Drug-sensitive Cancer Cells Research Articles

Association of genomic imbalances with drug resistance and thermoresistance in human gastric carcinoma cells.

Therapy resistance is the major obstacle to advances in successful cancer treatment. To characterize chromosomal alterations associated with different types of acquired MDR and thermoresistance, we applied CGH to compare a unique panel of human gastric carcinoma cells consisting of the parental, drug-sensitive and thermosensitive cancer cell line EPG85-257P, the atypical MDR variant EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. CGH with genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included various members of the families of ABC transporters and molecular chaperones. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.

Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene

The breast cancer resistance protein (BCRP) gene, formally known as ATP-binding cassette transporter G2 (ABCG2) gene, encodes an ABC half transporter that causes resistance to certain cancer chemotherapeutic drugs when transfected and expressed in drug sensitive cancer cells. Here we report the organization of the BCRP gene, and the initial characterization of the BCRP promoter. We identified the genomic sequence of BCRP and its promoter by screening a human genomic lambda phage library, as well as a BAC library, and by searching the human genome database. The BCRP gene spans over 66 kb and consists of 16 exons and 15 introns. The exons range in size from 60 to 532 bp. The translational start site is found in the second exon. The first exon contains the majority of the 5′ UTR. Promoter activity was characterized by a luciferase reporter assay using transient transfection of the human breast cancer cell line MCF7, and the human choriocarcinoma cell lines JAR, BeWo and JEG-3, which we find to have high endogenous expression of BCRP. The BCRP gene is transcribed by a TATA-less promoter with several putative Sp1 sites, which are downstream from a putative CpG island. The sequence 312 bp directly upstream from the BCRP transcriptional start site conferred basal promoter activity. The 5′ region upstream of the basal promoter is characterized by both positive and negative regulatory domains.

Leptin and high glucose induces cell proliferation in drug sensitive human breast cancer cells (MCF7) but not in multidrug-resistant cells (MCF7/ADR): Possible involvement of the changes of PKC-α expression treated with glucose and leptin

Leptin and high glucose induces cell proliferation in drug sensitive human breast cancer cells (MCF7) but not in multidrug-resistant cells (MCF7/ADR): Possible involvement of the changes of PKC-α expression treated with glucose and leptin

Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.

Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Effect of γ-linolenic acid on cellular uptake of structurally related anthracyclines in human drug sensitive and multidrug resistant bladder and breast cancer cell lines

This study investigated the effect on drug uptake in multidrug resistant cells by the incorporation of the essential fatty acid γ-linolenic acid (GLA). The cell lines used were the MCF-7/R resistant human breast cancer and MGH-U1/R bladder cancer. Uptake of drug (doxorubicin, epirubicin, mitoxantrone and idarubicin) after the incorporation of GLA was investigated quantitatively by flow cytometry and qualitatively by confocal microscopy. There was no observable overall increase in drug uptake due to GLA incorporation into the cells as shown by flow cytometry. However, an increase in uptake of the chemotherapeutic agent idarubicin was observed in GLA-treated resistant cells compared with untreated cells using the confocal microscope. This overall increase in cellular drug uptake was not accompanied by a change in cellular drug distribution. Only one drug, mitoxantrone, displayed a change in intracellular drug distribution due to GLA incorporation into MCF-7/R cells. This suggests that essential fatty acid incorporation into the cellular membranes of some resistant cells may cause a shift in the intracellular distribution of certain chemotherapeutic drugs.

