Proteomic analysis of proteins responsible for the development of doxorubicin resistance in human uterine cancer cells

Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.     

Synthesis and anticancer evaluation of vitamin K(3) analogues

Novel vitamin K(3) analogues were synthesized and evaluated for their anticancer activity. Compound 6, 9, 10, 11, 14, and (+/-)15 demonstrated a strong inhibitory activity against the tumor cells of A-549, Hep G2, MCF7, MES-SA, MES-SA/Dx5, MKN45, SW-480, and TW-039. Compound (+/-)15 displayed potent tumor cell cytotoxicity, and compound 14 selectively affected MCF7, even though it did not influence normal cells Detroit551 and WI-38. Compound (+/-)15 inhibited MES-SA and MES-SA/Dx5, and this specific result shows that compound (+/-)15 may become a good anticancer drug candidate.     

Quinoline derivative KB3-1 potentiates paclitaxel induced cytotoxicity and cycle arrest via multidrug resistance reversal in MES-SA/DX5 cancer cells

AIMS: The resistance to chemotherapeutic drugs is a major problem for successful cancer treatment. Multidrug resistance (MDR) phenotype is characterized by over-expression of P-glycoprotein (P-gp) on the cancer cell plasma membrane that extrudes drugs out of the cells. Therefore, novel MDR reversal agents are desirable for combination therapy to reduce MDR and enhance anti-tumor activity. Thus, the present study was aimed to evaluate the potent efficacy of novel quinoline derivative KB3-1 as a potent MDR-reversing agent for combined therapy with TAX. MAIN METHODS: MDR reversing effect and TAX combined therapy were examined by Rhodamine accumulation and efflux assay and Confocal immunofluorescence microscopy, Western blotting, TUNEL assay, and cell cycle analysis. KEY FINDINGS: The discovery of quinoline-3-carboxylic acid [4-(2-[benzyl-3[-(3,4-dimethoxy-phenyl)-propionyl]-amino]-ethyl)-phenyl]-amide (KB3-1) as a novel MDR-reversal agent. KB3-1 significantly enhanced the accumulation and retention of a P-gp substrate, rhodamine-123 in the P-gp-expressing MES-SA/DX5 uterine sarcoma cells but not in the P-gp-negative MES-SA cells at non-toxic concentrations of 1 microM and 3 microM. Similarly, fluorescence microscopy observation revealed that KB3-1 reduced the effluxed rhodamine-123 expression on the membrane of MES-SA/DX5 cells. Consistent with decreased P-gp pumping activity, confocal microscopic observation revealed that KB3-1 effectively diminished the expression of P-gp in paclitaxel (TAX)-treated MES-SA/DX-5 cells. Furthermore, Western blotting confirmed that KB3-1 reduced P-gp expression and enhanced cytochrome C release and Bax expression in TAX treated MES-SA/DX-5 cells. In addition, KB3-1 enhanced TAX-induced apoptotic bodies in MES-SA/DX5 cells by TdT-mediated-dUTP nick-end labeling (TUNEL) staining assay aswell as potentiated TAX- induced cytotoxicity, G2/M phase arrest and sub-G1 apoptosis in MES-SA/DX5 cells but not in MES-SA cells. Interestingly, KB3-1 at 3 microM had comparable MDR-reversal activity to 10 microM verapamil, a well-known MDR- reversal agent. SIGNIFICANCE: KB3-1 can be a MDR-reversal drug candidate for combination chemotherapy with TAX.     

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.     

Synthesis and cytotoxic activity of a new potent daunomycinone derivative

The preparation and cytotoxic activity of 4′-azido-3′-bromo-3′-deamino-4′-deoxydaunorubicin is described. The new compound was found to be less active in vitro than adriamycin against L1210 and the sensitive cell lines KB-3-1 and MES-SA, but retained interesting cytotoxicity against the adriamycin resistant subline KB-A1 and the multidrug resistant MES-SA/Dx5 subline.     

Design and synthesis of novel amino-substituted xanthenones and benzo[b]xanthenones: evaluation of their antiproliferative activity and their ability to overcome multidrug resistance toward MES-SA/D x 5 cells

A number of new xanthenone and benzo[b]xanthenone amino derivatives and their pyrazole-fused counterparts have been designed and synthesized possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine. The synthesis of the compounds proceeds through nucleophilic substitution of 1-chloro-4-nitroxanthenone or the corresponding benzo[b]xanthenone by the appropriately substituted amine or hydrazine, reduction of the nitro group, and conversion into the suitable dialkylaminoacetamides. This method cannot be applied for synthesis of the pyrazole-fused benzo[b]xanthenones, consequently a different, simple, and high-yielding synthetic procedure was developed for the preparation of the target molecules. In vitro cytotoxic potencies of the new derivatives toward the murine leukemia L1210 cell line, human colorectal adenocarcinoma (HT-29), and human uterine sarcoma (MES-SA and its 100-fold resistant to doxorubicin variant MES-SA/D x 5) cell lines are described and compared to those of reference drugs. The compounds exhibited significant cytotoxic activity against the tested cell lines and in addition they retain activity against the multidrug resistant MES-SA/D x 5 subline, showing resistant factors close to 1. A number of derivatives were found to possess DNA binding capacity, according to a standard ethidium bromide displacement assay. The majority of the studied compounds induce a G2/M arrest, although among them some G1 or S blockers have also been identified.     

