mRNA Expression Levels Of MDR1 Research Articles

Renal clearance of graphene oxide: glomerular filtration or tubular secretion and selective kidney injury association with its lateral dimension

BackgroundRenal excretion is one of the major routes of nanomaterial elimination from the body. Many previous studies have found that graphene oxide nanosheets are excreted in bulk through the kidneys. However, how the lateral size affects GO disposition in the kidneys including glomerular filtration, active tubular secretion and tubular reabsorption is still unknown.ResultsThe thin, two-dimensional graphene oxide nanosheets (GOs) was observed to excrete in urine through the kidneys, but the lateral dimension of GOs affects their renal clearance pathway and renal injury. The s-GOs could be renal excreted via the glomerular filtration, while the l-GOs were predominately excreted via proximal tubular secretion at a much faster renal clearance rate than the s-GOs. For the tubular secretion of l-GOs, the mRNA level of basolateral organic anion transporters Oat1 and Oat2 in the kidney presented dose dependent increase, while no obvious alterations of the efflux transporters such as Mdr1 and Mrp4 mRNA expression levels were observed, suggesting the accumulation of l-GOs. During the GO renal elimination, mostly the high dose of 15 mg/kg s-GO and l-GO treatment showed obvious kidney injuries but at different renal compartment, i.e., the s-GOs induced obvious glomerular changes in podocytes, while the l-GOs induced more obvious tubular injuries including necrosis of renal tubular epithelial cells, loss of brush border, cast formation and tubular dilatation. The specifically tubular injury biomarkers KIM1 and NGAL were shown slight increase with mRNA levels in l-GO administrated mice.ConclusionsThis study shows that the lateral size of GOs affected their interactions with different renal compartments, renal excretion pathways and potential kidney injuries.

Effect of hypoxia on HIF-1α/MDR1/VEGF expression in gastric cancer cells treated with 5-fluorouracil.

Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition. Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU. Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α. Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.

Comparison of effects of aminosalicylic acid, glucocorticoids and immunosuppressive agents on the expression of multidrug-resistant genes in ulcerative colitis

To compare the effects of aminosalicylic acid, glucocorticoids and immunosuppressants on the expression levels of multidrug resistance genes in patients with ulcerative colitis (UC), with the aim of providing a theoretical and therapeutic basis for the diagnosis, treatment, and prevention of UC. Fresh colonic mucosal tissues or postoperative pathological biopsies from 148 UC patients were collected, and the distribution sites and morphology of P-glycoprotein (P-gp) were detected using immunohistochemical staining. RT-PCR was used to quantify the expression levels of multidrug resistance gene (MDR1) mRNA before and after the corresponding treatment, and the effects of aminosalicylic acid, glucocorticoids and immunosuppressive drugs on P-gp were compared. In addition, the effects of the three drugs on MDR1 mRNA were analyzed. Administration of 5-aminosalicylic acid (5-ASA) drugs did not correlate with MDR1 expression in UC, whereas administration of glucocorticoids and immunosuppressive drugs was positively correlated with MDR1 expression profile. The expression levels of MDR1 mRNA and its product P-gp were significantly upregulated in patients who did not respond to glucocorticoids and immunosuppressive drugs. 5-ASA had no effect on the expression levels of MDR1 and its product P-gp in patients with a confirmed diagnosis of UC. However, the use of glucocorticoids and immunosuppressants can increase the expression level of MDR1.

Evaluation of the Effect of Gene Duplication by Genome Editing on Drug Resistance in Plasmodium falciparum.

The emergence and spread of drug-resistant Plasmodium falciparum have compromised antimalarial efficacy and threatened the global malaria elimination campaign using artemisinin combination therapies. The impacts of amino acid substitutions in antimalarial drug resistance-associated genes on drug susceptibility have been investigated; however, the effects of amplification of those genes remain unexplored due to the lack of robust genetic approaches. Here, we generated transgenic P. falciparum parasites with an additional copy of a drug resistance-associated gene using the highly efficient CRISPR/Cas9 system and investigated their drug response. Insertion of a drug resistance-associated gene expression cassette in the genome resulted in a roughly twofold increase of mRNA levels of the target gene mdr1, which encodes multidrug resistance protein 1. The gene duplication event contributed to resistance to mefloquine, lumefantrine, and dihydroartemisinin, while the duplication of a genomic region encoding plasmepsin 2 and plasmepsin 3 did not affect resistance to antimalarial drugs, including piperaquine. We further demonstrated that mdr1 mRNA expression levels are strongly associated with mefloquine resistance in several field-derived P. falciparum lines with various genetic backgrounds. This study provides compelling evidence that mdr1 could serve as a molecular marker for the surveillance of mefloquine-resistant parasites. Long DNA integration into parasite genomes using the CRISPR/Cas9 system provides a useful tool for the evaluation of the effect of copy number variation on drug response.

