Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

Regulatory effect of resveratrol and prednisolone on MDR1 gene expression in acute lymphoblastic leukemia cell line (CCRF-CEM): An epigenetic perspective.

Chemotherapy is the most common method to treat leukemia as well as other types of human cancers. However, drug resistance has remained as the main challenge against the efficacy of treatments. Furthermore, having various adverse effects, chemotherapy drugs are becoming replaced by natural modalities for cancer therapy. In this regard, herbal components such as resveratrol and prednisolone have been identified to sensitize the leukemic cells to programmed cell death through a set of complex processes. In this study, we have examined DNA methylation on the human multidrug resistance gene 1 (MDR1) as a well-known marker for cellular drug resistance. We evaluated the effect of resveratrol and prednisolone on DNA methylation patterns of MDR1 gene promoter in the CCRF-CEM cell line as a representative for acute lymphoblastic leukemia. The study was aimed to clarify whether the MDR1 gene expression is regulated via DNA promoter methylation as a potential underlying mechanism, following exposure to resveratrol and prednisolone. Our data revealed that despite a strong influence to down-regulate the MDR1 expression, Resveratrol and Prednisolone did not alter the methylation pattern, suggesting other regulatory mechanisms in controlling the MDR1 expression in CCRF-CEM cell line. Unchanged status of DNA methylation of MDR1 gene may suggest that Resveratrol and Prednisolone causes the gene expression changes through a distinct mechanism which requires further studies to be understood. A more detailed understanding of the mechanisms beyond the regulation of the genes involved in cancer formation will help to design novel therapeutic strategies to fight the human cancers.

The effects of Astragalus polysaccharides, tragacanthin, and bassorin on methotrexate-resistant acute lymphoblastic leukemia.

Background and purpose: One strategy to overcome methotrexate (MTX) resistance in acute lymphoblastic leukemia is suppressing MDR1 expression. It has been proved Astragalus polysaccharides (APS) exert their anticancer effect by reversing drug resistance. Due to the structural similarity of tragacanthin and bassorin with APS, we aimed to investigate the effects of the aforementioned polysaccharides on the expression of the MDR1 gene in the MTX-treated CCRF-CEM cells. Experimental approach: Cytotoxicity of APS, bassorin, and tragacanthin on CCRF-CEM, CCRF-CEM/MTX (cells treated with MTX at IC50), and CCRF-CEM/R cells (CCRF-CEM cells resistant to MTX) was evaluated by MTT assay. The effect of all three compounds on MDR1 expression was evaluated using RT-PCR. Findings/Results: All the concentrations of tragacanthin, bassorin, and APS (except at 0.8-100 µg/mL in CCRF-CEM) decreased the viability of all the cells compared to the negative control group; and against the positive control (MTX-treated cells), only bassorin at 20-100 µg/mL in CCRF-CEM/R and tragacanthin at 50 and 100 µg/mL in CCRF-CEM/MTX and at 2-100 µg/mL in CCRF-CEM/R decreased cell viability. Tragacanthin diminished MDR1 expression in CCRF-CEM/MTX and CCRF-CEM/R cells, which MTX had already induced. Conclusion and implication: According to the results of this study, tragacanthin was a potent cytotoxic agent against CCRF-CEM cells and enhanced the chemosensitivity of CCRF-CEM/MTX and CCRF-CEM/R cells to MTX by down-regulation of MDR1 gene expression. Therefore, it could be a promising compound against cancer. Other possible mechanisms of action of tragacanthin should be evaluated and further in vitro and in vivo investigations are required.

[Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin].

