therapeutic index of BCNU chemotherapy for gliomas.     

18b-甘草次酸诱导人乳腺癌细胞凋亡及与细胞内Ca2+释放的关系

Objective: To study the effects of 18-glycyrrhetinic acid (GA) on proliferation inhibition, apop- totic induction, and the relationship between GA-induced apoptosis and intracellular Ca²⁺ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 mol/L to 250 mol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 mol/L, 150 mol/L, and 200 mol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 mol/L GA for 24 h, intracellular Ca²⁺ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 mol/L to 250 mol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose- dependent fashion. IC50 of the proliferation inhibition was 234.33 mol/L. Treated with 100 mol/L, 150 mol/L, and 200 mol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca²⁺ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca²⁺ level may be depended on apoptosis induced by GA in MCF-7 cells.