Schedule dependent potentiation of antitumor drug effects by α-difluoromethylornithine in human gastric carcinoma cells in vitro

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with -difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.     

Heterogeneous responses of an in vitro model of human stomach cancer to anticancer drugs

Four permanent clones of a human adenocarcinoma of the stomach and the parent line from which they were isolated were used as an in vitro model system to evaluate the effects of 8 anticancer agents on cell survival. The drugs tested were actinomycin D (Act-D), Bleomycin (Bleo), adriamycin (adria), melphalan, chlorambucil, 5 Fluorouracil (5FU), 1,2:5,6-Dianhydrogalactitol (DAG), and 1-(2-chloroethyl)-3-(4-methyl cyclohexyl-1-nitrosourea) (MeCCNU). Although the cell lines had similar growth properties, morphologies and modal chromosome numbers, the clones expressed heterogeneous survival responses to each of six drugs tested. A comparison of the doses lethal to 90% of a clonal population (LD90) for each drug indicated large differences between the most sensitive and least sensitive clones. For chlorambucil there was a 160% difference between the LD90 values of the most and least sensitive clones. For MeCCNU the difference was 200%; for adria, 230%; Bleo, 280%; 5FU, 360%; and melphalan, 600%. Despite the heterogeneity in response among the clones to these agents, no particular clone was always the most sensitive or resistant. Of particular interest was the finding that these stomach cancer clones demonstrated uniform responses to both Act-D and DAG. Since the differential drug sensitivities expressed by heterogeneous tumor populations could be a cause of treatment failure in the patient, the demonstration of uniform sensitivities to Act-D and DAG are encouraging and suggest that other anticancer drugs which produce uniform cell killing may be identified and tested. Act-D and adria were the most effective of the drugs tested when compared on a dose for dose basis. Both agents killed more than 99.9% of the parent cell line with doses below 3 micrograms/ml (1-h treatments). The cells were least sensitive to 5FU, with only 30% of the cells killed at 100 micrograms/ml. The studies reported here indicate that this human stomach cancer model can provide valuable insight into the design of clinical protocols for treatment of gastric carcinoma in man.     

Changes in drug sensitivity of a human astrocytoma clone previously treated with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea in vitro

Summary We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3–4) to MeCCNU (10 g/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 g/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 g MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.Abbreviations DAG Galactitol - Adria Adriamycin - PCNU 1-(2-Chloroethyl)-3-(2,6 dioxo-3-piperidyl-1-nitrosourea) - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - FMF Flow microfluorometry Dedicated to the memory of Benjamin Drewinko, Physician, Scientist and friend.     

Heterogeneous survival responses of human gastric cancer clones to αDifluoromethylornithine in vitro

The effects of Difluoromethylornithine (DFMO)² on survival of human gastric cancer clones were studied in vitro. The responses were dose and time dependent. Treatments which lasted for less than 12 h were cytotoxic at only the highest doses used. The greatest effects on survival were observed only when cells were treated for 48 and 72 h. The effects on the clones produced by such prolonged treatment durations were heterogeneous, with survival values differing by as much as 460%. By contrast, the clonal survival responses to short DFMO treatments (12 h) were very uniform (dose differential of only 19%); however, this uniformity in response could be achieved only by using non-pharmacological doses of DFMO. The heterogeneity in survival responses in the clones might be slightly associated with their levels of intracellular spermidine. Clones with the smallest amounts of intracellular spermidine at the start of treatment were most sensitive to DFMO. However, this association may not hold up with further testing in other gastric cancer clones or when studied in other cancer lines in vitro.Abbreviations LD₉₀ dose lethal to 90% of the cell population - D₀ dose required to reduce survival by 63% on exponential portion of the survival curve - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - Bleo Bleomycin - 5FU 5 fluorouracil - DFMO Difluoromethylornithine - ODC ornithine decarboxylase     

Gastroprotective activity of a new semi-synthetic solidagenone derivative in mice

