PKC Modulators Research Articles

Extraction optimization and reactivity of 7α-acetoxy-6β-hydroxyroyleanone and ability of its derivatives to modulate PKC isoforms

Protein kinase C is a family of kinases that play important roles in carcinogenesis. Medicinal plants from Plectranthus spp. (Lamiaceae) are a well-known source of interesting abietanes, such as 7α-acetoxy-6β-hydroxyroyleanone (Roy). This study aimed to extract and isolate Roy from P. grandidentatus Gürke, comparing two extraction methods (CO2 supercritical and ultrasound-assisted acetonic extraction), and design new royleanone derivatives for PKC modulation focusing on breast cancer therapy. The concentration of Roy in the extracts was determined by HPLC–DAD. The supercritical extraction method yielded 3.6% w/w, with the presence of 42.7 μg mg−1 of Roy (yield of 0.13%), while ultrasound-assisted acetonic extraction yielded 2.3% w/w, with the presence of 55.2 μg mg−1 of Roy (yield of 0.15%). The reactivity of Roy was investigated aiming at synthetizing new ester derivatives through standard benzoylation and esterification reactions. The benzoylated (Roy-12-Bz) and acetylated (Roy-12-Ac) derivatives in the C12 position were consistently prepared with overall good yields (33–86%). These results indicate the 12-OH position as the most reactive for esterification, affording derivatives under mild conditions. The reported di-benzoylated (RoyBz) and di-acetylated (RoyAc) derivatives were also synthesized after increasing the temperature (50 °C), reaction time, and using an excess of reagents. The cytotoxic potential of Roy and its derivatives was assessed against breast cancer cell lines, with RoyBz emerging as the most promising compound. Derivatization at position C-12 did not offer advantages over di-esterification at positions C-12 and C-6 or over the parent compound Roy and the presence of aromatic groups favored cytotoxicity. Evaluation of royleanones as PKC-α, βI, δ, ε, and ζ activators revealed DeRoy’s efficacy across all isoforms, while RoyPr showed promising activation of PKC-δ but not PKC-ζ, highlighting the influence of slight structural changes on isoform selectivity. Molecular docking analysis emphasized the importance of microenvironmental factors in isoform specificity, underscoring the complexity of PKC modulation and the need for further exploration.

Hydrocephalus: Current State of Diagnostics and Treatment

Hydrocephalus, the accumulation of cerebrospinal fluid in the brain’s ventricles, has numerous difficulties regarding diagnosis, treatment, and patient care. This article provides a comprehensive analysis of the condition, including the areas of current therapeutic options, surgical intervention (implementation of shunts/ third ventriculostomies), and novel therapeutic approaches including inhibition of NKCC1 and modulation of PKC. This paper also discusses noninvasive treatments like gene therapy and “smart shunt” systems. From a global perspective, it is revealed that there are numerous disparities regarding hydrocephalus and its treatment modalities. This very aspect forms the basis for special healthcare programs to cater to these global inequalities. This study bedevils us with the fact of how intricate hydrocephalus management is, a complication that is mostly determined by a specific country’s healthcare infrastructure. The region's funding, health facilities, and cultural issues also are used to showcase hydrocephalus causes and effects. Furthermore, there is a large focus on patient care and also a meticulous presentation of subsequent care strategies and rehabilitation with the family's support to improve the results for those who are afflicted with the disease. This article concludes with a call to go beyond research in this area with the international cooperation that will lead to comprehending this challenge and the multidisciplinary problems and opportunities that come along with it. This research study provides an indispensable contribution to the fields of neurology and healthcare, and this adds value in terms of the care and treatment of hydrocephalus by facilitating its understanding on a scientific basis for students and healthcare professionals around the world.

An Update on Protein Kinases as Therapeutic Targets-Part II: Peptides as Allosteric Protein Kinase C Modulators Targeting Protein-Protein Interactions.

