Epigenetic approaches to cancer therapy

2004 ◽  
Vol 32 (6) ◽  
pp. 1095-1097 ◽  
Author(s):  
J.A. Plumb ◽  
N. Steele ◽  
P.W. Finn ◽  
R. Brown
Keyword(s):  
Dna Methylation ◽  
Cell Line ◽  
Gene Promoter ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Tumour Suppressor Genes ◽  
Control Of Gene Expression ◽  
Hmlh1 Gene

Histone deacetylation and DNA methylation have a central role in the control of gene expression, including transcriptional repression of tumour suppressor genes. Loss of DNA mismatch repair due to methylation of the hMLH1 gene promoter results in resistance to cisplatin in vitro and in vivo. The cisplatin-resistant cell line A2780/cp70 is 8-fold more resistant to cisplatin than the non-resistant cell line, and has the hMLH1 gene methylated. Treatment with an inhibitor of DNA methyltransferase, DAC (2-deoxy-5′-azacytidine), results in a partial reversal of DNA methylation, re-expression of MLH1 (mutL homologue 1) and sensitization to cisplatin both in vitro and in vivo. PXD101 is a novel hydroxamate type histone deacetylase inhibitor that shows antitumour activity in vivo and is currently in phase I clinical evaluation. Treatment of A2780/cp70 tumour-bearing mice with DAC followed by PXD101 results in a marked increase in the number of cells that re-express MLH1. Since the clinical use of DAC may be limited by toxicity and eventual re-methylation of genes, we suggest that the combination of DAC and PXD101 could have a role in increasing the efficacy of chemotherapy in patients with tumours that lack MLH1 expression due to hMLH1 gene promoter methylation.

scholarly journals GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4069-4069
Author(s):  
Wenyu Shi ◽  
Jian-Yong Li
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Large Cell ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Expression Level ◽  
Downstream Signaling ◽  
Single Drug

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control>GSK single drug 30 mg/kg>GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genotoxic Effects of Green Tea Extract on Human Laryngeal Carcinoma Cells In Vitro

2011 ◽  
Vol 62 (2) ◽  
pp. 139-146 ◽  
Author(s):  
Ksenija Durgo ◽  
Sandra Kostić ◽  
Katarina Gradiški ◽  
Draženka Komes ◽  
Maja Osmak ◽  
...  
Keyword(s):  
Cell Line ◽  
Green Tea ◽  
Laryngeal Carcinoma ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Green Tea Extract ◽  
Carcinoma Cells ◽  
Tea Extract ◽  
Human Laryngeal Carcinoma

Genotoxic Effects of Green Tea Extract on Human Laryngeal Carcinoma Cells In VitroGreen tea (Camellia sinensis) contains several bioactive compounds which protect the cell and prevent tumour development. Phytochemicals in green tea extract (mostly flavonoids) scavenge free radicals, but also induce pro-oxidative reactions in the cell. In this study, we evaluated the potential cytotoxic and prooxidative effects of green tea extract and its two main flavonoid constituents epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) on human laryngeal carcinoma cell line (HEp2) and its cross-resistant cell line CK2. The aim was to see if the extract and its two flavonoids could increase the sensitivity of the cisplatin-resistant cell line CK2 in comparison to the parental cell line. The results show that EGCG and green tea extract increased the DNA damage in the CK2 cell line during short exposure. The cytotoxicity of EGCG and ECG increased with the time of incubation. Green tea extract induced lipid peroxidation in the CK2 cell line. The pro-oxidant effect of green tea was determined at concentrations higher than those found in traditionally prepared green tea infusions.

Detection of the 170 kDa P-Glycoprotein in Neoplastic and Normal Tissues

1989 ◽  
Vol 75 (6) ◽  
pp. 542-546 ◽  
Author(s):  
Enrico Ronchi ◽  
Ornella Sanfilippo ◽  
Giovanni Di Fronzo ◽  
Maria Rosa Bani ◽  
Gabriella Della Torre ◽  
...  
Keyword(s):  
Protein A ◽  
Resistant Cell ◽  
Nodal Metastasis ◽  
Resistant Cell Line ◽  
Resistant Variant ◽  
Normal Tissues ◽  
Immunoblotting Assay ◽  
Human Solid Tumors

A membrane purification procedure and an immunoblotting assay have been designed to allow screening of human solid tumors for overexpression of the GP170 glycoprotein without employing a disaggregation method to obtain cell suspensions. The electrophoresed membrane proteins were probed, after Western Blotting, with the C219 monoclonal antibody and iodinated Protein A. The labeling intensity of the bands on the autoradioimmunoblots were quantified by densitometry. To test for the presence of GP170, we used membranes from the UV 2237 fibrosarcoma line and its adriamycin-resistant variant ADMR, grown in vitro or as solid tumor in mice. Membranes of human normal and tumor tissues obtained from previously untreated patients were also tested. An immunoreaction was observed in the adriamycin-resistant UV 2237 lines grown in vitro or in vivo. Quantitatively, the binding of the resistant cell line grown in vitro was higher than that observed in cells grown in mice. Bands in the GP 170 region were observed in 4/7 normal and in 7/7 tumor colon tissues and in the normal medulla from 2 patients with cancer of the renal cortex. No reaction could be found in samples from normal tissue, primary tumor or nodal metastasis from 7 patients with breast cancer.

