Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways.

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

The protective effects of puerarin in cardiomyocytes from anoxia/reoxygenation injury are mediated by PKCε.

Puerarin is an isoflavone isolated from traditional Chinese medicine Ge-gen (Radix Puerariae). Clinical studies have confirmed the cardioprotective effects of puerarin; however, the mechanisms underlying these effects are still unclear. On the basis of previous findings, we hypothesized that puerarin protects cardiomyocytes from ischemia-reperfusion injury via the protein kinase C epsilon (PKCε) (a critical cardioprotective protein) signalling pathway. Neonatal rat primary cardiomyocytes were preconditioned with puerarin or puerarin plus εV1-2, a selective PKCε inhibitor, prior to anoxia/reoxygenation (A/R) treatment. Western blot analysis showed that expression and activity of PKCε protein in puerarin preconditioned group were both increased compared with the control or A/R group. Subsequent assays showed that preconditioning with puerarin could increase the viability of neonatal rat primary cardiomyocytes treated with A/R, decreased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, cell necrosis and apoptosis induced by A/R injury. However, the protective effects of puerarin completely disappeared in the group pretreated with puerarin plus εV1-2. Thus, for the first time, we revealed the protective effects of puerarin in cardiomocytes from anoxia/reoxygenation injury are mediated by PKCε.

Plumbagin inhibits prostate cancer development in TRAMP mice via targeting PKCε, Stat3 and neuroendocrine markers.

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Cardoguard, an Ayurvedic antihypertensive formulation, prevents cardiac remodeling in spontaneously hypertensive rats by inhibition of ERK and PKCε signaling pathways.

Ayurveda is an Indian system of medicine. Despite clinical efficacy, lack of scientific validation has limited the effective use of Ayurvedic drugs. Cardoguard is an Ayurvedic antihypertensive drug formulated by Nagarjuna Herbal Concentrates Ltd., Kerala, India. Left ventricular hypertrophy (LVH) is a modifiable risk factor, and regression of LVH reduces the propensity for adverse cardiovascular events. This study was taken up with the objective of evaluating the efficacy of Cardoguard in the prevention of cardiac remodeling. Cardoguard was administered orally to 2-month-old spontaneously hypertensive rats for 4 months at a dose of 5 mg·day(-1). The dose corresponds to the therapeutic dose calculated on the basis of body surface area. Lower hypertrophy index, decrease in cardiomyocyte area, and reduction of interstitial fibrosis in treated spontaneously hypertensive rats indicate amelioration of cardiac hypertrophy by Cardoguard. Cardiac output increased in response to treatment. Immunostaining for the phosphorylated components of major signaling pathways associated with hypertrophy suggests that prevention of LVH by Cardoguard is possibly mediated through inhibition of extracellular signal-regulated kinases and protein kinase C-ε signaling pathways. Reduced expression of 3-nitrotyrosine in response to the treatment suggests that prevention of cardiac remodeling by Cardoguard is mediated by reduction of oxidative stress.

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

St. John's Wort reduces neuropathic pain through a hypericin-mediated inhibition of the protein kinase Cgamma and epsilon activity.

Current pharmacological treatments for neuropathic pain have limited efficacy and severe side-effect limitations. St. John's Wort (SJW) is a medicinal plant, mainly used as antidepressant, with a favourable side-effect profile. We here demonstrate the ability of SJW to relieve neuropathic pain in rat models. The antihyperalgesic profile and mechanism of action of SJW and its main components were studied in two rat models of neuropathic pain: the chronic constriction injury and the repeated administration of oxaliplatin. SJW, acutely administered at low doses (30-60 mg kg(-1) p.o.), reversed mechanical hyperalgesia with a prolonged effect, being effective up to 180 min after injection. Further examinations of the SJW main components revealed that hyperforin and hypericin were responsible for the antihyperalgesic properties whereas flavonoids were ineffective. The effect of SJW on the PKC expression and activation was investigated in the periaqueductal grey (PAG) area by immunoblotting experiments. Mechanistic studies showed a robust over-expression and hyperphosphorylation of the PKCgamma (227.0+/-15.0% of control) and PKCepsilon (213.9+/-17.0) isoforms in the rat PAG area. A single oral administration of SJW produced a significant decrease of the PKCgamma (131.8+/-10.0) and PKCepsilon (105.2+/-12.0) phosphorylation in the PAG area due to the presence of hypericin. Furthermore, SJW showed a dual mechanism of action since hyperforin antinociception involves an opioid-dependent pathway. Rats undergoing treatment with SJW and purified components did not show any behavioural side effects or signs of altered locomotor activity. Our results indicate SJW as a prolonged antihyperalgesic treatment through inhibition of PKC isoforms and their phosphorylation.