Human protein kinases are highly-sought-after drug targets, historically harnessed for treating cancer, cardiovascular disease, and an increasing number of autoimmune and inflammatory conditions. Most current treatments involve small molecule protein kinase inhibitors that interact orthosterically with the protein kinase ATP-binding pocket. As a result, these compounds are often poorly selective and highly toxic. Part I of this series reviews the role of PKC isoforms in various human diseases, featuring cancer and cardiovascular disease, as well as translational examples of PKC modulation applied to human health and disease. In the present Part II, we discuss alternative allosteric binding mechanisms for targeting PKC, as well as novel drug platforms, such as modified peptides. A major goal is to design protein kinase modulators with enhanced selectivity and improved pharmacological properties. To this end, we use molecular docking analysis to predict the mechanisms of action for inhibitor-kinase interactions that can facilitate the development of next-generation PKC modulators.

Protein Kinase C at the Crossroad of Mutations, Cancer, Targeted Therapy and Immune Response.

The frequent PKC dysregulations observed in many tumors have made these enzymes natural targets for anticancer applications. Nevertheless, this considerable interest in the development of PKC modulators has not led to the expected therapeutic benefits, likely due to the complex biological activities regulated by PKC isoenzymes, often playing ambiguous and protective functions, further driven by the occurrence of mutations. The structure, regulation and functions of PKCs have been extensively covered in other publications. Herein, we focused on PKC alterations mostly associated with complete functional loss. We also addressed the modest yet encouraging results obtained targeting PKC in selected malignancies and the more frequent negative clinical outcomes. The reported observations advocate the need for more selective molecules and a better understanding of the involved pathways. Furthermore, we underlined the most relevant immune mechanisms controlled by PKC isoforms potentially impacting the immune checkpoint inhibitor blockade-mediated immune recovery. We believe that a comprehensive examination of the molecular features of the tumor microenvironment might improve clinical outcomes by tailoring PKC modulation. This approach can be further supported by the identification of potential response biomarkers, which may indicate patients who may benefit from the manipulation of distinctive PKC isoforms.

PKC Modulation by GIRK Channels is Involved in the Pathophysiology of Atrial Fibrillation

Impaired GIRK channel activity in the cardiac atria has been implicated in the development of atrial fibrillation. GIRK channels are essential to the maintenance of the resting membrane potential and inhibitory post-synaptic potentials in the body. In the cardiac atria, GIRK1/4 channels play a role in the down-stroke of the atrial action potential during atrial re polarization. The over-activity of GIRK channels is hence implicated in the shortening of the atrial action potential and the effective refractory period. Both these electrophysiological changes allow for an increased firing rate by the atria and a de-synchrony between the upper and lower chambers of the heart. The resulting irregular heart rate is a hallmark of atrial fibrillation. We show that GIRK1/4 channel activity is enhanced by the novel isoform of PKC, PKCε, and inhibited by the conventional isoform, PKCα. Both PKCε and PKCα also modulate the activity of GIRK1 and GIRK4 subunits, individually. Additionally, we demonstrate that this change in channel activity due to PKC is a result of direct and specific phosphorylation of each channel subunit and its subsequent alteration of channel-PIP2 interactions. Existing therapies for AF are non-specific and are fraught with their own limitations. The mechanistic insight into GIRK1/4 pathological activity is guiding design of therapeutic small-molecule channel inhibitors.

Mechanism of a methylxanthine drug theophylline-induced Ca2+ signaling and cytotoxicity in AML12 mouse hepatocytes.

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca2+ concentration ([Ca2+]i) was measured by the Ca2+-sensitive fluorescent dye fura-2. Theophylline (25-125μM) induced [Ca2+]i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca2+]i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca2+]i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca2+]i rises. In Ca2+-containing medium, modulators of store-operated Ca2+ channels inhibited 30% of the [Ca2+]i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca2+-associated cytotoxicity and induced Ca2+ entry through PLC-independent Ca2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats.

PKC and PKA phosphorylation inhibit TREK-1 channels downstream of Gs protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.

