Delivery and cytotoxicity of RS-1541 in St-4 human gastric cancer cells in vitro by the low-density-lipoprotein pathway

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. RS-1541 has been shown to bind preferentially to plasma lipoproteins and to exhibit selective and sustained uptake by tumors in mice. To elucidate a mechanism of RS-1541 cytotoxicity, the cellular uptake and the cytotoxicity of a complex of RS-1541 with human low-density lipoprotein (RS-1541/LDL complex) were investigated in cultured St-4 human gastric cancer cells. Both the cellular uptake and the cytotoxicity of the RS-1541/LDL complex were greater in cells with higher LDL-receptor activities than in control cells. Excess amounts of LDL or 1 M of monensin, a proton ionophore, significantly inhibited both the uptake and the cytotoxicity of the complex. Chloroquine, an inhibitor of lysosomal enzymes, decreased the intracellular level of rhizoxin liberated from RS-1541 and suppressed the cytotoxicity of the RS-1541/LDL complex. However, a detergent-aided solution of RS-1541 showed very low cellular uptake and cytotoxicity, irrespective of the LDL-receptor activities of these cells. These results demonstrate that the RS-1541/LDL complex is incorporated into the cells via the LDL receptor and that it manifests its cytotoxic activity after forming rhizoxin, the original antitumor agent, in the lysosomes.     

Characterization of the Interaction of TZT-1027, a Potent Antitumor Agent, with Tubulin

TZT‐1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μM with IC₅₀ values of 2.2±0.6 and 2.3±0.7 μM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μM with IC₅₀ values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μM or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μM, with an IC₅₀ of 1.2 μM, whereas VLB was only effective at 0.3–3 μM, with an IC₅₀ of 0.6 μM, and caused so‐called “aggregation” of tubulin at 10 μM. Scatchard analysis of the binding data for [³H]VLB suggested one binding site (K_d 0.2±0.04 μM and B_max 6.0±0.26 nM/mg protein), while that for [³H]TZT‐1027 suggested two binding sites, one of high affinity (K_d 0.2±0.01 μM and B_max 1.7±0.012 nM/mg protein) and the other of low affinity (K_d 10.3±1.46 μM, and B_max 11.6±0.83 nM/mg protein). [³H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [³H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [³H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [³H]guanosine 5′‐triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration‐dependently to a lesser extent than TZT‐1027, but no inhibitory effect of VLB on [³H]GTP binding to tubulin was evident even at 100 μM. Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.     

A phase I study of rhizoxin (NSC 332598) by 72-hour continuous intravenous infusion in patients with advanced solid tumors

Background:Rhizoxin (NSC 332598) is a novel macrolide antitumorantibiotic that inhibits microtubule assembly and also depolymerizes preformedmicrotubules. In preclinical evaluations, rhizoxin demonstrated broadantitumor activityin vitroand in vivoincluding bothvincristine- and vindesine-resistant human lung cancers. Prolonged exposureschedules in xenograft models demonstrated optimal efficacy indicatingschedule-dependent antitumor activity. The early phase I and II evaluationsa five-minute bolus infusion schedule was studied, however, only modestanti-tumor activity was noted, possibly due to rapid systemic clearance. Toovercome these limitations and to exploit the potential for schedule-dependentbehavior of rhizoxin, the feasibility of administering rhizoxin as a 72-hourcontinuous intravenous (i.v.) infusion was evaluated. Patients and methods:Patients with advanced solid malignancieswere entered into this phase I study, in which both the infusion duration anddose of rhizoxin were increased. The starting dose was 0.2 mg/m²over 12 hours administered every 3 weeks. In each successive dose level, thedose and infusion duration were incrementally increased in a stepwise fashion.Once a 72-hour i.v. infusion duration was reached, rhizoxin dose-escalationsalone continued until a maximum tolerated dose (MTD) was determined. Results:Nineteen patients were entered into the study. Rhizoxinwas administered at doses ranging from 0.2 mg/m² i.v. over 12 hoursto 2.4 mg/m² i.v. over 72 hours every 3 weeks. The principaldose-limiting toxicities (DLT) were severe neutropenia and mucositis, and theincidence of DLT was unacceptably high at rhizoxin doses above 1.2mg/m², which was determined to be the MTD and dose recommended forphase II studies. At these dose levels, rhizoxin could not be detected in theplasma by a previously validated and sensitive high-performance liquidchromatography assay with a lower limit of detection of 1 ng/ml. No antitumorresponses were observed. Conclusions:Rhizoxin can be safely administered using a 72-houri.v. infusion schedule. The toxicity profile is similar to that observedpreviously using brief infusion schedules. Using this protracted i.v. infusionschedule the maximum tolerated dose is 1.2 mg/m²/72 hours.     

