Cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles in rat glioma cell lines using different assays

The cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles (Dox-PBCA-NP) was investigated in the rat glioma cell lines GS-9L, F-98 and RG-2. MTT and LDH assays were used as cytotoxic assays. In general, the cytotoxicity of nanoparticle-bound doxorubicin (Dox) was enhanced compared to the free drug in solution. However, responses of the cell lines towards the drug effects were different. In the case of free Dox in solution, this difference correlated with different intracellular concentrations of Dox, which in turn, depended on the level of P-glycoprotein (P-gp) expression in these cell lines. Accordingly, the 9L gliosarcoma (GS-9L) cells, which appeared to be most resistant towards Dox, were characterized by the highest P-gp expression.

Additionally, the influence of surfactants on the cytotoxic effect was investigated at different Dox concentrations. It was shown that the presence of polysorbate 80 (Tween® 80) in the nanoparticle formulation significantly enhanced the cytotoxicity, whereas poloxamer 188 (Pluronic® F68) and poloxamine 908 (Tetronic® 908) had a negligible influence.     

A thermally targeted elastin-like polypeptide-doxorubicin conjugate overcomes drug resistance

The ability of cancer cells to become simultaneously resistant to different drugs, a trait known as multidrug resistance, remains a major obstacle for successful anticancer therapy. One major mechanism of resistance involves cellular drug efflux by expression of P-glycoprotein (P-gp), a membrane transporter with a wide variety of substrates. Anthracyclines are especially prone to induction of resistance by the P-gp mechanism. P-gp mediated resistance is often confronted by use of P-gp inhibitors, synthesis of novel analogs, or conjugating drugs to macromolecular carriers in order to circumvent the efflux mechanism. In this report, the effect of free and Elastin-like polypeptide (ELP) bound doxorubicin (Dox) on the viability of sensitive (MES-SA and MCF-7) and multidrug resistant (MES-SA/Dx5 and NCI/ADR-RES) human carcinoma cells was studied in vitro. The resistant MES-SA/Dx5 cells demonstrated about 70 times higher resistance to free Dox than the sensitive MES-SA cells, and the NCI/ADR-RES cells were about 30 fold more resistant than the MCF-7 cells. However, the ELP-bound Dox was equally cytotoxic in both sensitive and resistant cell lines. The ELP-bound Dox was shown to accumulate in MES-SA/Dx5 cells, as opposed to free Dox, which was rapidly pumped out by the P-gp transporter. Since ELP is a thermally responsive carrier, the effect of hyperthermia on the cytotoxicity of the ELP-Dox conjugate was investigated. Both cytotoxicity and apoptosis were enhanced by hyperthermia in the Dox resistant cells. The results suggest that ELP-Dox conjugates may provide a means to thermally target solid tumors and to overcome drug resistance in cancer cells.     

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5?µM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.     

Increase in doxorubicin cytotoxicity by carvedilol inhibition of P-glycoprotein activity

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR1 (multidrug resistance) gene encoding for the transmembrane efflux pump, P-glycoprotein (P-gp). The calcium channel blocker verapamil has been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether the new antihypertensive drug carvedilol influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil (10 micromol/L), and even more markedly, carvedilol (10 micromol/L) increased cellular uptake of P-gp-transported calcein of a P-gp-expressing breast cancer cell line (Hs578T-Dox). In the subline (Hs578T) not expressing P-gp, no effects of carvedilol or verapamil on calcein uptake were seen. Carvedilol and verapamil (10 micromol/L) reduced the LD50 (dose which results in the death of half the number of cells) of the Hs578T-Dox subline from 200 mg/L to approx. 10 mg/L Dox, whereas the LD50 of the Hs578T subline was only marginally affected. Carvedilol (10 micromol/L) reduced P-gp activity approximately twice as effectively as verapamil at an equimolar concentration. Carvedilol did not affect pyrogallol cytotoxicity and pyrogallol was without effect on calcein accumulation of the Hs578T-Dox cell line, indicating the lack of antioxidative properties affecting P-gp activity and associated toxicity of the drug. The results suggest that carvedilol has the clinical potential to reverse tumour MDR involving the efflux protein P-gp.     

