Telomere dynamics determine episodes of anticancer drug resistance in rat hepatoma cells

Clinical and experimental observations indicate that resistance to anticancer drugs may be spontaneously reversible over time, but the mechanisms of this reversal are unknown. The resistance of cultured hepatoma cells to methotrexate (MTX) and cisplatin was followed for 9 months. Cells were exposed to three treatments: MTX 200 nM for 24 h or 15 nM continuously and cisplatin 50 microM for 2 h. We investigated the relation between the temporal pattern of cell resistance and the previously reported fluctuations in cell proliferation rate, telomere length and telomerase activity. Spontaneous major peaks in resistance to each drug fell in time windows of 2-3 months (60-70 population doublings) and were at different times for each drug. The frequency of the fluctuations in drug resistance was the same as that of variations in cell growth rate, but amplitudes were unrelated. By contrast, resistance was directly related to telomere length dynamics in the same cells. MTX resistance occurred when telomeres shortened and cisplatin resistance when they were elongated. Furthermore, peaks of resistance to the continuous treatment with MTX were observed at 350-bp intervals of mean telomere length (9.06, 9.41, and 9.76 kbp) during the two 2-month phases of telomere shortening. Statistical analysis demonstrates the sinusoidal relationship between intermittent MTX resistance and telomere length. Possibly, erosion of telomeres encroaches on periodically spaced nucleosomal proteins, defining the onset of resistance phases. This evidence that resistance of tumoral cells to anticancer drugs may be intermittent and that onset of resistance is dictated by telomere length has major implications for clinical practice.     

Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.     

P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines. Evidence for a saturation of active daunorubicin transport.

We studied the resistance of colon tumors to anticancer agents in vitro. Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e. HT29, SW1116 and COLO 320, and the murine colon cell line C-26. The sensitive/MDR human ovarian cancer cell line couple A2780/2780AD was used as a reference. The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 less than or equal to SW1116 less than C26 less than or equal to COLO 320 less than 2780AD. The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present. A flow-through system was used to study the mechanism of DN transport. For the first time, evidence for saturation of an active transport of DN from the cells is reported. We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.     

Direct determination of unbound intrinsic drug clearance in the microsomal stability assay.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CL(in,u)) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CL(in,u) from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CL(in,u) of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay.     

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells.

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.     

Acquired and intrinsic glycopeptide resistance in enterococci

Enterococci are Gram-positive cocci responsible for severe human infections, such as endocarditis, meningitis, and septicemia and constitute an increasingly frequent cause of nosocomial infections. Enterococci are resistant to nearly all classes of drugs including, since 1986, glycopeptides. Vancomycin and teicoplanin act by blocking cell wall formation and resistance is due to synthesis of modified late peptidoglycan precursors. Glycopeptide resistance can be intrinsic or acquired and strains may be resistant to vancomycin and teicoplanin, or to vancomycin only. Five types of glycopeptide resistance and their biochemical mechanisms have been described in enterococci. Clinical isolates that are dependent on vancomycin for growth have been isolated. Data suggest a dual origin for resistance: glycopeptide-producing organisms or enterococcal species intrinsically resistant to these drugs.     

A kojic acid derivative promotes intrinsic apoptotic pathway of hepatocellular carcinoma cells without incurring drug resistance

Hepatocellular carcinoma is one of the most pervasive cancers with low prognosis, high frequency of recurrence, and metastasis. Studies conducted have focused on extricating novel strategies for successful treatment. Kojic acid and its derivatives are already proven to have depigmenting, anti‐inflammatory, and anti‐neoplastic properties. In the present study, kojic acid and its 10 distinct derivatives were tested on HEPG2 cell line for their possible anti‐cancer effect and seven of them were observed to be cytotoxic. Compound 6 was chosen to proceed as the IC₅₀ dosage for HEPG2 cells was lower in comparison with the other derivatives and kojic acid itself. Further experiments pointed out that intrinsic apoptotic pathway was triggered with the exposure of the cells to IC₅₀ concentration of the derivative as the treatment led to escalation of intracellular ROS, induction of TP53 gene, and activation of caspase 3/7. Pro‐apoptotic Jnk and Bax genes were not triggered suggesting that the apoptotic pathway advance through an alternative route. Complementary experiments are in need; howbeit, the current findings suggest that the derivative offers a novel promising approach against hepatocellular carcinoma as it is not detrimental to healthy cells within the concentrations applied, and it does not induce drug resistance.     