Insulin effects on methotrexate polyglutamate synthesis and enzyme binding in cultured human breast cancer cells

Summary Previous studies have demonstrated that insulin augments methotrexate transport and enhances its cytotoxicity to human breast cancer cells. We therefore investigated the effects of insulin on methotrexate polyglutamate synthesis and binding to dihydrofolate reductase (DHFR) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Cells were exposed to 2 M [³H]MTX and varying insulin concentrations for the desired time before determination of the polyglutamate content by high-performance liquid chromatography (HPLC). DHFR-bound drug was separated from free intracellular drug by chromatography on DEAE-Sephacel minicolumns prior to HPLC analysis. Incubation of MCF-7 cells with 2.5 nM insulin for 48 h before exposure to 2 M [³H]MTX for a further 24 h resulted in a significant increase in both total drug and total polyglutamates compared with control cells. Increasing the insulin concentration in the medium yielded further increases in polyglutamylation so that at 250 n M insulin and above total polyglutamates were increased by 64% compared with control cells. Further evaluation of the effects of physiologic insulin levels on polyglutamate synthesis revealed that 2.5 n M insulin caused an increase in the net glutamylation rate for each polyglutamate derivative during the final 12 h of a 24 h exposure to MTX. Analysis of the effects of insulin on polyglutamate binding to DHFR. revealed that exposure to 2.5 n M insulin resulted in the preferential binding of higher polyglutamates to DHFR. In MDA-231 cells, a breast cancer cell line with a poor capacity for polyglutamate synthesis, insulin exposure resulted in an increase in the cellular accumulation of each polyglutamate derivative, with the greatest proportionate increases occurring in the cellular levels of higher polyglutamates. These data suggest that insulin augmentation of MTX polyglutamate synthesis may account for its previously observed ability to enhance MTX cytotoxicity.Abbreviotions DHFR dihydrofolate reductase - FCS fetal calf senum - HPLC high performance liquid chromatography - MTX 4-amino-4-deoxy-N-10-methylpteroyl glu 1 - PBS phosphate buffered saline - TCA trichloroacetic acid RLS was supported by the Veterans Administration and an american Cancer Society Junior Faculty Clinical Fellowship     

Porfiromycin disposition in oxygen-modulated P388 cells

Summary The cytotoxicity, metabolism, and DNA alkylation of porfiromycin (PFM) under aerobic and hypoxic conditions were evaluated in P388 murine leukemia cells. Clonogenic assays showed that the IC₅₀ value for a 1-h exposure to PFM was 4 m for aerobic cells and 0.5 m for hypoxic cells. After a 1-h exposure to concentrations of 1.5, and 10 m [¹⁴C]-PFM, the accumulation of total radioactivity in hypoxic cells was 10 to 20 times that in aerobic cells. The disposition of radioactivity in cells that had been treated for 1 h with 5 m PFM under aerobic or hypoxic conditions showed that (a) under either condition, internal free-PFM concentration equalled the external drug concentration; (b) DNA-, RNA-, and protein-bound radioactivity were at least 10 times greater in hypoxic cells than in aerobic cells; and (c) known metabolites and unidentified radioactive products were also generated in greater amounts in hypoxic cells than in aerobic cells. thus, the increased amounts of radioactivity accumulated by hypoxic P388 cells after exposure to [¹⁴C]-PFM resulted from the accumulation of nonexchangeable protein and nucleic-acid adducts and metabolites rather than free PFM. Determinations of DNA adducts formed in P388 cells revealed five possible adducts: (1) N²-(2-deoxyguanosyl)-7-methylaminomitosene, (2) a second monofunctional PFM-guanine adduct, (3) a PFM cross-linked dinucleotide, (4) possibly a nucleoprotein-related adduct, and (5) an unknown. We conclude that the enhancement of PFM-induced cytotoxicity by hypoxia appears to be primarily due to increased alkylation of macromolecules.Abbreviations MMC mitomycin C - PFM porfiromycin - t-AHME trans-7-amino-1-hydroxy-2-methylaminomitosenet - c-AHME cis-7-amino-1-hydroxy-2-methylaminomitosene - AME 7-amino-2-methylaminomitosene - N2-GM N²-(2-deoxyguanosyl)-7-amino-1-methylaminomitosene - PBS phosphate-buffered saline - PK proteinase K This work was supported by grant CH-412 from the American Cancer Society, grant CA 33 697 from the National Institutes of Health, and an award from the Bressler fund of the School of Medicine at the University of Maryland     

Modulation of Adriamycin cytotoxicity and transport in drug-sensitive and multidrug-resistant Chinese hamster ovary cells by hyperthermia and cyclosporin A

Purpose: Chemosensitizers such as cyclosporin A can increase intracellular accumulation of chemotherapeutic agents such as Adriamycin in certain multidrug-resistant (MDR) cell lines with overexpression of P-glycoprotein. It is likely that, when combined with cyclosporin A, hyperthermia could increase membrane permeability to Adriamycin and enhance its cytotoxic effects. The ability of both hyperthermia and cyclosporin A to modulate the cytotoxicity, transport and subcellular distribution pattern of Adriamycin was studied in a pleiotropic MDR Chinese hamster ovary cell line (CH^RC5) and in the drug-sensitive parent line (AuxB1). Methods: Adriamycin cytotoxicity was evaluated by clonogenic cell survival, drug transport using [¹⁴C]-labeled Adriamycin and intracellular drug distribution by fluorescence microscopy. Results: Adriamycin cytotoxicity was increased in both drug-sensitive and MDR cells by cyclosporin A (5 μM) alone, and by hyperthermia alone (41–43 °C) only in sensitive cells. However, when cyclosporin A and 42 °C hyperthermia were used in combination, a large increase in drug cytotoxicity occurred in both cell lines. This effect increased with time and was temperature-dependent. The increase in Adriamycin cytotoxicity caused by cyclosporin A and hyperthermia was accompanied by alterations in membrane permeability to the drug. Cyclosporin A increased [¹⁴C]Adriamycin uptake, while drug efflux decreased, for both AuxB1 and CH^RC5 cells and nuclei. For AuxB1 cells only, drug distribution studies showed that cyclosporin A promoted an increase in both nuclear and cytoplasmic drug accumulation. Hyperthermia, combined with cyclosporin A, increased [¹⁴C]Adriamycin uptake. This effect was seen as an increase in intensity of nuclear and cytosolic drug fluorescence in both cell lines. Cyclosporin A alone diminished drug efflux and caused Adriamycin to remain firmly bound in the nucleus of AuxB1 cells, while it remained primarily in the cytoplasm of CH^RC5 cells. Conclusions: Hyperthermia alone had little effect on Adriamycin cytotoxicity and transport in MDR cells, in contrast to drug-sensitive cells. This suggests that P-glycoprotein is fully functional in these MDR cells. Our findings suggest that cyclosporin A and hyperthermia could be beneficial by increasing intracellular drug accumulation, thus improving the effectiveness of Adriamycin against both drug-sensitive and MDR cells within a localized target region. Received: 17 February 1999 / Accepted: 26 August 1999     

Relationship between the cellular accumulation and the cytotoxicity of S12363, a new vinca alkaloid derivative

Summary S 12363, a new vinca alkaloid derivative, was considerably more cytotoxic to murine L1210 cells and five human tumor cell lines (HL60, HT-29, COLO 320DM, NCI-H460, and PANC-1) than was vincristine (VCR) or vinblastine (VLB). S 12363 bound to tubulin in crude extracts from brain or L1210 cells with an affinity similar to that of VLB and VCR (apparentK _d value: 1.1–1.6, 1.2–1.7, and 0.6–0.8 M, respectively). After 1 h exposure, the accumulation of 20 nM [³H]-S 12363 by L1210 cells was 4-to 18-fold that of [³H]-VLB and [³H]-VCR, respectively. After the cells had been preloaded for 1 h with the labeled drugs and then incubated for 3 h in drug-free medium, 37%–55% of the [³H]-S 12363 was retained by the cells vs 36%–47% of the [³H]-VCR and <6% of the [³H]-VLB. Similar results were obtained for the five human cell lines tested. The accumulation factors (intracellular vs extracellular concentrations) found for [³H]-S 12363 (54- to 167-fold) were significantly higher than those observed for [³H]-VCR (5- to 14-fold) or [³H]-VLB (19- to 41-fold). >90% of the radioactivity extracted from L1210 cells that had been treated with [³H]-S 12363 was recovered as unmodified drug, demonstrating that [³H]-S 12363 was not metabolized by these cells. S 12362, which differs from S 12363 only in the absolute configuration of the asymmetric carbon atom of its -aminophosphonic side chain, was 300 times less cytotoxie, bound to tubulin with a lower affinity (apparentK _d value, 4.9–9.6 m), and was neither accumulated nor retained by the cells. Taken together, these results demonstrate that the potency of S 12363 is due at least in part to its cellular accumulation and retention.     