The gastroprotective activity of the new semi-synthetic solidagenone derivative 15,16-epoxy8(9),13(16),14-labdatrien-7 beta-methoxy-6 beta-ol (ELMO) has been assessed on the model of HCl/EtOH-induced gastric lesions in mice. Human gastric epithelial cells (AGS) and fibroblasts (MRC-5) were used to determine its mode of action. The effect of ELMO on the prostaglandin E2 content, cellular reduced glutathione (GSH) and protection against damage induced by sodium taurocholate was assessed against AGS cells. The effect of ELMO on the growth of AGS and fibroblast cultures was evaluated. The superoxide anion scavenging capacity of the compound was studied also. The cytotoxicity of ELMO, expressed as cell viability, was assessed using two independent endpoints: neutral red uptake (NRU) and the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for MRC-5 fibroblasts and NRU for AGS cells. A single oral dose of ELMO (10 and 20 mg kg(-1)) inhibited the appearance of gastric lesions in mice displaying similar values to lansoprazole at 20 mg kg(-1). At 40 microM ELMO increased the prostaglandin E2 content but not GSH in AGS cells. The compound showed no effect on sodium taurocholate-induced damage and was devoid of superoxide anion scavenging activity. Concentrations of 0.5, 1, 2 and 4 microM stimulated fibroblast but not AGS cell proliferation. The compound showed weak cytotoxicity with values (IC50) of 411 (NRU) and 418 microM (MTT) for fibroblasts and 261 microM (NRU) for AGS cells. The results support further pharmacological study of this compound as a potential new anti-ulcerogenic drug.     

Overcoming Multidrug Resistance in 2D and 3D Culture Models by Controlled Drug Chitosan‐Graft Poly(Caprolactone)‐Based Nanoparticles

The principal limitations of chemotherapy are dose‐limiting systemic toxicity and the development of multidrug‐resistant phenotypes. The aim of this study was to investigate the efficiency of a new sustained drug delivery system based on chitosan and ε‐caprolactone to overcome multidrug resistance in monolayer and drug resistance associated with the three‐dimensional (3D) tumor microenvironment in our established 3D models. The 5‐fluorouracil (5‐FU)‐loaded nanoparticles (NPs) were characterized by transmission electron microscope and dynamic light scattering, and its released property was determined at different pH values. 5‐FU/NPs exhibited well‐sustained release properties and markedly enhanced the cytotoxicity of 5‐FU against HCT116/L‐OHP or HCT8/VCR MDR cells in two‐dimensional (2D) and its parental cells in 3D collagen gel culture with twofold to threefold decrease in the IC₅₀ values, as demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, Hoechst/propidium iodide staining and flow cytometry analysis. Furthermore, the possible mechanism was explored by high‐performance liquid chromatography and rhodamine 123 accumulation experiment. Overall, the results demonstrated that 5‐FU/NPs increase intracellular concentration of 5‐FU and enhance its anticancer efficiency by inducing apoptosis. It was suggested that this novel NPs are a promising carrier to decrease toxic of 5‐FU and has the potential to reverse the forms of both intrinsic and acquired drug resistance in 2D and 3D cultures. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1064–1074, 2014     

Overexpression of glutathione S-transferase and elevation of thiol pools in a multidrug-resistant human colon cancer cell line.

A human colon cancer cell line with acquired multidrug resistance (MDR) was assayed for the intracellular GSH level and the activity of GSH-S-transferase (GST), which catalyzes the conjugation reaction of electrophilic drugs with GSH. The GSH level and GST activity (as measured with 1-chloro-2,4-dinitrobenzene) were elevated in the resistant cells by 1.7-fold and 2-fold, respectively. This elevated catalytic activity of the resistant cells was reflected in a 2-fold increase in GST-pi mRNA, which was not the result of gene amplification. In addition, buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased Adriamycin sensitivity in both the MDR and the parental cells, affecting the former more than the latter. The effects seen with buthionine sulfoximine were not seen with puromycin and actinomycin D. A dramatic overexpression of mdr1, a P-glycoprotein gene responsible for the MDR phenotype, was also observed in the MDR cells. In contrast, none of these products (i.e., mdr P-glycoprotein, GSH level, total GST activity, GST-pi gene copy, and GST-pi mRNA level) was elevated in HeLa cells resistant to cisplatin and some alkylating agents, supporting the notion that the acquisition of cisplatin resistance differs from the mechanism of MDR. These results indicate that the intrinsic GSH level and GST-pi activity affect anthracycline resistance per se and not MDR in the human colon cancer cells.     

Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin-dependent monooxygenase 3