A shift from normal to high glucose levels stimulates cell proliferation in drug sensitive MCF-7 human breast cancer cells but not in multidrug resistant MCF-7/ADR cells which overproduce PKC-?II

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)-betaII in regulating MCF-7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC-betaII-specific inhibitor (CGP53353) were examined in cultures of MCF-7 human breast cancer cell line and its multidrug resistant variant (MCF-7/ADR). Cell proliferation and DNA synthesis of MCF-7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF-7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC-betaII protein and the corresponding mRNA levels were 4- to 5-fold higher in MCF-7/ADR than in MCF-7 cells. High glucose-induced decreases of PKC-betaII protein and mRNA levels were observed during the DNA synthesis phase in MCF-7 but not in MCF-7/ADR cells. Decreases in PKC-betaII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose-stimulated proliferation of MCF-7 cells. Cultures of multidrug resistant MCF-7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose-induced PKC-betaII isozyme down-regulation with concomitant acceleration of cell cycle progression in MCF-7 cells.

Physiologic and Pathologic Drug Resistance in Ovarian Carcinoma: A Hypothesis Based on a Clonal Progression Model

Conventional models for successful chemotherapy suggest that early-stage ovarian cancer should be more responsive to cytotoxic drugs than advanced disease, that multiple drugs will prove more efficacious than single agents and that more intensive chemotherapy will prove superior to conventional doses. The purpose of numerous clinical studies has been to test these predictions and the results often fall short of the expectations. In trials to date, adjuvant chemotherapy for high-risk, early-stage ovarian cancer has provided only a modest, equivocal increase in disease-free survival. Combination chemotherapy produces higher response rates but this has not consistently resulted in prolonged survival. Dose intensification with high-dose chemotherapy increases rate of response further, but most responses are of short duration. The objective of this review is to integrate clinical and molecular biological observations into a novel model of drug resistance. We hypothesize that drug sensitivity is an acquired characteristic of neoplastic cells, and that there are two broad cellular forms of resistance to cytotoxic drugs. 'Physiological drug resistance' is a cellular state when drug sensitivity of cancer cells is similar to that of the corresponding normal tissue. This form of drug resistance can precede the acquisition of drug sensitivity and may be predominant in the early stages of neoplastic progression. A distinct, 'pathological drug resistance' can reappear later during tumor progression or during chemotherapy as a result of increased detoxification, upregulation of repair pathways or defective apoptosis. Tumors are formed of heterogeneous cell populations and in terms of drug sensitivity three distinct cell types may be present: drug-sensitive, physiologically drug-resistant and pathologically drug-resistant. Clinical response to chemotherapy is determined by the relative contribution of these different cell populations to the total cellular mass. Depending on the predominant type of surviving population, distinct patterns of clinical failure may develop that require different treatment strategies for optimal management. For chemosensitive cells that survived insufficient chemotherapy, consolidation with further, perhaps, high-dose chemotherapy is a rational option. For pathologically drug-resistant cells, pharmacological manipulation of drug resistance, and signaling pathways in combination with chemotherapy could be exploited. For physiologically drug-resistant cells, non-chemotherapy-based, 'chemopreventive' strategies to arrest tumor progression may prove beneficial.

Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-alpha (PKC-alpha) pseudosubstrate peptides through a P-glycoprotein-independent mechanism.

