Vickram Ramkumar

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.

Direct interaction of adenosine with the TRPV1 channel protein.

Vanilloid receptor 1 (TRPV1), a nonspecific cation channel expressed primarily in small sensory neurons, mediates inflammatory thermal pain sensation. The function and expression of TRPV1 are enhanced during inflammation and certain neuropathies, leading to sustained hyperalgesia. Activation of TRPV1 in the spinal cord and periphery promotes release of adenosine, which produces analgesia by activating A1 and A2A adenosine receptor (AR) on central and peripheral neurons. This study provides evidence of a direct interaction of AR analogs with TRPV1. Adenosine analogs inhibit TRPV1-mediated Ca2+ entry in human embryonic kidney (HEK293) cells stably expressing TRPV1 (HEK/TRPV1) and DRG neurons. This inhibition was independent of A2AAR activation. Specific binding of [3H]resiniferatoxin (RTX) in plasma membrane preparations was inhibited by CGS21680, an A2AAR agonist. Similar degrees of inhibition were observed with both agonists and antagonists of ARs. Adenosine analogs inhibited [3H]RTX binding to affinity-purified TRPV1, indicative of a direct interaction of these ligands with the receptor. Furthermore, specific capsaicin-sensitive binding of [3H]CGS21680 was observed in Xenopus oocyte membranes expressing TRPV1. Capsaicin-induced inward currents in DRG neurons were inhibited by adenosine and agonist and antagonist of A2AAR at nanomolar concentrations. Increasing the concentrations of capsaicin reversed the inhibitory response to capsaicin, suggesting a competitive inhibition at TRPV1. Finally, exposure of HEK/TRPV1 cells to capsaicin induced an ∼2.4-fold increase in proapoptotic cells that was abolished by adenosine analogs. Together, these data suggest that adenosine could serve as an endogenous inhibitor of TRPV1 activity by directly interacting with the receptor protein.

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.

Multiple components of the A1 adenosine receptor-adenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion.

The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.

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.

Up-regulation of adenosine receptors in the cochlea by cisplatin

In a previous study, we have demonstrated the presence of two adenosine receptor (AR) subtypes, namely A1 and A3AR, in the chinchilla cochlea. One or both of these receptors couple to activation of antioxidant enzymes, with resulting decreases in lipid peroxidation. The chemotherapeutic agent, cisplatin, was shown to produce ototoxicity within a few days of administration presumably by generating reactive oxygen species (ROS) and thereby increasing lipid peroxidation. In this study, we focused on whether lipid peroxidation induces hearing loss by assessing the cochlear antioxidant defense system over a shorter time period (24 h) following cisplatin administration. Cisplatin was administered to anesthetized chinchillas by round window membrane application and hearing loss was determined by compound action potential (CAP) and endocochlear potential (EP) 24 and 72 h post-treatment. Elevations in CAP thresholds in response to click and to 2, 4, 8 and 16 kHz tones and decreases in EP were obtained within 24 h of cisplatin treatment. These changes persisted for at least up to 72 h. Measurements of antioxidant enzymes indicate no change in the activities of superoxide dismutase, catalase or glutathione peroxidase, either 24 or 72 h following cisplatin treatment. The levels of malondialdehyde obtained at these time points were equivalent to those obtained from the controls. Furthermore, no difference in cochlear morphology was detectable by scanning electron microscopy at the basal, middle or apical turns of the cochlea within 24 h. By 72 h, however, losses in both inner and outer hair cells were observed in the basal and middle turns of the cochlea. A major finding of this study is that exposure to cisplatin led to a 5-fold up-regulation of [125I]N6-2-[4-amino-3-phenyl]ethyladenosine binding in the cochlea within 24 h, reflecting increases in expression of AR(s) in this tissue. These data indicate a dissociation between cisplatin acute (within 24 h) ototoxicity and lipid peroxidation. Furthermore, up-regulation of AR(s) may represent a rapid compensatory mechanism by the cochlea to counter the toxic effects of increased ROS generated by cisplatin.