肝肿瘤靶向给药的研究进展

本文通过查阅近年国内外关于肝肿瘤靶向的文献,系统地阐述了肝肿瘤靶向给药系统的新靶点、靶向原理和靶向制剂类型3个方面的内容,旨在为肝靶向制剂的进一步研制提供理论依据。     

Cyclosporins as drug resistance modifiers.

Cyclosporin A (CsA), a cyclic peptide of 11 amino acids isolated from the fungus Tolypoclodium inflatum Gams, is the principle drug used for immunosuppression in organ transplant patients. It is known to have a very specific effect on T-cell proliferation although the precise mechanism remains unclear. Following internalization, CsA binds to a cytosolic protein, cyclophilin, which has been shown to possess peptidyl-prolyl cis-trans isomerase activity. CsA is an effective modifier of multidrug resistance in human and rodent cells at doses in the range of 1 to 5 micrograms/mL. Although it reverses the drug accumulation deficit associated with multidrug resistance in some cell types, this is not always the case. CsA has P-glycoprotein binding activity but less specific membrane effects and inhibition of protein kinase C may also be involved in its resistance modifier action. A number of non-immunosuppressive analogues of CsA have been shown to have resistance modifier activity and some are more potent than the parent compound. One analogue from Sandoz, PSC-833, has been shown to be approximately 10-fold more potent than CsA and is expected to enter clinical trial in the near future. The use of such agents may allow a full test of the hypothesis that reversal of multidrug resistance will prove a useful clinical strategy.     

Deferoxamine toxicity in hepatoma and primary rat cortical brain cultures.

Deferoxamine is commonly used for treatment of iron intoxication. Because the usual dose is unable to chelate sufficient iron before severe injury occurs, "high-dose" deferoxamine treatment has been proposed. However, several authors have reported severe toxicity after deferoxamine therapy. Although the hemodynamic effects are well described, the cellular toxicity of deferoxamine is unknown. Accordingly, we investigated the cellular toxicity of deferoxamine using in vitro techniques in two cell lines. Brain cells were harvested from fetal rats and cultured for 14-21 days before deferoxamine exposure. Using similar techniques, rat hepatoma cells were grown until confluent. Deferoxamine was added to the cultures to achieve final concentrations of 200-800 microg/ml, corresponding to in vivo infusion rates of 15-60 mg/kg/h. Deferoxamine was removed after 3 or 6 days by changing the medium. Subtoxic FeCl3 (500 mg/dl) was concurrently added to identical cultures to determine if deferoxamine potentiated iron toxicity. Cell viability was measured by a colorimetric assay. The addition of deferoxamine (0.2, 0.4, 0.8 mg/ml) significantly decreased cell viability in both cell groups. The effect of deferoxamine on primary cortical brain cultures was similar for the three concentrations used, and was similar when examined either 72 h or 6 days later. In contrast, hepatoma cell cultures evidenced a dose- dependent cell loss that increased with the length of exposure. The addition ofsubtoxic amounts of FeCl3 (500 microg/dl) in the presence of deferoxamine was protective in all cultures, and abolished deferoxamine-induced cell loss. Interestingly, the addition of serum albumin significantly reduced the amount of iron present in cells, suggesting its potential use to treat iron toxicity. These results suggest that deferoxamine, in the absence of iron, is toxic to cortical brain and hepatoma cells in vitro.     