scholarly journals Cryoprotective agents as inducers of erythroleukemic cell differentiation in vitro

Blood ◽  
1976 ◽  
Vol 47 (3) ◽  
pp. 363-368 ◽  
Author(s):  
HD Preisler ◽  
G Christoff ◽  
E Taylor
Keyword(s):  
Cell Line ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Chemical Induction ◽  
Cryoprotective Agents ◽  
Differentiated Cells ◽  
Friend Leukemia ◽  
A Cell ◽  
Erythroleukemic Cell

Abstract The ability of families of compounds with known and potential cryoprotective properties to induce the differentiation of Friend leukemia cells in vitro was studied. For each agent, both the proportion of differentiated cells in the culture and the total amount of heme/10(7) cells were determined. Within each family of compounds there was a direct correlation between a compound's cryoprotective ability, its ability to donate electron pairs for hydrogen bonding (basicity), and its ability to induce differentiation. While individual agents differed with respect to the proportion of cells which were induced to differentiate, the biology of the process of differentiation appeared to be similar, regardless of the agent used. A cell line which was unresponsive to DMSO was responsive to other inducers, suggesting that this DMSO-resistant cell line differed from its parent DMSO- responsive cell line either in its metabolism of the inducers or in the ability of the inducers to enter the cell. Alternatively, there may be more than one mechanism involved in the chemical induction of differentiation.

scholarly journals Non-canonical AR activity facilitates endocrine resistance in breast cancer

2019 ◽  
Vol 26 (2) ◽  
pp. 251-264 ◽  
Author(s):  
KeeMing Chia ◽  
Heloisa Milioli ◽  
Neil Portman ◽  
Geraldine Laven-Law ◽  
Rhiannon Coulson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Clinical Trials ◽  
Endocrine Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Breast Cancer Cell Lines ◽  
Microarray Gene Expression ◽  
Pdx Model

The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro, even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.

scholarly journals Chemical Genomics Reveals JAK STAT Activation As a Mechanism of Resistance to HDAC Inhibitors in B Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 271-271
Author(s):  
Matthew S. McKinney ◽  
Anne W Beaven ◽  
Andrea Moffitt ◽  
Jason Landon Smith ◽  
Eric Lock ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Resistant Cell ◽  
Hdac Inhibition ◽  
Stat Pathway

Abstract Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics. Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p < 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P < 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines. We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher's exact test). Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P < 0.001 and FDR < 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice. Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Role of metalloproteinase-7 in oxaliplatin acquired resistance in colorectal cancer cell lines

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20042-20042 ◽  
Author(s):  
V. Almendro ◽  
J. Maurel ◽  
J. Augé ◽  
G. Laus ◽  
J. Domingo-Domenech ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Fasl Expression ◽  
Ic50 Values

20042 Background: Mechanisms responsible for acquired resistance in colorectal cancer (CRC) tumors are not well understood. Anticancer drugs have been shown to enhance FASL expression by NF-kB induction. Additionally Metalloproteinase (MMP)-7 is over-expressed in CRC and has been shown to inhibit apoptosis by cleavage of FASL. We have previously shown in vivo that, sFASL increment was associated with acquired chemoresistance. Therefore we speculate that inhibition of MMP-7 or NF-kB can reverse chemoresistance in CRC cell lines. Methods: We generated an oxaliplatin-resistant cells (HT29R) from a p53 mutated (HT29) cell line. Both cell lines were cultured for 72h with different concentrations of oxaliplatin, BAY11–7085 (inhibitor of NF-kB activation), 0.01 mM of the MMP-7 inhibitor 1,10-Phenanthroline monohydrate (1,10-PM) and 100 ng/ml of DX2 monoclonal antibody. Different drug combinations were performed. Citotoxicity was determined by the MTS method, and cell cycle was analysed at 72h. Cell lines were characterized for MMP-7 expression (ELISA), NF-KB (Western-Blot), Fas expression (immunohistochemistry) and FasL expression (Western-Blot). Results: FAS was down-expressed in HT29R compared to HT29. The HT29R cells showed a IC50 for oxaliplatin 2-fold higher than normal cells. Treatment with 1,10-PM decrease MMP-7 levels (p < 0.005) compared with untreated cells. Additionally, inhibition of MMP-7, restore IC50 values after oxaliplatin treatment in HT29R without changes in NF-KB expression. This oxaliplatin-resistant cell line, presents also sensibility for BAY11–7085, without affecting MMP-7 levels. Finally the addition of oxaliplatin to the MMP-7 inhibitor, increase FAS-mediated apoptosis (induced by DX2 antibody), suggesting that FASL cleavage is responsable of sensitivity. Conclusions: Reversal of oxaliplatin chemo-resistance can be obtained either by MMP-7 or NF-kB inhibition. Both drugs induced sFASL decrement, by inhibiting cleavage or expression, respectively. No significant financial relationships to disclose.