Human flavin-dependent monooxygenase (FMO) isoforms 1 and 3 were expressed by retroviral gene transfer in mouse C3H/10T1/2 cells. FMO function was determined by the sulfoxidation of p-tolylmethylsulfide (TMS). Enzyme activity ranged from 4 to 30 nmol p-tolylmethylsulfoxide (TMSO)/30 min/mg cell protein for FMO 3 clones; for FMO 1 clones, the range was 1-6 nmol TMSO/30 min/mg. Cytotoxicity in these clones after exposure to thiocarbamate compounds was assessed by clonogenic assay. Thiourea (TU), phenylthiourea (PTU), and alpha-naphthylthiourea (ANTU) were toxic to FMO 3 cells but not to parental and FMO 1 clones; 50% toxicity was attained at 1x10(-4) M TU, 5x10(-6) M PTU, and 1x10(-6) M ANTU. Toxicity was observed after a minimum exposure time of 6 hr. Parental cells were resistant to toxicity for exposure times spanning the entire clonogenic assay period (10 days). Ethylene thiourea (ETU) was not toxic to FMO 3 cells, but preincubation with 1x10(-3) M ETU blocked TU toxicity. Reducing GSH levels by preincubation with 1x10(-5) M buthionine sulfoxime (BSO) increased TU sensitivity in FMO 3 cells from 1x10(-4) to 1x10(-6) M to achieve 50% toxicity. BSO also increased the sensitivity of "low expressor" FMO 3 clones to TU, but did not alter the refractoriness of either parental or FMO 1 expressing cells to TU. N-Acetylcysteine afforded modest protection to TU toxicity by shifting 50% cytotoxicity for TU from 5x10(-5) to 1x10(-3) M. TU mutagenicity was assayed by the development of ouabain resistance in parental and FMO 3 C3H/10T1/2 cells. Exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, direct acting mutagen) and TU was executed with and without prior sensitization with BSO. The mutation frequency for MNNG was 76/1x10(6) surviving cells, whereas no mutants were observed for TU-exposed cultures. The results of this study show that, in isolation, the major human hepatic form of FMO is capable of promoting thiocarbamate toxicity. Consistent with the known reactivity of thiocarbamate intermediates with GSH, treatments that alter GSH levels also altered toxicity in either the protective or sensitizing direction. These cell lines expressing variable levels of FMO 3 and TU sensitivity should prove useful as in vitro systems for dissecting the thiocarbamate toxicity pathway.     

Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells.

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.     

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5-25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5-30 μM) and DOX (0.025-3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC₅₀ values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.     

Reduced cellular transport and activation of fluoropyrimidine nucleosides and resistance in human lymphocytic cell lines selected for arabinosylcytosine resistance

Arabinofuranosylcytosine (araC) resistant H9-araC0.05 and H9-araC0.5 sublines were obtained following in vitro exposure of H9 cells to 0. 05 and 0.5 microM araC, respectively. These cell lines were 83.3- and 266.7-fold, 21- and 80-fold, and 2.4- and 4.0-fold more resistant to 5-fluorouridine (FUR), 5-fluoro-2'-deoxyuridine (FdUR), and 5-fluorouracil (FU), respectively. Compared with H9 cells, the cellular accumulation of FUR was 2.2 and 0.2%, FdUR 15.6 and 0.9%, and FU 56.9 and 66.5% in H9-araC0.05 and H9-araC0.5 cells, respectively. An araC resistant HL60 cell line (promyelocytic cell line) was 5.0- and 1.7-fold resistant to FUR and FdUR, respectively, but displayed no resistance to FU. The lower FUR and FdUR nucleotide levels in the resistant cells were a result of reduced cellular transport and uridine kinase (UR kinase) and thymidine kinase (TK) activities. Compared with the parental cell line, the p-nitrobenzyl thioinosine (an inhibitor of nucleoside transport) binding sites also were lower in the araC resistant cells. There was no difference in the expression of multidrug-resistant protein and thymidylate synthase mRNA in the parental and the resistant cell lines. Data presented here suggest that araC exposure of H9 cells, in addition to araC resistance, induced/selected cells that were resistant to FUR and FdUR. These cells had altered cellular drug transport and lower TK and UR kinase activities. Further studies to understand molecular mechanisms of this phenomenon are warranted.     

Characterisation of a newly isolated Caco-2 clone (TC-7), as a model of transport processes and biotransformation of drugs

