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Intra-neuronal accumulation of hyper-phosphorylated tau as neurofibrillary tangles (NFT) is a hallmark of Alzheimer's disease (AD). To prevent the aggregation of phosphorylated tau in neurons, decreasing the phosphorylated tau protein levels is important. Here, we examined the biological effects of rottlerin, a phytochemical compound extracted from the Kamala tree, Mallotus philippinensis, on phosphorylated tau levels. Notably, rottlerin decreased the levels of intracellular phosphorylated and total tau. A marked increase in the LC3-II, a hallmark of autophagy, was observed in these cells, indicating that rottlerin strongly induced autophagy. Interestingly, rottlerin induced the phosphorylation of Raptor at S792 through the activation of adenosine-monophosphate activated-protein kinase (AMPK), which likely inhibits the mammalian target of rapamycin complex 1 (mTORC1), thus resulting in the activation of transcription factor EB (TFEB), a master regulator of autophagy. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) activity increased in the presence of rottlerin. The decrease of phosphorylated tau levels in the presence of rottlerin was ameliorated by the knockdown of TFEB and partially attenuated by the knockout of the Nrf2 gene. Taken together, rottlerin likely enhances the degradation of phosphorylated tau through autophagy activated by TFEB and Nrf2. Thus, our results suggest that a natural compound rottlerin could be used as a preventive and therapeutic drug for AD. [ABSTRACT FROM AUTHOR]

Abstract Candida species have been responsible for a high number of invasive infections worldwide. In this sense, Rottlerin has demonstrated a wide range of pharmacological activities. Therefore, this study aimed to evaluate the antifungal, antibiofilm and antivirulence activity of Rottlerin in vitro against Candida spp. and its toxicity and antifungal activity in vivo. Rottlerin showed antifungal activity against all yeasts evaluated, presenting Minimum Inhibitory and Fungicidal Concentration (MIC and MFC) values of 7.81 to > 1000 µg/mL. Futhermore, it was able to significantly inhibit biofilm production, presenting Biofilm Inhibitory Concentration (MICB50) values that ranged from 15.62 to 250 µg/mL and inhibition of the cell viability of the biofilm by 50% (IC50) from 2.24 to 12.76 µg/mL. There was a considerable reduction in all hydrolytic enzymes evaluated, with emphasis on hemolysin where Rottlerin showed a reduction of up to 20%. In the scanning electron microscopy (SEM) analysis, Rottlerin was able to completely inhibit filamentation by C. albicans. Regarding in vivo tests, Rottlerin did not demonstrate toxicity at the therapeutic concentrations demonstrated here and was able to increase the survival of C. elegans larvae infected. The results herein presented are innovative and pioneering in terms of Rottlerin’s multipotentiality against these fungal infections.

Simple Summary: Adrenocortical tumors are rare neoplasms with an uncertain prognosis. A greater understanding of the biology of these tumors will allow new therapeutic targets to be identified and the prognosis of these patients to be better understood, enabling more precise therapeutic targeting. Our study sought to evaluate the expression of the PRKAB2 gene as a prognostic biomarker in 63 cases of pediatric adrenocortical tumors and to analyze how efficient Rottlerin is in altering the tumorigenic profile of the NCI-H295R adrenocortical carcinoma (ACC) cell line. Pediatric adrenocortical tumors (ACTs) are rare, highly heterogeneous neoplasms with limited therapeutic options, making the investigation of new targets with potential therapeutic or prognostic purposes urgent. The PRKAB2 gene produces one of the subunits of the AMP-activated protein kinase (AMPK) complex and has been associated with cancer. However, little is known about the role AMPK plays in ACTs. We have evaluated how PRKAB2 is associated with clinical and biological characteristics in 63 pediatric patients with ACTs and conducted in vitro studies on the human NCI-H295R ACC cell line. An analysis of our cohort and the public ACC pediatric dataset GSE76019 showed that lower PRKAB2 expression was associated with relapse, death, metastasis, and lower event-free and overall survival rates. Multivariate analysis showed that PRKAB2 expression was an independent prognostic factor when associated with age, tumor weight and volume, and metastasis. In vitro tests on NCI-H295R cells demonstrated that Rottlerin, a drug that can activate AMPK, modulated several pathways in NCI-H295R cells, including AMPK/mTOR, Wnt/β-catenin, SKP2, HH, MAPK, NFKB, and TNF. Treatment with Rottlerin decreased cell proliferation and migration, clonogenic capacity, and steroid production. Together, these results suggest that PRKAB2 is a potential prognostic marker in pediatric ACTs, and that Rottlerin is promising for investigating drugs that can act against ACTs. [ABSTRACT FROM AUTHOR]

Neuroblastoma is one of the most common solid tumors in children younger than 1 year of age, with poor prognosis and survival rates. Therefore, novel molecular targets and therapeutic strategies are needed to prolong patient survival. For this purpose, we investigated the effects of rottlerin and genistein separately and in combination on neuroblastoma cells (SH-SY5Y, Kelly). First, the effects of rottlerin and genistein were investigated on cell proliferation. Different rottlerin (1–50 µM) and genistein (5–150 µM) doses were used as experimental groups compared to the control (DMSO/vehicle). The IC50 dose was found to be 5 µM for rottlerin and 30 µM for genistein (P < 0.0001). Other analyses, such as colony formation assays, annexin V/propidium iodide staining, matrigel invasion assays, and Western blot analysis, were performed with these doses and their combinations. To assess statistical significance, statistical analysis was conducted using the one-way ANOVA with the post hoc Tukey test. Our results showed that IC50 doses of rottlerin and genistein induced a significant reduction in cell proliferation, colony formation, and invasion in neuroblastoma cells (P < 0.0001). The combination of these doses increased the levels of inhibition of cell proliferation and invasion while decreasing the level of apoptosis (P 0.0001). Furthermore, these agents caused G1-cell cycle arrest in these cells. Our western blot data showed that rottlerin and genistein treatments markedly inhibit elongation factor 2 kinase (EF2K) and other pro-tumorigenic, metastatic proteins in neuroblastoma cells. These agents probably showed their anti-proliferative, anti-metastatic, and pro-apoptotic effects through EF2K downregulation. Our results suggested that rottlerin and genistein have inhibitory effects on cancer cell proliferation, invasion, and cell cycle and induce apoptosis in both cell lines. Combined treatment with rottlerin and genistein may be a viable approach and beneficial to neuroblastoma patients as the combined effect significantly suppresses the above-mentioned pathways. [ABSTRACT FROM AUTHOR]

