Novel drug interaction for improving intracellular accumulation and efficacy of chemotherapeutics in retinoblastoma and mitochondrial localization of transporters in corneal epithelial cells

Chemoreduction is the key treatment for retinoblastoma management. However, anticancer agents suffer from poor cell permeability and chemoresistance due to major interaction with multidrug resistant (MDR) efflux proteins (MDR1 and MRP2) over-expressed on retinoblastoma tumor. Ocular cells have shown good tolerability against antimicrobial moxifloxacin (fourth generation fluoroquinolone) at higher dose level. As majority of fluoroquinolones become ineffective due to efflux mediated acquired drug resistance therefore, we have first determined an interaction of moxifloxacin with efflux (MDR1 and MRP2) transporters. Our results provides direct evidence that moxifloxacin is an overlapping substrate of MDR1 and MRP2 efflux transporters. Since, literature has evident co-administration of overlapping drug substrate as a viable strategy to overcome MDR. Therefore, we have tested moxifloxacin potential to overcome chemoresistance in retinoblastoma. Our results showed that 2-2.5 fold increased uptakes of three anticancer drugs (etoposide, topotecan and vinblastine) were observed in the presence of moxifloxacin across model cell lines suggesting moxifloxacin mediated evasion of efflux pumps. Significant reductions in efflux ratio of these anticancer agents were also observed in the presence of moxifloxacin indicating moxifloxacin mediated improved anticancer transport. Following cytotoxicity study, tenfold reduction in IC50 value of topotecan and etoposide and twofold reduction in IC50 value of vinblastine was observed in the presence of moxifloxacin. Significant enhancement of anticancer mediated caspase-3 enzyme activity and reduction in proinflammatory cytokines (IL-8 and IL-6) release were also observed in the presence of moxifloxacin. There is a need to further explore this finding to confirm its clinical feasibility, if proven, ultimately it will aid to reduce the chemotherapeutic dose and associated dose-limiting toxicities. Considering the fact that most drug targets are indeed contained within specific intracellular compartments, the ability of a drug to accumulate into these sites is a critical determinant in the observed drug response. Mitochondrion is an attractive target for drug-delivery because there is a growing confirmation to support an association between mitochondrial dysfunctions and a number of ocular diseases. A range of possibilities exist for the selective drug delivery to the mitochondria one among such is a transporter targeted strategy. Localization of various influx and efflux transporters is generally considered to be restricted to cell surface level. However, cellular compartments may also be a potential site for these transporters. The functional localization of both efflux (MDR1) and influx (PepT-1) transporters in the mitochondrial membrane of primary corneal epithelial cells was studied. Isolation and purification of mitochondria was performed by optimized cell fractionation method. Functional activity of MDR1 and PepT-1 transporter was assessed by performing in vitro uptake studies on isolated mitochondria. Molecular characterization of both transporters was confirmed by western blot and confocal analysis. This knowledge of mitochondrial existence of MDR1 and PepT-1transporter may aid in the development of subcellular ocular drug delivery strategies.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Pharmacy (UMKC)

Grantor dc:publisher

University of Missouri--Kansas City

Year dc:date.issued

2013

Author dc:creator

• Barot, Megha

Advisor dc:contributor.advisor

• Mitra, Ashim K., 1954-

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10355/33232

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/33232