Phorbol ester protein kinase C (PKC) activators and PKC isozyme over-expression have been shown to significantly reduce intracellular accumulation of chemotherapeutic drugs, in association with the induction of multidrug resistance (MDR) in drug-sensitive cancer cells and enhancement of drug resistance in MDR cancer cells. These observations constitute solid evidence that PKC plays a significant role in the MDR phenotype of cancer cells. PKC-catalyzed phosphorylation of the drug-efflux pump P-glycoprotein was recently ruled out as a contributing factor in MDR. At present, the sole drug transport-related event that has been identified as a component of the role of PKC in MDR is PKC-induced expression of the P-glycoprotein-encoding gene mdr1. The objective of this study was to test the hypothesis that PKC can modulate the uptake of chemotherapeutic drugs in cancer cells independently of P-glycoprotein. We analyzed the effects of selective PKC activators/inhibitors on the uptake of radiolabelled cytotoxic drugs by cultured human colon cancer cells that lacked P-glycoprotein activity and did not express the drug efflux pump at the level of message (mdr1) or protein. We found that the selective PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly reduced uptake of [14C] Adriamycin and [3H] vincristine in human colon cancer cells devoid of P-glycoprotein activity, and that PKC-inhibitory N-myristoylated PKC-alpha pseudosubstrate synthetic peptides potently and selectively induced uptake of the cytotoxic drugs in the phorbol ester-treated and non-treated colon cancer cells. TPA treatment of the cells did not induce expression of either P-glycoprotein or its message mdr1. In contrast with [14C]Adriamycin and [3H] vincristine uptake, [3H] 5-fluorouracil uptake by the cells was unaffected by TPA and reduced by the PKC-inhibitory peptides. These results indicate that PKC activation can significantly reduce the uptake of multiple cytotoxic drugs by cancer cells independently of P-glycoprotein, and that N-myristoylated PKC-alpha pseudosubstrate peptides potently and selectively induce uptake of multiple cytotoxic drugs in cultured human colon cancer cells by a novel mechanism that does not involve P-glycoprotein and may involve PKC isozyme inhibition. Thus, N-myristoylated PKC-alpha pseudosubstrate peptides may offer a basis for the development of agents that reverse intrinsic drug resistance in human colon cancer.

Increasing drug resistance in human lung cancer cells by mutant-type p53 gene mediated by retrovirus

Human mutant-type (mt) p53 cDNA was synthesized and cloned from human lung cancer cell line GL containing mt-p53 gene by using polymerase chain reaction (PCR). It was confirmed that the mt-p53 cDNA contained the complete coding sequence of p53 gene but mutated at codon 245 (G-->T) and resulted in glycine to cysteine by sequencing analysis. The retroviral vector pD53M of the mt-p53 was constructed and introduced into the drug-sensitive human lung cancer cells GAO in which p53 gene did not mutate. The transfected GAO cells strongly expressed mutant-type p53 protein by immunohistochemistry, showing that pD53M vector could steadily express in GAO cells. The drug resistance to several anticancer agents of GAO cells infected by pD53M increased in varying degrees, with the highest increase of 4-fold,in vitro andin vivo. By quantitative PCR and flow cytometry (FCM) analyses, the expression of MDR1 gene and the activity of P-glycoprotein (Pgp) did not increase, the expression of MRP gene and the activity of multidrug resistance-related protein (Mrp) increased slightly. These results indicated that the drug resistance associated with mt-p53 gene might be somewhat correlated with MRP/Mrp but not with MDR1/Pgp. It was possible to modify the tumor drug resistance by changing status of p53 gene.

Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography

Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography

Morphonuclear characterization of drug resistance by means of digital cell-image analysis: an in vitro assessment.

The prediction of tumor resistance to antineoplastic drugs remains an important challenge in cancer chemotherapy. Several methods have been proposed in this connection, but they present a number of problems such as clinical relevance and applicability. In the present work we put forward an original methodology to assess the drug sensitivity of cancer cells. For this purpose we submitted chemosensitive and chemoresistant cell lines to different anticancer drugs and monitored the cell growth and the drug-induced morphonuclear effects by means of digital cell-image analysis of Feulgen-stained nuclei. The results showed that drug-induced effects at the morphonuclear level correlated statistically with the effects produced at the cell proliferation level. For example, the mean nuclear size value increased as a function of the drugs' efficiency recorded at the cell proliferation level. In the same way, the frequency of large dense chromatin clumps also increased in accordance with the drugs' efficiency. The present work thus demonstrates that digital cell-image analysis can be applied to monitor the efficiency of chemotherapeutic treatment carried out on cell lines in vitro. The present methodology could possibly be used on solid tumors, from which biological material can be obtained serially by means of fine-needle aspiration. As evidence of this, the present methodology can also be applied to hematological cancers.

Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography.

