Vincristine-resistant Sublines Research Articles

Selective Cytotoxic Effects of 5-Trifluoromethoxy-1H-indole-2,3-dione 3-Thiosemicarbazone Derivatives on Lymphoid-originated Cells.

The present study aims to identify the anticancer effect of novel 1H-indole-2,3-dione 3- thiosemicarbazone derivatives. These compounds could be promising anticancer agents in leukemia treatment. Conventional chemotherapeutic agents accumulate in both normal and tumor cells due to nonspecificity. For effective cancer treatment, new drugs need to be developed to make chemotherapeutics selective for cancer cells. The ultimate goal of cancer treatment is to reduce systemic toxicity and improve the quality of life. In this study, the anticancer effects of 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives (A-L) were investigated in chronic myelogenous leukemia K562, Burkitt's lymphoma P3HR1, acute promyelocytic leukemia HL60 cells, and vincristine-resistant sublines of K562 and P3HR1 cells. Additionally, the compounds were tested on lymphoid-derived cells from ALL patients. In order to investigate the particular mechanism of death caused by the cytotoxic effects of the compounds, immunohistochemical caspase 3 staining was performed in P3HR1 cells, and the resulting apoptotic activities were demonstrated. All tested compounds have been found to have cytotoxic effects against lymphoma cells at submicromolar concentrations (IC50= 0.89-1.80 μM). Most compounds show significant selectivity for the P3HR1 and P3HR1 Vin resistance. The most effective and selective compound is 4-bromophenyl substituted compound I (IC50=0.96 and 0.89 μM). Cyclohexyl and benzyl substituted compounds D and E have also been found to have cytotoxic effects against K562 cell lines (IC50=2.38 μM), while the allyl substituted compound C is effective on all cell lines (IC50=1.13-2.21 μM). 4-Fluorophenyl substituted F compound has been observed to be effective on all cells (IC50=1.00-2.41 μM) except K562 cell. Compound C is the only compound that shows inhibition of HL-60 cells (IC50= 1.13 μM). Additionally, all compounds exhibited cytotoxic effects on lymphoidderived cells at 1μM concentration. These results are in accordance with the results obtained in lymphoma cells. All compounds tested have submicromolar concentrations of cytotoxic effects on cells. These compounds hold potential for use in future treatments of leukemia.

556 - Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status

The PKCβ inhibitor enzastaurin was tested in parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Enzastaurin IC50s ranged from 3.3 to 9.5 μM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125 μM interfered with ABCB1-mediated drug transport. PKCα and PKCβ may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ≥ 1.25 μM. The investigated cell lines did not express PKCβ. PKCα depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates.

Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status.

The PKCβ inhibitor enzastaurin was tested in parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Enzastaurin IC50s ranged from 3.3 to 9.5 μM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125 μM interfered with ABCB1-mediated drug transport. PKCα and PKCβ may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ≥ 1.25 μM. The investigated cell lines did not express PKCβ. PKCα depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates.

Different types of cell cycle- and apoptosis-related gene expressions alter in corticosteroid-, vincristine-, and melphalan-resistant u-266 multiple myeloma cell lines.

Objective: Deregulation of the cell cycle and apoptosis mechanisms in normal cells causes many problems, including cancer. In this study, a genome-wide expression analysis of cell cycle- and apoptosis-related genes in corticosteroid-, vincristine-, and melphalan-resistant U-266 multiple myeloma cell lines was conducted. Materials and Methods: Resistant U-266 sublines were induced by application of each drug by stepwise dose increments. Resistance gained by the cells was confirmed with XTT cytotoxicity assay and microarray analyses were carried out. Among the cell cycle- and apoptosis-related gene expressions, alterations of more than 2-fold were considered significant. Results: Cyclin E2 was drastically overexpressed in the vincristine-resistant subline and a general upregulation was observed for various cyclin-dependent kinases. Some of the cyclin-dependent kinase inhibitor encoding genes were downregulated in resistant sublines in general. Tumor necrosis factor receptor genes were generally downregulated in corticosteroid- and melphalan-resistant U-266 sublines. Different types of effector caspases were downregulated in all resistant sublines. Ceramide metabolism genes seemed to be changed in favor of survival, especially in the melphalan-resistant subline. Conclusion: This report shows that different types of chemotherapeutic drugs alter different apoptotic and cell cycle-related gene expressions and, as a result, may cause drug-resistant phenotypes in U-266 multiple myeloma cell lines. Among those gene expressions, the most drastic increase in cyclin E2 could be important for the survival of vincristine-resistant U-266 cell lines, whereas alteration of ceramide metabolism genes could be important in melphalan resistance.