Three clones isolated from early (clone PD-7 and PF-11) and late (TC-7) passages of the parental Caco-2 epithelial cell line, were characterized for their ability to transport and metabolize endogenous compounds as well as xenobiotics. All three clones were able to form a homogeneous well-differentiated epithelial monolayer as demonstrated by the presence of microvilli at the apical pole of the cells and a high transepithelial electrical resistance. These cell monolayers were further characterized for their ability to transport different probes such as mannitol for the paracellular route, testosterone for passive diffusion and taurocholic acid for the presence of active biliary acids transporters. Only small differences were observed between the parental cell line Caco-2 and the different clones in terms of transepithelial electrical resistance, mannitol paracellular transport and testosterone passive diffusion. However, large differences were observed in the active transport of taurocholic acid with V_max/K_m values of 0.037, 0.048, 0.060 and 0.178 for Caco-2 parental cell line, clones PD-7, PF-11 and TC-7, respectively. Among transport processes, clones were also characterized for the expression of various enzyme systems involved in the biotransformation of endogenous compounds and xenobiotics, such as cytochromes P450 and UDP-glucuronosyltransferases. All cell types expressed cytochrome P450IA1, as demonstrated by the O-deethylation of 7-ethoxyresorufin. However, a 3 day β-naphthoflavone pretreatment induced 10.1 ± 3.0− and 10.4 ± 5.9− fold increases in 7-ethoxyresorufin O-deethylation in Caco-2 cells and PF-11 clone, respectively, while 24.7 ± 9.6− and 22.7 ± 8.1− fold increases were observed for PD-7 and TC-7 clones, respectively. These two clones also exhibited a much higher catalytic activity towards 1-naphthol, a substrate for UDP-glucuronosyltransferases. Since the intestinal epithelium plays an important role in the rate of absorption of intact drugs following their oral administration, both transport and metabolic characteristics make the Caco-2/TC-7 clone a suitable in vitro model for studying the intestinal disposition of drugs.     

Antioxidant and anti-inflammatory activities of lycopene against 5-fluorouracil-induced cytotoxicity in Caco2 cells

5-fluorouracil (5FU) is widely used to treat colorectal cancer (CC) and its main mechanisms of anticancer action are through generation of ROS which often result in inflammation. Here, we test the effect of Lycopene against 5FU in Caco2 cell line. Caco2 cells were exposed to 3 µg/ml of 5FU alone or with 60, 90, 120 µg/ml of lycopene. This was followed by assessment of cytotoxicity, oxidative stress, and gene expression of inflammatory genes. Our findings showed that Lycopene and 5FU co-exposure induced dose-dependent cytotoxic effect without compromising the membrane integrity based on the LDH assay. Lycopene also significantly enhanced 5FU-induced SOD activity and GSH level compared to control for all mixture concentrations (p < 0.01). Lycopene alone and combination with 5FU-induced expression of IL-1β, TNF-α, and IL-6. Furthermore, IFN-γ expression was significantly enhanced by only mixture of lycopene (90 µg/ml) and 5FU (p < 0.05). In conclusion, Lycopene supplementation with 5FU therapy resulted in improvement in antioxidant parameters such as catalase and GSH levels giving the cell capacity to cope with 5FU-mediated oxidative stress. Lycopene also enhanced IFN-γ expression in the presence of 5FU, which may activate antitumor effects further enhancing the cancer killing effect of 5FU.     

Reduced topoisomerase II and elevated alpha class glutathione S-transferase expression in a multidrug resistant CHO cell line highly cross-resistant to mitomycin C.

We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin. This cell line, designated CHO-Adrr, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adrr cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adrr cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adrr cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene (mdr) sequences and overexpressed (mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adrr cells expressed a 4-fold reduced level of topo II protein but overexpressed an alpha class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adrr hybrids compared to CHO-Adrr/CHO-Adrr hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adrr cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.     

Glutathione modifies the toxicity of triethyltin and trimethyltin in C6 glioma cells

It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6. Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC₅₀ values for cell death of c. 0.02 μM and 0.8 μM respectively. Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell. Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure. This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism. To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity. Adding GSH to the culture media did not protect the cells. Depletion of intracellular GSH with buthionine-[S,R] sulphoximine did not alter cytotoxicity of TET or TMT. However, pre-treatment with (−)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds. The EC₅₀ for TMT was increased from 0.77 to 1.8 μM, a 2.3-fold shift, whereas the EC₅₀ for TET was increased >20-fold, from 0.022 to 0.47 μM. One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent. Under conditions where GSH is depleted, additional protective mechanisms may be active. Received: 26 May 1997 / Accepted: 21 October 1997     

Development of resistance to glutathione depletion-induced cell death in CC531 colon carcinoma cells: association with increased expression of bcl-2

The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.     

hERG potency estimates based upon dose solution analysis: What have we learned?

Introduction

Measurement of drug-induced inhibition of potassium current flow through the hERG channel is used to determine potency at the channel, which is used as an in vitro risk assessment for QTc interval prolongation in vivo. In the hERG assay, test solutions of varying strength are prepared to construct a concentration–response curve based upon the nominal drug concentration (NOM). Dose–solution analysis (DSA) is an analytical approach to confirm the test concentration achieved in an in vitro assay (Herron, Towers, & Templeton, 2004), and can be included as a component of hERG channel study to confirm drug concentration in the assay buffer to determine potency using the “actual” drug level in solution (ACT). Thus, DSA could be helpful in confirming test article concentrations. This study examined whether inclusion of DSA improved the accuracy of potency estimates based upon the ACT compared to the NOM concentration during hERG voltage clamp assays (non-GLP) for 99 diverse agents.