Studies on responsiveness of hepatoma cells to catecholamines. IV. Lack of adrenergic activation of phosphorylase in rat ascites hepatoma cells

Glycogen phosphorylase a activity in 7 rat ascites hepatoma cell lines treated with adrenergic agents, phenylephrine, epinephrine and isoproterenol, was investigated as compared with that in freshly isolated rat hepatocytes. Basal phosphorylase activities in hepatoma cells except AH7974 cells were lower than that in hepatocytes. Phosphorylase in hepatoma cells was not activated by any of the agents, while the enzyme activity in hepatocytes was clearly increased in a dose- and time-dependent manner. Phosphorylase in hepatocytes was sensitive to glucagon, but it was found to be insensitive to glucagon in all hepatoma cells. The present results suggest that rat ascites hepatoma cells may escape the glycogenolytic regulation by catecholamines and glucagon.     

Comparative drug exsorption in the perfused rat intestine.

The factors affecting drug exsorption into the gastrointestinal tract are uncertain. In this study, the intestinal clearance (CLi) of compounds which vary in their lipophilicity, serum protein binding, molecular weight and ionic charge at physiological pH, has been measured. Male Sprague-Dawley rats with ligated bile ducts were infused with the test compounds through the jugular vein. The small intestine was intubated and perfused with Tyrode solution at 20 mL h-1. The CLi of the compounds investigated (urea, polyethylene glycol, inulin, albumin, dextran, barbituric acid, salicylic acid, thiobarbital, thiopental, thioseconal, theophylline, S-disopyramide and quinidine) was determined under anaesthesia by dividing the rate of a component's appearance rate in the perfusate by its carotid arterial concentration. Serum protein binding of the compounds was determined by equilibrium dialysis. The n-octanol-water partition coefficients of the compounds were measured as indices of lipophilicity. The CLi values of dextran, albumin, inulin, polyethylene glycol and urea were 0.56, 1.03, 4.5, 4.8 and 12.0 mL h-1, respectively. The larger the molecular weight of a compound, the smaller its CLi. The molecular weight is apparently one of the major determinants of CLi. Thiobarbital, thiopental and thioseconal are compounds of similar structure with increasing lipophilicity and serum protein binding. The CLi of thiobarbital, thiopental and thioseconal was proportional to the unbound fraction in serum. The unbound clearance (CLui) of three thiobarbiturates were similar (approximately 11 mL h-1). The unbound fraction of drug in serum appears to be a factor determining their CLi. Barbituric acid and salicylic acid, two acidic compounds, showed a low CLi (less than 1 mL h-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on responsiveness of hepatoma cells to catecholamines. I. Lack of beta-adrenergic responsiveness in rat ascites hepatoma AH13 cells

The beta-adrenoceptor density and the activities of adenylate cyclase and cyclic AMP phosphodiesterase were examined to compare AH13 cells having lower beta-adrenergic responsiveness with other rat ascites hepatoma cells and normal rat liver cells. Normal rat liver cells used were cultured for 24 hr after the collagenase digestion of liver. The density of binding sites of 3H-dihydroalprenolol in AH13 cell plasma membrane was very similar to the density in AH44 and normal liver cell membrane, but that in AH130 cell plasma membrane was about 10-fold greater than those in the other three cell lines. The activity of cyclic AMP phosphodiesterase was about 2.5- to 7-fold higher in hepatoma cells than in rat liver cells, but this enzyme activity of AH13 cells was not especially high among the hepatoma cells examined. The basal adenylate cyclase activity was lower in AH44 cells, but was higher in AH13 and AH130 cells than in rat liver cells. However, adenylate cyclase of AH13 cells was hardly activated by isoproterenol, while the enzyme of the other cells was activated 3- to 5-fold. On the other hand, adenylate cyclase of each cell line including AH13 was activated 4- to 14-fold by NaF. From these results, it is suggested that AH13 cells can hardly produce cyclic AMP by the beta-adrenergic stimulation because of the disordered interaction of beta-adrenoceptors with adenylate cyclase.     