Antitumor agents 292. Design, synthesis and pharmacological study of S- and O-substituted 7-mercapto- or hydroxy-coumarins and chromones as potent cytotoxic agents

Thirty-five S- and O-substituted 7-mercaptocoumarin (9–23) and 7-hydroxy- or 7-mercapto-chromone (24–43) analogs were designed, synthesized and evaluated in vitro against four human tumor cell lines [KB (nasopharyngeal), KB-vin (vincristine-resistant subline), A549 (lung) and DU145 (prostate)] with paclitaxel as the positive control. Many of the synthesized compounds exhibited potent cytotoxic activity. Among them, compounds 10 and 18 showed broad spectrum activity with GI50 values ranging from 0.92 to 2.11 μM and 2.06–14.07 μM, respectively. However, 33, a 3-brominated compound, displayed significant and selective inhibition against MDR KB-vin with a GI50 of 5.84 μM. Regardless of the size of the 7-alkoxy group, 2-α-bromoethyl-8-bromomethyl compounds (40–43) exhibited increased cytotoxicity compared with 2-ethyl-8-bromomethyl compounds (36–39). Moreover, in a preliminary pharmacological study, 10 not only remarkably increased cellular apoptosis in a concentration-dependent manner, but also clearly induced A549 cell cycle arrest at the G2/M phase. Thus, these coumarin derivatives merit investigation as novel potential antitumor agents with further structural modification to produce an optimal lead compound and elucidate the detailed pharmacological mechanism(s).

Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.

Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF.

Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

Increased tubulin acetylation accompanies reversion to stable ploidy in vincristine-resistant CCRF-CEM cells

The T-cell leukemia line CCRF-CEM is unstable with respect to ploidy, whereas a vincristine-resistant subline, CEM/VCR R, maintains a stable pseudodiploid karyotype. Ploidy change in the parental cells requires the involvement of two cell cycle lesions. The first, in mitosis, prevents cell division after S-phase. The second, in G1, allows a cell with 4N DNA content to re-enter S-phase. We examined differences in expression of tubulin, a major component of the mitotic spindle and the cellular target for vincristine, between the two cell lines. Levels of the β III isotype were decreased and levels of acetylated α-tubulin, a marker for microtubule stability, were increased in the CEM/VCR R cells relative to the parental line, which suggests that the CEM/VCR R cells have a more stable mitotic spindle. Both cell lines exhibit some level of constitutive expression of p53 and c-myc. Constitutive expression of and mutant p53 would contribute to the failure of these cells to recognise G1 checkpoints. Therefore, G1 checkpoint failure and the intrinsically less stable mitotic spindle in the CCRF-CEM cells may contribute to the observed ploidy instability. Conversely, the presence of markers of microtubule stability in the CEM/VCR R cells would predispose them to maintain their ploidy.

Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells.

Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed.

Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells.

We have isolated from Chinese hamster ovary cells, 30 sublines resistant to vincristine, doxorubicin or etoposide and 43 sublines evading treatment with a pair of these drugs. Isolated in one step and under low selective pressure, sublines were 3- to 25-fold more resistant to their selecting drug(s) than the parental cells. Possible P-glycoprotein-associated multidrug resistance was investigated through pgp gene copy number and mRNA expression level. DNA topoisomerase II alteration was evaluated from the ability of nuclear extracts to form cleavable complexes. Vincristine (all sublines) and doxorubicin (6/7 sublines) preferentially selected for pgp gene amplification and mRNA overexpression, whereas selection with etoposide resulted in a decrease of cleavable complex formation in 11 out of 13 sublines. A common pgp gene-mediated resistance was found in the 13 doxorubicin plus vincristine-selected sublines, whereas all but one of the 12 etoposide plus vincristine-resistant sublines displayed both pgp mRNA overexpression and decreased ability to form cleavable complexes. Among the 18 doxorubicin plus etoposide selected sublines, five exhibited a decreased ability to form cleavable complexes only, six exhibited pgp mRNA overexpression only and six exhibited both alterations. Overall, drug resistance could not be attributed to either mechanism in three of the 73 sublines. We conclude that under low selective pressure it is possible to find a combination of drugs which require simultaneous selection of more than one resistance mechanism; such cells emerge with very low frequency.

Cellular control of human multidrug resistance 1 (mdr-1) gene expression in absence and presence of gene amplification in human cancer cells.

