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Rottlerin Inhibits ROS Formation and Prevents NFκB Activation in MCF-7 and HT-29 Cells

Rottlerin, a polyphenol isolated from Mallotus Philippinensis, has been recently used as a selective inhibitor of PKC δ, although it can inhibit many kinases and has several biological effects. Among them, we recently found that Rottlerin inhibits the Nuclear Factor κB (NFκB), activated by either phorbol esters or H2O2. Because of the redox sensitivity of NFκB and on the basis of Rottlerin antioxidant property, we hypothesized that Rottlerin could prevent NFκB activation acting as a free radicals scavenger, as other natural polyphenols. The current study confirms the antioxidant property of Rottlerin against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in vitro and against oxidative stress induced by H2O2 and by menadione in culture cells. We also demonstrate that Rottlerin prevents TNFα-dependent NFκB activation in MCF-7 cells and in HT-29 cells transfected with the NFκB-driven plasmid pBIIX-LUC, suggesting that Rottlerin can inhibit NFκB via several pathways and in several cell types.


Introduction
Rottlerin (also called mallotoxin or kamala) is a pigmented plant product isolated from MallotusPhilippinensis ( Figure 1), used in India in ancient times as a remedy for tapeworm [1][2][3].
Since 1994, Rottlerin has been used as a PKCδ inhibitor based on in vitro studies that have shown that the IC50 for PKCδ was 3-6 μM compared to 30-100 μM for the other PKC isozymes [4], although the selectivity of Rottlerin in inhibiting PKC δ isoform has been recently questioned [5].
Several phloroglucinol derivatives, which are similar to Rottlerin in structure, have been demonstrated to have antiinflammatory and antiallergic properties [6,7].
Moreover, Rottlerin can target mitochondria, interfering in the respiratory chain as an uncoupler of oxidative phosphorylation [8], and activates the large conductance voltage and Ca ++ activated K + channel (BK) in human vascular smooth muscle; its use has been patented (patent WO/2006/060196) in the therapy of hypertension and related disorders [9].
Although the underlying molecular mechanisms have not been yet investigated in details Rottlerin interference in the NFκB activation process was likely achieved, in MCF-7 cells, through inhibition of CaMKII, without involvement of the AKT/PKB pathway [10].
The transcription factor NFκB consists of p50 and p65 heterodimer, which is retained in the cytoplasm by masking nuclear localization signal (NLS) by the inhibitor IκBα.  Upon activation, IκBα kinase (IKK) phosphorylates IκBα, promotes its ubiquitination and degradation, thus allowing p50-p65 to translocate to the nucleus, bind to its consensus sequence, and induce transcription of genes essential for cell proliferation and survival [20]. Consistently, Rottlerin inhibition of NFκB caused downregulation of cyclin D1 and growth arrest in both MCF-7 and in HaCaT cells [10,11].
Because NFκB can be activated by a number of signaling molecules [21], we performed further experiments to support the idea that Rottlerin inhibits NFκB in different cell types and under different stimuli.
Although it could have been deduced from the Rottlerin structure (known at least since 1994), only recently antioxidant properties of Rottlerin have been started to be described [30].
In the current paper, we demonstrate that Rottlerin has antioxidant properties and that the observed inhibitory effect on NFκB might be the result of Rottlerin hydrogen donating ability and free radical scavenger activity, in addition to its biological inhibition of signaling molecules. We also provide evidence that Rottlerin inhibits NFκB activation in MCF-7 and HT-29 cells and transcriptional activity in cells transfected with the NFκB-driven plasmid pBIIX-LUC. Na-Pyruvate and L-glutamine and 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and menadione were purchased from Sigma (Milan, Italy), dihydrorhodamine 123 (DHR-123) from Invitrogen, geneticin (G418 sulfate) from Alexis (San Diego, CA), Rottlerin from Calbiochem (Milan, Italy).

Cell
Treatments. MCF-7 and HT-29 cells, pretreated with or without 20 μM Rottlerin for 30 min, were stimulated with 10 ng/mL TNFα for 4 hr. As to concern the vitamin E samples, 24 hr preincubation time with 50 μM α-tocopherol (dissolved in DMSO at the final concentration in the cells of 0.1%) was selected on the basis of uptake experiments performed on cells (data not shown).