Preclinical and Clinical Studies on Bryostatins, A Class of Marine-Derived Protein Kinase C Modulators: A Mini-Review

Bryostatins are complex macrolactones isolated from marine organisms Bryozoan Bugula neritina. They are potent modulators of protein kinase C isozymes (PKCα: ki = 1.3-188 nM), and are one of the most extensively investigated marine natural products in clinical trials. Although ~21 natural bryostatins have been isolated, however only bryostatin-1 (1) has received much interest among medicinal chemists and clinicians. The structure-activity relationship of bryostatins has been well established, with the identification of key pharmacophoric features important for PKC modulation. The low natural abundance and the long synthetic route have prompted medicinal chemists to come-up with simplified analogs. Bryostatin skeleton comprises three pyran rings connected to each other to form a macrocyclic lactone. The simplest analog 27 contains only one pyran, which is also able to modulate the PKCα activity; however, the cyclic framework appears to be essential for the desired level of potency. Another simplified analog 17 ("picolog") exhibited potent and in-vivo efficacy against lymphoma. Bryostatin-1 (1) has shown an acceptable intravenous pharmacokinetic profile in mice and displayed promising in-vivo efficacy in mice models of various cancers and Alzheimer's disease. Bryostatin-1 was investigated in numerous Phase I/II oncology clinical trials; it has shown minimal effect as a single agent, however, provided encouraging results in combination with other chemotherapy agents. FDA has granted orphan drug status to bryostatin-1 in combination with paclitaxel for esophageal cancer. Bryostatin-1 has also received orphan drug status for fragile X syndrome. Bryostatin-1 was also investigated in clinical studies for Alzheimer's disease and HIV infection. In a nutshell, the natural as well as synthetic bryostatins have generated a strong hope to emerge as treatment for cancer along with many other diseases.

PP 2.10 - Barcoded HIV reveals effects of PKC modulation on viral reservoir

PP 2.10 - Barcoded HIV reveals effects of PKC modulation on viral reservoir

A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos

BackgroundThe segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively.ResultsWe show that SOX17 starts to be expressed in 16–32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor.ConclusionsThis study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

Characterization of designed, synthetically accessible bryostatin analog HIV latency reversing agents

HIV latency in resting CD4+ T cell represents a key barrier preventing cure of the infection with antiretroviral drugs alone. Latency reversing agents (LRAs) can activate HIV expression in latently infected cells, potentially leading to their elimination through virus-mediated cytopathic effects, host immune responses, and/or therapeutic strategies targeting cells actively expressing virus. We have recently described several structurally simplified analogs of the PKC modulator LRA bryostatin (termed bryologs) designed to improve synthetic accessibility, tolerability in vivo, and efficacy in inducing HIV latency reversal. Here we report the comparative performance of lead bryologs, including their effects in reducing cell surface expression of HIV entry receptors, inducing proinflammatory cytokines, inhibiting short-term HIV replication, and synergizing with histone deacetylase inhibitors to reverse HIV latency. These data provide unique insights into structure-function relationships between A- and B-ring bryolog modifications and activities in primary cells, and suggest that bryologs represent promising leads for preclinical advancement.

Protein kinase C activator bryostatin‐1 modulates proteasome function

Proteasome activity in ubiquitin-proteasome pathway plays a pivotal role in degradation and clearance of aggregated, oxidized, damaged, and misfolded unwanted proteins to control protein homeostasis or proteostasis. Proteasome activity decreases with cellular senescence, aging, and age-related diseases. Therefore, enhancement of impaired proteasome function by molecular biological and/or pharmacological intervention is an active area of research. Bryostatin-1, a naturally occurring macrocyclic lactone, activates PKC isozymes (specifically, -α and -ϵ) at sub-nanomolar concentrations, but downregulates at higher concentrations. Here, we present bryostatin-1 increased chymotrypsin-like proteasome activity of 20S assembly at sub-nanomolar to nanomolar concentrations (0.3-30 nM). However, proteasome activity decreased at a micromolar concentration of bryostatin-1 (AG08044 cultured skin: P < 0.005; differentiated SH-SY5Y cells: P < 0.02). Modulation of proteasome function by bryostatin-1 was studied in six dermal fibroblast primary cell lines developed both from freshly taken biopsies from healthy donors (n = 2) and obtained from well-characterized cell repositories (n = 4; without any diseases). Bryostatin-1 enhanced proteasome activity in cultured skin fibroblasts obtained from banked and freshly isolated skin fibroblasts from skin biopsies at the sub-nanomolar concentration (P < 0.015). Modulation of proteasome function by bryostatin-1 was confirmed in neuron-like differentiated SH-SY5Y cells. Direct additions of bryostatin-1 into cell lysates prepared from neuron-like differentiated SH-SY5Y, Jurkat cells, and cultured skin fibroblasts were unable to increase proteasome activity indicating that bryostatin-1 can only modulate proteasome activity when added to live cell culture systems. Standard PKC inhibitors blocked bryostatin-1 induced proteasome activity modulation suggesting that enhancement of proteasome activity was mediated by PKC modulation.