Preclinical development of a bifunctional cancer cell homing, PKCepsilon inhibitory peptide for the treatment of head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide, comprising approximately 50% of all malignancies in some developing nations. Our recent work identified protein kinase Cepsilon (PKCepsilon) as a critical and causative player in establishing an aggressive phenotype in HNSCC. In this study, we investigated the specificity and efficacy of HN1-PKCepsilon, a novel bifunctional cancer cell homing, PKCepsilon inhibitory peptide, as a treatment for HNSCC. HN1-PKCepsilon peptide was designed by merging two separate technologies and synthesized as a capped peptide with two functional modules, HN1 (cancer cell homing) and PKCepsilon (specific PKCepsilon inhibitory), connected by a novel linker module. HN1-PKCepsilon preferentially internalized into UMSCC1 and UMSCC36 cells, two HNSCC cell lines, in comparison with oral epithelial cells: 82.1% positive for UMSCC1 and 86.5% positive for UMSCC36 compared with 1.2% positive for oral epithelial cells. In addition, HN1-PKCepsilon penetrated HNSCC cells in a dose- and time-dependent manner. Consistent with these in vitro observations, systemic injection of HN1-PKCepsilon resulted in selective delivery of HN1-PKCepsilon into UMSCC1 xenografts in nude mice. HN1-PKCepsilon blocked the translocation of active PKCepsilon in UMSCC1 cells, confirming HN1-PKCepsilon as a PKCepsilon inhibitor. HN1-PKCepsilon inhibited cell invasion by 72 +/- 2% (P < 0.001, n = 12) and cell motility by 56 +/- 2% (P < 0.001, n = 5) in UMSCC1 cells. Moreover, in vivo bioluminescence imaging showed that HN1-PKCepsilon significantly (83 +/- 1% inhibition; P < 0.02) retards the growth of UMSCC1 xenografts in nude mice. Our work indicates that the bifunctional HN1-PKCepsilon inhibitory peptide represents a promising novel therapeutic strategy for HNSCC.

Perspectives of protein kinase C (PKC) inhibitors as anti-cancer agents.

Although the role of serine/threonine protein kinase C (PKC) in malignant transformation is known from decades, an anti-PKC based approach in cancer therapy was hampered for the difficulties in developing pharmacological compounds able to selectively inhibit specific PKC isoforms. In this review, the role of PKC-epsilon and PKC-delta in promoting and counteracting tumor progression in different types of cancer, respectively, will be discussed in relationship with promising therapeutic perspectives based either on small molecule inhibitors or on natural compounds. Among a myriad of molecules able to modulate PKC activity, we will focus on the role of the enzastaurin and briostatin-1, which already entered clinical trials for several human cancers.

Identification of a PKCepsilon-dependent regulation of myocardial contraction by epicatechin-3-gallate.

In this study, the effects of tea catechins and tea theaflavins on myocardial contraction were examined in isolated rat hearts using a Langendorff-perfusion system. We found that both tea catechins and theaflavins had positive inotropic effects on the myocardium. Of the tested chemicals, epicatechin-3-gallate (ECG) and theaflavin-3,3'-digallate (TF(4)) appear to be the most effective tea catechin and theaflavin, respectively. Further studies of ECG-induced positive inotropy revealed the following insights. First, unlike digitalis drugs, ECG had no effect on intracellular Ca(2+) level in cultured adult cardiac myocytes. Second, it activated PKCepsilon, but not PKCalpha, in the isolated hearts as well as in cultured cells. Neither a phospholipase C (PLC) inhibitor (U73122) nor the antioxidant N-acetyl cysteine (NAC) affected the ECG-induced activation of PKCepsilon. Third, inhibition of PKCepsilon by either chelerythrine chloride (CHE) or PKCepsilon translocation inhibitor peptide (TIP) caused a partial reduction of ECG-induced increases in myocardial contraction. Moreover, NAC was also effective in reducing the effects of ECG on myocardial contraction. Finally, pretreatment of the heart with both CHE and NAC completely abolished ECG-induced inotropic effects on the heart. Together, these findings indicate that ECG can regulate myocardial contractility via a novel PKCepsilon-dependent signaling pathway.