Anthracenyl amino acid/dipeptide conjugates (AADC) represent novel structures rationally designed for their DNA-binding properties. A high-performance liquid chromatography method is described for simultaneous determination of five compounds that exhibit novel mechanisms of action as topoisomerase I and II inhibitors. The method uses an Apex ODS-2 column and a mobile phase of 0.25 M ammonium acetate/trifluoroacetic acid (pH 3) in methanol with gradient elution. Selective detection is achieved by monitoring at 545 nm, with limits of detection ranging between 2 and 4 ng on the column. AADC are recovered from cell sonicates by solid-phase extraction using C2 cartridges, with extraction efficiencies ranging from 84% to 95%. Drug uptake studies were performed with three active compounds in the human ovarian cancer cell line A2780 and its multi-drug-resistant counterpart 2780AD. Marked differences were observed in the pattern of cellular accumulation produced by each compound. NU/ICRF 505 (tyrosine derivative) was taken up most avidly, reaching plateau levels of 4000 pmol/10(6) cells after 2 h, with no difference being apparent between A2780 and 2780AD. NU/ICRF 510 (arginine derivative) accumulated slowly in A2780, failing to achieve an equilibrium after 4 h, and appeared to be completely excluded from 2780AD. NU/ICRF 500 (serine derivative) was most rapidly taken up by A2780, producing a plateau of 800 pmol/10(6) cells after only 30 min with approximately 3-fold less accumulation in 2780AD. These results are correlated to the chemosensitivity of the two cell lines to the three compounds.

Elemental mapping of fluorine using electron spectroscopic imaging for the intracellular localisation of an anthracycline drug (ME2303) in human ovarian cancer cells.

A major limitation to the clinical usefulness of cancer chemotherapy is the onset of acquired drug resistance, in which the effectiveness of a drug such as Doxorubicin (DOX), used in a wide variety of neoplasms, diminishes following repeated administration. Resistance is associated with drug exclusion from tumour cell nuclei. New analogues of DOX have been synthesised to reduce patient cardiotoxicity and to increase anti-tumour activity. More recently, a 2-fluoroglycoside of DOX (ME2303) has been shown to be more resistant to glycolysis and has marked anti-proliferative effects on DOX-resistant tumours. The aim of the current study was therefore to determine the intracellular localisation of ME2303 in drug sensitive and resistant human ovarian cancer cell aggregates by mapping fluorine, as a means of understanding the complex mechanisms of drug resistance. Cell aggregates of the human ovarian cell line A2780 and its DOX-resistant subline 2780AD were cultured for 1hr in 5μM of the drug ME2303, then chemically fixed and embedded in Lowicryl K4M using alcohol dehydration at -20°C. Ultrathin sections (40-50nm) were examined on a Zeiss TEM 902 energy filtering electron microscope using 80Kv at a magnification of 12,000x. Fluorine maps were generated using the two window method by collecting two integrated images above and two integrated images below the K ionisation edge for fluorine (685eV) with a 15eV window. Image sequences were collected within 20sec to minimise the effects of mass loss from the specimen via a Dage SIT 66 video camera connected to a DT 2861 video interface board (512x512 pixels with 256 grey levels) within a 486 PC. In the A2780 cell line, fluorine was found to be distributed diffusely within the cytoplasm and at discrete sites within the nucleus. In contrast, fluorine in the 2780AD subline (co-cultured with 1.4μM DOX to maintain resistance) was found to be largely associated with the peri-nuclear Golgi region and with mitochondria, but was also found within cell nuclei, along the inner nuclear envelope and in the nucleolus. The intra-nuclear localisation of fluorine suggests that even in the presence of DOX, ME2303 can mediate anti-proliferative activity in DOX resistant human ovarian cancer cells by effective nuclear translocation.

Modulation of drug sensitivity of cancer cells by the stimulation and inhibition of protein kinase C

Modulation of drug sensitivity of cancer cells by the stimulation and inhibition of protein kinase C