Panax notoginseng saponins reverse P-gp-mediated steroid resistance in lupus: involvement in the suppression of the SIRT1/FoxO1/MDR1 signalling pathway in lymphocytes

BackgroundP-glycoprotein (P-gp)-mediated steroid resistance (SR) has been suggested to play a significant role in lupus nephritis (LN) treatment failure. Panax notoginseng saponins (PNS), the main effective components of the traditional Chinese medicine notoginseng, exhibited potent reversal capability of P-gp-mediated SR, but its mechanism remains unknown. This study aimed to investigate the effect of PNS on reversing SR in lupus and its underlying mechanism in vivo and in vitro.MethodsIn this study, an SR animal and splenic lymphocyte model were established using low-dose methylprednisolone (MP). Flow cytometry was used to detect the effect of PNS on reversing P-gp-mediated SR and the expression of P-gp in different T-cells phenotypes. Serum levels of ANA and dsDNA in lupus mice were measured by ELISA. Apoptosis was identified by Annexin V-FITC/PI staining. RT–PCR and Western blotting were used to detect the protein and mRNA expression levels of SIRT1, FoxO1, and MDR1 in SR splenic lymphocytes from lupus mice (SLCs/MPs).ResultsPNS could reverse the SR in lupus mice. Simultaneously, PNS increased the apoptotic effect of MP on SLCs/MP cells. The increased accumulation of rhodamine-123 (Rh-123) indicated that intracellular steroid accumulation could be increased by the action of PNS. Moreover, PNS decreased the expression of P-gp levels. Further experiments elucidated that the SIRT1/FoxO1/MDR1 signalling pathway existed in SLCs/MP cells, and PNS suppressed its expression level to reverse SR. The expression of P-gp in Th17 from SLCs/MP cells was increased, while PNS could reduce its level in a more obvious trend.ConclusionThe present study suggested that PNS reversed P-gp-mediated SR via the SIRT1/FoxO1/MDR1 signalling pathway, which might become a valuable drug for the treatment of SR in lupus. Th17 might be the main effector cell of PNS reversing SR.

SH003 overcomes drug resistance and immune checkpoints by inhibiting JAK-STAT3 signaling in MCF7/ADR cells

BackgroundBreast cancer has the most commonly diagnosed malignancy cancer worldwide in women and has a high mortality. Various anticancer drugs to treat breast cancer have been developed and tested but have failed because of drug resistance. ObjectivesThe efficacy of SH003 against doxorubicin-resistant MCF/ADR cells has not been evaluated. In this study, we aimed to examine whether SH003 could efficiently prevent the proliferation of MCF7/ADR cells. MethodsCell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by SH003 was measured by an annexin V-FITC/PI assay. Levels of p-STAT3, p-PD-L1, MDR and caspases were measured by western blot analysis. mRNA expression levels of MDR1, MRP1, -2, -3, -4, -5, -6, -9, BCRP and PD-L1 were measured by RT-PCR. Nuclear staining of STAT3 was measured by immunocytochemistry. The expression levels of VEGF, MMP-2 and MMP-9 were measured by ELISA. ResultsSH003 inhibited the proliferation of MCF7/ADR cells and induced their sub-G1 cell cycle arrest. SH003 also induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in MCF7/ADR cells. Furthermore, SH003 suppressed STAT3 transcriptional activity and, more importantly, reduced the cytokine levels of VEGF, MMP-2 and MMP-9 in MCF7/ADR cells. SH003 decreased the protein expression of PD-L1 and MDR1. Additionally, SH003 reduced the mRNA expression of PD-L1; MDR1; MRP1, -2, -3, -4, -5, -6, and -9; and BCRP. ConclusionOur results clearly demonstrate that SH003 inhibits cell growth and induces apoptosis by inhibiting STAT3 signaling. Additionally, SH003 decreased the levels of MDR1 and PD-L1 by inhibiting STAT3 signaling in MCF7/ADR breast cancer cells. These results support the possibility that SH003 could be useful as an herbal medicine to treat doxorubicin-resistant breast cancer.