Rottlerin is isolated from Mallotus japonicus, a plant rich in polyphenols. Rottlerin is a selective PKCδ-inhibitor and is also known as an uncoupler of oxidative phosphorylation and anti-neoplastic agent. However, its anti-obesity effect is yet to be established. Therefore, this study tested whether rottlerin inhibits adipogenesis and de novo lipogenesis via the LRP6/mTOR/SREBP1C pathway in 3T3-L1 adipocytes. Rottlerin dramatically decreased lipid accumulation assessed by Oil Red O as evidence to support the cellular phenotype (p < 0.001). Pivotal messenger RNA and protein expressions associated with de novo lipogenesis (SREBP1C, ACC1, FAS, and SCD1) and adipogenesis (PPARγ and C/EBPα) were subsequentially verified by rottlerin in a dose-dependent manner (p < 0.05). Further investigation revealed that rottlerin reduced the AKT/mTOR pathway via diminished total protein of LRP6 (p < 0.05). Collectively, these findings establish a causal link between rottlerin, LRP6, and the altered nutrient-sensing mTOR pathway, in which rottlerin regulates de novo lipogenesis and adipogenesis in white adipocytes. [ABSTRACT FROM AUTHOR]

Simple Summary: Rottlerin, a type of natural extract, can inhibit the activity of various viruses. Feline coronavirus (FCoV) causes a devastating disease in cats. In this study, we investigated whether rottlerin has inhibitory effects on FCoV. We demonstrated that rottlerin inhibits FCoV replication and FCoV-induced PKCδ phosphorylation. We showed that rottlerin affects the early (endocytosis) and late (syncytium formation) stages of FCoV replication. We also observed that the liposome encapsulation technique overcomes the drawbacks of rottlerin and enhances its efficacy. Therefore, we suggest that rottlerin-liposome is worth further investigation as a potential treatment for FCoV. Rottlerin (R) is a natural extract from Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV. [ABSTRACT FROM AUTHOR]

Background: Rottlerin, a natural polyphenolic ketone is a major constituent of Mallotus philippensis (Lam.) Mull. Arg. plant. Although, less explored it has diverse array of therapeutic applications. Objectives: The current work aimed to develop novel, simple, speedy, accurate, potent High Performance Thin Layer Chromatography Method (HPTLC) to separate, and quantify rottlerin from the herbal topical patch In-House formulation. Materials and Methods: This was the foremost attempt to develop and validate HPTLC method using Analytical Quality by design (AQbD) approach. Box Behnken design (BBD) coupled with response surface methodology approach was followed for the systematic optimization. The end results of toluene content, chamber conditioning time, and solvent front on Rf value of rottlerin was studied. The optimised method was validated as per ICH guidelines. Results: For HPTLC, stationary phase used was aluminium plates 60F254 coated with silica gel as. AQbD optimised conditions were mobile phase proportion namely toluene: ethyl acetate: methanol: ammonia (5:4:2:0.2), development distance 80 mm, band width 6mm, and chamber saturation time 10 min. The method generated well resolved and compact bands at Rf of 0.41±0.05. The developed method exhibited high accuracy, precision, robustness, and recovery. The quantity of Rottlerin in developed herbal patch was found to be 134.8 ng/band. Conclusion: In a nutshell, the present study undeniably vouches for a QbD based method development approach for Rottlerin and can be extrapolated to estimate it in other herbal extracts, or marketed formulations. [ABSTRACT FROM AUTHOR]

Abstract Background It has been demonstrated that reactive astrocytes can be polarized into pro-inflammatory A1 phenotype or anti-inflammatory A2 phenotype under neurotoxic and neurodegenerative conditions. Microglia have been suggested to play a critical role in astrocyte phenotype polarization by releasing pro- and anti-inflammatory mediators. In this study, we examined whether trimethyltin (TMT) insult can induce astrocyte polarization in the dentate gyrus of mice, and whether protein kinase Cδ (PKCδ) plays a role in TMT-induced astrocyte phenotype polarization. Methods Male C57BL/6 N mice received TMT (2.6 mg/kg, i.p.), and temporal changes in the mRNA expression of A1 and A2 phenotype markers were evaluated in the hippocampus. In addition, temporal and spatial changes in the protein expression of C3, S100A10, Iba-1, and p-PKCδ were examined in the dentate gyrus. Rottlerin (5 mg/kg, i.p. × 5 at 12-h intervals) was administered 3–5 days after TMT treatment, and the expression of A1 and A2 transcripts, p-PKCδ, Iba-1, C3, S100A10, and C1q was evaluated 6 days after TMT treatment. Results TMT treatment significantly increased the mRNA expression of A1 and A2 phenotype markers, and the increased expression of A1 markers remained longer than that of A2 markers. The immunoreactivity of the representative A1 phenotype marker, C3 and A2 phenotype marker, S100A10 peaked 6 days after TMT insult in the dentate gyrus. While C3 was expressed evenly throughout the dentate gyrus, S100A10 was highly expressed in the hilus and inner molecular layer. In addition, TMT insult induced microglial p-PKCδ expression. Treatment with rottlerin, a PKCδ inhibitor, decreased Iba-1 and C3 expression, but did not affect S100A10 expression, suggesting that PKCδ inhibition attenuates microglial activation and A1 astrocyte phenotype polarization. Consistently, rottlerin significantly reduced the expression of C1q and tumor necrosis factor-α (TNFα), which has been suggested to be released by activated microglia and induce A1 astrocyte polarization. Conclusion We demonstrated the temporal and spatial profiles of astrocyte polarization after TMT insult in the dentate gyrus of mice. Taken together, our results suggest that PKCδ plays a role in inducing A1 astrocyte polarization by promoting microglial activation and consequently increasing the expression of pro-inflammatory mediators after TMT insult.