Evaluation of SRC-family kinases in gastrointestinal stromal tumors

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 9529-9529
Author(s):  
S. Bauer ◽  
G. D. Demetri ◽  
J. A. Fletcher
Keyword(s):  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Src Family Kinases ◽  
Inhibition Of Proliferation ◽  
Variable Expression ◽  
Primary Gist

9529 Background: Inhibition of KIT oncoproteins by imatinib mesylate (IM) induces clinical responses in most GIST patients. However, many patients develop IM-resistance and novel KIT-Inhibitors show clinical benefit in only a minority of patients. SRC-family kinases (SFK) are important participants in kinase oncoprotein signaling pathways in ALL and CML, and dual-specificity SRC/ABL-inhibitors show promising activity in IM-resistant CML in vitro. However, little is known about SFK functional roles in IM-resistant GIST. Methods: GIST were analyzed for SFK expression and activation by western blotting. Biological consequences of KIT inhibition alone (IM), SFK inhibition (SU6656) and combined KIT/SFK inhibition (PP1, PP2, SU6656+IM) were determined by immunoblotting for protein activation, and—in GIST cell lines—by luminescence assays for cell proliferation. Results: Primary GIST and GIST cell lines showed weak SFK activation compared to Jurkat (T-cell ALL) controls. Variable expression of the SFK proteins FYN, LYN and SRC (but not BLK and LCK) was shown in the cell lines and primary GIST. Expression of SFK proteins HCK and FGR was found in 2 primary GIST only. KIT and KIT signaling pathways (pAKT, pS6, pSTAT3) were affected by IM and combined KIT inhibitors but not by SU6656 at 1μM in GIST882 (IM-sensitive cell line) and GIST48 (IM-resistant cell line). SRC phosphorylation (Y418) was not affected by IM or SFK-inhibitors at 1μM. IM showed superior inhibition of proliferation compared to PP1 and PP2 (1μM) in GIST882 (IM: 67%, PP1: 1%, PP2: 0%) and comparable inhibition in GIST48 (IM:30%, PP1: 29%, PP2: 29%). No effects on proliferation were seen with SU6656 alone or in combination with IM; no antagonistic effects were observed. Conclusions: Our analysis showed expression and weak activation of several SFK in GIST. Expression of heme-associated SFKs in primary GIST may be explained by lymphocellular infiltration. Biochemical inhibition of SFK alone does not have antiproliferative effects in GIST cell lines, and no synergies or antagonisms were found in combination with IM or with combined KIT/SFK inhibitors. Therefore, targeting of SFK may be of less therapeutic value in GIST than in kinase-driven haematological cancers. No significant financial relationships to disclose.

scholarly journals Hypermethylation involved in DNA profiles of lung cancer specific tumour suppressor genes and epigenetic modification caused by an ultra-highly diluted homeopathic drug, Condurango 30C, in vitro and in vivo

2021 ◽  
Vol 13 (47) ◽  
pp. 99-99
Author(s):  
Anisur Rahman Khuda-Bukhsh ◽  
Sourav Sidkar
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Epigenetic Modification ◽  
Tumour Suppressor ◽  
Tumour Suppressor Genes ◽  
Dna Hypermethylation ◽  
Suppressor Genes ◽  
Homeopathic Drug

Background and objectives: DNA hyper-methylation is an important aspect involved in carcinogenesis and cancer progression, which affects mainly CpG islands of DNA and causes inactivation of tumour suppressor genes. Therefore DNA hypermethylation status of the genomic DNA in both the transformed cancerous cell lines and in carcinogen-induced lung cancer was ascertained by analysis of expressions of certain major lung cancer specific tumour suppressor genes. The other objective was to examine if ultra highly diluted homeopathic drug, Condurango 30C, had ability to modulate DNA methylation. Methods: DNA methylation activity, if any, has been ascertained in H460-NSCLC cells in vitro and in BaP-induced lung cancer of rats in vivo, in respect of tumour suppressor genes like p15, p16, p18 and p53 by using PCR-SSCP analyses. The ability of modulation of DNA methylation, if any, by Condurango 30C was also verified against placebo control in a blinded manner. Results: Condurango 30C-treated DNA showed significant decrease in band-intensity of p15 and p53 genes especially in methylated condition, in vitro, at the IC50 dose (2.43µl/100µl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also supported ability of Condurango 30C to modulate methylation state, in vitro. Inhibition of p15 hypermethylation was observed after post cancer treatment of rat with Condurango 30C. SSCP results gave a better indication of differences in band-position and single strand separation of p15 and p53 in Condurango 30C treated samples. Conclusion: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation but placebos could not.

Mitotracker Green Is a P-Glycoprotein Substrate

2003 ◽  
Vol 23 (4) ◽  
pp. 199-212 ◽  
Author(s):  
Luis F. Marques-Santos ◽  
José G. P. Oliveira ◽  
Raquel C. Maia ◽  
Vivian M. Rumjanek
Keyword(s):  
Cell Line ◽  
Fluorescent Dyes ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Normal Tissues ◽  
P Glycoprotein ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line ◽  
Mitotracker Green

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.

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