Cellular pharmacology ofd-3-azido-3-deoxy-myo-inositol,an inhibitor of phosphatidylinositol signaling having antiproliferative activity

d-3-Azido-3-deoxy-myo-inositol (3AMI) is an inhibitor of the growth of v-sis-transformed NIH 3T3 cells but not of wild-type NIH 3T3 cells, whose effects may be mediated through the phosphatidylinositol-3-kinase pathway. We studied some properties of the cellular pharmacology of 3AMI using high-specific-activity [³H]-3AMI. The uptake of [³H]-3AMI by wild-type NIH 3T3 and v-sis NIH 3T3 cells was similar. [³H]-3AMI was a substrate for phosphatidylinositol synthetase, with the maximal velocity (V_max) being 1.0 nmol min^–1 mg^–1 and the Michaelis constant (K _m) being 23 mM. Corresponding values obtained for [³H]-myo-inositol as a substrate were 5.5 nmol min^–1 mg^–1 and 3.2 mM. [³H]-3AMI was incorporated into the cellular inositol lipids of v-sis NIH 3T3 cells to a similar extent as that observed for [³H]-myo-inositol but was not incorporated into the inositol lipids of wild-type NIH 3T3 cells. The [³H]-3AMI incorporated by the v-sis NIH 3T3 cells was present in the phosphatidylinositol and phosphatidylinositol phosphate fractions but not in bisphosphorylated phosphatidylinositol.myo-Inositol antagonized the growth-inhibitory effects of 3AMI. The v-sis NIH 3T3 cells were found to be more sensitive than the wild-type NIH 3T3 cells to growth inhibition (without 3AMI) caused by the removal ofmyo-inositol from the medium. The results of the study suggest that 3AMI is an antimetabolite ofmyo-inositol. The relative sensitivity of v-sis NIH 3T3 and some other cells to 3AMI may be a reflection of increasedmyo-inositol requirements for the growth of these cells as compared with wild-type NIH 3T3 cells.Abbreviations 3AMI d-3-azido-3-deoxy-myo-inositol - 3AmMI d-3-amino-3-deoxy-myo-inositol - PIPLC phosphoinositide-selective phospholipase - C PtdIns(4,5)P₂, phosphatidylinositol(4,5)bisphosphate - Ins(1,4,5)P₃ myo-inositol(1,4,5)trisphosphate - Ins(1,3,4,5)P₄ myo-inositol(1,3,4,5)tetrakisphosphate - PKC protein kinase C - PtdIns3 K phosphatidylinositol-3-kinase - PDGF plateletderived growth factor - EGF epidermal growth factor - CDP diglyceride, cytidine dipalmitoyl diphosphodiglyceride - DMEM Dulbecco's modified Eagle's medium - PBS Dulbecco's phosphate-buffered saline - HCS heat-inactivated calf serum - IC₅₀ concentration required to cause 50% inhibition - PtdIns synthetase; phosphatidylinositol synthetase - DAG diacylglycerol     

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     

Doxorubicin binds in a cooperative manner to myocardial cells

Summary Experimental evidence indicates that the anthracycline antibiotic doxorubicin (adriamycin) localizes mainly in cell nuclei of cardiac cells and has a high affinity to several cellular constituents in addition to DNA. In the present study the cellular kinetics of doxorubicin in cultured rat myocardial cells were determined by measuring its uptake, its binding pattern over a concentration range of 0.1 mM to 80 M, and the cellular release by means of [14-¹⁴C]doxorubicin. The binding kinetics of doxorubicin were compared with the doxorubicin-induced inhibition of [methyl-³H]thymidine incorporation into DNA. It is demonstrated that at micromolar concentrations doxorubicin is readily taken up by myocardial cells and that myocardial cells have the ability to bind doxorubicin at two specific binding sites and that a noncooperative high-affinity/low-capacity type and a positive cooperative type of binding are involved, as indicated by the positive slope in the initial region of the binding isotherm (Scatchard plot). A dose-dependent inhibition of [methyl-³H]thymidine incorporation into DNA is demonstrated. It is suggested that this is associated with the positive cooperative binding of doxorubicin. The cellular release of doxorubicin appeared to be biphasic, with estimated half-lives of about 5–6 h for the initial phase and 50–60 h for the terminal phase. The results of this study indicate that doxorubicin preferably binds to sites within myocardial cells and that the positive cooperative binding pattern is due to DNA as one of the binding sites. A relationship between the noncooperative high-affinity/low capacity binding and the pharmacological activity has yet to be determined.     

