“Atypical” multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3)

Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are crossresistant to various other topoisomerase-II-targeted drugs but not to vinblastine. The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide (VP16)-induced DNA single-strand breaks, under deproteinizing conditions. In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity. No significant alterations in cellular drug uptake were observed in DX3 cells. Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantroneor VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance. Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal. However, an assay that specifically measures generation of 5-protein-linked breaks in³²P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16. These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drugcytotoxicity rather than loss of normal topoisomerase catalytic function. These data support the hypothesis that A2780-DX3 cells display an atypical multidrug resistance.Abbreviations MDR multidrug resistance - SSB Single-strand break     

Association between ABCB1, ABCG2 carrier protein and COX-2 enzyme gene polymorphisms and breast cancer risk in a Turkish population

AimBreast cancer is the most common cancer and the second leading cause of cancer-related deaths among women. Several genetic and environmental factors are known to be involved in breast cancer pathogenesis, but the exact etiology of this disease is complicated and not completely understood. We aimed to investigate whether the gene polymorphisms of ABCB1 and ABCG2 carrier proteins and COX-2 enzyme affect breast cancer risk.MethodABCG2 C421A (rs2231142), ABCB1 C3435T (rs1045642), COX-2 T8473C (rs5275) and COX-2 G306C (rs5277) were genotyped 104 breast cancer patients and 90 healthy controls using a real-time PCR for breast cancer susceptibility.ResultsPatients carrying ABCG2 C421A, the CC genotype, had a higher risk of disease compared with patients carrying any A allele (OR = 3.06; 95% CI = 1.49–6.25, p = 0.0019). The other variants showed no association with breast cancer (p > 0.05). Comparing the pathological parameters with the variants, only, the frequency of C allele of ABCB1 C3435T was significantly lower in the estrogen receptor-α (ERα) (OR = 2.25; 95% CI: 0.75–6.76; p = 0.041) and progesterone receptor (PgR) (OR = 3.67; 95% CI: 1.34–10.03; p = 0.008) positive breast cancer patients.ConclusionABCB1 C3435T and ABCG2 C421A might represent a potential risk factor for breast cancer for Turkish women.     

MDR1 gene expression and drug resistance of AML cells

We investigated the cellular drug resistance to aclarubicin (Acla), cytosine arabinoside (Ara-C), daunorubicin (Dau), doxorubicin (Dox), etoposide (Etop) and mitoxantrone (Mitox) using the MTT assay at time of disease presentation in 93 cases of acute myeloid leukaemia (AML). In 31 cases we concomitantly investigated MDR1 (multiple drug resistance 1 gene) expression (semi-quantitative competitive RT-PCR) of the leukaemic cells. Drug resistance towards Dau, Dox and Etop was correlated to the MDR1 expression of the AML cells ( P  < 0.05) with high MDR1 expression being associated with high drug resistance towards these drugs. Although the data did not allow firm conclusions to be drawn on the correlation between MDR1 expression and drug resistance towards Ara-C and Mitox, the drug resistance towards Acla clearly was not correlated to, or dependent on, the MDR1 expression level of the AML blast cells. In addition, when examining the cross-activities among the six drugs distinct patterns emerged. Thus, high to very high degrees of cross-activity were found to exist between Dau, Dox, Etop and Mitox, whereas Ara-C had moderate cross-activity with the other drugs except Acla, which showed absent to moderate cross-activity with the other drugs. We conclude that MDR1 gene expression is of significance for cellular drug resistance towards specific (MDR1-related) drugs in AML, whereas it is not of significance regarding drug resistance towards other drugs, which is the case with the anthracycline Acla. We suggest that in the place of other more or less complicated ways to circumvent MDR1-mediated drug resistance, Acla may be used to replace Dau, Dox and other MDR1-related drugs if proven as potent as the drug it is to substitute.     

Relationship between the expression of MDR1 in hepatocellular cancer and its biological behaviors

Objective: By the detection of HBV infection, AFP and AST, the targets of biological behavior and the gene expression of multi-drug resistance gene 1 (MDR1) in hepatocellular carcinoma (HCC), we investigate characteristics of the expression of MDR1 in HCC and its relationship with HCC biological behavior. Methods: Using real-time fluorescence quantitative PCR (FQ-PCR) to detect the expressions of MDR1 in 102 samples of HCC tissue and 20 samples of non-cancerous tissue, we analyze the relationship between expressions of MDR1 and biological characteristics of HCC. Results: The expression of MDR1 in HCC is 0.55±0.27, and in normal liver tissues is 0.23±0.10, respectively. The expression in HCC is higher than it in normal liver tissue, the difference is statistically significant (P<0.05) and the difference between the expression and the HCC envelopes is statistically significant, and the expression increases along with the increase of Edmondson classification (P<0.05). HBV infection, AFP positive, the rise of AST, all these factors have positive correlations with the expression (r=0.463, 0.473, 0.299). In MDR1 expressions of HCC patients, the survival curve of the negative is higher than that of the positive, but the difference is not statistically significant. Conclusion: There are drug resistance phenomena in HCC, MDR1 expression may play an important role in primary HCC drug resistance. HBV infection can be detected as a reference indicator of HCC chemotherapy resistance, plasma levels of AFP, AST can be used as a reference index change dynamic monitoring of MDR1 expression.     

