Paclitaxel inhibits the activity and membrane localization of PKCα and PKCβI/II to elicit a decrease in stimulated calcitonin gene-related peptide release from cultured sensory neurons

Peripheral neuropathy is a dose-limiting and debilitating side effect of the chemotherapeutic drug, paclitaxel. Consequently, elucidating the mechanisms by which this drug alters sensory neuronal function is essential for the development of successful therapeutics for peripheral neuropathy. We previously demonstrated that chronic treatment with paclitaxel (3–5 days) reduces neuropeptide release stimulated by agonists of TRPV1. Because the activity of TRPV1 channels is modulated by conventional and novel PKC isozymes (c/nPKC), we investigated whether c/nPKC mediate the loss of neuropeptide release following chronic treatment with paclitaxel (300 nM; 3 and 5 days). Release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Following paclitaxel treatment, cultured dorsal root ganglia sensory neurons were stimulated with a c/nPKC activator, phorbol 12,13-dibutyrate (PDBu), or a TRPV1 agonist, capsaicin, in the absence and presence of selective inhibitors of conventional PKCα and PKCβI/II isozymes (cPKC). Paclitaxel (300 nM; 3 days and 5 days) attenuated both PDBu- and capsaicin-stimulated release in a cPKC-dependent manner. Under basal conditions, there were no changes in the protein expression, phosphorylation or membrane localization of PKC α, βI or βII, however, paclitaxel decreased cPKC activity as indicated by a reduction in the phosphorylation of cPKC substrates. Under stimulatory conditions, paclitaxel attenuated the membrane translocation of phosphorylated PKC α, βI and βII, providing a rationale for the attenuation in PDBu- and capsaicin-stimulated release. Our findings suggest that a decrease in cPKC activity and membrane localization are responsible for the reduction in stimulated peptide release following chronic treatment with paclitaxel in sensory neurons

Role of the DNA base excision repair protein, APE1 in cisplatin, oxaliplatin, or carboplatin induced sensory neuropathy.

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN

Platinum-induced phosphorylation of H2AX in sensory neuronal cultures is increased by reducing APE1 expression.

<p>The top panels show representative Western blots of phospho-H2AX (P-H2AX) and actin from cultures prior to and after 8, 24 and 48 hours of exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). Cultures were exposed to SCsiRNA or APE1siRNA as indicated. The bottom panels represent the densitometry of P-H2AX expression normalized to actin from three independent experiments. The columns represent the mean ± SEM from cultures treated with SCsiRNA (lightly shaded columns) or APE1siRNA (heavy shaded columns) prior to or after exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). An asterisk indicates a statistically significant increase in P-H2AX density in cells treated with APE1siRNA compared to those treated with SCsiRNA. Cisplatin data can be found in our previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106485#pone.0106485-Jiang1" target="_blank">[24]</a>.</p

Reducing APE1 expression enhances the ability of oxaliplatin but not carboplatin to reduce capsaicin-evoked release of CGRP from sensory neurons in culture.

<p>Each column represents the mean ± SEM of CGRP release in fmol/well/min for untreated sensory neurons in culture (controls) or cultures treated with SCsiRNA or APE1siRNA as indicated. Cells were exposed to 30 µM oxaliplatin (A) or 300 µM carboplatin (B) for 24 hours prior to release experiments. For release, wells of cells from three independent harvests were exposed for 10 min to HEPES alone (basal; open columns), or HEPES in the presence of 30 nM capsaicin (solid columns) as indicated. An asterisk indicates a significant difference in capsaicin-stimulated release compared to untreated cells, whereas a cross indicates a significant difference in cultures treated with APE1siRNA versus those treated with SCsiRNA using Student's <i>t</i>-test. Cisplatin analyses can be found in our previous publication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106485#pone.0106485-Jiang1" target="_blank">[24]</a>.</p

Systemic administration of cisplatin, oxaliplatin, or carboplatin decreases capsaicin-induced vasodilatation in the rat hindpaw.

<p>Each column represents the mean ± SEM of the capsaicin-evoked changes in blood flow over 15 minutes (capsaicin-stimulated blood flow minus basal blood flow) normalized to the vehicle treated controls. Animals are injected with vehicle (lightly shade columns) or platinum drugs (dark shaded columns) once a week for three weeks. Blood flow is measured each week 4 days after dosing with 3 mg/kg cisplatin (A), 3 mg/ml oxaliplatin (B) or 30 mg/kg carboplatin(C) and for 1–3 weeks after dosing is discontinued. An asterisk indicates statistical significance between the platinum-treated group and the corresponding vehicle-injected group using the statistical analysis software SPSS 11.0; post hoc analysis is LSD (Fisher's least significant difference) and Student-Newman-Keuls.</p

Platinum-induced oxidative DNA damage measured by 8-oxoG DNA adduct immunocytochemistry is increased by reducing APE1 expression in cisplatin and oxaliplatin treated sensory neuronal cultures.

<p>The top panels (A) show representative immunohistochemical staining for 8-oxoG DNA adducts in control (media), scrambled (SCsiRNA) or APE1 knockdown (APEsiRNA) from cultures after 24 hrs treatment with cisplatin (50 µM), oxaliplatin (300 µM) or carboplatin (500 µM). The bottom panels (B) represent the quantitation of the 8-oxoG positive staining cells as described in ”Methods.” The columns represent the mean ± SEM from cultures treated with media along (lightly shaded columns), SCsiRNA (medium shaded columns) or APE1siRNA (heavy shaded columns). An asterisk indicates a statistically significant increase in 8-oxoG in cells treated with APE1siRNA compared to those treated with SCsiRNA.</p

Apoptosis induced by cisplatin and oxaliplatin, but not carboplatin, is increased by reducing the expression of APE1 in sensory neuronal cultures.

<p>Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell apoptosis was detected by Annexin-V and PI staining and FACS analyses after cells were grown for 12 days. The left panels show representative fluorescence-activated cell sorting (FACS) for cells treated with various concentrations of cisplatin (A), oxaliplatin (B), or carboplatin (C) and scrambled siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from five independent harvests. Each column represents the mean ± SEM of the percent of apoptotic cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's <i>t</i>-test.</p

Production of reactive oxygen species (ROS) by cisplatin and oxaliplatin, but not carboplatin is increased by reducing the expression of APE1 in sensory neuronal cultures.

<p>Neuronal cultures were exposed to siRNAs on days 3-5 in culture then exposed to various concentrations of platins for 24 hours starting on day 11 in culture. ROS generation was measured by Carboxy-H2DCFDA and FACS analysis. The left panels show representative FACS for cells treated with various concentrations of cisplatin (A), oxaliplatin (B) or carboplatin (C) and scramble siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from 4-6 independent harvests. Each column represents the mean ± SEM of the percent of ROS positive cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and treated with various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in the number of ROS positive cells in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's <i>t</i>-test.</p

Reducing the expression of APE1 augments the ability of cisplatin and oxaliplatin, but not carboplatin to reduce cell viability in sensory neuronal cultures.

<p>Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell viability as measured by trypan blue exclusion was determined on day 12 in culture from three independent harvests. Each column represents the mean ± SEM of percent survival of cells from cultures treated with scrambled siRNA (SCsiRNA; lightly shaded columns) or with APE1si RNA (heavy shaded columns), then exposed to various concentrations of cisplatin (panel A), oxaliplatin (panel B), or carboplatin (panel C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using ANOVA and Tukey's post hoc test.</p