The role of bioreductive activation of antitumour anthracycline drugs in cytotoxic activity against sensitive and multidrug resistant leukaemia HL60 cells

Clinical usefulness of anthracyclines belonging to bioreductive antitumour drugs is limited by the occurrence of multidrug resistance (MDR). The aim of this study was to examine the role of structural factors of antitumour anthracycline drugs in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially in regard to MDR tumour cells. It was evidenced that at high NADPH concentration (500 μM) anthracyclines having non-modified quinone structure: doxorubicin (DOX), daunorubicin (DR) and idarubicin (IDA) were susceptible upon CPR catalysis to undergo a multi-stage chemical transformation concerning their chromophore part. Additionally, it was evidenced that the modification of the sugar moiety of DOX did not disturb the susceptibility of the obtained derivative (4'-O-tetrahydropyranyl-doxorubicin, pirarubicin, PIRA) to undergo CPR reductive activation. It was also evidenced that the derivatives having modified quinone groupment (5-iminodaunorubicin, 5-Im-DR) were not able to undergo reductive activation by CPR. The high impact of CPR-dependent reductive activation of anthracycline drugs on increasing the activity in regard to sensitive leukaemia HL60 cell line and its MDR sublines overexpressing P-glycoprotein (HL60/VINC) and MRP1 (HL60/DOX) was evidenced. Furthermore, significant changes in binding manner of activated compounds to naked DNA and cellular nucleus in comparison to their non-activated forms were also observed. It could prevent the export of formed adducts out of the cell by MDR proteins and may explain significant increases in intracellular accumulation of these compounds in HL60/VINC and HL60/DOX cells.     

Anthraquinone antitumour agents, doxorubicin, pirarubicin and benzoperimidine BP1, trigger caspase-3/caspase-8-dependent apoptosis of leukaemia sensitive HL60 and resistant HL60/VINC and HL60/DOX cells

We examined the effect of selected anthraquinone antitumour agents - doxorubicin (DOX), pirarubicin (PIRA) and benzoperimidine BP1 - on inducing apoptosis of the sensitive leukaemia HL60 cell line and its multidrug resistance sublines overexpressing P-glycoprotein (HL60/VINC) and multidrug resistance-associated protein 1 (HL60/DOX). All agents used at IC50 and IC90 were able to influence the cell cycle of sensitive HL60 and resistant cells and induce apoptosis. Interestingly, it was seen that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by the studied anthraquinone compounds compared with HL60 and HL60/DOX cells. However, the examined agents did not change the expression of Fas receptors on the surface of HL60-sensitive and-resistant cells.     

R-dl-型维拉帕米调低KBv200肿瘤细胞对长春新碱和多柔比星的多药抗药性

目的:研究R-型维拉帕米对多药耐药的降低作用及其急性动物毒性,并与消旋维拉帕米的相应结果作比较。方法:细胞毒性的测定用MTT法;细胞内多柔比星积累的测定用萤光分光光度计法。急性毒性试验用BALB/c小鼠腹腔注射法。结果:R-型维拉帕米部分调低KBv200细胞对长春新碱和多柔比星的耐药性,其调低效应与作用浓度和作用时间有关。1.25μmol·L~(-1)的R-型维拉帕米与长春新碱对细胞作用24h,能够显著增加KBv200细胞对长春新碱的敏感性。在增敏和增加细胞内多柔比星累积方面,R-型维拉帕米与消旋维拉帕米效果一样,但R-型维拉帕米的急性动物毒性明显低于消旋维拉帕米。结论:R-型维拉帕米1.25μmol·L~(-1)提高耐药肿瘤细胞对长春新碱和多柔比星的敏感性,增加对长春新碱敏感性所需的药物作用时间可缩短至24h。     

R—dl—型维拉帕米调低KBv200肿瘤细胞对长春新碱和多柔比星的多？…

目的：研究Ｒ－型维拉帕米对多药耐药的降低和及其急性动物毒性,并与消旋维闰帕米的相应结果作比较。方法：细胞毒性的测定用ＭＴＴ法;细胞内多柔比星积累的测定用萤光分光光度计法。急性毒性试验用ＢＡＬＢ－ｃ小鼠腹腔注射法。结果：Ｒ－型维拉帕米部分调低ＫＢｖ２００细胞对长春新碱和多柔比星的耐区性,其调低效应与作用肖度和作用时间有关。     

Role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase: implications for increasing the activity against sensitive and multidrug-resistant leukaemia HL60 cells

The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 μmol/l), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined.     

Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL60) and human peripheral blood mononuclear cells

Dehydrocrotonin (DHC) is a diterpene lactone obtained from Croton cajucara (Sacaca). Dimethylamide-crotonin (DCR), a DHC derivative, has a similar inhibitory effect on leukemic HL60 cells than its parent compound evaluated by different endpoints of cytotoxicity. No cytotoxicity or morphological alterations associated with apoptosis were detected in human peripheral blood mononuclear cells (PBMC) after treatment with up to 400 micro M DCR in presence of phytohemaglutinin (5 micro g/ml). Based on morphological changes and the pattern of DNA fragmentation, DHC and DCR were found to induce apoptosis and terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells, but these compounds did not show any toxic effect in PBMC. Thus, DCR and DHC inhibit HL60 cell growth in vitro partly by inducing apoptosis and cell differentiation, but does not cause serious damage to immune cells according to our experimental conditions.     

The cytotoxic effect of mistletoe lectins I, II and III on sensitive and multidrug resistant human colon cancer cell lines in vitro

Multidrug resistance glycoprotein1 (MDR-1) eliminates amphiphilic chemotherapeutic agents out of tumour cells leading to therapeutic failures. The aim of this study was to investigate the cytotoxic effect of mistletoe lectins (MLs) I, II and III on the sensitive human colon cancer cell line HT 29(mdr-), its multidrug resistant variant HT 29(mdr+), the variant HT 29(SF1m) transfected with the MDR-1 gene and its sensitive control cell line HT 29(deltaSF). Both cell proliferation and ML binding pattern were analysed. Marked quantitative differences concerning the cytotoxic effect of the three MLs on the different cell lines were observed. All MLs showed the greatest cytotoxicity towards the HT 29(mdr+) cells, in which multidrug resistance (MDR) was induced by increasing concentrations of a MDR inducing agent. In contrast, MDR-1 and mock-transfected cells showed almost the same sensitivity towards the three MLs as the control cells (HT 29(mdr-)). FACS analysis showed that the HT 29(mdr+) cells were the cells with the highest density of ML binding sites. Thus, higher sensitivity of HT 29(mdr+) cells are not caused by the overexpression of MDR-1, but are caused by the general changes of the cellular glycosylation during the acquisition of the MDR phenotype.     

Cadmium-induced alterations in RNA metabolism in cultures of Chinese hamster cells sensitive to and resistant to the cytotoxic effects of cadmium.

A variant population (CdR) of cultured Chinese hamster cells (line CHO) was derived that is more than 100 times as resistant to the cytotoxic effects of Cd2+ than is the parent population. The effects on RNA metabolism of exposure to sublethal concentrations of Cd2+ were studied in CHO and CdR. Exposure to 2 X 10(-7) M CdCl2 for 24 h resulted in increased polysome content (1.2 times) and increased uridine or adenosine incorporation into heterogeneous nuclear RNA (1.2-1.4 times) and messenger RNA ((1.5-1.7 times) in both populations. Measurement of ATP pool specific activity following exposure to radiolabeled adenosine showed that increased incorporation reflects increased synthesis. The equivalence of CHO and CdR in dose-response in terms of stimulated RNA synthesis and their disparity in dose-response in terms of cytotoxic effects indicate that the systems involved in conferring protection against the lethal effects of Cd2+ are not similarly involved in attenuating the effects on RNA metabolism.     

Influence of serum protein binding on the uptake and retention of idarubicin by sensitive and multidrug resistant human leukemic cells

Objective: The objectives of the investigations were (1) to determine the binding characteristics of idarubicin (IDA) in human serum and cell culture solutions, (2) to determine the effect of protein binding on the uptake and retention of IDA by human leukemic cell lines in culture and the extent to which R-verapamil (R-VRP), an inhibitor of the P-glycoprotein (P-gp) transporter, can modulate these processes, and (3) to assess the importance of protein binding on cytostatic and chemosensitizer action in vivo. Methods: The protein binding of IDA was determined using equilibrium dialysis. Cell uptake of IDA was measured using sensitive and P-gp-containing resistant human leukemic cell lines (HL-60 and HL-60-Vinc) in vitro. IDA was assayed spectrophotofluorometrically. Results: In the incubation media examined, the free fraction of IDA varied more than seven-fold from approximately 60% in 15% fetal calf serum (FCS)/PBS to only 8% in human serum. Cellular uptake of IDA was approximately three times higher in medium containing low protein concentrations. R-VRP eliminated the difference in IDA uptake between resistant and sensitive cell lines and this was the case when the cells were incubated in solutions containing both high and low protein concentrations. However, R-VRP did not overcome the effect of high protein concentrations on IDA uptake. Conclusions: Plasma protein binding is an important determinant for cellular uptake of IDA in vitro. This should be taken into account when interpreting results of in vitro functional assays with patient material. Chemosensitizers such as R-VRP are effective in both high and low protein solutions. Investigations like these may be useful for evaluating cytostatic efficacy and chemosensitizer action in vivo. Received: 10 August 1998 / Accepted in revised form: 15 January 1999     