N,N-diphenyl-N-picrylhydrzyl (DPPH) Radical Scavenging Activity.
To evaluate the anti-oxidant activity of Rottlerin, the model of scavenging the stable free radical DPPH by hydrogen donating compounds was used.
As stock solutions, 20 μM DPPH (A) and 10 μM Rottlerin (B), were prepared in absolute ethanol and mixed by adding increasing volumes of the Rottlerin solution to that of DPPH in a final volume of 3 ml to obtain DPPH/Rottlerin molar ratios 10 : 1, 4 : 1, and 2 : 1. The reaction mixtures were shaken vigorously and kept in the dark for 30 min. The absorbance of the remaining DPPH was determined at 516 nm by UV/Vis Kontron 941 spectrophotometer.
For electron paramagnetic resonance (EPR) spectroscopy, the same DPPH/Rottlerin solutions were transferred to glass capillary tubes and then placed in the EPR cavity of a Varian E4 ESR spectrometer for spectral measurements. The level of ROS was indicated by median of DHR-123 fluorescence. Debris and dead cells were discriminated from the analysis using FSC versus SSC gating.

Luciferase Reporter Construct and Stable HT-29 Cell
Transfection. The reporter construct pBIIX-LUC was kindly donated by Dr. Kalle Saksela (Institute of Molecular Medicine, University of Tampere, Finland). The NF-κBdriven plasmid pBIIX-LUC was constructed by inserting a synthetic fragment with two copies of the sequence ACA GAG GGG ACT TTC CGA GAG separated by four nucleotides (ATCT) in front of the mouse fos promoter in plasmid pfLUC [18].

HT-29 Cell Treatment and Luciferase Activity Assay.
Stably transfected cells were seeded in 12-well plates and treated, 24 hr later, with Rottlerin for 30 min followed by TNFα (10 ng/mL) for 4 hr. After the treatment, cells were rinsed with PBS and then lysed with 100 μl of reagent (Luciferase Assay System; Promega Corp., Madison, WI). Fifty μl of the extracts was mixed with 50 μl of luciferase substrate, and luminescence was quantified by luminometer.
2.9. Statistical Analysis. Results are reported as means ± SEM. Statistical analysis was performed by using Student's t-test. P < .05 was considered statistically significant. Figure 2, nuclear extracts from nonactivated MCF-7 and HT-29 cells (C) contained a basal or constitutive NFκB/DNA binding activity which was not affected by 20 μM Rottlerin treatment (R) nor by Vitamin E supplementation (V). Vitamin E was chosen as control since it is a well-known liposoluble antioxidant with the ability to inhibit NFκB. Treatment with 10 ng/ml TNFα (T) caused an elevated level of NFκB/DNA binding and the pretreatment of cells with Rottlerin or Vitamin E (T+R and T+V) inhibited TNFα-induced NFκB/DNA binding. The unlabeled NFκB consensus oligomer was used as the specific competitor and unlabeled NF-IL6 consensus oligomer as the nonspecific competitor.

Antioxidant Activity of Rottlerin: Reaction with DPPH.
In the case of the UV measurements, the DPPH and Rottlerin spectra do not overlap and the complete disappearance of the typical absorption of DPPH at 514 nm was observed when equal volume of the DPPH and Rottlerin solutions, A and B, respectively, were mixed ( Figure 3). Similar results were obtained by EPR, which directly measures the free radical concentration; by mixing the two solutions into the EPR cavity, the typical EPR signal of DPPH disappeared when equal volumes of the reagents solutions were mixed, that is in a 2 : 1 molar ratio (Figur 4), because two molecules of DPPH are reduced by one molecule of Rottlerin. Figure 5, both 5 and 20 μM Rottlerin significantly decreases MCF-7 basal ROS levels and largely prevent ROS generated by a 30-min treatment with either 100 μM H 2 O 2 or 100 μM menadione. Figure 6, Rottlerin pretreatment for 30 min prevented activation of NFκB in a dose-dependent manner, both basal and after a 4 hr TNFα treatment in HT-29 cells transfected with the NFκB-driven plasmid pBIIX-LUC.

Effect of Rottlerin on NFκB Activation in HT-29 Transfected Cells. As shown in
These results confirm that Rottlerin is a general inhibitor of NFκB nuclear migration and transcriptional activity.

Discussion
DPPH is a stable free radical, which is widely used to test the hydrogen donation of antioxidants [33]. Recently, the ability of Rottlerin to scavenge DPPH in comparison to other phenolic phytochemicals has been described. Empirically established concentrations of phytochemicals and DPPH showed that the Rottlerin scavenging ability was lower than that of resveratrol and genistein, higher than that of quercetin and epigallocatechin gallate, and roughly of the same order of that of curcumin [30].
In the present study, in order to further evaluate the hydrogen donation power of Rottlerin, we reacted one molecule of Rottlerin with two molecules of DPPH and followed the reaction by UV measurement and EPR spectroscopy (Figures 2 and 3). This last reaction is thermodynamically supported by the fact that the Bond Dissociation Enthalpy of the N-H group of the reduced DPPH is 80 Kcal/mol [34] and that of the O-H group of Vit-E is 77.9 Kcal/mol [35]. On the basis of this data it may be assumed that the hydrogen donation power of Rottlerin is of the same order of that of Vit-E, which is recognized like the best antioxidant for lipid systems [36]. The radical scavenging ability of polyphenols depends mainly on the positioning of the O-H groups rather than on their number [37], and this could be evident in Rottlerin, which possesses five phenolic O-H all conjugated to the same −CH 2 − group. This may suggest a higher antioxidant activity than that of the two frequently used ROS scavengers, namely, butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT), which show BDEs of 81.1 and 79.1 Kcal/mol, respectively [38]. Because DPPH is an artificial radical and may not reproduce the in vivo situation, we also tested the Rottlerin antioxidant properties on cultured cells. According to the DPPH radical-reducing activity and its fast reactivity, Rottlerin neutralized hydrogen peroxide added to the cells and prevents free radicals generation ( Figure 4). These finding are consistent with our previous report showing that Rottlerin quickly inhibited NFκB activation triggered by hydrogen peroxide in HaCaT keratinocytes [11] and are in accordance with the study by Longpre and Loo [30], showing that 20 μM Rottlerin prevented deoxycholate-induced ROS generation in HCT-116 cells.
Rottlerin-mediated inhibition of NFκB is not restricted to PMA [10] and hydrogen peroxide [11] because it also blunts TNFα-mediated NFκB activation. The current study indeed showed that NFκB nuclear migration and transcriptional activity, both basal and after stimulation by TNFα, are strongly inhibited by the drug in native MCF-7 cells and in HT-29 cells, and in HT-29 transfected with the NFκBdriven plasmid pBIIX-LUC, (2). Our results also parallel with a study by Zhang et al. where has been shown that Rottlerin inhibits NFκB activation in MCF7 sensitized cells to TRAILinduced apoptosis [39].
Taking in account the current results and previous data on PMA-stimulated MCF-7 cells [10] and H 2 O 2 -stimulated-HaCaT cells [11], it can be safely stated that Rottlerin is By integrating these findings, we conclude that the inhibitory effect of Rottlerin on NFκB is likely the result of a convergent double activity, free radical scavenging, and inhibition of key molecules, such as CaMKII [10] and/or PKC [40,41] involved in the NFκB activation process.
Activation of NFκB is regulated by a bewildering number of signaling pathways, the contribution of which, in particular the cascade that mediates oxidative stress-induced NFκB, is not always known in detail. The only target common to the several positive or negative signals to NFκB is the IKK complex, but this, representing the final converging step of the different cascades, does not give information on the upstream mediators.
It is likely that Rottlerin acts at a step in which all the stimuli used (PMA, H 2 O 2 , menadione, TNFα) converge in the signal transduction pathway leading to NFκB activation.
It has been reported that 100 μM Resveratrol blocks NFκB activation through inhibition of the upstream PKC δ/PKD cascade in HeLa cells [41]. Since Rottlerin, at doses between 5 and 20 μM, is a PKCδ inhibitor, we initially thought that the Resveratrol's mechanism of action could explain also the Rottlerin effects on NFκB in MCF-7 cells. However, a preliminary experiment showed that 20 μM Rottlerin did not inhibit PMA-stimulated PKD phosphorylation on Ser 744 and Ser 748 (unpublished observation), indicating that Resveratrol and Rottlerin, although both are antioxidant with similar potency [30] and PKC δ inhibitors, act with different mechanisms in NFκB inhibition. 6 Journal of Biomedicine and Biotechnology As far as the Rottlerin antioxidant effect is concerned, the interference of other natural antioxidants in the NFκB activation process has been already described. For example, mangiferin, a natural polyphenol with antioxidant properties, and melatonin, another potent antioxidant, block TNFinduced NFκB activation [42,43].
In closing, the antioxidant effect of Rottlerin surely plays a main role in the NFκB activation process triggered by PMA or H 2 O 2 or menadione or TNFα, but further studies will be necessary to establish the relative contribution of the two (chemical and biological) mechanisms and to clarify whether they act in parallel or in interdependent ways.