First Sequencing of Caprine Mdr1 (Abcb1) mRNA Due to Suspected Neurological Adverse Drug Reaction in a Thuringian Goat Following Extra-Label Use of Doramectin.

The multidrug resistance gene MDR1 encodes for an efflux transporter called P-glycoprotein (P-gp). In the canine Mdr1 gene, a nonsense mutation was identified in certain dog breeds causing increased drug sensitivity to various P-gp substrates such as antiparasitic macrocyclic lactones. Symptoms of neurologic toxicity include ataxia, depression, salivation, tremor, apparent blindness, and mydriasis. In the current report, a Thuringian goat developed similar neurological signs after treatment with doramectin, a compound from the macrocyclic lactone class. Therefore, Mdr1 might be defective in this individual goat. For diagnostic purposes, sequencing of the complete mRNA transcript coding for caprine Mdr1 was performed to investigate a potential missense mutation. The Mdr1 transcripts of two related goats without drug sensitivity were also sequenced to allow differential diagnosis and were compared to the suspected drug-sensitive goat. The only position where the Mdr1 sequence from the suspected drug-sensitive goat differed was in the 3′-untranslated region, being a heterozygous single nucleotide polymorphism c.3875C>A. It can be suspected that this variant affects the expression level, stability, or translation efficiency of the Mdr1 mRNA transcript and therefore might be associated with the suspected drug sensitivity. To clarify this, further studies are needed, particularly investigating the Mdr1 mRNA and protein expression levels from brain material of affected goats. In conclusion, Mdr1 variants may exist not only in dogs, but also in individual goats. The current report provides the first Mdr1 mRNA transcript sequence of a goat and therefore represents the basis for more detailed Mdr1 sequence and expression analyses.

Anticarcinogenic Effects of Capsaicin-Loaded Nanoparticles on In vitro Hepatocellular Carcinoma

Background:: Hepatocellular Carcinoma (HCC) is the fifth most frequent cancer worldwide with a low overall survival due to high metastasis and recurrence rates. The aim of this study is to assess and compare the possible anti-neoplastic effect of capsaicin and nanoformulated capsaicin on in vitro HCC human cell line HepG2. The source of the cell line, including when and from where it was obtained. Whether the cell line has recently been authenticated and by what method. Whether the cell line has recently been tested for mycoplasma contamination. Materials and Methods:: Capsaicin-loaded Trimethyl Chitosan Nanoparticles (CL TMCS NPs) were synthesized by ionotropic gelation of cationic TMCS with capsaicin. The synthesized nanoparticles were characterized through TEM, and zeta analyzer. Human hepatocarcinoma HepG2 cell lines were cultured and treated with 50, 75 &amp; 100 μM of Capsaicin (CAP), plain TMCS NPs and CL-NPs as well as ethanol (control) for 24h and 48h. The induced effects were investigated by flow cytometry, immunocytochemistry assay for Bcl-2, Bax, and caspase proteins and evaluating gene expression levels of Bcl-2, Bax, and MDR-1 mRNA by real-time PCR. Results:: Our results demonstrated that capsaicin- loaded NPs had the potential to significantly increase capsaicin bioactivity compared with the plain capsaicin formulation either in inducing apoptosis through altering expression of apoptotic regulators or modifying MDR-1 expression. Conclusions:: TMCs nanoparticles investigated in this study may be a good drug delivery vehicle for capsaicin. Application of capsaicin-loaded NPs in HCC management as an adjunct therapeutic approach may be a novel strategy to improve the treatment efficacy and resistance of the conventionally used chemotherapy.