The microtubule-active antitumor compound TTI-237 has both paclitaxel-like and vincristine-like properties

Purpose  To compare TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1, 2, 4]triazolo[1,5-a]pyrimidin-7-amine butanedioate) with paclitaxel and vincristine in order to better understand the properties of this new anti-microtubule agent. Methods  Tubulin polymerization and depolymerization were followed by turbidimetric assays. Effects of compounds on the binding of [³H]guanosine triphosphate ([³H]GTP) to tubulin were studied by competition binding assays. Effects of compounds on the phosphorylation of a panel of intracellular proteins were determined by flow cytometry using phosphoprotein-specific antibodies. Results  At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca⁺⁺ at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [³H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells. Conclusions  TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds.     

Paclitaxel/carboplatin administration along with antiangiogenic therapy in non-small-cell lung and breast carcinoma models

Introduction: With the introduction of new drugs, the efficacy of chemotherapy in non-small-cell lung cancer has been improving. The combination of paclitaxel and carboplatin has shown activity in this disease but is far from curative. Methods: The antiangiogenic agent regimen of TNP-470/minocycline was added to treatment with paclitaxel and carboplatin alone and in combination in animals bearing the Lewis lung carcinoma. Results: Administration of the antiangiogenic regimen prior to, during and after the cytotoxic therapy increased the tumor growth delay 1.6-fold and decreased the number of lung metastases to 20% of the number observed in the control animals. [¹⁴C]Paclitaxel, platinum from carboplatin and [¹⁴C]albumin levels were determined over a 24-h time course in tumors and normal tissues of animals bearing the Lewis lung carcinoma and pretreated with TNP-470/minocycline or not pretreated. There were higher levels of [¹⁴C]paclitaxel, platinum from carboplatin and [¹⁴C]albumin in the tumors and some normal tissues of the animals that had received TNP-470/minocycline compared with those that had not received TNP-470/minocycline, especially at the earlier time points. Administration of TNP-470/minocycline to animals bearing the EMT-6 mammary carcinoma increased the cytotoxicity of high-dose paclitaxel toward EMT-6 tumor cells and toward bone marrow CFU-GM. Administration of TNP-470/minocycline to animals bearing the EMT-6 mammary carcinoma also increased the cytotoxicity of carboplatin toward the EMT-6 tumor cells but did not affect the toxicity of carboplatin toward the bone marrow CFU-GM. Conclusions: The addition of TNP-470/minocycline to treatment with paclitaxel and carboplatin resulted in increased antitumor activity and efficacy and further investigation of this combination is warranted. Received: 11 June 1997 / Accepted: 8 October 1997     

The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology

Summary The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of highdose ara-C clinical protocols. In this study, the incorporation of [³H]-dThd and [³H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [³H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [³H]-dThd incorpration, DNA synthesis was not completely inhibited since [³H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [³H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [³H]-dThd to [³H]-dTTP. Within 30 min exposure to 10 M ara-C, the rate of cellular [³H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [³H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [³H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [³H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [³H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 M ara-C (60 M and 200 M, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP. The ara-C-induced decline in dCTP noted above was apparently a secondary effect resulting from the inhibition of ribonucleotide reductase by the elevated dTTP and dATP. CDP reductase activity in the presence of dATP and dTTP at the concentrations found in ara-C-treated cells was 58% of the activity observed in the presence of nucleotide levels found in control cells. The decrease in dCTP levels was associated with a reciprocal increase in the rate of [³H]-ara-C phosphorylation following subsequent exposure to unlabeled ara-C. Thus, ara-C self-potentiated its own uptake in these cells. These observations of the self-limiting and self-potentiating effects of high concentrations of ara-C may be relevant to the selection of the optimal dose and the duration of exposure in the clinical use of high-dose ara-C infusions.Abbreviations ara-C I--d-arabinofuranosyl, cytosine (cytosine arabinoside) - ara-CTP ara-C triphosphate - NTP unspecified nucleoside 5-triphosphate - dNTP deoxynucleoside 5-triphosphate - PBS phosphate-buffered saline This research was supported by grants from the American Cancer Society (CH35J), the National Institutes of Health (CA 12197), the Gaston County Cancer Society, and Dr. George Royer of the Upjohn Company     

