Rottlerin

Synergism between PKCdelta regulators hypericin and rottlerin enhances apoptosis in U87 MG glioma cells after light stimulation.[Pubmed:28373118]

Photodiagnosis Photodyn Ther. 2017 Jun;18:267-274.

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 PKCdelta 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 PKCdelta 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.

Rottlerin-induced autophagy leads to apoptosis in bladder cancer cells.[Pubmed:28101215]

Oncol Lett. 2016 Dec;12(6):4577-4583.

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.

The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels.[Pubmed:27997884]

Cell Physiol Biochem. 2016;40(6):1549-1558.

BACKGROUND/AIMS: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. METHODS: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. RESULTS: We show that currents carried by Kv7.1 (EC50 = 1.48 muM), Kv7.1/KCNE1 (EC50 = 4.9 muM), and Kv7.4 (EC50 = 0.148 muM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. CONCLUSION: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.

Rottlerin exhibits antitumor activity via down-regulation of TAZ in non-small cell lung cancer.[Pubmed:27999199]

Oncotarget. 2017 Jan 31;8(5):7827-7838.

Rottlerin, a polyphenolic compound derived from Mallotus philipinensis, has been reported to exhibit anti-tumor activities in a variety of human malignancies including NSCLC (non-small cell lung cancer). TAZ (transcriptional co-activator with PDZ-binding motif), one of the key activators in Hippo pathway, has been characterized as an oncoprotein. Therefore, inhibition of TAZ could be useful for the treatment of human cancers. In the current study, we aimed to explore whether Rottlerin inhibits the expression of TAZ in NSCLC, leading to its anti-cancer activity. Multiple approaches were applied for determining the mechanism of Rottlerin-mediated anti-tumor function, including cell growth assay, Flow cytometry, wound healing assay, invasion assay, Western blotting, and transfection. We found that Rottlerin inhibited cell growth, triggered apoptosis, arrested cell cycle, and retarded cell invasion in NSCLC cells. Moreover, our results showed that overexpression of TAZ enhanced cell growth, stimulated apoptosis, and promoted cell migration and invasion. Consistently, inhibition of TAZ exhibited anti-tumor activity in NSCLC cells. Notably, we validated that Rottlerin exerted its tumor suppressive function via inactivation of TAZ in NSCLC cells. Taken together, our study indicates that inhibition of TAZ by Rottlerin could be a promising strategy for the prevention and therapy of NSCLC.

Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.[Pubmed:19771169]

PLoS One. 2009 Sep 22;4(9):e7124.

BACKGROUND: Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS: Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (Rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or Rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by Rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE: The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.

Neuroprotective effect of protein kinase C delta inhibitor rottlerin in cell culture and animal models of Parkinson's disease.[Pubmed:17565007]

J Pharmacol Exp Ther. 2007 Sep;322(3):913-22.

Recent studies from our laboratory demonstrated that the protein kinase C (PKC) delta isoform is an oxidative stress-sensitive kinase and a key mediator of apoptotic cell death in Parkinson's Disease (PD) models (Eur J Neurosci 18:1387-1401, 2003; Mol Cell Neurosci 25:406-421, 2004). We showed that native PKC delta is proteolytically activated by caspase-3 and that suppression of PKC delta by dominant-negative mutant or small interfering RNA against the kinase can effectively block apoptotic cell death in cellular models of PD. In an attempt to translate the mechanistic studies to a neuroprotective strategy targeting PKC delta, we systematically characterized the neuroprotective effect of a PKC delta inhibitor, Rottlerin, in 1-methyl-4-phenylpyridinium (MPP(+))-treated primary mesencephalic neuronal cultures as well as in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. Rottlerin treatment in primary mesencephalic cultures significantly attenuated MPP(+)-induced tyrosine hydroxylase (TH)-positive neuronal cell and neurite loss. Administration of Rottlerin, either intraperitoneally or orally, to C57 black mice showed significant protection against MPTP-induced locomotor deficits and striatal depletion of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. Notably, Rottlerin post-treatment was effective even when MPTP-induced depletion of dopamine and its metabolites was greater than 60%, demonstrating its neurorescue potential. Furthermore, the dose of Rottlerin used in neuroprotective studies effectively attenuated the MPTP-induced PKC delta kinase activity. Importantly, stereological analysis of nigral neurons revealed Rottlerin treatment significantly protected against MPTP-induced TH-positive neuronal loss in the substantia nigra compacta. Collectively, our findings demonstrate the neuroprotective effect of Rottlerin in both cell culture and preclinical animal models of PD, and they suggest that pharmacological modulation of PKC delta may offer a novel therapeutic strategy for treatment of PD.

Specificity and mechanism of action of some commonly used protein kinase inhibitors.[Pubmed:10998351]

Biochem J. 2000 Oct 1;351(Pt 1):95-105.

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Rottlerin, a novel protein kinase inhibitor.[Pubmed:8123051]

Biochem Biophys Res Commun. 1994 Feb 28;199(1):93-8.

Rottlerin, a compound from Mallotus philippinensis, is shown to inhibit protein kinases with some specificity for PKC. To some extent, the novel inhibitor is able to differentiate between PKC isoenzymes, with IC50 values for PKC delta of 3-6 microM, PKC alpha,beta,gamma of 30-42 microM and PKC epsilon,eta,zeta of 80-100 microM. Inhibition of PKC appears, at least in part, to be due to a competition between Rottlerin and ATP. Among the protein kinases tested, only CaM-kinase III is suppressed by Rottlerin as effectively as PKC delta. The chemical structure of Rottlerin might serve as a basis for the development of novel inhibitors with improved selectivity for a distinct PKC isoenzyme, such as PKC delta, or for CaM-kinase III.

Rottlerin, a natural product purified from Mallotus Philippinensis, is a specific PKC inhibitor, with IC50 values for PKCδ of 3-6 μM, PKCα,β,γ of 30-42 μM, PKCε,η,ζ of 80-100 μM. Rottlerin acts as a direct mitochondrial uncoupler, and stimulates autophagy by targeting a signaling cascade upstream of mTORC1. Rottlerin induces apoptosis via caspase 3 activation.

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