In vitro cellular accumulation and cytotoxicity of liposomal and conventional formulations of daunorubicin and doxorubicin in resistant K562 cells

Previous investigations have indicated the possibility to circumvent multidrug resistance (MDR) by incorporation of an anthracycline into liposomes. We examined the in vitro cytotoxicity and cellular drug accumulation of the anthracyclines daunorubicin and doxorubicin compared with the commercially available liposomal formulations DaunoXome and Caelyx in human myelogenous leukemia K562 cells. The drug-sensitive parental K562/K line was compared with the P-glykoprotein (P-gp)-expressing cell lines K562/Dnr and K562/Vcr. Two cell lines with reduced levels of topoisomerase II (K562/Nov and K562/Ida) were also included. The cytotoxicity was determined by fluorometric microculture cytotoxicity assay and the cellular drug levels were determined by high performance liquid chromatograghy. There was a strong inverse correlation between P-gp levels and cellular drug accumulation (rho = -0.83, p = 0.04) and cytotoxicity (rho = -0.95, p = 0.01) of daunorubicin. Also the cytotoxicity of DaunoXome and doxorubicin was related to P-gp levels (rho = -0.96, p = 0.01 and rho = -0.90, p = 0.07, respectively). Caelyx did not show any cytotoxic effect due to impaired cellular uptake of the pegylated liposome. Regardless of the P-gp levels of the treated cells, DaunoXome showed the same cytotoxic effect despite lower intracellular accumulation (range 22-47%), compared with conventional daunorubicin.     

Inhibition of P-Glycoprotein by D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS)

Purpose. To investigate whether d--tocopheryl polyethylene glycol 1000 succinate (TPGS) functions as an inhibitor of P-glycoprotein (P-gp), the multidrug resistance transporter. Methods. Two assays were used to measure the function of TPGS on P-gp function. First, we examined the ability of TPGS to modulate the cytotoxicity of established, cytotoxic, P-glycoprotein substrates. Parental NIH 3T3 cells and NIH 3T3 cells transfected with the human MDR1 cDNA (G185) were exposed to doxorubicin, paclitaxel, colchicine, vinblastine and 5-fluorouracil (5FU) in the presence or absence of TPGS. Cytotoxicity was assessed with the MTT assay. Second, polarized transport of the P-gp substrates rhodamine 123 (R123), paclitaxel and vinblastine was measured using the human intestinal HCT-8 and Caco-2 cell lines grown in Transwell dishes. Drug flux was measured by liquid scintillation counting or fluorescence spectroscopy of the media. Results. G185 cells were 27–135 fold more resistant to the cytotoxic drugs doxorubicin, vinblastine, colchicine and paclitaxel than the parental NIH 3T3 cells. In contrast 5FU, which is not a P-gp substrate, is equally cytotoxic to parental and G185 cells. Co-administration of TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel, and colchicine in the G185 cells to levels comparable to the parental cells. TPGS did not increase the cytotoxicity of 5FU in the G185 cells. Using a polarized epithelial cell transport assay, TPGS blocked P-gp mediated transport of Rl 23 and paclitaxel in a dose responsive manner. Conclusions. These data demonstrate that TPGS acts as a reversal agent for P-glycoprotein mediated multidrug resistance and inhibits P-gp mediated drug transport. These results suggest that enhanced oral bioavailability of drugs co-administered with TPGS may, in part, be due to inhibition of P-glycoprotein in the intestine.     