Reversal of acquired cisplatin resistance by nicotinamide in vitro and in vivo

At a concentration of 2.5 mM, nicotinamide (NA), an inhibitor of poly(ADP-ribose) polymerase (PARP), significantly potentiated the cytotoxicity of cisplatin (DDP) in a DDP-resistant rat ovarian tumor cell line (O-342/DDP) in vitro, whereas the same treatment had no substantial effect on DDP's cytotoxic activity against the DDP-sensitive parental line (O-342). Furthermore, in a nude mouse model where the O-342/DDP tumor grew intraperitoneally, whereas DDP given alone at 1 mg/kg ×3 exhibited no antitumor activity as compared with control values due to the resistance, NA given at a nontoxic dose (5 mmol/kg ×3) significantly increased the mean survival time (MST) of the tumor-bearing NMRI nude mice from 20.7 days in the DDP-treated group to 29.0 days in the combination group. Mechanism studies showed that endogenous PARP activity (incorporation of tritiated nicotinamide adenine dinucleotide, [³H]-NAD) was 2.6 times higher in O-342/DDP than in O-342 cells and that the presence of 2.5 mM NA during the incubation with the isotope resulted in 73.3% inhibition of the enzyme activity in O-342/DDP cells but in only about 30% inhibition in the sensitive line. However, treatment with NA during and after DDP exposure failed to produce any significant effect on the formation of DNA single-strand breaks (SSB) but decreased the induction of DNA interstrand cross-links (ISCL) by DDP in the sensitive and resistant cell lines. These results suggest that NA might have some clinical potential in reversing DDP resistance, and further studies are therefore warranted to confirm the resistance-reversing effect of NA in other DDP-resistant cell lines.Abbreviations DDP cisplatin,cis-diamminedichloroplatinum(II) - ID₅₀ 50% inhibitory dose - ISCL DNA interstrand cross-links; MST, mean survival time - NAD nicotinamide adenine dinucleotide - PARP poly(ADP-ribose) polymerase (formerly ADPRT; ADP-ribosyl transferase) - SSB DNA single-strand breaks - TCA trichloroacetic acid     

The effects ofO 6-benzylguanine and hypoxia on the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea in nitrosourea-resistant SF-763 cells

O ⁶-Alkylguanine-DNA alkyltransferase (AGT) activity is associated with resistance of brain tumor cell lines to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). SF-763 cells exhibit high AGT activity and are resistant to BCNU. In this study, we compared the effects of the AGT inhibitorO ⁶-benzylguanine (BG) on the cytotoxicity of BCNU in oxic and hypoxic SF-763 cells; we also measured AGT activity, ornithine decarboxylase (ODC) activity, and polyamine levels to determine if there was any correlation with cell survival as determined by colony-forming efficiency assay. Exponentially growing monolayer cells were pretreated with 10 M BG for 2 h under oxic or hypoxic (95% nitrogen/5% CO₂) conditions and then exposed to graded concentrations of BCNU for 1 h. BG significantly lowered AGT activity but had no cytotoxic effect in oxic or hypoxic cells; hypoxia alone was not cytotoxic. The cytotoxicity of BCNU was 4 times higher in BG-treated hypoxic cells than in oxic cells treated with BCNU alone; the BCNU doses required for a 1-log cell kill were 75 and 300 M, respectively. ODC activity was lowered by hypoxia alone but was not significantly affected by BG in either hypoxic or oxic cells. Polyamine levels were not significantly affected by hypoxia or BG. These results indicate that pretreatment with BG dramatically lowers AGT activity and increases the cytotoxicity of BCNU in both oxic and hypoxic SF-763 cells. The mechanism of this enhanced cytotoxicity is apparently unrelated to ODC activity or polyamine levels.Abbreviations CENU chloroethylnitrosourea - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - AGT O ⁶-alkylguanine-DNA alkyltransferase - BG O ⁶-benzylguanine - ODC ornithine decarboxylase - CMEM complete minimal essential medium - PE plating efficiency This study was supported by NIH grants CA-42779, CA-13525, CA-09215, CA-18137, CA-47228, and CA-57725     

Original article: Preclinical antitumour activity and animal toxicology studies of rhizoxin, a novel tubulin-interacting agent