γ-Tocotrienol reverses multidrug resistance of breast cancer cells through the regulation of the γ-Tocotrienol-NF-κB-P-gp axis

The problem of multidrug resistance (MDR) presents a major obstacle in the chemotherapy of cancer. The MDR phenotype is often linked to the overexpression of ATP-binding cassette (ABC) transporters, that pumps out and decreased intracellular drug accumulation. γ-Tocotrienol, an unsaturated tocopherol belonging to the vitamin E family, has been shown to reverse the MDR of MCF-7/Adr cell. To reveal the role of γ-tocotrienol-NF-κB-P-gp axis in the reversal process, the expression level of mdr1/P-gp was determined by real-time PCR and western blot, while NF-κB activity was detected by immunofluorescence and NF-κB transcriptional activity reporter assay. Besides, mdr1 promoter activity and P-gp transport capacity were measured with the effect of γ-tocotrienol and NF-κB agonist/antagonist. Results showed that γ-tocotrienol effectively inhibited the expression levels of mdr1 mRNA and P-gp protein. It is demonstrated that γ-tocotrienol also suppressed mdr1 promoter activity and the efflux activity of P-gp. In addition, the activation of NF-κB signaling pathway and the transcriptional activity of NF-κB were both reduced by γ-tocotrienol. Evidences also showed that the NF-κB pathway is really involved in the regulation of the expression and function of mdr1/P-gp. Taken together, we confirmed that γ-tocotrienol reversed the MDR of MCF-7/Adr through the signaling pathway of NF-κB and P-gp.

Cytotoxic effect of 6-Shogaol in Imatinib sensitive and resistant K562 cells.

Chronic Myeloid Leukemia (CML) is a clonal hematopoietic malignancy characterized by the formation of BCR-ABL fusion protein. Imatinib (IMA) is a BCR-ABL tyrosine kinase inhibitor (TKI), which exhibited a high rate of response for newly diagnosed CML patients. Emergence of IMA resistance considered as a major challenge in CML therapy. Recent studies reported the anti-cancer effect of natural extracts such as 6-Shogaol (6-SG) which is extracted from ginger and the mechanisms involved in targeting of cancer cells. In the present study, we aimed to explore the potential anticancer effect of 6-SG on K562S (Imatinib sensitive) and K562R (Imatinib resistant) cells. K562S and K562R cells were incubated with increasing concentrations of 6-SG (5μM- 50μM) to determine its cytotoxic and apoptotic effects. Cell viability and apoptosis were investigated with spectrophotometric MTT assay and flow cytometric Annexin V staining, respectively. The mRNA expression levels of apoptotic related genes (BAX and BCL-2) and drug transporter (MDR-1 and MRP-1) genes were evaluated with qRT-PCR. According to our results, 6-SG treatment inhibited cell viability, induced apoptosis in both K562S and K562R cells. Based on our RT-PCR results, 6-SG enhanced pro-apoptotic BAX gene and decreased anti-apoptotic BCL-2 gene expression levels significantly in both treated K562S and K562R cells. Furthermore, 6-SG increased MDR-1 mRNA expression level in K562S and K562R cells in comparison with their control counterparts. Whereas, 6-SG decrease MRP-1 mRNA expression level in K562S cells significantly. It is the first study that reveals the apoptotic effect of 6-SG in CML cell line and IMA resistance. Therefore, 6-SG treatment can be suggested as a promising strategy for CML therapy.

Bioactive Hexapeptide Reduced the Resistance of Ovarian Cancer Cells to DDP by Affecting HSF1/HSP70 Signaling Pathway.

Ovarian cancer is the leading cause of death in gynecologic malignancies. Ovarian cancer as a metastatic malignant tumor is highly recurrent and prone to drug resistance. Bioactive peptides are an emerging area of biomedical research in reducing resistance of tumor cell to drugs. In this paper, we investigated the effects and mechanisms of bioactive hexapeptide (PGPIPN) derived in milk protein on the sensitivity of ovarian cancer cells to cis-dichlorodiammine platinum (DDP). Human ovarian cancer cell lines (SKOV3 and COC1), their DDP-resistant sublines (SKOV3/DDP and COC1/DDP) and human primary ovarian cancer cells were cultured in vitro under the combined treatment of DDP (close to IC50) and different concentrations of PGPIPN. The viabilities, apoptosis and cell cycle changes were respectively measured by WST-8 and flow cytometry. The mRNA and protein expression levels of HSF1, HSP70, MDR1, ERCC1 and β-actin gene were respectively assayed by RT-qPCR and western blotting. The results showed that PGPIPN significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro. But the effects in sensitive cells were lower than DDP-resistant cells. PGPIPN significantly changed the cell cycles in all human ovarian cancer cells, which leaded to a significant increase in the percentage of cells blocked at G2/M phase and decrease the percentage of cells at G1 phases in a dose-dependent manner. PGPIPN affected the expression levels of HSF1, HSP70, MDR1 and ERCC1 genes. Compared with cells in DDP treatment alone, the expression levels of HSF1 and HSP70 in human ovarian cancer cells treated with DDP and PGPIPN together significantly decreased in dose-dependent manner. PGPIPN significantly decreased MDR1 and ERCC1 of drug-resistant ovarian cancer cell lines and human primary ovarian cancer cell in a dose-dependent manner. Pifithrin-μ (PFTμ, HSP70 inhibitor) decreased or removed the effects of peptide in increasing the sensitivity of ovarian cancer cells to DDP. This suggests that PGPIPN enhanced the sensitivity of ovarian cancer cells to DDP partially via reducing the activity of HSF1/HSP70 signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage.