Drugs and drug resistance in African trypanosomiasis.

Despite the many decades of use of most of the current trypanocides, we know little of their mode of action. This may in part be because most of these will act on multiple targets once inside the cell, and they derive their selective action on the parasite from selective accumulation by the pathogen. Loss of this capacity for drug uptake by the trypanosome would thus be a major cause for drug resistance. We here discuss the use of current drugs against human and veterinary African trypanosomiasis, the prevalence, causes and mechanisms of drug resistance and new developments in trypanosomiasis therapy such as the introduction of nifurtimox and DB289.     

Mechanism of resistance to tiazofurin in hepatoma 3924A

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) has shown potent cytotoxic and antitumor activity against hepatoma 3924A carried in the rat [Lui et al. J. biol. Chem. 259, 5078 (1984)]. However, eventually the tumor emerged, proliferated and killed the host. To throw light on the factors that play a role in the resistance to this drug, a tiazofurin-induced resistant hepatoma 3924A line in culture was produced, and its biochemical and pharmacological pattern was examined. Resistance in hepatoma cells was expressed by a reprogramming of gene expression that entailed the display of a program of multiple biochemical alterations. In the resistant cells the activity of IMP dehydrogenase, the target enzyme of tiazofurin, was increased 2- to 3-fold. The steady-state guanylate pools were elevated 3-fold, and there was a decrease in the de novo synthesis of guanylate. There was an expansion of guanylate salvage, which could circumvent inhibition of de novo guanylate synthesis by tiazofurin. For the first time in studies on the resistance of different cell lines to tiazofurin, reduced tiazofurin transport (to 50%) in resistant hepatoma cells was identified which might account for the decreased concentration (50%) of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), in these cells. NAD pyrophosphorylase activity also decreased to 53% of that of the sensitive line, which was responsible, in part at least, for the decreased TAD concentration of the resistant cells. When resistant cells were cultured in the absence of tiazofurin, resistance to the drug gradually decreased, and by 50 passages sensitivity returned. Resistance to tiazofurin in hepatoma cells appears to be a drug-induced metabolic adaptation which involves alterations in the activity of the target enzyme, in the transport and concentration of the drug and the active metabolite, and an increase of guanylate concentration and guanine salvage capacity.     

Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells

The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.     

Biochemical pharmacology of acivicin in rat hepatoma cells

The antiglutamine agent acivicin, l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, inhibited the growth of hepatoma 3924A cells in culture. After 7 days of incubation with the drug, an lc₅₀ of 1.4 μM was observed by determination of colony forming ability. A combination of cytidine (1 mM), deoxycytidine (10 μM) and guanosine (10 μM) completely protected the hepatoma cells against the cytotoxic action of acivicin, but each nucleoside by itself had no effect. Acivicin (0.1 mM) inhibited the incorporation of uridine and thymidine into macromolecules, but not that of leucine. Acivicin depressed the pools of CTP, GTP, dCTP, dGTP and dTTP to 46, 62, 40, 64 and 53%, respectively, but it increased UTP level to 152% of the values of untreated cancer cells. The activity of a highly purified CTP synthetase (EC 6.3.4.2) from rat liver and hepatoma 3924A was inhibited by acivicin. The inhibition was competitive with respect to l-glutamine, and the K_i values with liver and hepatoma enzymes, determined by Dixon and reciprocal plots, were 1.1 and 3.6 μM respectively. The hydroxy analog of acivicin was also a competitive inhibitor, but it was less effective than acivicin, with a K_i value of 1.8 mM for the hepatoma enzyme. Our observations on the impact of acivicin on the behavior of pools of ribonucleotides and deoxyribonucleotides and the competitive inhibition of purified CTP synthetase from hepatoma cells suggest that a major mechanism of action for this drug is the inhibition of CTP synthetase and GMP synthetase (EC 6.3.5.2).