Modulation of copper transporters in protection against cisplatin-induced cochlear hair cell damage

Cisplatin belongs to platinum-based drugs and is widely used in cancer chemotherapy.Ototoxicity is one of the major dose limiting side-effects of cisplatin.For toxicity to occur cisplatin must first be transported from the bloodstream into cochlear cells.Three copper transporters are considered pathways for regulating the uptake and translocation of cisplatin into cells:Ctr1,ATP7A and ATP7B.Our recent study with cochlear organotypic cultures shows that cochlear hair cells can be destroyed by cisplatin at low concentrations from 10μm to 100μn.However,high doses of cisplatin cannot damage hair cells,maybe due to intrinsic feedback reactions that increase export of platinum by ATP7B when the platinum concentration is high in extracellular space.Cimitidine is a specific copper transporter inhibitor that can block the entrance of copper and platinum,and may prevent cisplatin-induced cochlear hair cell injury.To evaluate this hypothesis,we treated cochlear organotypic cultures with cisplatin (10 μm or 50 μm) alone,or cisplatin combined with cimitidine at concentrations ranging from 10-2000 μm for 48 hours.cisplatin at 10 μm damaged about 20% hair cells.In contrast,when cimitidine (10 μm,100 μm and 2000 μm) was added to the culture,near 100% cochlear hair cell survived.At higher concentration (50 μm),cisplatin destroyed about 80% of cochlear hair cells.However,100 μmcimitidine rescued about 50% hair cells from cisplatin damage,and 2000μm cimitidine protected about 80% hair cells.The data of western blot showed that CTR1 and ATP7B expressions were increased in cisplatin treated cochlear tissue,but cimitidine significantly reduced CTR1 and ATP7B.In addition,ATP7A expression was depressed a little after cisplatin treatment.Considering that Ctr1 is involved in copper and platinum influx,but the ATP7A and ATP7B are copper export transporters,the results suggest that cimitidine can effectively block the entrance by copper transporters and stop the influx of cisplatin.

OTOTOXIC MODEL OF OXALIPLATIN AND PROTECTION FROM NICOTINAMIDE ADENINE DINUCLEOTIDE

Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initial-ly increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demon-strated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20 mM NAD+. Treatment with 10 μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20 mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apop-totic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20 mM NAD+treatment great-ly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+provides signifi-cant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply.

Cisplatin-induced vestibular hair cell lesion-less damage at high doses

Cisplatin, a widely used anticancer drug, damages hair cells in cochlear organotypic cultures at low doses, but paradoxically causes little damage at high doses resulting in a U-shaped dose-response function. To determine if the cisplatin dose-response function for vestibular hair cells follows a similar pattern, we treated vestibular organotypic cultures with doses of cisplatin ranging from 10 to 1000 μM. Vestibular hair cell lesions progressively increased as the dose of cisplatin increased with maximum damage occurring around 50-100 μM, but the lesions progressively decreased at higher doses resulting in little hair cell loss at 1000 μM. The U-shaped doseresponse function for cisplatin-treated vestibular hair cells in culture appears to be regulated by copper transporters, Ctrl, ATP7A and ATP7B, that dose-dependently regulate the uptake, sequestration and extrusion of cisplatin.

Different gap junction-propagated effects on cisplatin transfer result in opposite responses to cisplatinin normal cells versus tumor cells

OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.

Pharmacogenomics of Cisplatin Sensitivity in Non-small Cell Lung Cancer

Cisplatin, a platinum-based chemotherapeutic drug, has been used for over 30 years in a wide variety of cancers with varying degrees of success. In particular, cisplatin has been used to treat late stage non-small cell lung cancer （NSCLC） as the standard of care. However, therapeutic outcomes vary from patient to patient. Considerable efforts have been invested to identify biomark- ers that can be used to predict cisplatin sensitivity in NSCLC. Here we reviewed current evidence for cisplatin sensitivity biomarkers in NSCLC. We focused on several key pathways, including nucleotide excision repair, drug transport and metabolism. Both expression and germline DNA variation were evaluated in these key pathways. Current evidence suggests that cisplatin-based treatment could be improved by the use of these biomarkers.