Abstract
Background: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. Conclusions: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.
Highlights
Cisplatin (CDDP) is a well-known platinum coordination compound used as a drug in cancer therapy usually in combination with other drugs
We demonstrated that RAB7A is downregulated in resistant cells and that RAB7A
Intensity quantification revealed that C13 cells have about 35% and 63% reduction of LysoSensor DND-160 and DND-167 staining, respectively, compared to 2008 cells (Figure 1B)
Summary
Cisplatin (CDDP) is a well-known platinum coordination compound used as a drug in cancer therapy usually in combination with other drugs. Cancers 2019, 11, 52 bladder and lung cancer Side effects such as nephrotoxicity and peripheral neurotoxicity, and intrinsic or acquired resistance greatly limit its use [2]. For these reasons, numerous platinum derivatives have been developed, their success has been restricted to few compounds [2]. The main obstacle to an effective reduction of toxicity and resistance is the complexity, and the still limited knowledge, of uptake, efflux, trafficking and mechanisms of biodistribution and action of CDDP and platinum derivatives [1,3]. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases
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