The Kst-6 cell line is a human KB carcinoma cell line that has a stably integrated chloramphenicol acetyl-transferase (CAT) reporter gene under the control of the human mdr-1 promoter. Using Kst-6 cells as the parental cell line, five vincristine-resistant sublines, designated Kst-V5, -V25, -V50, -V75, and -V100, were isolated by exposure to increasing concentrations of drug. These sublines showed increased resistance to vincristine when compared with parental Kst-6 cells. Two sublines, V25-1 and V25-2, were further isolated from Kst-V25 after culture in the presence of vincristine, and V100-1 and V100-2 were also isolated from Kst-V100. Southern analysis demonstrated mdr-1 gene amplification in Kst-50, Kst-V75, Kst-V100, V100-1, and V100-2 cells, respectively, but not in Kst-V5, Kst-V25, V25-1, and V25-2 cells. In contrast, increased mdr-1 expression was documented by Northern analysis in Kst-V25, V25-1, and V25-2 cells and five cell lines with mdr-1 amplification. Southern blot analysis utilizing a CAT probe demonstrated a stable copy number in all vincristine-resistant sublines. However, Northern analysis documented increased CAT expression in Kst-V5, Kst-V25, V25-1, and V25-2 cells but reduced mRNA levels in the cell line with amplification of the endogenous mdr-1 gene. Expression of the CAT gene was increased along with the endogenous mdr-1 gene in the early steps of the selection but decreased with the onset of gene amplification. There appeared almost similar mRNA levels of two trans-acting factor genes, SP-1 and MDR-NF1/YB-1, which are supposed to be involved in mdr-1 gene promoter activation, among all cell lines used in this study. These findings suggest that transcription of both the CAT gene fused to the mdr-1 promoter and the endogenous mdr-1 gene is enhanced through activation by trans-acting factors in the early steps of drug selection. However, the quantity of trans-activating factor is limiting, and with the onset of gene amplification, less is available for activation of the CAT gene, resulting in decreased expression.

Antigenic properties associated with vinca alkaloid resistance in ovarian cancer cells: identification of a 92,000 DA protein

In order to characterize the membrane changes related to Vinca alkaloid resistance, we raised monoclonal antibodies (mAbs) against a Vincristine resistant subline (OV1/VCR) derived from a human ovarian adenocarcinoma cell line (OV1/p). Among three monoclonal antibodies selected for a higher binding to OV1/VCR than to OV1/p cells, one designated OVR09, recognized a Mr 92,000 protein. This protein appears to be gradually overexpressed along the drug resistance establishment in vitro, and to decrease slowly in absence of drug. Further, mAb OVR09 showed a much higher binding to the vinblastine resistant epidermoid tumor cell line KbV1 than to its parental counterpart. The Mr 92,000 protein was also detected in various tumor cell lines and in an ovarian carcinoma surgical sample.

Drug-related chromosomal changes in chemoresistant human ovarian carcinoma cells

Four resistant sublines were derived from the sensitive human ovarian carcinoma IGROV 1 (OV1-P) cell line by exposure to increasing concentrations of vincristine, doxorubicin, and cisplatinum. The vincristine-resistant sublines expressed the MDR phenotype associated with a complete reversion of malignant properties. Cytogenetic studies of sensitive and resistant cells have been repeatedly performed over a 1-year period. Consistent and stable drug-related chromosomal structural rearrangements have been observed in each resistant population affecting chromosomes 3, 4, 6, 8, 11, 22, and X. The most significant result was the presence in OV1/P cells of a minor subclone with a del(11)(p13) marker that represented the whole OV1/VCR population. This result suggests a possible role of this deletion either in the drug-selection process, or in the malignant reversion.

Lectin staining patterns of plasma membranes of daunorubicin and vincristine resistant Ehrlich ascites tumour cells

The plasma membrane is considered to play a major role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). Previous studies have reported an increase in plasma membrane carbohydrates in pleiotropic resistant cells compared with wild-type cells. The present study has utilized a panel of 11 lectins and the streptavidin-biotin histochemical technique in order to compare plasma membrane carbohydrates from wild-type Ehrlich ascites tumour cells with cells from daunorubicin and vincristine resistant sublines. While the lectins ConA, LCA, PSA, PNA after neuraminidase and WGA stained plasma membranes of daunorubicin-resistant cells to a significantly greater degree than those of wild-type cells, no difference was apparent between vincristine-resistant and wild-type cells. PWM and WGA after neuraminidase pretreatment showed similar staining of the wild-type and both resistant sublines, while SBA with and without neuraminidase pretreatment, HPA, DBA, LTA and UEA I demonstrated either very weak or negative reactions with all sublines. We conclude that the observed increase in plasma membrane carbohydrate found in anthracycline-resistant cells is possibly due to drug action during acquisition and maintainance of resistance, and, though conceivably of importance in the development of resistance towards anthracyclines, is without significance for the pleiotropic resistance phenotype itself.

Cross-resistance of vincristine-resistant sublines of P388 leukemia to mitoxantrone with special emphasis on the relationship between in vitro and in vivo cross-resistance.

In vitro and in vivo cross-resistance to mitoxantrone of two vincristine-resistant sublines of P388 leukemia with different degrees of resistance in vitro was compared with that to adriamycin. A subline with a lower degree of resistance to vincristine exhibited approximately the same responses in vivo to mitoxantrone and adriamycin as the original P388 leukemic cell line, although it was evidently cross-resistant in vitro to these agents. Another subline having a higher degree of resistance to vincristine showed a 25-fold cross-resistance in vitro and gave no response in vivo to adriamycin. With mitoxantrone, on the other hand, this subline was solidly resistant as compared with the sensitive line but still retained significant responsiveness in vivo irrespective of a 150-fold cross-resistance in vitro. These results suggest that cross-resistance on a cellular basis does not necessarily correspond to in vivo cross-resistance. The relationship between in vitro and in vivo cross-resistance is discussed.