OBJECTIVE: To investigate the effects of down-regulating MDR1 gene expression by CRISPRi on enhancing the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin. METHODS: The potential CRISPRi interference sites on the MDR1 gene promoter were predicted by bioinformatics software, and the interference fragments were designed and constructed. The mRNA and protein expression levels of MDR1 gene in each group of cells were detected by qRT-PCR and Western blot methods, and the recombinant vectors with high interference efficiency were screened. Human lung cancer A549/DDP cells were divided into three groups: A549/DDP, Scrambed and sgRNA-MDR1-1, with three multiple holes in each group. After each vector was transfected into the cells for 48 h, the efflux of cells in each group was detected by flow cytometry, the IC50 value of cells in each group was detected by MTT method, and the cell morphology of cells treated with cisplatin was observed under laser confocal microscope. RESULTS: After sequencing and comparison, two kinds of CRISPRi recombinant vectors interfering with MDR1 gene transcription were constructed successfully. After transfection of A549/DDP cells, the mRNA and protein levels of MDR1 gene in all transfection groups were decreased significantly (P＜ 0.01). Among them, the interference efficiency of sgRNA-MDR1-1 was the highest, and the interference efficiency of mRNA and protein was 60% and 51%, respectively. After transfection of sgRNA-MDR1-1 vector, compared with the control group, the efflux ability of cells was decreased (P＜0.01), the IC50 value of cells to cisplatin was decreased significantly (P＜0.01), and the intracellular chromatin gathered and marginalized, and apoptotic bodies appeared. CONCLUSION: CRISPRi interference with MDR1 gene in drug-resistant A549/DDP cells can significantly enhance the sensitivity to cisplatin.

The Correlation Between MDR1 Gene Polymorphism and Clopidogrel Resistance in People of the Hui and Han Nationalities.

To investigate the differences in the correlation between multidrug resistance protein 1 (MDR1) (ABCB1) gene polymorphism and clopidogrel resistance in patients of the Hui and Han nationalities with percutaneous coronary intervention (PCI). A total of 377 subjects (154 people of Hui nationality, 223 people of Han nationality) with PCI were enrolled in the study. Each patient's platelet aggregation rate was induced by adenosine diphosphate and measured using light turbidimetry. Based on the results, the patients were divided into two groups: a clopidogrel resistance (CR) group and a non-clopidogrel resistance (NCR) group. Restrictive fragment-length polymorphism polymerase chain reaction technology was then used to determine the genotype and alleles at two loci (C3435 T[rs1045642] and C1236 T[rs1128503]), calculate the frequencies of the genotype and alleles at these two loci, and conduct correlation analysis. The incidence rate of clopidogrel resistance was 23.4%, and the frequencies of the TT genotype and T allele at C3435 T for patients of both nationalities were significantly higher in the CR group than in the NCR group (P < 0.05). There were no significant differences between the two groups in genotype or allele frequency at C1236 T. There was a significant difference in the distribution of C1236 T polymorphism between the two nationalities (P < 0.05), but there was no significant difference between the two nationalities in C3435 T polymorphism. Patients with a T allele at MDR1 C3435 T are more likely to show clopidogrel resistance, and no significant differences were identified in C3435 T gene polymorphism between the two nationalities.

Assessment of potential drug-drug interactions among outpatients in a tertiary care hospital: focusing on the role of P-glycoprotein and CYP3A4 (retrospective observational study).

Background: Selecting a medicine has a significant impact on the quality of therapy including efficacy and safety. P-glycoprotein and CYP3A4 share several common substrates known as bi-substrates. Both play major role in the pharmacokinetics and pharmacodynamics when over or under expressed. Objective: The study aimed to assess the Drug-Drug Interaction (DDI) related to P-glycoprotein (P-gp) and Cytochrome P450-3A4 (CYP3A4), to predict their clinical outcomes and also to discover prospective predictors of pDDIs. Methods: The subjects in this retrospective study ranged in age from 18 to 95 years with polypharmacy prescriptions. Information was gathered through patient medical records. Based on Micromedex and previous literature studies, medications prescribed to the patients were observed for pDDIs according to risk rating scale for drug interactions. Results: A total of 504 patients (160 males and 344 females) were included in the study. The mean of pDDI seen in the patients was 1.66 ± 1.48 and total 825 pDDIs were discovered. The factors significantly associated with having ≥1 pDDIs included: taking ≥5 medicines (OR 1.747), increased age (OR 1.026) increased comorbidities (OR 1.73). Conclusion: In prescriptions, a considerable number of probable DDI were discovered. Therefore, careful selection of drugs and identification of mechanisms for DDI is needed to lower the frequency of pDDI.

Reversion of Multidrug-Resistance by Proteasome Inhibitor Bortezomib in K562/DNR Cell Line.