Methods

We examined the correlation of hERG IC₅₀ derived from NOM with hERG IC₅₀ derived from ACT, and analyzed potential mechanisms of deviation between ACT and NOM potency values, including solubility, cLogP, PKa, and molecular weights.

Results

Seventy-four (74) of 99 agents (73.7%) had NOM- and ACT-derived IC₅₀ values within 3-fold, 87 of 99 (87.8%) had an IC₅₀ ratio within 10-fold, and 12 (12.1%) had a > 10-fold difference in their NOM IC₅₀ and ACT IC₅₀ values. On average, these 12 compounds had less soluble, more lipophilic (high cLogP values), and more basic characters (high pKa values).

Discussion

Our investigation indicated that DSA did not alter hERG potency estimation for the majority of compounds in this dataset, i.e., DSA confirmed the NOM concentration within 3-fold. For poorly soluble agents or agents with high cLogP and pKa values, however, DSA did not clarify or improve hERG potency estimates.     

Role of glutathione and glutathione S-transferase in chlorambucil resistance.

A chlorambucil (CLB)-resistant cell line, N50-4, was developed from the established mouse fibroblast cell line NIH 3T3, by multistep drug selection. The mutant cells exhibited greater than 10-fold resistance to CLB. Alterations in GSH and glutathione S-transferase (GST) were found in CLB-resistant variants. A 7-10-fold increase in cellular GSH content and a 3-fold increase in GST activity were detected in N50-4 cells, compared with parental cells, as determined by enzymatic assays. An increase in steady state levels of the GST-alpha isozyme mRNA was found in the CLB-resistant cells, as analyzed by Northern blotting. No GST gene amplification or rearrangement was shown by Southern blot analysis. To test the relative roles of GSH and GST in CLB resistance, a number of GSH- and GST-blocking agents were used. The CLB toxicity was significantly enhanced in N50-4 cells by administration of either the GSH-depleting agent buthionine sulfoximine or the GST inhibitors ethacrynic acid or indomethacin. The resistance to CLB cytotoxicity in N50-4 cells, however, was still significantly higher than that of parental cells. The resistance of N50-4 cells to CLB was almost completely abolished by combination pretreatment yielding both GSH depletion and GST inhibition. The results indicate that both increased cellular GSH content and increased GST activity play major roles in CLB resistance in N50-4 mutant cells.     

Antiproliferative effects of ZD0473 (AMD473) in combination with 5-Fluorouracil or SN38 in human colorectal cancer cell lines

PURPOSE: ZD0473 (AMD473) [cis-amminedichloro(2-methylpyridine) platinum(II)] is a novel platinum agent of proven activity in vitro against a variety of human tumor-derived cell lines even with intrinsic or acquired resistance to CDDP. The aim of this study is to provide the basis for a rational design of ZD0473-based combination in colon cancer. EXPERIMENTAL DESIGN: We evaluated the cytotoxic effect of ZD0473 administered alone or in combination with 5-Fluorouracil (5FU) or SN38 in a panel of sensitive and 5FU-resistant colorectal cell lines (HT29/HT29-5FUR and LoVo/LoVo-5FUR). We analyzed four sequential schedules of administration: ZD0473 --> 5FU, 5FU --> ZD0473, ZD0473 --> SN38 and SN38 --> ZD0473. MTT-assay and isobologram analyses were performed to determine the synergism/antagonism. RESULTS: The pattern of response towards ZD0473, administered as single agent, was similar in all cases and independent of the 5FU-resistance phenotype (IC50 from 48.1 to 76.6 microM) and/or p53 status. No differences in sensitivity to ZD0473 alone or in combination were observed between DNA-mismatch repair-proficient (HT29/HT29-5FUR) and -deficient (LoVo/LoVo-5FUR) cells. ZD0473 administered prior to 5FU leads to synergistic/additive effect in all cell lines, while the 5FU --> ZD047 schedule was only synergistic in HT29 cells. Exposure to ZD0473 prior to SN38 leads to a synergistic/additive schedule in LoVo/LoVo-5FUR cells, while SN38 --> ZD0473 schedule was only synergistic in parental cell lines. CONCLUSIONS: The combinations of ZD0473 and 5FU or SN38 have shown to be active in sensitive and 5FU-resistant colorectal cell lines when a correct schedule of administration is applied. These results may be further exploited to promote new schedules of administration for advanced colorectal cancer treatment.