After prostatectomy due to prostate carcinoma, patients often develop metastases. Although prostate cancer is susceptible to hormonal manipulation, many patients become castration-resistant. Therefore, new therapies are the focus of investigations. We analyzed the effect of the tyrosine kinase inhibitors (TKIs), sorafenib and sunitinib, in combination with rottlerin, a PKCd inhibitor, on metastatic mechanisms in prostate carcinoma cells. LNCaP and PC-3 prostate carcinoma cells were treated with sorafenib or sunitinib alone at various concentrations (1-20 µM) or in combination with rottlerin (10 µM) for 24 h. Then, cell toxicity (MTT test) and cell proliferation (BrdU incorporation assay) were quantified. The study demonstrated a dose-dependent inhibitory effect of sorafenib and sunitinib on PC-3 and LNCaP cell activity and proliferation. Both agents showed significantly stronger cytotoxic effects in LNCaP cells. At the highest concentrations, sorafenib and sunitinib inhibited the viability of LNCaP cells up to 2 % and 31 %, respectively, and the viability of PC-3 cell line up to 20 % and 43 %, respectively. The proliferation of both cell lines was significantly stronger inhibited by sorafenib than by sunitinib. In LNCaP cells, sorafenib and sunitinib at the highest concentrations inhibited cell proliferation up to 46 % and 49 %, respectively, and the proliferation of PC-3 line up to 40 % and 47 %, respectively. Rottlerin reduced the viability and proliferation of PC3 cells to 81 % and 42 %, whereas the viability and proliferation of LNCaP cells were reduced to 25 % and 57 %, respectively. Sorafenib and sunitinib at low concentrations partly neutralized the inhibitory effect of rottlerin on cell viability and proliferation. On the other hand, in PC-3 cells, rottlerin reduced the inhibitory effects of sorafenib and sunitinib at the highest concentrations on cell viability from 20 % to 30 % and from 43 % to 61 %, respectively. An additive effect on cell activity was observed after treating LNCaP cells with both sunitinib at high concentrations and rottlerin. This combination increased the cytotoxic effect from 31 % to 13 % at the highest sunitinib concentration. Our results showed that monotherapy with sorafenib was the most efficient in both PCa cell lines. A marginally additive effect of rottlerin was only observed in LNCaP cells treated with sunitinib at a high concentration. Sorafenib and sunitinib reduced cell migration in PC-3 cells to 10 % and 32 % of untreated cells, respectively. Co-treatment with sorafenib/sunitinib and rottlerin did not result in a significantly stronger anti-migratory effect than the treatment with each TKI alone. Given the strong cytotoxic effect of TKIs, especially sorafenib, on LNCaP cells, the results of the migration assay in this line were severely biased and not considered in the analysis. Unlike in other malignancies, combination therapy with TKI and rottlerin seems not beneficial in prostate cancer. More promising seems to be monotherapy with rottlerin, but further studies are needed to confirm this observation.

Rottlerin (R) is a natural extract from Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV.

Background: It has been demonstrated that reactive astrocytes can be polarized into pro-inflammatory A1 phenotype or anti-inflammatory A2 phenotype under neurotoxic and neurodegenerative conditions. Microglia have been suggested to play a critical role in astrocyte phenotype polarization by releasing pro- and anti-inflammatory mediators. In this study, we examined whether trimethyltin (TMT) insult can induce astrocyte polarization in the dentate gyrus of mice, and whether protein kinase Cδ (PKCδ) plays a role in TMT-induced astrocyte phenotype polarization.Methods: Male C57BL/6 N mice received TMT (2.6 mg/kg, i.p.), and temporal changes in the mRNA expression of A1 and A2 phenotype markers were evaluated in the hippocampus. In addition, temporal and spatial changes in the protein expression of C3, S100A10, Iba-1, and p-PKCδ were examined in the dentate gyrus. Rottlerin (5 mg/kg, i.p. × 5 at 12-h intervals) was administered 3-5 days after TMT treatment, and the expression of A1 and A2 transcripts, p-PKCδ, Iba-1, C3, S100A10, and C1q was evaluated 6 days after TMT treatment.Results: TMT treatment significantly increased the mRNA expression of A1 and A2 phenotype markers, and the increased expression of A1 markers remained longer than that of A2 markers. The immunoreactivity of the representative A1 phenotype marker, C3 and A2 phenotype marker, S100A10 peaked 6 days after TMT insult in the dentate gyrus. While C3 was expressed evenly throughout the dentate gyrus, S100A10 was highly expressed in the hilus and inner molecular layer. In addition, TMT insult induced microglial p-PKCδ expression. Treatment with rottlerin, a PKCδ inhibitor, decreased Iba-1 and C3 expression, but did not affect S100A10 expression, suggesting that PKCδ inhibition attenuates microglial activation and A1 astrocyte phenotype polarization. Consistently, rottlerin significantly reduced the expression of C1q and tumor necrosis factor-α (TNFα), which has been suggested to be released by activated microglia and induce A1 astrocyte polarization.Conclusion: We demonstrated the temporal and spatial profiles of astrocyte polarization after TMT insult in the dentate gyrus of mice. Taken together, our results suggest that PKCδ plays a role in inducing A1 astrocyte polarization by promoting microglial activation and consequently increasing the expression of pro-inflammatory mediators after TMT insult. [ABSTRACT FROM AUTHOR]

Exosomes/microvesicles originate from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of rottlerin, a polyphenol, on exosome/microvesicle secretion in a model of intracellular lipid trafficking impairment, and elucidated the mechanism of action. In a model of lipid trafficking impairment in C6 glia cells, rottlerin increased ceramide levels, while decreasing hexosylceramide content. This was accompanied by increased exosome/microvesicle secretion, thereby reducing the concentration of lipids in the endolysosomal compartment. The reduction of hexosylceramide levels by rottlerin was attributed to the increase of β-glucosidase (glucosylceramidase) activity, and the effects of rottlerin were abrogated by β-glucosidase inhibitors such as isofagomine D-tartrate and AMP-deoxynojirimycin. Moreover, treatment with ML-266, a potent activator of the β-glucosidase enzyme, recapitulated the effects of rottlerin on the sphingolipid profile and exosome/microvesicle secretion. Finally, inhibition of AMPK (AMP-activated protein kinase) using compound C prevented both exosome/microvesicle secretion and the elimination of endolysosome lipids, which were promoted by rottlerin. The results showed that the decrease in intracellular lipid deposition induced by rottlerin was mediated by β-glucosidase activation and exosome/microvesicle release via the AMPK pathway. Rottlerin consumption could represent an additional health benefit in lysosomal deposition diseases. [ABSTRACT FROM AUTHOR]

Kamala tree (Mallotus philippensis) is traditionally used by different ethnic groups to treat a variety of diseases and health ailments. However, these traditional uses need to be scientifically investigated and validated in order to develop drugs from this tree. Therefore, the present article is aimed to review the scientifically validated knowledge on the pharmacology and phytochemistry of the tree. To accomplish this, we extensively surveyed the available databases like Scopus, Web of Science, Google Scholar, ScienceDirect, NCBI including PubMed and PubChem, etc. by using keywords ‘Mallotus philippensis’, ‘Mallotus phillippinensis’ and ‘Mallotus philippinensis’. Our results indicated that the tree possesses more than 50 different types of important phytochemicals of natural origin. The wide array of phytochemicals possesses fascinating biological activities like anthelmintic, antibacterial, anti-inflammatory, anti-oxidant, anti-cancerous, anti-tuberculosis, anti-parasitic, analgesic, anti-urolithiatic and anti-viral activities. Thus, pharmacological activities and isolation of active phytochemicals make the tree a promising candidate for drug discovery. However, pharmacological activities such as antibacterial and anti-oxidant activities are often tested with crude extracts and in vitro rudimentary methods that can be sometimes misleading and non-specific. Thus, more sophisticated techniques may be applied for the isolation of active chemicals and elucidating their mechanism of actions.