The Influence and Mechanism of Paeoniflorin and Albiflorin on Strychnine and Brucine Transport Through Madin-Darby Canine Kidney/Multidrug Resistance1 Cells In Vitro Model

This research sought to study the influence and potentialmechanism of paeoniflorin and albiflorin on strychnine and brucine transport in MDCK-MDR1 cells regulated by P-gp. Cytotoxicity of drugs was tested by MTT assay, and the transport studies were performed in both directions in MDCK-MDR1 cells. The influence of drugs on P-gp ATPase, and the efflux function of P-gp, the expression levels of P-gp and MDR1 mRNA were also estimated. Strychnine and brucine showed well absorption, and the main transport mechanism might be passive diffusion. Verapamil could significantly decrease the efflux rate (ER) of strychnine and brucine, while the ER of strychnine and brucine was increased significantly when co-administrated with paeoniflorin or albiflorin, indicating that paeoniflorin and albiflorin could promote the efflux of these two alkaloids. Strychnine and brucine could activate the activity of P-gp ATPase, suppress the efflux function of P-gp, but have no significant effect on the expression of P-gp. In addition, strychnine could upregulate the expression of MDR1 mRNA. Paeoniflorin and albiflorin could increase the transmembrane transport of strychnine and brucine mediated by P-gp when co-administrated with strychnine or brucine via stimulating the activity of P-gp ATPase, enhancing the efflux function of P-g, increasing the expression levels of MDR1 mRNA and P-gp.

Reversal of P-glycoprotein-mediated multidrug resistance by novel curcumin analogues in paclitaxel-resistant human breast cancer cells.

Multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy, and the main cause of MDR has been attributed to overexpression of P-glycoprotein (P-gp). In this present study, four P-gp modulators (E,E)-4,6-bis(styryl)-2-(substituted amino)-pyrimidines were evaluated for their activity in a breast cancer cell line overexpressing P-gp (LCC6MDR). The four modulators displayed significantly better P-gp modulating activity compared with the positive control verapamil (RF = 5.4), with a relative fold (RF) increase in activity ranging from 33.3 to 86.0. In contrast to compounds a and c that exhibited lower cytotoxicity, compounds b and d were nontoxic towards both cancer cells and normal cells, with IC50 values greater than 100 μmol/L. The qRT-PCR results demonstrated that after exposure to 2 μmol/L of compounds a, b, c, and d, the mRNA expression level of MDR1 in LCC6MDR cells decreased to 45%, 50%, 38%, and 51%, respectively. However, the Western-blot results indicated that compound c could reverse P-gp mediated MDR, but not via decreases in protein expression. DOX and Rh123 accumulation and efflux results further confirmed that the reversal of MDR activity happens via inhibition of P-gp efflux and increases in intracellular drug accumulation. These results demonstrated that compound c has low toxicity and is an efficient P-gp modulator, highlighting its potential as a promising candidate for P-gp-mediated reversal of MDR.

Multidrug resistance affects the prognosis of primary epithelial ovarian cancer.

Multidrug-resistant tumor cells can tolerate different structures, functions and antidrug action mechanisms, therefore, allowing these cells to respond to various structurally unrelated mechanisms of different chemotherapy drugs and to exhibit cross-resistance. The present study aimed to investigate the role of Multi-drug resistance gene (MDR1), Placental glutathione S-transferase-P1 (GSTP1), Lung resistance protein (LRP) and Ras association domain family member 1 (RASSF1A) in primary epithelial ovarian cancer (PEOC). The mRNA (protein) expression levels of MDR1, product P glycoprotein, LRP and GSTP1 were evaluated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in all tissue samples, ovarian cancer cell line A2780 and A2780/DDP. Methylation-specific PCR (MSP) was used to detect RASSF1A gene methylation in all tissue samples. The resistance genes/proteins were either poorly or not expressed in A2780, however were highly expressed in A2780/DDP cell line. The expression of resistance genes/proteins decreased following different concentrations of zebularine-stimulated A2780/DDP. Hypermethylation and low expression of RASSF1A gene were detected in PEOC and A2780/DDP. Subsequent to being exposed to different concentrations of zebularine-stimulated A2780/DDP, the RASSF1A methylation level was decreased, while the unmethylation level was increased. The expression of RASSF1A gene/protein was gradually restored, and the gene/protein expression was enhanced with the increase in drug concentration. Multivariate logistic regression indicated that the expression level of gene LRP and GSTP1 was a risk factor for PEOC prognosis. Furthermore, the expression of LRP and GSTP1 in the negative-group survival curves was higher compared with the positive group. High expression of resistance genes may serve an important role in cancer primary resistance. Low expression caused by hyper-methylation of RASSF1A gene may serve an important role in cancer-acquired resistance in PEOC. The present study suggested that resistant gene expression may be a potential prognostic biomarker.

PB1696 EVALUATION OF TUMOR CELL SENSITIVITY TO DAUNORUBICIN USING WST1 TEST IN ACUTE MYELOBLASTIC LEUKEMIA PATIENTS

Background:In recent years the MTT test for the assessment of the sensitivity of tumor cells to cytotoxic drugs in leukemia patients were used. In present study, the possibility of the WST1 test as a screening method of drug sensitivity of tumor cells of leukemia patients using peripheral blood and bone marrow samples was evaluated.Aims:The aim of this work was to study the effectiveness of the WST1 test for the initial estimation of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the treatment response of patients with acute myeloblastic leukemia.Methods:The study included 42 patients with acute myeloblastic leukemia (AML). The average age of the patients was 53.3 ± 12.7 years. Leukemia cells were obtained from peripheral blood and bone marrow of patients. The sensitivity of tumor cells of patients with AML to daunorubicin in vitro using the WST1 test was made prior to initiating of cytotoxic therapy. Confirmation of multidrug resistance by evaluation of MDR1 mRNA expression levels in tumor cells of AML patients was performed on bone marrow and/or peripheral blood samples using quantitative real‐time reverse transcription‐polymerase chain reaction (real‐time qPCR).In all patients, the diagnosis was confirmed by the study of myelogram, flow cytofluorimetry, immunocytochemistry, cytogenetic analysis, as well as standard laboratory and instrumental studies that are included in the standard examination of patients with leukemia. Based on immunopenotyping and cytogenetic analysis, each patient was assigned to a risk group and prognosis. The obtained results were compared with the clinical and hematological response of these patients to cytotoxic therapy, as well as with the MDR1 mRNA expression level.Also we evaluated the survival of leukemia patients depending on the sensitivity to daunorubicin and MDR1 mRNA expression levels in tumor cells of AML patients.All statistical analyses were done with computer software MS Excel, OriginPro 7.5, Statistica 10.0.Results:Regardless of the class of chemotherapeutic drugs administered to the patients, the survival rate was higher in the group of patients with high sensitivity to daunorubicin and low expression levels of the MDR1 mRNA in tumor cells. Patients received daunorubicin within the “7 + 3” regimen (7 days of cytarabine and 3 days of daunorubicin) showed a high correlations (r = 0.8, p &lt;0.05) between the expression levels of MDR1 mRNA and sensitivity to daunorubicin, moderate correlations (r = 0.63, p &lt; 0.05) between the response to chemotherapy and sensitivity to daunorubicin, and a moderate correlations between the prediction (r = 0.48, p &lt; 0.05) and the risk group (r = 0.49, p &lt; 0.05) and sensitivity to daunorubicin. It is important to note that a high correlations (r = 0.86, p &lt; 0.05) was found between the expression levels of MDR1 mRNA and the response to therapy.Summary/Conclusion:The obtained data demonstrate that the WST1 test can be effectively used as a screening method for the initial assessment of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the response to treatment of AML patients.

Intercellular transfer of P-glycoprotein mediates the formation of stable multi-drug resistance in human bladder cancer BIU-87 cells.