Mussel blood cells,resistant to the cytotoxic effects of okadaic acid,do not express cell membrane p-glycoprotein activity (multixenobiotic resistance)

Okadaic acid (OA) is a dinoflagellate toxin, accumulating in shellfish and causing diarrhetic shellfish poisoning (DSP) in humans. OA is a highly cytotoxic agent in most cell lines because of its inhibiting properties of protein phosphatases. So far, the cytotoxicity of OA in mussels, the main vectors of DSP, has not been investigated. In this paper, the viability of mussel (Mytilus edulis) blood cells incubated in 10 nM-1 microM OA was studied. After 72 h of exposure, viability was reduced to 54% in 1 microM OA compared with 88% in control cells. This yielded a LC50 of >1 microM for OA, which is 30-1000-times higher compared with other cell types. It was hypothesised that P-glycoprotein (p-gp) activity (multixenobiotic resistance, MXR) contributed to the resistance to OA. Vincristine and rhodamine B was used as p-gp substrates and verapamil or staurosporine (ST) as inhibitors of p-gp transport. However, no indications of cell membrane p-gp activity were detected. Instead, experimental observations led to the conclusion that a MXR transport system was present within lysosomal membranes. Various concentrations of OA did not affect the dynamics of vincristine in blood cells. As a positive control for the assay, p-gp activity was measured in mussel gill tissue. The efflux of rhodamine B was reduced by verapamil, which is, considered evidence for cell membrane p-gp activity, thus the accuracy of the method was confirmed. Rhodamine B efflux was also reduced by OA in gill tissue, which suggested that OA is either a competitive substrate or inhibitor of p-gp activity. When the volume of the lysosomal compartment was measured in blood cells pre-exposed to OA, a significant increase was detected compared with control cells. It was proposed that uptake and storage of OA within the lysosomal system might protect mussel blood cells from the cytotoxic effects of this compound.     

Anthrapyridones, a novel group of antitumour non-cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells

1. Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP-binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation. 2. The search of new compounds able to overcome MDR is of prime importance. 3. Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain. 4. The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells. 5. It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P-gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P-gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake. 6. For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P-gp resistant cells in comparison to sensitive cells. 7.We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.     

The novel pyrrolo-1,5-benzoxazepine,PBOX-6, synergistically enhances the apoptotic effects of carboplatin in drug sensitive and multidrug resistant neuroblastoma cells

Neuroblastoma, a malignancy of neuroectoderrmal origin, accounts for 15% of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat. A major obstacle in the effective treatment of neuroblastoma is the development of multidrug resistance (MDR). There is thus a compelling demand for new treatment strategies for this cancer that can bypass such resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various cancer cell lines, ex vivo patient samples and in vivo cancer models. In this study we examined the ability of two members, PBOX-6 and -15, to exhibit anti-cancer effects in a panel of drug sensitive and MDR neuroblastoma cell lines. The PBOX compounds potently reduced the viability of all neuroblastoma cells examined and exhibited a lower fold resistance in MDR cells when compared to standard chemotherapeutics. In addition, the PBOX compounds synergistically enhanced apoptosis induced by etoposide, carboplatin and doxorubicin. Exposure of drug sensitive and resistant cell lines to PBOX-6/carboplatin induced cleavage of Bcl-2, a downregulation of Mcl-1 and a concomitant increase in Bak. Furthermore, activation of caspase-3, -8 and -9 was demonstrated. Finally, gene silencing of Mcl-1 by siRNA was shown to sensitise both drug sensitive and multidrug resistant cells to carboplatin-induced apoptosis demonstrating the importance of Mcl-1 downregulation in the apoptotic pathway mediated by the PBOX compounds in neuroblastoma. In conclusion, our findings indicate the potential of the PBOX compounds in enhancing chemosensitivity in neuroblastoma.     

抗肿瘤药物对~(125)I-纤维蛋白原与白血病细胞P388和HL60结合的抑制作用

肿瘤转移是肿瘤患者致死的关键。肿瘤转移涉及很多复杂的过程和机制[1] 。对它们的深入研究将有助于我们了解肿瘤转移过程 ,找出有针对性的治疗方法 ,对提高临床肿瘤的治疗效果有很重要的作用。肿瘤转移与血液系统 ,尤其是与纤维蛋白和纤维蛋白原关系已引起人们长期的关注。纤维蛋白与纤维蛋白原是肿瘤组织的主要基质 ,已经被认为通过下列 3条途径帮助肿瘤转移形成 :①凝结成自身网络帮助肿瘤细胞在上面吸附 ,以利于肿瘤细胞扩增和转移 ;②在肿瘤细胞表面形成包被以抵消宿主淋巴细胞对肿瘤细胞的攻击 ;③帮助肿瘤血管的形成[2 ,3 ] 。因此 ,…     

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.