Abstract Background Upregulation of matrix metalloproteinase-9 (MMP-9) has been indicated as one of the inflammatory biomarkers. In the central nervous system (CNS), the MMP-9 is induced by several proinflammatory mediators and participates in the CNS disorders, including inflammation and neurodegeneration. In addition, protein kinase Cs (PKCs) has been shown to be involved in regulation of various inflammatory factors like MMP-9 by several stimuli in many cell types. Several phytochemicals are believed to reduce the risk of several inflammatory disorders including the CNS diseases. The rottlerin, a principal phenolic compound of the Kamala plant Mallotus philippinensis, has been shown to possess an array of medicinal properties, including anti-PKC-δ, antitumor, anti-oxidative, and anti-inflammatory activities. Methods Herein, we used rat brain astrocytes (RBA) to demonstrate the signaling mechanisms of phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression by zymographic, RT-PCR, subcellular isolation, Western blot, ROS detection, and promoter reporter analyses. Then, we evaluate the effects of rottlerin on PMA-induced MMP-9 expression in RBA and its influencing mechanism. Results We first demonstrated that PMA stimulated activation of various types of PKC, including PKC-δ in RBA. Subsequently, PMA induced MMP-9 expression via PKCδ-mediated reactive oxygen species (ROS) generation, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, and then induced c-Fos/AP-1 signaling pathway. Finally, upregulation of MMP-9 by PMA via the pathway may promote astrocytic migration, and the event could be attenuated by rottlerin. Conclusions These data indicated that rottlerin may have anti-inflammatory activity by reducing these related pathways of PKC-δ-dependent ROS-mediated MMP-9 expression in brain astrocytes.

In this work, we have extracted natural dye from the pericarp of Mallotus phillipensis (kamala tree) fruit which is commonly found in East Asia. The natural dye extract has been used such a photosensitizer for dye-sensitized solar cells. Density Functional calculations on one of the major constituent natural dye rottlerin, have been performed to check the properties and applicability of the proposed dye as a photosensitizer and manifested high electron injection efficiency. The best performing device exhibited a power conversion efficiency of 0.55% under 100 mW/cm2. This kind of non-toxic and biodegradable natural dye can easily be extracted from an inedible source using a very low-cost technique and therefore it can provide the best alternative to the synthetic dye for solar cells. [ABSTRACT FROM AUTHOR]

Exosomes/microvesicles originate from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of rottlerin, a polyphenol, on exosome/microvesicle secretion in a model of intracellular lipid trafficking impairment, and elucidated the mechanism of action. In a model of lipid trafficking impairment in C6 glia cells, rottlerin increased ceramide levels, while decreasing hexosylceramide content. This was accompanied by increased exosome/microvesicle secretion, thereby reducing the concentration of lipids in the endolysosomal compartment. The reduction of hexosylceramide levels by rottlerin was attributed to the increase of β-glucosidase (glucosylceramidase) activity, and the effects of rottlerin were abrogated by β-glucosidase inhibitors such as isofagomine D-tartrate and AMP-deoxynojirimycin. Moreover, treatment with ML-266, a potent activator of the β-glucosidase enzyme, recapitulated the effects of rottlerin on the sphingolipid profile and exosome/microvesicle secretion. Finally, inhibition of AMPK (AMP-activated protein kinase) using compound C prevented both exosome/microvesicle secretion and the elimination of endolysosome lipids, which were promoted by rottlerin. The results showed that the decrease in intracellular lipid deposition induced by rottlerin was mediated by β-glucosidase activation and exosome/microvesicle release via the AMPK pathway. Rottlerin consumption could represent an additional health benefit in lysosomal deposition diseases.

Rationale and objective: As a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor and a mitochondrial uncoupler, oncologists have extensively studied rottlerin. Neuroscientists, however, have accumulated scarce data on the role of rottlerin in affective and cognitive functions. Only two prior studies have, respectively, documented its antidepressant-like effect and how it impairs psychostimulant-supported memory. Whether or not rottlerin would affect aversive memory remains unknown. Hence, we sought to investigate the effects of rottlerin on aversive memory in the inhibitory avoidance (IA) task in mice. Materials and methods: Male C57BL/6J mice were trained to acquire the IA task. Rottlerin (5 mg/kg, i.p. or 3 μg bilaterally in the hippocampus) or the vehicle was administered before footshock training (acquisition), after footshock training (consolidation), after the memory reactivation (reconsolidation), and before the test (retrieval) in the IA task. Results: Systemic and intrahippocampal rottlerin impaired the acquisition, consolidation, and retrieval of IA memory, without affecting the reconsolidation process. Rottlerin (5 mg/kg, i.p.) induced a fast-onset and long-lasting increase in the brain-derived neurotrophic factor (BDNF) protein levels in the mouse hippocampus. Systemic injection of 7,8-dihydroxyflavone (7,8-DHF, 30 mg/kg), a BDNF tropomyosin receptor kinase B (TrkB) agonist impaired IA memory consolidation, and treatment with K252a (5 μg/kg), a Trk receptor antagonist, reversed the suppressing effect of rottlerin on IA memory consolidation. Conclusion: Rottlerin impairs IA memory consolidation through the enhancement of BDNF signaling in the mouse hippocampus. Excessive brain BDNF levels can be detrimental to cognitive function. Rottlerin is likely to affect the original memory-associated neuroplasticity. Thus, it can be combined with exposure therapy to facilitate the forgetting of maladaptive aversive memory, such as post-traumatic stress disorder (PTSD). [ABSTRACT FROM AUTHOR]

Rottlerin, a chromenochalcone accuring naturally in Mallotus philippensis, processes a variety of biological activities. The compound has a pseudodimeric structure, as two phloroacetophenone derived aryl cores are linked by a methylene bridge. In this paper, we described a La(OTf)3‐catalyzed activated arene‐formaldehyde condensation strategy to construct the methylene bridge, affording the desired product rottlerin and its analogue rottlerone. Moreover, with Eschenmoser's salt, we accomplished the synthesis of rottlerin and rottlerone in one pot. [ABSTRACT FROM AUTHOR]