Rhizoxin is a 16-membered antifungal macro-cyclic lactone isolatedfrom the plant pathogenic fungus Rhizopus chinensis. The compoundbinds to tubulin, preventing microtubule formation, and inhibitingmitosis. It possesses antitumour activity in vivo against variouspreclinical murine models, both leukaemias and solid tumoursmodel, as well as in vincristine- and doxorubicin-resistantleukaemia lines. In the present study, cytotoxic activity wasobserved in human tumour cell lines in vitro at very low concentrations(±10–¹⁰M) particularly against melanoma, colon,renal, non-small cell and small cell lung cancer. In vivo antitumouractivity was demonstrated in murine P388 and L1210 murine leukaemias,solid tumour models B16 melanoma and M5076 sarcoma, and in 5out of 9 human solid tumour xenografts: LOX melanoma, MX-1 breastcancer, non-small cell lung cancer A549, and small cell lungcancers LXFS 605 and LXFS 650. The absence of cross-resistanceto vinca alkaloids was confirmed in vivo against the vincristine-resistantP388 leukaemia subline and the vincristine-resistant human smallcell lung cancer LXFS 650. In addition, the antitumour activityof rhizoxin was improved by prolonged or repeated drug administrationindicating a schedule dependency. In animal toxicology studies,transient changes in erythrocyte and leukocyte numbers, localphlebitis, diarrhea, and sper-matogenic arrest were observed.The LD₁₀value of rhizoxin after a single intravenous injectionwas 2.8 mg/kg (8.4 mg/ m²). One-tenth of the mouse equivalentLD₁₀ (0.84 mg/m²), the starting dose for clinical phase I studies,was considered to be safe in rats. The antitumour activity ofrhizoxin, its unique interactions with tubulin and the absenceof non-manageable toxic effects in the animal toxicologicalstudies have led to rhizoxin's selection for clinical trials.A phase 1clinical trial has been completed showing leukopenia,muco-sitis and diarrhea to be the dose-limiting toxicities.In some cases phlebitis was observed. These toxicities werepredicted from the animal toxicological studies. In addition,rhizoxin caused minor responses in three heavily pretreatedpatients with recurrent breast cancer. Phase II clinical trialswill start soon within the framework of the EORTC and CRC. rhizoxin, tubulin binder, antitumour activity, NCI in vitro screen, vinca alkaloid sensitives/resistants, animal toxicology     

Cytotoxic and biochemical implications of combining AZT and AG-331

 We have reported that noncytotoxic concentrations of 3′-azido-3′-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5’-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N ⁶-[4-(Morpholinosulfonyl)benzyl]-N ⁶-methyl-2,6-diaminobenz[cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 μM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 μM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 μM AG-331 plus 1.8 μM [³H]-AZT produced as much as a 68%±7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells. Received: 20 May 1994/Accepted: 22 September 1994     

Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes

Purpose The purpose of the present study was to investigate the effects of phenothiazines (at clinically relevant doses) on the viability and proliferation of leukemic cell lines and normal lymphocytes, and to investigate the possibility of specific induction of apoptosis in leukemic cells.Methods Phenothiazines with different chemical structure and hydrophobicity were used: chlorpromazine (CPZ); levomepromazine (LVPZ); prometazine (PMZ); trifluoperazine (TFPZ); thioridazine (TRDZ). The leukemic cell lines used were: Daudi and Raji (derived from Burkitts lymphoma), K-562 (derived from myelogenous leukemia), and BALL-1, MOLT-4, HPB-ALL and CCRF-HSB-2 (derived from acute lymphoblastic leukemia). The cytotoxicity of the phenothiazines was determined by a CellTiter-Glo luminescent cell viability assay, using ATP bioluminescence as a marker of cell viability as well as a marker of mitochondrial activity. The proliferation of leukemic cells was determined using a CellTiter-AQ cell proliferation assay which is based on the reduction of a methyl-tetrazolium compound to the formazan product. Apoptosis induction was estimated using phosphatidylserine (PSer) translocation to the cell surface and DNA fragmentation as characteristics of the process.Results Phenothiazines (at concentrations in the range 0.1–10 M) did not affect the viability of normal lymphocytes during a 24-h incubation. Moreover, about 15–20% increase in ATP bioluminescence was observed in normal cells during treatment with 40 M phenothiazines. In contrast, the phenothiazines manifested strong cytotoxicity and antiproliferative activity against leukemic cells. The most powerful drugs were TFPZ and TRDZ, followed by CPZ. They showed a significant cytotoxic effect against leukemic cells even at 5–10 M. The most sensitive cell lines were MOLT-4 and Raji, and the most resistant were HPB-ALL and CCRF-HSB-2. All phenothiazines induced PSer exposure on the surface of leukemic cells, but not of normal lymphocytes. TFPZ, TRDZ and CPZ also induced DNA fragmentation in almost all leukemic cell lines during a 48-h incubation. The strongest apoptotic agent was TRDZ. The apoptosis induction was not accompanied by a significant release of cytochrome c from the mitochondria into the cytoplasm of native cells. Moreover, the drugs markedly suppressed Ca²⁺-induced cytochrome c release in isolated mitochondria of leukemic cells.Conclusions The results suggest that in clinically relevant doses (up to 20 M) some phenothiazines (TFPZ, TRDZ, CPZ) expressed a selective cytotoxicity and antiproliferative activity, and induced apoptosis in leukemic cells without any influence on the viability of normal lymphocytes. It is considered that the mechanism of apoptosis induction in phenothiazine-treated leukemic cells is associated with inhibition of mitochondrial DNA polymerase and decreased ATP production, which are crucial events for the viability of cancer cells.Abbreviations CPZ Chlorpromazine - LVPZ Levomepromazine - PBS(–) Phosphate-buffered saline (Ca²⁺- and Mg²⁺-free) - PMZ Prometazine - PSer Phosphatidylserine - ROS Reactive oxygen species - TFPZ Trifluoperazine - TRDZ Thioridazine     