Surfactant-polymer nanoparticles overcome P-glycoprotein-mediated drug efflux

Nanoparticles enhance the therapeutic efficacy of an encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. We have previously demonstrated that Aerosol OT (AOT)-alginate nanoparticles, a novel formulation developed recently in our laboratory, significantly enhance the therapeutic efficacy of encapsulated drugs like doxorubicin in drug-sensitive tumor cells. The purpose of this study is to evaluate the drug delivery potential of AOT-alginate nanoparticles in drug-resistant cells overexpressing the drug efflux transporter, P-glycoprotein (P-gp). AOT-alginate nanoparticles were formulated using an emulsion-cross-linking process. Rhodamine 123 and doxorubicin were used as model P-gp substrates. Cytotoxicity of nanoparticle-encapsulated doxorubicin and kinetics of nanoparticle-mediated cellular drug delivery were evaluated in both drug-sensitive and -resistant cell lines. AOT-alginate nanoparticles enhanced the cytotoxicity of doxorubicin significantly in drug-resistant cells. The enhancement in cytotoxicity with nanoparticles was sustained over a period of 10 days. Uptake studies with rhodamine-loaded nanoparticles indicated that nanoparticles significantly increased the level of drug accumulation in resistant cells at nanoparticle doses higher than 200 microg/mL. Blank nanoparticles also improved rhodamine accumulation in drug-resistant cells in a dose-dependent manner. Nanoparticle-mediated enhancement in rhodamine accumulation was not because of membrane permeabilization. Fluorescence microscopy studies demonstrated that nanoparticle-encapsulated doxorubicin was predominantly localized in the perinuclear vesicles and to a lesser extent in the nucleus, whereas free doxorubicin accumulated mainly in peripheral endocytic vesicles. Inhibition of P-gp-mediated rhodamine efflux with AOT-alginate nanoparticles was confirmed in primary brain microvessel endothelial cells. In conclusion, an AOT-alginate nanoparticle system enhanced the cellular delivery and therapeutic efficacy of P-gp substrates in P-gp-overexpressing cells.     

A New Polymer–Lipid Hybrid Nanoparticle System Increases Cytotoxicity of Doxorubicin Against Multidrug-Resistant Human Breast Cancer Cells

Purpose This work is intended to develop and evaluate a new polymer–lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride (Dox) and enhance Dox toxicity against multidrug-resistant (MDR) cancer cells. Methods Cationic Dox was complexed with a new soybean-oil-based anionic polymer and dispersed together with a lipid in water to form Dox-loaded solid lipid nanoparticles (Dox–SLNs). Drug loading and release properties were measured spectrophotometrically. The in vitro cytotoxicity of Dox–SLN and the excipients in an MDR human breast cancer cell line (MDA435/LCC6/MDR1) and its wild-type line were evaluated by trypan blue exclusion and clonogenic assays. Cellular uptake and retention of Dox were determined with a microplate fluorometer. Results Dox–SLNs were prepared with a drug encapsulation efficiency of 60–80% and a particle size range of 80–350 nm. About 50% of the loaded drug was released in the first few hours and an additional 10–20% in 2 weeks. Treatment of the MDR cells with Dox–SLN resulted in over 8-fold increase in cell kill when compared to Dox solution treatment at equivalent doses. The blank SLN and the excipients exhibited little cytotoxicity. The biological activity of the released Dox remained unchanged from fresh, free Dox. Cellular Dox uptake and retention by the MDR cells were both significantly enhanced (p < 0.05) when Dox was delivered in Dox–SLN form. Conclusions The new polymer–lipid hybrid nanoparticle system is effective for delivery of Dox and enhances its efficacy against MDR breast cancer cells.     

Ability of Doxorubicin-Loaded Nanoparticles to Overcome Multidrug Resistance of Tumor Cells After Their Capture by Macrophages

Purpose. Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. Methods. The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. Results. Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. Conclusions. Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/ Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantagein vivo by confining both drugs in the same compartment, hence reducing the adverse effects.     

Increase in doxorubicin cytotoxicity by inhibition of P-glycoprotein activity with lomerizine

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR (multidrug resistance)1 gene encoding the transmembrane efflux pump, P-glycoprotein (P-gp). Calcium channel blockers such as verapamil, nifedipine and nicardipine have been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether a new calcium channel blocker, lomerizine, influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil, and even more markedly, lomerizine, increased cellular uptake of calcein transported by P-gp in a P-gp-expressing erythroleukemia cell line, K562-Dox. Ten microM of lomerizine reduced the IC50 of doxorubicin in the K562-Dox from 60000 ng/ml to 800 ng/ml, whereas the IC50 of doxorubicin in the K562 subline was only marginally affected by these drugs. Lomerizine showed greater reduction in P-gp efflux than verapamil at an equimolar concentration. These results suggest that lomerizine has the clinical potential to reverse tumor MDR involving the efflux protein P-gp.     