Background: Upregulation of matrix metalloproteinase-9 (MMP-9) has been indicated as one of the inflammatory biomarkers. In the central nervous system (CNS), the MMP-9 is induced by several proinflammatory mediators and participates in the CNS disorders, including inflammation and neurodegeneration. In addition, protein kinase Cs (PKCs) has been shown to be involved in regulation of various inflammatory factors like MMP-9 by several stimuli in many cell types. Several phytochemicals are believed to reduce the risk of several inflammatory disorders including the CNS diseases. The rottlerin, a principal phenolic compound of the Kamala plant Mallotus philippinensis, has been shown to possess an array of medicinal properties, including anti-PKC-δ, antitumor, anti-oxidative, and anti-inflammatory activities.Methods: Herein, we used rat brain astrocytes (RBA) to demonstrate the signaling mechanisms of phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression by zymographic, RT-PCR, subcellular isolation, Western blot, ROS detection, and promoter reporter analyses. Then, we evaluate the effects of rottlerin on PMA-induced MMP-9 expression in RBA and its influencing mechanism.Results: We first demonstrated that PMA stimulated activation of various types of PKC, including PKC-δ in RBA. Subsequently, PMA induced MMP-9 expression via PKCδ-mediated reactive oxygen species (ROS) generation, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, and then induced c-Fos/AP-1 signaling pathway. Finally, upregulation of MMP-9 by PMA via the pathway may promote astrocytic migration, and the event could be attenuated by rottlerin.Conclusions: These data indicated that rottlerin may have anti-inflammatory activity by reducing these related pathways of PKC-δ-dependent ROS-mediated MMP-9 expression in brain astrocytes. [ABSTRACT FROM AUTHOR]

Rottlerin is a natural product consisting of chalcone and flavonoid scaffolds, both of which have previously shown quorum sensing (QS) inhibition in various bacteria. Therefore, the unique rottlerin scaffold highlights great potential in inhibiting the QS system of Pseudomonas aeruginosa. Rottlerin analogues were synthesised by modifications at its chalcone- and methylene-bridged acetophenone moieties. The synthesis of analogues was achieved using an established five-step synthetic strategy for chalcone derivatives and utilising the Mannich reaction at C6 of the chromene to construct morpholine analogues. Several pyranochromene chalcone derivatives were also generated using aldol conditions. All the synthetic rottlerin derivatives were screened for QS inhibition and growth inhibition against the related LasR QS system. The pyranochromene chalcone structures displayed high QS inhibitory activity with the most potent compounds, 8b and 8d, achieving QS inhibition of 49.4% and 40.6% and no effect on bacterial growth inhibition at 31 µM, respectively. Both compounds also displayed moderate biofilm inhibitory activity and reduced the production of pyocyanin.

Rottlerin is a cytostatic and cytotoxic drug in a variety of cancer cells. Our previous experience demonstrated that depending upon the genetic/biochemical background of cancer cells, rottlerin is able to induce both apoptotic and autophagic cell death, or dramatically disturb protein homeostasis leading to lethal cellular atrophy. In the current study, we investigated the cytotoxic effects and mechanisms of rottlerin against human amelanotic A375 melanoma cells. In this cell line, rottlerin exhibits its main and newest cytotoxic properties, that is, growth arrest, apoptosis induction, and translation shutoff. In fact, the drug, time‐, and dose‐dependently, markedly inhibited cell proliferation through cyclin D1 downregulation and induced apoptotic cell death as early as after 18 h treatment. Mechanistically, rottlerin triggered apoptosis by both intrinsic and extrinsic pathways. Both pathways are likely activated by the downregulation of the antiapoptotic B‐cell lymphoma 2 (Bcl‐2) protein, which simultaneously affects mitochondrial and endoplasmic reticulum (ER) membranes stability. Concomitantly to extrinsic apoptosis induction, the rottlerin‐activated ER stress/eukaryotic initiation factor 2 (eIF2) α axis blocked the translational apparatus. The altered proteostasis precluded the complete cells' rescue from death in the presence of apoptosis inhibitors. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still kills up to 59,000 people worldwide every year. To date, only preventive or post-exposure vaccination protects against the disease but therapeutics are missing. After screening a library of 80 kinases inhibitors, we identified two compounds as potent inhibitors of RABV infection: tyrphostin 9 and rottlerin. Mechanism of action studies show that both inhibitors interfere with an early step of viral cycle and can prevent viral replication. In presence of tyrphostin 9, the viral entry through endocytosis is disturbed leading to improper delivery of viral particles in cytoplasm, whereas rottlerin is inhibiting the transcription, most likely by decreasing intracellular ATP concentration, and therefore the replication of the viral genome. • A library of 80 kinase inhibitors was screened for anti-rabies virus infection. • Tyrphostin 9 and Rottlerin inhibit early steps of rabies infection in vitro. • Tyrphostin 9 inhibits viral entry by blocking endosome acidification. • Rottlerin inhibits viral transcription, most likely by reducing intracellular ATP content. [ABSTRACT FROM AUTHOR]

Abstract Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ 1–40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC 50 and DC 50 values were calculated in the range of 1.3–36.4 μM and 15.6–25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ 1–40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation. Graphical abstract Unlabelled Image Highlights • " In silico " studies on ligand-amyloidogenic proteins interactions of selected polyphenols recommended rottlerin for experimental analysis • The computational studies indicate easily adjustment of rottlerin conformational shape to match the binding site of amyloidogenic proteins • Rottlerin is able to inhibit amyloid fibril formation and to disassemble pre-formed amyloid fibrils for all of the amyloidogenic proteins tested • In vivo assays indicated concentration and time-dependent effects on cell viability and morphology [ABSTRACT FROM AUTHOR]

Flavonoids have been chronicles of the history of a long way journey in the cure of physiological or pathophysiological conditions in various diseases including cancer. Our previous findings suggest the extensive mechanism of quercetin (QUE) mediated regression of cell survival, cell proliferation, oxidative stress, inflammation, and angiogenesis via modulating PI3K and PKC signaling in lymphoma as well as hepatocellular carcinoma. PI3K-PKC pathway is a key monitor of mammalian cells regulated by its different isoenzymes, which may exert similar or opposite cellular effects by differential coupling of signaling pathways. Put forward the invention of selective inhibitors against various isoenzymes is beneficial to reduce the burden of inclusive deleterious effects of drug for normal physiological process. Therefore, we hypothesized the improved anticancer efficacy of QUE in combination with isoenzyme inhibitors—rottlerin (ROT-PKCδ inhibitor), G0 6983 (PKCα inhibitor), and PI-103 (p110α-class I PI3K inhibitor) in MCF-7 and RAW 264.7 cells. QUE significantly improves the cytotoxicity of ROT + G0 6983 ranged 30-55% and PI-103 ranged 24-63% after 24-48 h against MCF-7 cells. Additionally in the presence of QUE, the improved cytotoxicity of ROT + G0 6983 is observed to range 69-75% and PI-103 ranged 45-88% after 24-48 h in RAW 264.7 cells. This increment in cell deaths are positively correlated with enhanced morphological alteration observed in MCF-7 cells. Further, QUE significantly increases the attenuation of PKCα level approximately by 50% in combination with PI-103. Overall results of the current study suggested that QUE improves the synergistic anticancer efficacy in combination with PI-103, ROT, and G0 6983 in MCF-7 and RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii , can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti- T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis. • Rottlerin controls T. gondii infection in human trophoblast cells and villous explants. • Rottlerin strongly inhibits T. gondii proliferation with an irreversible manner. • Rottlerin caused parasite cell cycle arrest in G1 phase. • Rottlerin treatment upregulates IL-6 and MIF levels in BeWo cells. • Rottlerin is able to affect lipid droplet formation and mitochondrial function. [ABSTRACT FROM AUTHOR]

Abstract A variety of drugs targeting monoamine receptors are routinely used in human pharmacology. We assessed the effect of these drugs on the viability of tumor‐initiating cells isolated from patients with glioblastoma. Among the drugs targeting monoamine receptors, we identified prazosin, an α1‐ and α2B‐adrenergic receptor antagonist, as the most potent inducer of patient‐derived glioblastoma‐initiating cell death. Prazosin triggered apoptosis of glioblastoma‐initiating cells and of their differentiated progeny, inhibited glioblastoma growth in orthotopic xenografts of patient‐derived glioblastoma‐initiating cells, and increased survival of glioblastoma‐bearing mice. We found that prazosin acted in glioblastoma‐initiating cells independently from adrenergic receptors. Its off‐target activity occurred via a PKCδ‐dependent inhibition of the AKT pathway, which resulted in caspase‐3 activation. Blockade of PKCδ activation prevented all molecular changes observed in prazosin‐treated glioblastoma‐initiating cells, as well as prazosin‐induced apoptosis. Based on these data, we conclude that prazosin, an FDA‐approved drug for the control of hypertension, inhibits glioblastoma growth through a PKCδ‐dependent mechanism. These findings open up promising prospects for the use of prazosin as an adjuvant therapy for glioblastoma patients.

Ethambutol (EMB), an effective first-line antituberculosis agent, can cause serious visual impairment or irreversible vision loss in a significant number of patients. However, the mechanism underlying this ocular cytotoxicity remains to be elucidated. In this study, we found that there were statistically significant dose- and time-dependent increases in the number of cytoplasmic vacuoles and the level of cell death in EMB-treated RGC-5 cells (retinal ganglion cells). The protein kinase C (PKC)δ inhibitor rottlerin markedly reduced the EMB-induced activation of caspase-3 and the subsequent apoptosis of RGC-5 cells. Western blot analysis revealed that the expression levels of class III PI3K, Beclin-1, p62 and LC3-II were upregulated, and LC3 immunostaining results showed activation of the early phase and inhibition of the late stage of autophagy in retinas of the EMB-intraperitoneal (IP)-injected rat model. We further demonstrated that exposure to EMB induces autophagosome accumulation, which results from the impaired autophagic flux that is mediated by a PKCδ-dependent pathway, inhibits the PI3K/Akt/mTOR signaling pathway and leads to apoptotic death in retina neuronal cells. These results indicate that autophagy dysregulation in retinal neuronal cells might play a substantial role in EMB-induced optic neuroretinopathy.

Purpose: Autophagy, as a highly conserved cellular degradation and recycling process, plays an important part in maintaining cellular homeostasis. PKC signaling is involved in multiple pathways including cell cycle progression, tumorigenesis, migration and autophagy.Methods: Literatures about PKC and autophagy from PubMed databases were reviewed in this study.Results: Studies regarding the association of PKC and autophagy remain debatable. Different duration of the stimulation of autophagy and distinct cell contexts result in different function of PKC in regulating autophagy. The subcellular localization of PKCs and their downstream regulators may influence the autophagy regulation as well. As important intracellular components, the mitochondria play an important role in regulating autophagy, by metabolic modulation and structural derangement.Conclusion: Phase II studies regarding PKC-β inhibitor, enzastaurin, showed promising results in MCL, DLBCL and recurrent high-grade gliomas. However, the detailed mechanism is still in need. The mechanism of PKC-β in mediating autophagy in lymphoma and high-grade gliomas remains elusive as well. Moreover, several studies were in agreement that rottlerin enhanced autophagy in breast cancer cells, which warrants further clinical studies to verify PKC-δ as a therapeutic target. Thus, identifying the function of PKC in modulating autophagy and conducting related clinical studies help find novel target for chemotherapy. [ABSTRACT FROM AUTHOR]

Rottlerin as a natural agent, which is isolated from Mallotus philippinensis, has been identified to play a critical role in tumor inhibition. However, the molecular mechanism of rottlerin-mediated anti-tumor activity is still ambiguous. It has been reported that EZH2 exhibits oncogenic functions in a variety of human cancers. Therefore, inhibition of EZH2 could be a promising strategy for the treatment of human cancers. In this study, we aim to explore whether rottlerin could inhibit tumorigenesis via suppression of EZH2 in prostate cancer cells. Multiple approaches such as FACS, Transwell invasion assay, RT-PCR, Western blotting, and transfection were performed to determine our aim. We found that rottlerin treatment led to inhibition of cell growth, migration and invasion, but induction of apoptosis in prostate cancer cells. Importantly, we defined that rottlerin decreased the expression of EZH2 and H3K27me3 in prostate cancer cells. Moreover, overexpression of EZH2 abrogated the rottlerin-induced inhibition of cell growth, migration, and invasion in prostate cancer cells. Consistently, down-regulation of EZH2 enhanced rottlerin-triggered anti-tumor function. Collectively, our work demonstrated that rottlerin exerted its tumor suppressive function via inhibition of EZH2 expression in prostate cancer cells. Our findings indicated that rottlerin might be a potential therapeutic compound for treating patients with prostate cancer. [ABSTRACT FROM AUTHOR]