Design, synthesis and antiproliferative activity of novel aminosubstituted benzothiopyranoisoindoles

The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their in vitro cytotoxic activity evaluation against HCT-116, MES-SA, and MES-SA/Dx cancer cell lines is reported. The majority of the derivatives possessed noticeable cytotoxicity in a low μM range indicating an interesting structure-activity relationship.     

Synthesis and anticancer evaluation of bis(benzimidazoles), bis(benzoxazoles), and benzothiazoles

Four classes of UK-1 analogues were synthesized and their cytotoxicity testing against human A-549, BFTC-905, RD, MES-SA, and HeLa carcinoma cell lines was determined. The results revealed that UK-1 and four of these analogues (15-18) are potent against the cancer cell lines. In particular, compound 16 is more potent than UK-1 against A-549 and HeLa cell lines, and compounds 15, 17, and 18 selectively exhibit potent cytotoxic activity against the BFTV-905 cells (IC50 9.6 microM), A-549 cells (IC50 6.6 microM), and MES-SA cells (IC50 9.2 microM), respectively.     

Lysosomal accumulation of drugs in drug-sensitive MES-SA but not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells

Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR. Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation. Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9. However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment. While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH. We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation. These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR.     

The effect of the plasticizer diethylhexyl phthalate on transport activity and expression of P-glycoprotein in parental and doxo-resistant human sarcoma cell lines

Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 μM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 μM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 μM doxo in the presence of the lowest concentration of DEHP (3 μM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 μM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 μM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.     

Ultrasound-Induced New Cellular Mechanism Involved in Drug Resistance

The acoustic effects in a biological milieu offer several scenarios for the reversal of multidrug resistance. In this study, we have observed higher sensitivity of doxorubicin-resistant uterine sarcoma MES-SA/DX5 cells to ultrasound exposure compared to its parent counterpart MES-SA cells; however, the results showed that the acoustic irradiation was genotoxic and could promote neotic division in exposed cells that was more pronounced in the resistant variant. The neotic progeny, imaged microscopically 24 hr post sonication, could contribute in modulating the final cell survival when an apoptotic dose of doxorubicin was combined with ultrasound applied either simultaneously or sequentially in dual-treatment protocols. Depending on the time and order of application of ultrasound and doxorubicin in combination treatments, there was either desensitization of the parent cells or sensitization of the resistant cells to doxorubicin action.     

Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells

The multidrug-resistant cancer cell lines NCI/AdR(RES) and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.     

Design, synthesis, and antiproliferative activity of some novel aminosubstituted xanthenones, able to overcome multidrug resistance toward MES-SA/Dx5 cells

A series of novel xanthenone aminoderivatives and their pyrazole-fused counterparts possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine (PZA) reported. These compounds exhibited an interesting cytotoxic activity against a panel of cell lines. Most noticeably, they retain activity against the multidrug resistant MES-SA/Dx5 subline, showing resistant factors close to 1.     

Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5

The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans--one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing beta(1-4)-N-acetyl-D-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galbeta(1-4)-GlcNAc(x) component found in other Pgps.     

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability – Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) – to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.     

Effects of proteolysis of the extending parts of the high-molecular-weight microtubule-associated proteins on interactions between microtubules

Digestion of assembled microtubules with agarose-bound trypsin was performed to obtain microtubules which lack the extending projections, the non-tubulin-binding part of the high-molecular-weight microtubule-associated proteins. The assembly kinetics and the minimum protein concentration for assembly were the same for these trypsinated microtubules as for normal, untreated microtubules. Furthermore, the digested microtubules gave rise to the same change in turbidity per polymer mass as that found for normal microtubules. However, electron microscopy of pelleted microtubules revealed a closer packing after trypsin treatment. A substantially lower increase in specific viscosity was found upon assembly. At concentrations of above approx. 1.5 mg/ml, the viscosity of trypsin-treated microtubules was almost independent of the protein concentration, in contrast to the turbidity, which still increased. Both microtubules and the trypsin-digested microtubules were easily oriented by shear, although the flow linear dichroism signal for the microtubules after trypsin treatment was only half of that found for perfectly oriented normal microtubules. At higher shear force gradients, digested microtubules aggregated side by side as shown by electron microscopy. This was not found for normal microtubules. Even although the extending parts of the high-molecular-weight proteins are not needed for assembly, they were found to play an important role in microtubule orientation and interactions between microtubules, probably by acting as spacers between microtubules.     

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.     

Binding of adenovirus to microtubules. II. Depletion of high-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding.

A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Characterisation of uterine sarcoma cell lines exhibiting MDR phenotype by vibrational spectroscopy

Multidrug resistance (MDR) enables cancer cells to escape cytotoxic insults of anticancer drugs. Rapid identification of cells exhibiting the MDR phenotype is very important since it can lead to an effective and individual patient based treatment plan. We have investigated a combined vibrational spectroscopic approach, using both micro-Raman and FTIR techniques, in order to characterise a sensitive human uterine sarcoma cell line MES-SA and its multidrug-resistant derivative Garf. In this study, these two complementary methods have been evaluated via the use of principal components analysis (PCA), for discrimination of cells exhibiting the MDR phenotype. Our results indicate that, though they inherently have different sensitivities, both Raman and IR methods can provide a good differentiation of cell phenotypes.