Improved cytotoxicity and preserved level of cell death induced in colon cancer cells by doxorubicin after its conjugation with iron-oxide magnetic nanoparticles

A promising strategy for overcoming the problem of limited efficacy in antitumor drug delivery and in drug release is the use of a nanoparticle-conjugated drug. Doxorubicin (Dox) anticancer chemotherapeutics has been widely studied in this respect, because of severe cardiotoxic side effects. Here, we investigated the cytotoxic effects, the uptake process, the changes in cell cycle progression and the cell death processes in the presence of iron-oxide magnetic nanoparticles (Nps) and doxorubicin conjugates (Dox-Nps) in human colon HT29 cells. The amount of Dox participated in biological action of Dox-Nps was determined by cyclic voltammetry and thermogravimetric measurements. The cytotoxicity of Dox-Nps was shown to be two/three times higher than free Dox, whereas Nps alone did not inhibit cell proliferation. Dox-Nps penetrated cancer cells with higher efficacy than free Dox, what could be a consequence of Dox-Nps aggregation with proteins in culture medium and/or with cell surface. The treatment of HT29 cells with Dox-Nps and Dox at IC₅₀ concentration resulted in G2/M arrest followed by late apoptosis and necrosis. Summing up, the application of iron-oxide magnetic nanoparticles improved Dox-Nps cell penetration compared to free Dox and achieved the cellular response to Dox-Nps conjugates similar to that of Dox alone.     

Pegylated phosphotidylethanolamine inhibiting P-glycoprotein expression and enhancing retention of doxorubicin in MCF7/ADR cells

The failure of the clinical treatment of cancer patients is often attributed to drug resistance of the tumor to chemotherapeutic agents. P-glycoprotein (P-gp) contributes to drug resistance via adenosine 5'-triphosphate (ATP)-dependent drug efflux pumps and is widely expressed in many human cancers. Up to date, a few of nanomaterials have shown the effects on P-gp function by different ways. To study the mechanism of the increased cytotoxicity of doxorubicin (DOX) by pegylated phosphotidylethanolamine (PEG-PE) in drug-resistant cancer cells, a series of in vitro cell assays were performed, including identification of P-gp function, quantitative studies on uptake and efflux of DOX, inhibitory effects of blank PEG-PE micelles on mRNA and protein levels of P-gp, and intracellular ATP content alteration. Finally, combining MDR-1 RNA interference (siRNA) with DOX encapsulated in PEG-PE micelles (M-DOX) to improve cytotoxicity of DOX was also studied. M-DOX showed fivefold lower the concentration that caused 50% killing tumor cell than that of free DOX in the P-gp-overexpressing MCF-7 breast cancer (MCF-7/ADR) cells. M-DOX enhanced the cellular uptake and retention of DOX in MCF-7/ADR cells. PEG-PE block molecules can inhibit P-gp expression through downregulating MDR-1 gene. Cytotoxicity of M-DOX was further improved by knocking down the MDR-1 gene using siRNA in the multidrug-resistant cells. We conclude that the increased cytotoxicity of DOX encapsulated in PEG-PE micelle is due to the reduced P-gp expression by PEG-PE block molecules, and accordingly enhancing the cellular accumulation of DOX. To overcome drug resistance of tumor cells, the combination of nanotechnology and biotechnology could be an effective strategy such as PEG-PE formed micelles and siRNA.     

Solid Lipid Nanoparticles for Potential Doxorubicin Delivery in Glioblastoma Treatment: Preliminary In Vitro Studies

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood–brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.     

一种新番荔枝内酯单体atemoyacin—B克服肿瘤多药抗药性

目的 探讨ａｔｅｍｏｙａｃｉｎ－Ｂ（Ａｔｅ）克服肿瘤多药抗药性（ＭＤＲ）作用及机制。方法 Ｂｕｌｌａｔａｃｉｎ（Ｂｕｌ）为阳性对照物,细胞毒测定以ＭＴＴ法,Ｐｇｐ功能测定以Ｆｕｒａ２－ＡＭ法,细胞内药物积累测定以荧光分光光度计法;细胞凋亡测定以流式细胞仪法,结果：Ａｔｅ对ＭＣＦ－７／Ｄｏｘ,ＭＣＦ－７,ＫＢＶ２００和ＫＢ细胞的ＩＣ５０分别为１２２,１２０,１．３４,１．２７ｍｍｏｌ．Ｌ＾－１,Ａｔ     

一种针对mdr1基因的寡核苷酸-阿霉素偶联物:对KB-A-1细胞的毒性及其抑制P-gp蛋白表达的效果

目的 :癌细胞膜上P gp糖蛋白的过量表达是肿瘤多药抗性的主要机制。人体内编码P gp糖蛋白的基因中仅有mdr1涉及多药抗性。本研究中设计了针对mdr1的反义核酸与阿霉素的偶联物 ,并且对其细胞毒性进行了考察。同时对偶联物对人表皮癌细胞株KB A 1内的P gp蛋白的表达也做了分子水平上的研究。方法 :使用MTT法考察偶联物对KB A 1细胞的毒性。用HPLC考察偶联物对细胞内阿霉素的积累量的影响。对于P gp蛋白表达的变化 ,主要是通过RT PCR及WesternBlot方法进行了研究。结果 :偶联物的细胞毒性比寡核苷酸高。在低剂量的偶联物 (0 .5 μmol·L-1)的作用下 ,细胞对阿霉素的敏感性提高。偶联物能有效的提高细胞内阿霉素的积累量。并且从RT PCR及WesternBlot看 ,偶联物处理后的细胞内P gp表达最少。结论 :选用合适的基团 ,对反义核酸进行结构修饰能够较好的增强反义核酸的性能。采用阿霉素作为偶联基团尽管增强了细胞毒性 ,但是在更大程度上增强了其抑制P gp蛋白的效力 ,提高了肿瘤耐药性的逆转倍数 ,具有一定的潜在应用价值。     

An in vitro demonstration of overcoming drug resistance in SKOV3 TR and MCF7 ADR with targeted delivery of polymer pro-drug conjugates

Drug resistance is a common phenomenon that occurs in cancer chemotherapy. Delivery of chemotherapeutic agents as polymer pro-drug conjugates (PPDCs) pretargeted with bispecific antibodies could circumvent drug resistance in cancer cells. To demonstrate this approach to overcome drug resistance, Paclitaxel (Ptxl)-resistant SKOV3 TR human ovarian- and doxorubicin (Dox)-resistant MCF7 ADR human mammary-carcinoma cell lines were used. Pre-targeting over-expressed biotin or HER2/neu receptors on cancer cells was conducted by biotinylated anti-DTPA or anti-HER2/neu affibody – anti-DTPA Fab bispecific antibody complexes. The targeting PPDCs are either D-Dox-PGA or D-Ptxl-PGA. Cytotoxicity studies demonstrate that the pretargeted approach increases cytotoxicity of Ptxl or Dox in SKOV3 TR or MCF7 ADR resistant cell lines by 5.4 and 27 times, respectively. Epifluorescent microscopy – used to track internalization of D-Dox-PGA and Dox in MCF7 ADR cells – shows that the pretargeted delivery of D-Dox-PGA resulted in a 2- to 4-fold increase in intracellular Dox concentration relative to treatment with free Dox. The mechanism of internalization of PPDCs is consistent with endocytosis. Enhanced drug delivery and intracellular retention following pretargeted delivery of PPDCs resulted in greater tumor cell toxicity in the current in vitro studies.     

Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering thioxanthones

For many pathologies, there is a crescent effort to design multiple ligands that interact with a wide variety of targets. 1-Aminated thioxanthone derivatives were synthesized and assayed for their in vitro dual activity as antitumor agents and P-glycoprotein (P-gp) inhibitors. The approach was based on molecular hybridization of a thioxanthone scaffold, present in known antitumor drugs, and an amine, described as an important pharmacophoric feature for P-gp inhibition. A rational approach using homology modeling and docking was used, to select the molecules to be synthesized by conventional or microwave-assisted Ullmann C-N cross-coupling reaction. The obtained aminated thioxanthones were highly effective at inhibiting P-gp and/or causing growth inhibition in a chronic myelogenous leukemia cell line, K562. Six of the aminated thioxanthones had GI(50) values in the K562 cell line below 10 μM and 1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H-thioxanthen-9-one (37) had a GI(50) concentration (1.90 μM) 6-fold lower than doxorubicin (11.89 μM) in the K562Dox cell line. The best P-gp inhibitor found was 1-[2-(1H-benzimidazol-2-yl)ethanamine]-4-propoxy-9H-thioxanthen-9-one (45), which caused an accumulation rate of rhodamine-123 similar to that caused by verapamil in the K562Dox resistant cell line, and a decrease in ATP consumption by P-gp. At a concentration of 10 μM, compound 45 caused a decrease of 12.5-fold in the GI(50) value of doxorubicin in the K562Dox cell line, being 2-fold more potent than verapamil. From the overall results, the aminated thioxanthones represent a new class of P-gp inhibitors with improved efficacy in sensitizing a resistant P-gp overexpressing cell line (K562Dox) to doxorubicin.     

In-vitro toxicity of Ukrain against human Ewing tumor cell lines

Ukrain is advertised by the manufacturer as a drug for alternative cancer cures with high activity against progressive Ewing tumors. Using the MTT assay, we compared the cytotoxicity of Ukrain with the cytotoxicity of N,N',N'-triethylenethiophosphoramide (thioTEPA), Chelidonium majus L. alkaloids, doxorubicin, cyclophosphamide and etoposide against four human Ewing tumor cell lines. In addition, we studied the cytotoxicity of thioTEPA combined with C. majus L. alkaloids after 48, 72 and 96 h. All compounds reduced the growth of Ewing tumor cell lines in a dose-dependent manner. The concentrations that reduced cell growth by 50% ranged between 6.2 and 31.1 micromol/l for Ukrain, 1.9 and 26.1 micromol/l for C. majus L. extract, and 1.7 and 448 micromol/l for thioTEPA. The sensitivity profile of Ukrain was comparable to that of the C. majus L. alkaloids, and different from that of thioTEPA, cyclophosphamide, etoposide and doxorubicin. Overall, doxorubicin was the most cytotoxic drug followed by cyclophosphamide. Ukrain and the C. majus L. alkaloids were slightly more cytotoxic than etoposide, while thioTEPA showed the lowest cytotoxicity. Co-exposure of thioTEPA with C. majus L. alkaloids resulted in additive but not in synergistic cytotoxicity. The in-vitro results indicate that the cytotoxicity of Ukrain against Ewing tumors is comparable to that of etoposide. While the latter can be used on the basis of broad clinical experience and known risk-benefit ratio, Ukrain for the present might be considered as a candidate for subsequent drug development by xenograft studies followed by systematic clinical trials.     

New uses for old drugs: pharmacophore-based screening for the discovery of P-glycoprotein inhibitors

P-glycoprotein (P-gp) is one of the best characterized transporters responsible for the multidrug resistance phenotype exhibited by cancer cells. Therefore, there is widespread interest in elucidating whether existing drugs are candidate P-gp substrates or inhibitors. With this aim, a pharmacophore model was created based on known P-gp inhibitors and it was used to screen a database of existing drugs. The P-gp modulatory activity of the best hits was evaluated by several methods such as the rhodamine-123 accumulation assay using K562Dox cell line, and a P-gp ATPase activity assay. The ability of these compounds to enhance the cytotoxicity of doxorubicin was assessed with the sulphorhodamine-B assay. Of the 21 hit compounds selected in silico, 12 were found to significantly increase the intracellular accumulation of Rhodamine-123, a P-gp substrate. In addition, amoxapine and loxapine, two tetracyclic antidepressant drugs, were discovered to be potent non-competitive inhibitors of P-gp, causing a 3.5-fold decrease in the doxorubicin GI(50) in K562Dox cell line. The overall results provide important clues for the non-label use of known drugs as inhibitors of P-gp. Potent inhibitors with a dibenzoxazepine scaffold emerged from this study and they will be further investigated in order to develop new P-gp inhibitors.     

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.