Effects of the C1 domain-targeted PKC modulator HMI-1a3 on the viability of colon cancer cells in culture

Effects of the C1 domain-targeted PKC modulator HMI-1a3 on the viability of colon cancer cells in culture

Making Bryostatin 1 through Smart Chemistry

Scientists have come up with an efficient, scalable way to chemically synthesize bryostatin 1, a marine-derived natural PKC modulator that has been in scarce supply, impeding a thorough evaluation of its therapeutic potential.

PP3.7 - HIV latency reversal using designed PKC modulators

PP3.7 - HIV latency reversal using designed PKC modulators

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Evaluation of HIV latency reversal using designed PKC modulators in humanized BLT mice

Evaluation of HIV latency reversal using designed PKC modulators in humanized BLT mice

A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels.

The inwardly rectifying potassium 3.2 (Kir3.2) channel is found principally in neurons that regulate diverse brain functions, including pain perception, alcoholism, and substance addiction. Activation or inhibition of this channel leads to changes in neuronal firing and chemical message transmission. The Kir3.2 channel is subject to regulation by intracellular signals including sodium, G-proteins, ethanol, the phospholipid phosphatidylinositol bis-phosphate, and phosphorylation by protein kinases. Here, we take advantage of the recently published structure of Kir3.2 to provide an in-depth molecular view of how phosphorylation of a specific residue previously thought to be the target of PKC promotes channel gating and acts as an allosteric modulator of PKC-mediated inhibition.

Abstract 2136: Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal cancer

Abstract Protein Kinase C (PKC) is a family of serine/threonine kinases that are involved in almost every signal transduction cascade in cells. PKC enzymes are activated by second messangers and respond to specific environmental cues. The development of PKC modulators with anti-cancer therapeutic potential is a major target in cancer. However, this task is made difficult because PKC has an important role to play in normal processes and the PKC family consists of at least 9 different isozymes. Our hypothesis is that PKC expression, activity and localisation are altered as colon epithelial cells switch from normal to the transformed state. We are confident that being able to recognise these changes has the potential to be used as a biomarker and prognostic marker in the early detection of colon cancer. Real Time PCR was utilised to compare the gene expression profile of 9 PKC isozymes in matched normal and cancer tissue samples. We found differential expression of the conventional PKCs, with PRKCB (the gene coding for PKC Beta II) being significantly downregulated in the cancer tissue of over 90% of patients (p&amp;lt;0.01,n = 22). This change was closely linked to the progression of the disease. Using tissue microarrays, we also demonstrated significant alterations in both epithelial and stromal levels of PKC Beta II between the patients normal and diseased tissue. The level of PKC Beta II in the diseased tissue was significantly decreased across patients (p&amp;lt;0.01,n = 164) and we associate the decreased expression with tumour stage. In conclusion we suggest that differential expression of PKC Beta II may be an important mechanism regulating the change to the transformed phenotype in colon cancer. Citation Format: Catríona M. Dowling, James J. Phelan, Mary Clare Cathcart, Brian Mehigan, Paul McCormick, Tara Dalton, John C. Coffey, Jacintha O'Sullivan, Patrick A. Kiely. Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2015-2136

Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2–ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription–polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1µM to 1mM), in the presence of Ca2+ in the extracellular medium, induced a slow and progressive increase of [Ca2+]c toward a stable level. Melatonin did not inhibit the typical Ca2+ response induced by CCK-8 (1nM). When the cells were challenged with indoleamine in the absence of Ca2+ in the extracellular solution (medium containing 0.5mM EGTA) or in the presence of 1mM LaCl3, to inhibit Ca2+ entry, we could not detect any change in [Ca2+]c. Nevertheless, CCK-8 (1nM) was able to induce the typical mobilization of Ca2+. When the cells were incubated with the PKC activator PMA (1µM) in the presence of Ca2+ in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100µM. However, in the presence of Ro31-8220 (3µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca2+]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca2+. Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100µM) or PMA (1µM). On the contrary, pretreatment with Ro31-8220 (3µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca2+ signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells.