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Carcinogenesis Advance Access originally published online on May 13, 2008
Carcinogenesis 2008 29(6):1249-1257; doi:10.1093/carcin/bgn114
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Curcumin decreases 12-O-tetradecanoylphorbol-13-acetate-induced protein kinase C translocation to modulate downstream targets in mouse skin

Rachana Garg, Asha G. Ramchandani and Girish B. Maru*

Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India

* To whom correspondence should be addressed. Tel: +91 22 27405022; Fax: +91 22 27405085/+91 22 27415894; Email: gmaru{at}actrec.gov.in


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Curcumin has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumour promotion and some of the TPA-responsive markers in mouse skin. However, its mechanism of action is not fully elucidated. The present study focuses on understanding the role of protein kinase C (PKC), the major cellular receptor of TPA, in mediating TPA-induced biological responses in mouse skin and subsequently, elucidating the effects of curcumin on PKC and its downstream target molecules. As compared with controls, single topical application of TPA (5 nmol) to skin increased the translocation of PKC from cytosolic to particulate fraction, determined in terms of activity and protein levels. Ro-31- 8220 (PKC inhibitor, 1 nmol) when applied topically, alone or prior to TPA, inhibited PKC activity in both the compartments but did not affect the TPA-induced protein translocation. In contrast, though curcumin (10 µmol) alone did not alter the basal activity/levels, its pre-treatment decreased the TPA-induced translocation of PKC isozymes ({alpha}, β, {gamma}, {varepsilon}, {eta}), resulting in appropriate alterations in activity. Despite differences in modes of action of Ro-31-8220 (activity inhibition) and curcumin (decreasing translocation) in modulating PKC, their pre-treatment blunted the TPA-induced levels of mitogen-activated protein kinases and transcription factors (c-jun, c-fos and nuclear factor-kappa B) and downstream target proteins associated with cell proliferation (cyclin D1 and ornithine decarboxylase), cell death (Bax and Bcl2), inflammation (cyclooxygenase-2 and prostaglandin E2) and oxidative stress (8-hydroxy-2'-deoxyguanosine) in skin. These results demonstrate the crucial role of PKC in TPA-mediated cellular responses in skin and that curcumin modulates transmembrane signal transduction via PKC to affect TPA-induced biochemical and molecular alterations in mouse skin.

Abbreviations: AP-1, activator protein-1; COX-2, cyclooxygenase-2; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor-kappa B; ODC, ornithine decarboxylase; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; PGE2, prostaglandin E2; PKC, protein kinase C; p38, p38 protein kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Several phorbol esters are well-known tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) being the prototype for many years. Extensive studies on mode of action of TPA in mouse skin have shown that TPA-induced tumour promotion is accompanied by alterations in various cellular processes including cell growth, cell differentiation, cell death and inflammatory responses (1–4). Although a variety of intracellular signalling cascades are activated in response to TPA, studies have shown that the primary site of action of phorbol esters is located on cell surface membranes (5,6) and that protein kinase C (PKC) acts as the possible receptor protein for tumour-promoting phorbol esters (7,8).

Ca2+ and phospholipid-dependent PKCs represent a family of second messenger-dependent protein kinases that play a pivotal role in mediating cellular responses to extracellular stimuli. Upon stimulation, PKC translocates to the plasma membrane where it interacts with diacylglycerol and phosphatidylserine (9). In vitro and in vivo studies have documented the involvement of PKC in regulating target proteins associated with proliferation, differentiation, apoptosis, angiogenesis, invasion or metastasis (10–12). PKCs have, therefore, also been implicated in the promotion and/or progression phase of carcinogenesis (10,13).

