Abstract
BackgroundThe novel gene HA117 is a multidrug resistance (MDR) gene expressed by all-trans retinoic acid-resistant HL-60 cells. In the present study, we compared the multidrug resistance of the HA117 with that of the classical multidrug resistance gene 1 (MDR1) in breast cancer cell line 4T1.MethodsTransduction of the breast cancer cell line 4T1 with adenoviral vectors encoding the HA117 gene and the green fluorescence protein gene (GFP) (Ad-GFP-HA117), the MDR1 and GFP (Ad-GFP-MDR1) or GFP (Ad-GFP) was respectively carried out. The transduction efficiency and the multiplicity of infection (MOI) were detected by fluorescence microscope and flow cytometry. The transcription of HA117 gene and MDR1 gene were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis was used to detect the expression of P-glycoprotein (P-gp) but the expression of HA117 could not be analyzed as it is a novel gene and its antibody has not yet been synthesized. The drug-excretion activity of HA117 and MDR1 were determined by daunorubicin (DNR) efflux assay. The drug sensitivities of 4T1/HA117 and 4T1/MDR1 to chemotherapeutic agents were detected by Methyl-Thiazolyl-Tetrazolium (MTT) assay.ResultsThe transducted efficiency of Ad-GFP-HA117 and Ad-GFP-MDR1 were 75%-80% when MOI was equal to 50. The transduction of Ad-GFP-HA117 and Ad-GFP-MDR1 could increase the expression of HA117 and MDR1. The drug resistance index to Adriamycin (ADM), vincristine (VCR), paclitaxel (Taxol) and bleomycin (BLM) increased to19.8050, 9.0663, 9.7245, 3.5650 respectively for 4T1/HA117 and 24.2236, 11.0480, 11.3741, 0.9630 respectively for 4T1/MDR1 as compared to the control cells. There were no significant differences in drug sensitivity between 4T1/HA117 and 4T1/MDR1 for the P-gp substrates (ADM, VCR and Taxol) (P < 0.05), while the difference between them for P-gp non-substrate (BLM) was statistically significant (P < 0.05). DNR efflux assay confirmed that the multidrug resistance mechanism of HA117 might not be similar to that of MDR1.ConclusionsThese results confirm that HA117 is a strong MDR gene in both HL-60 and 4T1 cells. Furthermore, our results indicate that the MDR mechanism of the HA117 gene may not be similar to that of MDR1.
Highlights
Multidrug resistance (MDR) is a major cause of treatment failure and mortality in cancer patients
Production of recombinant adenoviruses in HEK 293 cells The recombinant adenoviruses Ad-green fluorescence protein (GFP)-HA117, AdGFP-multidrug resistance gene 1 (MDR1), and Ad-GFP were transducted into HEK 293 cells
The viral titers of Ad-GFPHA117, Ad-GFP-MDR1 and Ad-GFP ranged between 2.5-3.5 × 109 plaque forming units (PFU)/ml
Summary
Multidrug resistance (MDR) is a major cause of treatment failure and mortality in cancer patients. The novel gene HA117 (Gene Bank accession number: AY230154), which was screened and cloned from the ATRA-resistant acute myeloid leukemia cell line HL-60/ATRA using differential hybridization and gene chip assays [9], was shown to promote MDR in the chronic myelogenous myeloid leukemia cell line K562 [10]. The strength and mechanism of the MDR of HA117 have not yet been elucidated, especially in solid tumor cells. Our aim in the current study was to compare the MDR strength of HA117 to that of MDR1 and to examine the possible MDR mechanism(s) of HA117 in breast cancer cell line 4T1 which is a classical representation of solid tumor cell line. We compared the multidrug resistance of the HA117 with that of the classical multidrug resistance gene 1 (MDR1) in breast cancer cell line 4T1
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