Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein

Abstract

The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp). Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays. The results indicate that potentiation of the cytotoxicity of Dox by Ro44-5912 was accompanied by significant increases in both, Dox-induced DNA ssbs and dsbs in the resistant cells. Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges. The majority of the newly synthesized DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells. Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase. In contrast, exposure to Dox alone resulted in temporary blockage of cells in G2/M phase (approximately 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation of BrdUrd by flow cytometric analysis was inhibited by Dox in the presence of Ro44-5912, showing that there is a block of DNA replication. An increased damage in newly synthesized DNA could concur with a blocked DNA replication. Moreover, slowing progression through the S-phase in cells exposed to Dox in combination with Ro44-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro44-5912 in cells expressing Pgp. The results suggest that drug-induced augmentation of nascent DNA fragmentation and specific cell-cycle perturbation are potentially important molecular determinants for reversal of multidrug resistance in addition to restoration of intracellular drug retention.

Multidrug resistance-associated biomarkers PGP, GST-π, Topo-II and LRP as prognostic factors in primary ovarian carcinoma

“…The expression of PGP is positively related to the observed drug resistance. 5 In the present study, the positive expression rate of PGP in normal tissues and benign tumour tissues was 16.7% and 18.8%, respectively. According to the findings of Holzamayer et al 5 the ovary is not rich in PGP.…”

“…5 In the present study, the positive expression rate of PGP in normal tissues and benign tumour tissues was 16.7% and 18.8%, respectively. According to the findings of Holzamayer et al 5 the ovary is not rich in PGP. From the data presented here, the positive expression rate of PGP was 57.5% (46/80 ovarian carcinoma patients), which suggests that PGP does not play a key role in mediating primary drug resistance in ovarian carcinoma.…”

Establishment of intraperitoneal transplantation model of cisplatin-resistant ovarian carcinoma cell in scid mice

“…In ovarian carcinoma tissues, the expression of MDR1 can be found by determination of mRNA and protein and the study on the correlativity between the expression of MDR1 and the development of ovarian carcinoma is gaining more and more attention [12] . P-gp is the expression product of MDR1 and a dose dependent pump.…”

顺铂作用卵巢癌细胞株的蛋白质组学研究

“…(1) 样品制备: 当贴壁生长于RPMI1640 培养液 中的人卵巢癌SKOV3 细胞处于生长对数期时, 将顺 铂以 6 μg/mL [17] (2) 某些点中混杂有少量其他蛋白, 相对分子量 与等电点具有相似性, 但丰度较低. [20] , 谷 胱甘肽S-转移酶活性增加 [21] , 拓扑异构酶活性降低 [22] , 及P53 表达水平下降、 bcl-2 表达水平升高 [23] 等有 关. 但是这些关于顺铂抗肿瘤作用机制与耐药机制 研究都限于个别基因或蛋白质分子, 在蛋白质组学 水平, 相关研究目前尚未见报道.…”