ABSTRACTWe investigated the biological characteristics of acquired drug-resistant cells (AqMDRs) formed by intercellular P-glycoprotein (P-gp) transfer and whether AqMDRs can form stable drug-resistant strains. Drug-sensitive BIU-87 cells were co-cultured with doxorubicin (DOX)-resistant derivative BIU-87/DOX cells in transwell chambers for up to 96 h. The presence of P-gp in recipient cell membranes (AqMDRs) was detected by confocal microscopy, CCK-8, western blot, and RT-PCR were used to detect resistance index (RI), P-gp expression and MDR1 mRNA expression in AqMDRs after 0, 4, 8, 16, and 20 passages and frozen/resuscitated twentieth generation AqMDRs. There was an increase in P-gp transfer with longer co-culture times of drug-resistant and sensitive strains. Without DOX, although the AqMDR numbers increased with each passage, the RI and P-gp expression decreased gradually, and the expression level of MDR1 mRNA did not change significantly. With DOX, the RI and P-gp expression increased slightly, and the MDR1 mRNA expression level gradually increased to the BIU-87/DOX level. AqMDRs can grow stably at drug concentrations slightly higher than the IC50 of sensitive strains, which sensitive strains cannot survive. P-gp transfer between cells gradually increases with longer co-culturing of drug-resistant and sensitive strains. The drug resistance of AqMDRs decreases without drug intervention, but with drug intervention, cells can maintain resistance and gradually develop into stable drug-resistant cells. This article has an associated First Person interview with the first author of the paper.

Effects of anemoside B4 on pharmacokinetics of florfenicol and mRNA expression of CXR, MDR1, CYP3A37 and UGT1E in broilers.

Pulsatillae radix, a traditional Chinese medicine (TCM), is often used in combination with florfenicol for treatment of intestinal infection in Chinese veterinary clinics. Anemoside B4 (AB4) is the major effective saponin in Pulsatillae radix. This study aimed to investigate whether the pharmacokinetics of florfenicol in broilers was affected by the combination of AB4. In this study, broilers were given AB4 (50 mg/kg BW), or 0.9% sodium chloride solution by oral administration for 7 days. They were then fed florfenicol orally (30 mg/kg BW) on the eighth day. The results showed that the AUC(0-∞), MRT(0-∞), t1/2z and Cmax of florfenicol were significantly decreased, and the Vz/F and CLz/F were significantly increased by AB4; the mRNA expression levels of CXR, CYP3A37 and MDR1 (except CXR and CYP3A37 in the liver) were up-regulated by AB4. In conclusion, AB4 altered the pharmacokinetics of florfenicol, resulting in lower plasma concentrations of florfenicol, this was probably related to the mRNA expression of CXR, CYP3A37 and MDR1 in the jejunum and liver (except CXR and CYP3A37) increased by AB4. The implications of these findings on the effect of traditional Chinese medicine containing AB4 on the effectiveness of florfenicol in veterinary practice deserve study.

Effect of different concentrations of Gegen Qinlian decoction on expression of P-glycoprotein and multi-drug resistance protein transporter in Caco-2 cells

To investigate the effects of Gegen Qinlian decoction on the expression of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP) in epithelial cells of human colon adenocarcinoma Caco-2 cells．The effects of different concentrations of Gegen Qinlian decoction on the expression levels of p-gp and MRP1-6 mRNA in Caco-2 cells were detected by real time quantitative reverse transcriptase PCR (qRT-PCR).12 h after drug treatment (5.00 g·L⁻¹), the expression levels of MDR1 and MRP1-6 were significantly down-regulated at concentration of 5.00 g·L⁻¹; the mRNA expression levels of MDR1，MRP1，MRP2，MRP4，MRP5 and MRP6 were significantly down-regulated at concentration of 2.50 g·L⁻¹; only the expression levels of MRP2 and MRP5 were significantly affected at concentration of 1.00 g·L⁻¹. The results showed that the expression levels of MDR1 and MRP1-6 mRNA in Caco-2 cells could be down-regulated in a dose-dependent manner. Gegen Qinlian decoction may reduce drug efflux by down-regulating the mRNA expression of cell transporters in Caco-2 cell, and increase the time of drug action, thereby enhancing the bioavailability of chemotherapeutic drugs.