OBJECTIVE: To observe the reversion of multi-drug resistance by proteasome inhibitor bortezomib in K562/DNR cell line and to analyze the possible mechanism of reversion of multidrug-resistance. METHODS: MTT method was used to determine the drug resistance of K562/DNR cells and the cellular toxicity of bortezomib. K562/DNR cells were cultured for 12 hours, 24 hours and 36 hours with 100 µg/ml DNR only or plus 4 µg/L bortezomib. The expressions of NF-κB, IκB and P-gp of K562/DNR were detected with Western blot method, the activity of NF-κB was tested by ELISA method and the apoptosis rate was observed in each group respectively. RESULTS: The IC50 of DNR on cells of K562/S and K562/DNR groups were 1.16 µg/ml and 50.43 µg/mL, respectively. The drug-resistant fold was 43.47. The IC10 of PS-341 on Cell strain K562/DNR was 4 µg/L. Therefore, 4 µg/L was selected as the concentration for PS-341 to reverse drug-resistance in this study. DNR induced down-regulation of IκB expression, up-regulation of NF-κB and P-gp expression. After treatment with PS-341, a proteasome inhibitor, the IκB degradation was inhibited, IκB expression increased, NF-κB and P-gp expression decreased in a time dependent manner. Compared to DNR group, the NF-κB p65 activity of DNR+PS-341 group was decreased. Compared to corresponding DNR group, DNR induced apoptosis rate increases after addition of PS-341 in a time dependent manner. CONCLUSION: Proteasome inhibitor bortezomib can convert the leukemia cell drug resistance. The mechanism may be that bortezomib decreases the degradation of IκB and the expression of NF-κB and P-gp, therefore induces the apoptosis of multi-drug resistant cells.

Novel genotyping assay for the nt230 (del4) ABCB1 gene mutation and its allele frequency in Border Collie dogs in Mexico.

A 4-bp deletion in the ATP-binding cassette subfamily B member 1 (ABCB1) gene, also referred to as the multidrug resistance gene (MDR1), produces stop codons that cause premature termination of P-glycoprotein 1 (P-gp) synthesis. Dogs with the homozygous mutation do not express functional P-gp, which increases their sensitivity markedly to many common veterinary drugs. We detected the nt230 (del4) ABCB1 mutation in Border Collie dogs in western Mexico with a simple and affordable primer-introduced restriction analysis PCR (PIRA-PCR). PIRA-PCR clearly identified all genotypes in our sample of 104 dogs. Genotype frequencies were 0.952 (wild/wild), 0.029 (wild/mut) and 0.019 (mut/mut). Allele frequencies were 0.033 (mutant alleles) and 0.966 (wild-type alleles). In this small subset of the Mexican dog population, we found a higher prevalence of the nt230 (del4) MDR1/ABCB1 gene mutation than reported in other countries.

A Study on the Correlation Between MDR1 Polymorphism and Clopidogrel Resistance in Hui Patients Treated with Percutaneous Coronary Intervention.

OBJECTIVE: This study assesses the correlation between MDR1 gene polymorphism and clopidogrel resistance (CR) in Hui patients with coronary heart disease (CHD) who were treated with percutaneous coronary intervention (PCI). METHODS: The study includes 204 Ningxia Hui patients with CHD who were treated with PCI. These patients were divided into two groups: those who with CR and others were non-clopidogrel resistant (NCR), according to the results of the patients' platelet aggregation rate, which was tested by adenosine diphosphate-induced turbidimetry on the second postoperative day. C3435T and C1236T genotypes and alleles were tested by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The CR rate was 24.0%, and there were 3 genotypes of C3435T and C1236T. For C3435T, the distribution frequency of the 3435TT genotype and T allele was significantly higher in the CR group than in the NCR group. For C1236T, no significant difference was found between the two groups. CONCLUSION: Hui patients who had CHD were treated with PCI. CR was most likely to occur in those who had the T allele of MDR1 in gene C3435T.

Folate-Equipped Cationic Liposomes Deliver Anti-MDR1-siRNA to the Tumor and Increase the Efficiency of Chemotherapy.

In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O'-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action.