Highlights • Eukaryotic elongation factor-2 Kinase (eEF-2K) is significantly upregulated in lung cancer cells. • eEF-2K promotes tumor growth and invasion/metastasis ability of lung cancer cells. • eEF-2K expression is associated with shorter overall survival in patients with lung cancer. • High eEF-2K expression predicts short overall survival in lung cancer patients. • eEF-2K may be a novel potential therapeutic target in lung cancer. Abstract Objectives Lung cancer is the leading cause of cancer related deaths in worldwide. Despite recent advances in treatment options, patient survival has not improved substantially due to lack of commonly expressed molecular targets and effective targeted therapeutics. Thus, better understanding of the biology of lung cancer and identification of novel therapeutic targets are urgently needed for development of highly effective molecularly targeted therapies. Materials and methods Viability, proliferation and metastatic ability of lung cancer cells were evaluated using methylthiazoltetrazolium (MTT), colony formation and matrigel invasion assays, respectively. Western blotting, RT-PCR, and gene knockdown by siRNA transfections were carried out to investigate the effects of eEF-2K on lung cancer cells. Athymic Nu/Nu mice were treated with liposomal eEF-2KeEF-2K or control siRNA and tumor growth was evaluated in tumor xenograft models of lung cancer. Results and discussion Here, we report that Eukaryotic Elongation Factor-2 kinase (eEF-2K), a member of an atypical alpha kinases family, is significantly upregulated in lung cancer cell lines and its expression is associated with shorter overall patient survival in lung cancer. Inhibition eEF-2K expression by siRNA or a chemical inhibitorsignificantly suppressed lung cancer cell proliferation, colony formation, survival, migration/invasion and tumorigenesis by inhibiting cyclin D1, Src and Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinase (MAPK/ERK) signaling. In vivo targeting of eEF-2K by systemically injected nanoliposomal eEF-2K siRNA resulted in a significant inhibition of lung cancer tumor xenografts in nude mice. Our results suggest, for the first time, that expression of eEF-2K is associated with poor patient prognosis and involved in regulation of critical pathways, including Src and MAPK/ERK and cyclin D1, promoting tumor growth and progression, and thus may be a novel potential therapeutic target in lung cancer. [ABSTRACT FROM AUTHOR]

It is widely accepted that apoptosis is closely associated with cancer cell death. However, whether autophagy induces tumor cell death has not been fully elucidated. Various studies have discussed the antitumor properties of rottlerin in human malignancies. The current study aimed to investigate the effects of rottlerin, a natural product isolated from the kamala tree (Mallotus philipensis), on growth inhibition and autophagy in gastric cancer (GC) cell lines in vitro. The results of the present study demonstrated that rottlerin suppressed cell growth, induced autophagy and apoptosis, and reduced migration and invasion in the SGC-7901 and MGC-803 GC cell lines. Furthermore, rottlerin led to microtubule-associated protein 1 light chain 3β-II augmentation and the enrichment of autophagosomes. In addition, the protein expression levels of mechanistic target of rapamycin kinase and S-phase kinase-associated protein 2 were downregulated in GC cells following rottlerin treatment, which is associated with autophagy. The protein levels of caspase-3, cleaved-caspase-3, total poly (ADP-ribose) polymerase (PARP) and cleaved-PARP exhibited no marked alterations in the GC cells following rottlerin treatment, indicating that caspases were likely not involved in rottlerin-induced GC apoptosis. In summary, the results of the present study indicate that rottlerin may inhibit invasion and promote apoptosis in GC cells, which may be mediated by the activation of autophagy. Therefore, rottlerin may be of value in the treatment of GC. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells. [ABSTRACT FROM AUTHOR]

The glandular hair extracts from the fruit rind of Mallotus philippinensis Muell. is employed to treat various skin infections, however the anti-tyrosinase activity remains unknown. Hence the present study inspected on the anti-melanogenic activity of M. philippinensis constituents. Lineweaver Burk plot revealed mixed inhibition for Rottlerin; non-competitive type of inhibition for mallotophilippen A and B respectively. Thermodynamic studies resulted in static quenching forming ground state complex with higher binding constant temperature dependently. Fluorescence and circular dichroism study implicated conformational change in secondary and tertiary structure of tyrosinase. Molecular docking suggests rottlerin has high binding affinity to the active site pocket of tyrosinase. Simulation study further proved that the compactness of inhibitor with tyrosinase by hydrogen bonding influenced the stability of the enzyme. Depigmentation efficacy is further proved in Aspergillus niger spores. Thus our findings delineate that rottlerin could be utilized as a depigmentation agent in food pharmaceutical and agricultural industries. [ABSTRACT FROM AUTHOR]

Rottlerin, a polyphenolic compound, has been demonstrated to exhibit antitumor activity in various types of human cancer. Several studies have revealed that rottlerin exerts its anticancer function through PKC-dependent and independent pathways. The transcriptional co-activator with PDZ-binding motif (TAZ) oncopreotein is an important molecule in regulation of the Hippo pathway in human cancer. The present study investigated whether rottlerin has a tumor suppressive role via inhibiting the expression of TAZ, using cell viability assay, apoptosis and cell cycle analyses, western blot analysis and Tanswell invasion assay. The results demonstrated that rottlerin suppressed cell growth, triggered cell apoptosis and induced cell cycle arrest. In addition, rottlerin inhibited cell migration and invasion in hepatocellular carcinoma (HCC) cells. Mechanistically, the results demonstrated that rottlerin exerted its antitumor activity partly through the inhibition of TAZ. In addition, the depletion of TAZ led to inhibited cell growth and invasion, whereas the overexpression of TAZ enhanced cell growth and invasion in the HCC cells. Taken together, these findings indicated that the inhibition of TAZ by rottlerin may be a novel strategy for treating HCC. [ABSTRACT FROM AUTHOR]

• 16 phytochemicals from medicinal plants traditionally used for respiratory-related illnesses tested against coronaviruses. • Five compounds showed promising antiviral activity against HCoV-OC43. • Three of these compounds also suppressed SARS-CoV-2 replication. • One compound, rottlerin, was tested in vivo and reduced SARS-CoV2-induced mortality in human ACE2-expressing K18 mice. Several coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus OC43 (HCoV-OC43), can cause respiratory infections in humans. To address the need for reliable anti-coronavirus therapeutics, we screened 16 active phytochemicals selected from medicinal plants used in traditional applications for respiratory-related illnesses. An initial screen was completed using HCoV-OC43 to identify compounds that inhibit virus-induced cytopathic effect (CPE) and cell death inhibition. Then the top hits were validated in vitro against both HCoV-OC43 and SARS-CoV-2 by determining virus titer in cell supernatant and virus-induced cell death. Finally, the most active phytochemical was validated in vivo in the SARS-CoV-2-infected B6.Cg-Tg(K18-ACE2)2Prlmn/J mouse model. The phytochemicals lycorine (LYC), capsaicin, rottlerin (RTL), piperine and chebulinic acid (CHU) inhibited HCoV-OC43-induced cytopathic effect and reduced viral titres by up to 4 log. LYC, RTL and CHU also suppressed virus replication and cell death following SARS-CoV-2 infection. In vivo, RTL significantly reduced SARS-CoV-2-induced mortality by ∼40% in human angiotensin-converting enzyme 2 (ACE2)-expressing K18 mice. Collectively, these studies indicate that RTL and other phytochemicals have therapeutic potential to reduce SARS-CoV-2 and HCoV-OC43 infections. [ABSTRACT FROM AUTHOR]