Achyranthis bidentatae radix enhanced articular distribution and anti-inflammatory effect of berberine in Sanmiao Wan using an acute gouty arthritis rat model

Ethnopharmacological relevanceSanmiao Wan (SMW) has been a basic prescription employed for the treatment for gout in the clinic since Yuan dynasty. Achyranthis bidentatae radix (ABR) is designed as a lower-guiding drug in SMW to augment the articular accumulation of active ingredients and improve the anti­inflammatory effect. Aim of the studyPresent study was undertaken to investigate the dose-response relationship of berberine in SMW between the articular concentration and anti-inflammatory effect in the knee joint under the lower-guiding of ABR. Materials and methodsRats were divided into control group, model group and SMW without or with low, medium and high doses of ABR groups. Rat model of acute gouty arthritis (AGA) was established by intra-articular injection of 0.2 mL monosodium urate crystal (20 mg/mL) inside knee joint cavity on day 2 during drug treatment slots. Knee joint swelling, synovial hyperplasia and inflammatory cell infiltration were investigated for anti-inflammatory study. The concentrations of berberine in rat plasma and tissues were determined by UPLC–MS/MS method. The effect of ABR on the expression levels of P-glycoprotein (P-gp) and MDR1 mRNA in the synovial tissues of knee joints in AGA rats was examined by Western blot and RT-qPCR assay, respectively. ResultsThe distribution of berberine increased by 6.53%, 44.31% and 212.96% in the knee joint and 474.93%, 631.01% and 1063.3% in the ankle for SMW with low, medium and high doses of ABR groups, compared with SMW without ABR group. Similarly, the plasma level of berberine increased by 19.81%, 143.4% and 681.13%. On the contrary, the distribution of berberine evidently decreased 3.23, 10.61 and 46.21-fold in heart and 3.68, 6.74 and 24.78-fold in lung. SMW with different doses of ABR groups exhibited better efficiency than SMW without ABR group on ameliorating knee joint swelling, inhibiting synovial hyperplasia and alleviating inflammatory cell infiltration of AGA rats. The treatment with ABR could down-regulate the MDR1 mRNA and P-gp expressions of synovial tissues of knee joints in AGA rats. ConclusionsThe enhanced articular distribution of berberine in SMW was attributed to the lower-guiding effect of ABR, which could evidently increase the plasma concentration of berberine, improve the supply of blood of inflamed joint, reduce the distribution of berberine in heart and lung and significantly inhibit the MDR1 mRNA and P-gp expression of synovial tissues of knee joints in AGA rats. The dose-response relationship of berberine between the enhanced articular concentration and improved anti-inflammatory effect in the knee joint under the lower-guiding of ABR was observed for the first time.

TRIB2 knockdown as a regulator of chemotherapy resistance and proliferation via the ERK/STAT3 signaling pathway in human chronic myelogenous leukemia K562/ADM cells.

Acquired resistance to chemotherapy plays a critical role in human drug treatment failure in many tumor types. Multidrug resistance (MDR) to Adriamycin (ADM) also limits the efficacy of therapy in human chronic myelogenous leukemia (CML). The overexpression of drug efflux transporters is one mechanism uderlying MDR. In particular, the consistent activation of MDR1 and MDR‑associated protein 1 (MRP1) is involved in drug resistance. In the present study, ADM‑resistant human CML K562/ADM cells were stably transfected with a Tribbles homolog 2 (TRIB2)‑targeted vector. A CCK‑8 assay showed that the half maximal inhibitory concentration (IC50) of ADM and the cell proliferation were lower in the transfected cells compared with that in the parental K562/ADM cells. The mRNA and protein expression levels of MDR1 and MRP1 were determined by reverse transcription‑polymerase chain reaction (RT‑PCR), RT‑quantitative PCR and western blot analysis. The results showed that the expression of MDR1 and MRP1 was significantly reduced in K562/ADM cells transfected with pGPU6/GFP/Neo‑TRIB2. Due to the downregulation of MDR1 and MRP1, the intracellular accumulation of ADM was increased in the transfected cells compared with that in the parental K562/ADM cells. Therefore, the sensitivity of the K562/ADM cells to ADM was enhanced and proliferation was inhibited. Our research revealed that protein expression of the ERK signaling pathway was inhibited by downregulating TRIB2, indicating that the ERK pathway was involved in cell drug resistance and proliferation. Furthermore, we used the ERK‑specific blocker U0126 to demonstrate this phenomenon. In summary, our research suggested that knockdown of TRIB2 could slow cell growth and reverse resistance, implying that TRIB2 is a potential therapy target for resistant human CML.