Comparison of effects of growth factors and protein kinase C activators on cellular sensitivity to cis-diamminedichloroplatinum(II)

The anti-proliferative activity of the DNA-interactive anti-cancer agent cis-diamminedichloroplatinum(11) (cDDP) can be modulated by intracellular signaling systems. We have investigated the effects of growth factors on the sensitivity of human cervical carcinoma (HeLa) cells to cDDP. A 24-hr pretreatment of HeLa cells with 10 ng/ml epidermal growth factor (EGF) or transforming growth factor-α increased the anti-proliferatfve activity of cDDP by 2- to 4-fold. A similar pretreatment of HeLa cells with EGF did not alter cellular sensitivity to doxorubicin or vincristine. A brief exposure (15 min) to growth factors was not sufficient for cDDP sensitization. EGF caused a modest and transient increase in cellular diacylglycerol, the endogenous activator of protein kinase C. Bryostatin I, a partial agonist of protein kinase C, antagonized phorbol ester-mediated cDDP sensitization but had no effect on EGF-mediated sensitization to cODP. Both EGF and phorbol 12,13-dibutyrate (PDBu) enhanced the rate of [^195mPt]cDDP uptake but had no effect on the rate of [^195mPt]cDDP efflux in HeLa cells. Bryostatin I reversed the increase in [^195mPt]cDDP content by PDBu but failed to block EGF-induced increase in [^195mPt]cDDP accumulation. Therefore, although the mechanism of tDDP sensitization by both EGF and phorbol ester appears to involve enhanced drug uptake, they may utilize distinct signal transduction pathways.     

Use of V79 cells with stably transfected cytochrome P450 cDNAs in studying the metabolism and effects of cytotoxic drugs

Purpose: Studying the metabolism of cytotoxic drugs has become increasingly necessary to predict clinically significant drug-drug interactions and to understand the basis of interindividual variations in the pharmacokinetics of anticancer agents. The aim of this study was to determine the feasibility of using V79 Chinese hamster fibroblasts, which are stably transfected with cytochrome P450 (CYP) cDNAs, to study the metabolism of cytotoxic drugs in vitro. Methods: The 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell survival after incubation with drugs. Gas chromatography/mass spectroscopy was used for the quantitation of metabolites of cyclophosphamide and ifosfamide in culture medium. The coculture technique was used to study the generation of cytotoxic metabolites in culture medium. Results: After treatment with either cyclophosphamide or ifosfamide (100 μM to 1 mM ) cytotoxicity was demonstrated in only cytochrome CYP2B1- and cytochrome CYP3A4-expressing cells. Treatment of parental nontransfected cells that were cocultured with CYP-expressing cells with cyclophosphamide resulted in increased sensitivity to this drug. All active and inactive metabolites of cyclophosphamide and ifosfamide were detected in the culture medium. Cyclophosphamide-induced cytotoxicity in CYP2B1- and CYP3A4-expressing cells was abrogated by metyrapone and midazolam/ troleandomycin, respectively. Paclitaxel showed greater cytotoxicity against parental V79 cells than against the CYP2B1-, 2E1-, or 3A4-expressing cells, which was also influenced by cotreatment with CYP inhibitors. Conclusions: Stable expression of CYP cDNAs by V79 cells provided an in vitro system to study cytotoxic drug metabolism. Cell viability and metabolite assays were used to determine the differential metabolism and effects in different CYP-transfected cell lines treated with cytotoxic drugs. The potential use of this V79 cell expression system is in studying enzymes involved in the metabolism of cytotoxic drugs, especially early in drug development. In addition, this system may be used to determine drug interactions that may influence the outcome of therapy in patients with cancer. Received: 18 December 1997 / Accepted: 14 May 1998