CYP2C8 is a crucial CYP isoform responsible for the metabolism of xenobiotics and endogenous molecules. CYP2C8 converts arachidonic acid to epoxyeicosatrienoic acids (EETs) that cause cancer progression. Rottlerin possess significant anticancer actions. However, information on its CYP inhibitory action is lacking in the literature and therefore, we aimed to explore the same using in silico , in vitro, and in vivo approaches. Rottlerin showed highly potent and selective CYP2C8 inhibition (IC 50 < 0.1 μM) compared to negligible inhibition (IC 50 > 10 μM) for seven other experimental CYPs in human liver microsomes (HLM) (in vitro) using USFDA recommended index reactions. Mechanistic studies reveal that rottlerin could reversibly (mixed-type) block CYP2C8. Molecular docking (in silico) results indicate a strong interaction could occur between rottlerin and the active site of human CYP2C8. Rottlerin boosted the plasma exposure of repaglinide and paclitaxel (CYP2C8 substrates) by delaying their metabolism using the rat model (in vivo). Multiple-dose treatment of rottlerin with CYP2C8 substrates lowered the CYP2C8 protein expression and up-regulated & down-regulated the mRNA for CYP2C12 & CYP2C11 (rat homologs), respectively, in rat liver tissue. Rottlerin substantially hindered the EET formation in HLM. Overall results of rottlerin on CYP2C8 inhibition and EET formation insinuate further exploration for cancer therapy. [Display omitted] • Rottlerin: a selective, highly potent & reversible (mixed-type) CYP2C8 inhibitor. • Molecular docking depicted strong interactions: active site of CYP2C8 vs. rottlerin. • Rottlerin boosted plasma exposure of repaglinide & paclitaxel (CYP2C8 substrates). • Rottlerin attenuated CYP2C8-protein expression & its mRNA homologs in rats. • Rottlerin impeded CYP2C8-mediated EETs formation to be a cancer therapeutic target. [ABSTRACT FROM AUTHOR]

Background Gliomas belong to the most infiltrative types of tumors. Photodynamic therapy (PDT) can be applied to regulate glioma cell proliferation. The inhibitors of PKCs (Protein Kinase C) are very promising drugs that can mediate glioma cells apoptosis in PDT. Hypericin is one of PKCs regulators, and thanks to its physicochemical properties it can be used in PDT. Rottlerin is also considered to be the PKCδ inhibitor. Its implementation in PDT may significantly influence glioma cells response to PDT. Methods The viability of U87 MG glioma cells in the presence of rottlerin and hypericin was assessed by MTT assay and flow cytometry in the absence and presence of light. The flow cytometric data were analyzed through Shannon entropy. The oxidative stress and immunocytochemistry of PKCδ and phosphorylated Bcl-2 (the regulators of apoptosis) were observed using fluorescence microscopy. Results A pretreatment of glioma cells with rottlerin before hypericin induced PDT led to significant increase in apoptosis accompanied by the decrease of intracellular oxidative stress and increase of phosphorylated Bcl-2 in the cytoplasm of U87 MG cells. Conclusions In conclusion, we assume that the synergism between rottlerin and hypericin leads firstly to activation of rescue mechanisms in the glioma cells, but finally this cooperation triggers apoptosis rather than necrosis. [ABSTRACT FROM AUTHOR]

We have shown previously that prostratin, a non-tumor promoting phorbol ester, inhibits chikungunya virus (CHIKV)-induced cytopathic effects in vitro . Prostratin is a potent activator of protein kinases C (PKC), a family of related serine/threonine kinases that regulate many cellular processes such as proliferation and apoptosis. The objective of this study was to explore the mechanism of the anti-CHIKV activity of prostratin. Prostratin reduced the production of infectious virus particles and viral protein accumulation in a dose-dependent manner at a post-entry step during virus replication. The antiviral effect of the compound was cell-dependent, with potent antiviral activity observed in human skin fibroblasts cells, the primary target cells of CHIKV infection. The antiviral activity of prostratin was markedly reduced in the presence of PKC inhibitors, therefore confirming that the antiviral effect results from an activation of PKCs. Together these results showed that PKCs are potential host targets for the inhibition of CHIKV replication. [ABSTRACT FROM AUTHOR]

Abstract Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.

We investigated the relationship between phosphorylation of alphaB-crystallin (αBC) and retinal apoptosis in type 2 diabetes. The retinas of male Otsuka Long–Evans Tokushima fatty (OLETF) rats at 24 and 35 weeks were used as an animal model for type 2 diabetes and sex- and age-matched Long–Evans Tokushima Otsuka (LETO) rats were used as controls. In the retinas of 35-week OLETF rats, the interaction between αBC and protein kinase C delta (PKC δ) among the PKC isozymes, αBC phosphorylation at Ser45 (S45p-αBC), TUNEL-positive apoptotic ganglion cells, several apoptotic signs, and co-localization of S45p-αBC and TUNEL significantly increased as compared with other groups while the αBC–Bax interaction greatly decreased. These changes were abolished by rottlerin treatment, a highly specific PKC δ inhibitor. These results suggest that PKC δ is involved in regulation of anti-apoptotic function of αBC in the retina of type 2 diabetes.

It has been well-established that apoptosis contributes to cancer cell death; however, the role of autophagy in cancer cell death remains unclear. The aim of the present study was to investigate the effects of rottlerin, a traditional Indian medicine, on cell growth inhibition and autophagy in EJ human bladder carcinoma cells in vitro. Cell viability, measured by MTT assay, was found to be suppressed in a dose- and time-dependent manner. In addition, apoptosis was significantly increased in cells treated with rottlerin, as indicated by increased annexin V-fluorescein isothiocyanate/propidium iodide staining and changes in the cell cycle distribution that indicated blockage at G1 phase. Rottlerin treatment also enhanced the activation of autophagy, with increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II and the appearance of autophagosomes. The increased level of LC3-II and autophagosomes suggests that autophagy may contribute to apoptosis in these cells. In addition, no apparent alterations in the levels of pro-caspase-3, cleaved caspase-3, total poly (ADP ribose) polymerase (PARP) and cleaved-PARP were observed in cells treated with rottlerin, which indicates that caspases may not serve a key role during the process of apoptosis induced by rottlerin. Therefore, the results of the present study indicate that rottlerin promotes apoptosis and arrests the cell cycle in EJ cells, which may be caused by autophagy activation. [ABSTRACT FROM AUTHOR]