Debashree Mukherjea

Mechanisms of cisplatin-induced ototoxicity and prevention.

Cisplatin is a widely used chemotherapeutic agent to treat malignant disease. Unfortunately, ototoxicity occurs in a large percentage of patients treated with higher dose regimens. In animal studies and in human temporal bone investigations, several areas of the cochlea are damaged, including outer hair cells in the basal turn, spiral ganglion cells and the stria vascularis, resulting in hearing impairment. The mechanisms appear to involve the production of reactive oxygen species (ROS), which can trigger cell death. Approaches to chemoprevention include the administration of antioxidants to protect against ROS at an early stage in the ototoxic pathways and the application of agents that act further downstream in the cell death cascade to prevent apoptosis and hearing loss. This review summarizes recent data that shed new light on the mechanisms of cisplatin ototoxicity and its prevention.

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but independent of miR-21.These data suggest that resveratrol’s anti-tumor actions in prostate cancer could be explained, in part, through inhibition of Akt/miR-21 signaling pathway.

Transtympanic Administration of Short Interfering (si)RNA for the NOX3 Isoform of NADPH Oxidase Protects Against Cisplatin-Induced Hearing Loss in the Rat

Cisplatin produces hearing loss in cancer patients. Reactive oxygen species (ROS) in the cochlea leads to lipid peroxidation, death of outer hair cells (OHCs), and hearing loss. The cochlea expresses a unique isoform of NADPH oxidase, NOX3, which serves as the primary source of ROS generation in the cochlea. Inhibition of NOX3 could offer a unique protective target against cisplatin ototoxicity. Here, we document that knockdown of NOX3 using short interfering (si) RNA abrogated cisplatin ototoxicity, as evidenced by protection of OHCs from damage and reduced threshold shifts in auditory brainstem responses (ABRs). Transtympanic NOX3 siRNA reduced the expression of NOX3 in OHCs, spiral ganglion (SG) cells, and stria vascularis (SV) in the rat. NOX3 siRNA also reduced the expression of transient receptor potential vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1), biomarkers of cochlear damage. Also, transtympanic NOX3 siRNA reduced the expression of Bax, abolished the decrease in expression of Bcl2, and reduced apoptosis induced by cisplatin in the cochlea. These data suggest that NOX3 regulates stress-related genes in the cochlea, such as TRPV1 and KIM-1, and initiates apoptosis in the cochlea. This appears to be the first study of the efficacy of transtympanic delivery of siRNA attenuating cisplatin ototoxicity.

Short Interfering RNA against Transient Receptor Potential Vanilloid 1 Attenuates Cisplatin-Induced Hearing Loss in the Rat

Cisplatin, a chemotherapeutic agent of choice for the treatment of solid tumors, produces hearing loss in approximately half a million new cancer patients annually in the United States. The hearing loss is due, in part, to increased generation of reactive oxygen species (ROS) in the cochlea, leading to lipid peroxidation and damage or death of outer hair cells in the organ of Corti. The cochlea expresses the transient receptor potential vanilloid 1 (TRPV1), which are normally expressed on small diameter neurons in the peripheral nervous system and mediate thermal sensitivity, but whose role in the cochlea is unclear. In this study, we show that TRPV1 is coregulated along with the NADPH oxidase isoform, NOX3, by cisplatin. Induction of these proteins by cisplatin is dependent on ROS generation, since it is reversed by systemic lipoic acid administration. In organ of Corti hair cell cultures (UB/OC-1 cells), cisplatin activates and induces TRPV1 and NOX3, leading to apoptosis of these cells. Inhibition of TRPV1 by capsazepine or ruthenium red reduced the apoptosis, implicating TRPV1 in this process. Treatment of UB/OC-1 cultures with short interfering RNA (siRNA) against either TRPV1 or NOX3 reduced cisplatin-induced apoptosis, while round window application of TRPV1 siRNA to rats reduced TRPV1 expression, decreased damage to outer hair cells and reduced cisplatin-induced hearing loss. These data provide a link between NOX3 and TRPV1 in cisplatin-induced hearing loss and suggest that targeting these proteins for knockdown by siRNA could serve as a novel approach in treating cisplatin ototoxicity.

Adenosine receptors: expression, function and regulation.

Adenosine receptors (ARs) comprise a group of G protein-coupled receptors (GPCR) which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF)-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined.

