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ABSTRACT Our previous work revealed that chrysosplenetin in combination with artemisinin inhibited in vivo P-glycoprotein (P-gp, one of classic multi-drug resistance proteins) mediated digoxin transportation activity by reversing the upregulated P-gp/Mdr1 mRNA expression levels by artemisinin. Therefore, chrysosplenetin might be a potential artemisinin-resistance reversal agent as a P-gp inhibitor. But it still remains unknown if chrysosplenetin has an impact on another pivotal multi-drug resistance protein, breast cancer resistance protein (Bcrp), which is co-expressed with P-gp in apical membrane of intestinal epithelial cell and overlaps some of the substrates and inhibitors. This study, therefore, further addressed the impact of chrysosplenetin, per se or in combination with artemisin, on Bcrp/ABCG2 mRNA expression levels in mice small intestine determined by western blot and real time-quantitative polymerase chain reaction (RT-qPCR) assay. The drugs were intragastrically administrated once per day for 7 days. Novobiocin, a known Bcrp inhibitor, was observed to have no impact on Bcrp/ABCG2 levels with or without artemisinin versus vehicle. Interestingly, artemisinin alone attenuated Bcrp level while chrysosplenetin alone increased it (p < 0.05). Relative mRNA level was significantly decreased when co-used with artemisinin and chrysosplenetin in ratio of 1:2 (p < 0.05). The discrepant results for chrysosplenetin on Bcrp/ABCG2 mRNA expressions might be closely related to the transcriptional or posttranscriptional regulation.
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ABSTRACT The present study describes the impact of chrysosplenetin, in the absence and presence of artemisinin, on in vitro breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate. We observed that novobiocin, a known breast cancer resistance protein active inhibitor, increased Papp (AP-BL) of aristolochic acid I 3.13 fold (p < 0.05) but had no effect on Papp (BL-AP). Efflux ratio (PBA/PAB) declined 4.44 fold (p < 0.05). Novobiocin, consequently, showed a direct facilitation on the uptake of AAI instead of its excretion. Oppositely, both artemisinin and chrysosplenetin alone at dose of 10 µM significantly decreased Papp (BL-AP) instead of Papp (AP-BL). Chrysosplenetin alone attenuated the efflux ratio, which was suggestive of being as a potential breast cancer resistance protein suppressant. Oddly, Papp (BL-AP) as well as efflux ratio were respectively enhanced 2.52 and 2.58 fold (p < 0.05), when co-used with artemisinin and chrysosplenetin in ratio of 1:2. The potential reason remains unclear; it might be relative to binding sites competition between artemisinin and chrysosplenetin or the homodimer/oligomer formation of breast cancer resistance protein bridged by disulfide bonds, leading to an altered in vitro breast cancer resistance protein-mediated efflux transport function.
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ABSTRACT INTRODUCTION: Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. OBJECTIVE: The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. METHODS: A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(r) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNASerUCN gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. RESULTS: We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNASerUCN gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. CONCLUSION: Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype.
Resumo Introdução: Diversas mutações do DNA mitocondrial tem sido descritas, em diferentes famílias, associadas à deficiência auditiva não sindrômica. No entanto, pouco se sabe sobrea prevalência dessas mutações em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica. Objetivo: A finalidade do nosso estudo foi investigar a incidência dessas mutações no DNA mitocondrial nessa população. Método: No total, 178 pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica foram recrutados para participação no estudo. O DNA genômico foi extraído de amostra, de sangue periférico. Utilizamos o método de sequenciamento SNaPshot(r) para detecção de cinco mutações do DNA mitocondrial: A1555G e A827G no gene 12S rRNA e A7445G, 7472insCe T7511C no gene tRNASerUCN. Paralelamente, utilizamos a reação de polimerase em cadeia e sequenciamos os produtos para triagem das mutações no gene GJB2 nos pacientes portadores de mutações no DNA mitocondrial. Resultados: Em nossa população, não conseguimos detectar a presença da mutação A1555G no gene 12S rRNA e nem as mutações A7445G, 7472insC e T7511C no gene tRNASerUCN. Entretanto, constatamos que seis pacientes (3,37%) eram portadores da mutação homozigota A827G; e um deles também portava a mutação homozigota GJB2 235delC. Conclusão: Nossos achados no presente estudo indicam que, mesmo em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica, as mutações do DNA mitocondrial também podem contribuir para o fenótipo clínico.