Drug resistance pattern of Mycobacterium tuberculosis isolates from patients of five provinces of Iran

Objective:To determine the patterns of resistance to first line anti-tuberculosis(TB)drugs among a collection of Mycobacterium tuberculosis(MTB)isolates from 5 provinces of Iran.Methods:A total of the 6 426 clinical specimens from patients suspected of active TB were collected from March 2010 to June 2012.All specimens were subjected for microscopy and culture tests in the TB centers of studies provinces.Drug susceptibility testing to the first line anti-TB drugs for culture positive MTB was performed on Lwenstein-Jensen(LJ)medium using proportion method.Results:Of 6 426 clinical specimens,261 were culture positive for mycobacteria,of which 252 were MTB and 9 were MOTT(mycobacteria other than tuberculosis).Of 252 MTB isolates.211(83.7%)were pan-susceptible and 41(16.3%)were resistant to at least one drug.Resistance was most common to streptomycin.30 isolates(12.0%),followed by isoniuzid,20isolates(8.0%),rifampin,15 isolates(6.0%)and ethambutol,14 isolates(5.5%).Sixteen(6.3%)MTB isolates were MDR.A clear evidence of heterogeneity amongst the 5 provinces in the proportions with resistance to one or more drugs was observed[χ~2=12.209(4 degrees of freedom),P values=0.015 9].Conclusions:The prevalence of drug resistance in this study area underscoring the need for further enforcement of TB control strategies in the Iran.Drug susceptibility testing for all TB cases to provide optimal treatment,establishing advanced diagnostic facilities for rapid detection of MDR-TB and continuous monitoring of drug resistance are recommended for prevention and control of drug-resistant TB.     

多药耐药基因MDR1 C3435T基因多态性对根除幽门螺杆菌疗效的影响

目的研究多药耐药基因MDR1 C3435T基因多态性对质子泵抑制剂联合阿莫西林与克拉霉素三联1周疗法根除幽门螺杆菌（砀,）治疗的影响。方法选取101例却阳性的慢性胃炎或消化性溃疡患者,分成2组,分别进入埃索美拉唑联合阿莫西林与克拉霉素方案（EAC）或奥美拉唑联合阿莫西林与克拉霉素方案（OAC）进行1周根除治疗。采用聚合酶链反应-限制性内切片段长度多态性（polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP）的方法检测MDR1 C3435T基因型,比较不同基因型患者之间埤,根除率的差异。结果MDR1 CC3435、C3435T和3435TT的却根除率分别为72．4％、88．2％和81．0％。MDR1 C3435T各基因型组间却根除率比较均元显著性差异。结论MDR1 C3435T基因多态性与却根除疗效无显著相关性。     

Prediction of Human Brain Penetration of P-glycoprotein and Breast Cancer Resistance Protein Substrates Using In Vitro Transporter Studies and Animal Models

Four P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates with human cerebrospinal fluid (CSF) concentrations and preclinical neuropharmacokinetics were used to assess in vitro–in vivo extrapolation of brain penetration in preclinical species and the ability to predict human brain penetration. Unbound brain (C_b,u), unbound plasma (C_p,u), and CSF compound concentrations (C_CSF) were measured in rats and nonhuman primates (NHPs), and the unbound partition coefficients (C_b,u/C_p,u and C_CSF/C_p,u) were used to assess brain penetration. The results indicated that for P-gp and BCRP dual substrates, brain penetration was severally impaired in all species. In comparison, for P-gp substrates that are weak or non-BCRP substrates, improved brain penetration was observed in NHPs and humans than in rats. Overall, NHP appears to be more predictive of human brain penetration for P-gp substrates with weak or no interaction with BCRP than rat. Although C_CSF does not quantitatively correspond to C_b,u for efflux transporter substrates, it is mostly within 3-fold higher of C_b,u in rat and NHP, suggesting that C_CSF can be used as a surrogate for C_b,u. Taken together, a holistic approach including both in vitro transporter and in vivo neuropharmacokinetics data enables a better estimation of human brain penetration of P-gp/BCRP substrates.     

Effects of Various Pharmaceutical Excipients on the Intestinal Transport and Absorption of Sulfasalazine,a Typical Substrate of Breast Cancer Resistance Protein Transporter

Breast cancer resistance protein (BCRP) transporter is an efflux transporter that utilizes energy from adenosine triphosphate hydrolysis to push its substrates, regardless of the concentration gradient. Its presence on the apical membrane of the intestinal mucosa is a major obstacle for the intestinal absorption of its substrates. In this study, we examined the effects of various pharmaceutical excipients on the intestinal transport and absorption of sulfasalazine, a BCRP substrate. Four excipients, including 0.05% and 0.075% BL-9EX, 0.01% and 0.05% Brij 97, 0.075% Labrasol, and 0.05% and 0.1% Tween 20 decreased the secretory transport of sulfasalazine in an in vitro diffusion chamber. Further investigation in an in situ closed loop experiment in rats showed that 0.05% and 0.1% BL-9EX and 0.1% Brij 97 effectively enhanced the intestinal absorption of sulfasalazine while maintaining minimal toxicity to the intestinal mucosa. However, 0.1% Brij 97 also increased the intestinal absorption of 5(6)-carboxyfluorescein, a paracellular marker compound. These findings suggest that BL-9EX might effectively inhibit the BCRP-mediated efflux of sulfasalazine in vivo, indicating that BL-9EX could improve the intestinal absorption of sulfasalazine and other BCRP substrates.