Essential role of Rac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression

Nerve growth factor (NGF) regulates the nociceptive properties of a subset of small diameter sensory neurons by increasing the expression of the heat‐sensing transient receptor potential (TRP) channel, TRPV1. This action involves activation of the tyrosine kinase receptor (Trk) A/p38 MAPK pathway. Recent studies indicate that activation of TrkA promotes superoxide generation via NADPH oxidase. In this study, we determined whether the NADPH oxidase pathway is involved in NGF‐stimulated TRPV1 expression using a rat pheochromocytoma 12 line and rat dorsal root ganglion neurons. Treatment of these cells with NGF (100 ng/mL) increased TRPV1 protein expression (approx. twofold) but not mRNA. This increase was mimicked by H2O2 and attenuated by catalase and inhibitors of NADPH oxidase. NGF stimulated NADPH oxidase activity, while 24 h exposure further increased expression of the Rac1 and gp91phox subunits of the holoenzyme. Inhibition of NADPH oxidase by transient transfection of a dominant negative Rac1 mutant (RacN17) plasmid blocked NGF‐stimulated TRPV1 protein expression, while expression of a constitutively active Rac1 increased basal and NGF‐stimulated TRPV1 levels. Inhibition of NADPH oxidase activity also attenuated NGF‐dependent p38 MAPK activation. We conclude that the Rac1/NADPH oxidase pathway regulates p38 activation and TRPV1 expression which aids in the maintenance of peripheral neuron integrity and pain perception.

Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation.

Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients.

Expression of the kidney injury molecule 1 in the rat cochlea and induction by cisplatin

Cisplatin is a widely used chemotherapeutic agent whose dose-limiting side effects include ototoxicity and nephrotoxicity. Recent evidence indicates that cisplatin induces the expression of a novel protein, kidney injury molecule-1, in the renal proximal tubular epithelium to aid in regeneration. In this study, we determined whether kidney injury molecule-1 is expressed in the cochlea and is induced by cisplatin. Using reverse transcriptase polymerase chain reaction techniques, we have now identified kidney injury molecule-1 in the rat cochlea and in three different mouse transformed hair cell lines. Administration of cisplatin to rats produced hearing loss and induced kidney injury molecule-1 mRNA in the rat cochlea. Pretreatment of rats with lipoic acid, a scavenger of reactive oxygen species, significantly reduced cisplatin-induced hearing loss and kidney injury molecule-1 expression. Cisplatin also increased the expression of cochlear NOX3 mRNA, a member of the superoxide generating NADPH oxidase family of proteins recently identified in the cochlea, inhibition of which decreased kidney injury molecule-1 expression. Polymerase chain reaction performed on different regions of the cochlea indicated the presence of kidney injury molecule-1 mRNA in the lateral wall, organ of Corti and spiral ganglion. This distribution was confirmed by immunocytochemistry. Taken together, these data identify kidney injury molecule-1 as a novel cochlear injury molecule, whose expression is regulated by reactive oxygen species generated via the NADPH oxidase pathway.

Pharmacogenomics of cisplatin-induced ototoxicity

Cisplatin ototoxicity affects different individuals in a widely variable manner. These variations are likely to be explained by genetic differences among those affected. It would be highly advantageous to identify genetic variants that predispose to cisplatin ototoxicity in order to minimize the risk to susceptible subgroups. Although this area of research is very important, only a few studies have rigorously examined the genetic basis for cisplatin-induced susceptibility to hearing loss. This article addresses recent progress in clarifying the incidence of cisplatin ototoxicity and the risk factors and controversies regarding the identification of genetic variants associated with cisplatin-induced hearing loss.

TRPV1: A Potential Drug Target for Treating Various Diseases

Transient receptor potential vanilloid 1 (TRPV1) is an ion channel present on sensory neurons which is activated by heat, protons, capsaicin and a variety of endogenous lipids termed endovanilloids. As such, TRPV1 serves as a multimodal sensor of noxious stimuli which could trigger counteractive measures to avoid pain and injury. Activation of TRPV1 has been linked to chronic inflammatory pain conditions and peripheral neuropathy, as observed in diabetes. Expression of TRPV1 is also observed in non-neuronal sites such as the epithelium of bladder and lungs and in hair cells of the cochlea. At these sites, activation of TRPV1 has been implicated in the pathophysiology of diseases such as cystitis, asthma and hearing loss. Therefore, drugs which could modulate TRPV1 channel activity could be useful for the treatment of conditions ranging from chronic pain to hearing loss. This review describes the roles of TRPV1 in the normal physiology and pathophysiology of selected organs of the body and highlights how drugs targeting this channel could be important clinically.