TPA activates PKC by mimicking diacylglycerol, a natural ligand and activator of PKC, for binding to specific motifs in its regulatory domain (14). It has previously been shown that TPA treatment led to a relative increase in PKC activity in mouse epidermal particulate fraction (15); however, its association with TPA-induced molecular perturbations in mouse skin remains obscure. Earlier reports suggest that topical application of TPA resulted in increased expression of ornithine decarboxylase (ODC), the rate controlling enzyme in polyamine biosynthesis as well as cyclooxygenase-2 (COX-2) that catalyzes the rate-limiting step in prostaglandin biosynthesis and inflammation in mouse skin (16–18). TPA-induced COX-2 expression, in turn, is regulated by eukaryotic transcription factors, nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1) and upstream kinases including inhibitory kappa B kinase and mitogen-activated protein kinases (MAPKs) (19). Some of these TPA-triggered effects in mouse skin appear to be mediated, in part, by PKCs; hence, regulation of PKC activation by chemopreventives is likely to provide a rationale for controlling biological consequences elicited by tumour promoter.

The chemopreventive efficacy of turmeric/curcumin has been established in several experimental systems (20–22). Curcumin has also been shown to inhibit TPA-induced tumour promotion, ODC activity, cell proliferation and expression of oncogenes in mouse epidermis (23–26). An in vitro finding in NIH3T3 cells suggests that curcumin significantly inhibited TPA-induced PKC activity in particulate fraction with non-significant decrease in cytosolic fraction (27); however, the effects of curcumin on TPA-induced PKC in vivo largely remain unexplored.

The present study, therefore, aims to first investigate the role of PKC in TPA-induced molecular perturbation in mouse skin employing a PKC inhibitor and further to study the effects of curcumin on PKC and its downstream targets in TPA-treated mouse skin. In the present study, Ro-31-8220, a staurosporine analogue, has been employed as PKC inhibitor. It has been shown to be selective for PKC over both protein kinase A (PKA) and Ca2+/calmodulin-dependent kinase and inhibits PKC by competing with adenosine triphosphate for co-substrate site (28).

We demonstrate that though Ro-31-8220 (a known PKC inhibitor) and curcumin differ in their actions in modulating PKC in mouse skin, their topical application prior to TPA abrogated the activation of TPA-responsive markers (associated with oxidative damage, inflammation, proliferation and apoptosis) in skin. Thus, this study highlights the importance of PKC in TPA-mediated cellular actions in skin as well as demonstrates the role of a PKC-dependent mechanism in curcumin-mediated protective effects.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials
TPA, curcumin, aprotinin, leupeptin, phenylmethylsulphonyl fluoride and Ro-31-8220 were purchased from Sigma Chemical Company (St Louis, MO). Oligonucleotide probes were purchased from Sigma Chemical Company (Bangalore, India). Antibodies for COX-2, ODC, β-actin, β-tubulin, p38 protein kinase (p38), extracellular signal-regulated protein kinase (ERK) 1/2, c-Jun N-terminal protein kinase (JNK) 2, c-jun, c-fos, NF-{kappa}B p65, I{kappa}B{alpha}, Bax, Bcl2 and histone H1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho form of MAPKs (p38, ERK1/2 and JNK) were purchased from Cell Signalling Technology (Beverly, MA), whereas those for PKC isozymes ({alpha}, β, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}) were from BD Transduction Laboratories (San Jose, CA). {alpha}-32P deoxyadenosine triphosphate (specific activity >3800 Ci/mmol) was purchased from Board of Radiation & Isotope Technology (Mumbai, India).

Animal treatment
All animal studies were conducted after approval from the Institutional Animal Ethics Committee as per Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India guidelines. Female S/RVCri-ba or ‘bare’ mice—hairless mutants [that are highly susceptible to skin tumorigenesis (29)], were obtained from the animal colony of Advanced Centre for Treatment, Research and Education in Cancer (India). Bare mice (6–8 weeks old) were randomized and housed under standard conditions of 22 ± 2°C, 45 ± 10% relative humidity and 12 h light–dark cycles and were provided with standard pelleted diet and plain drinking water ad libitum. TPA (5 nmol) in 0.1 ml acetone was applied topically onto the dorsal skin of mice belonging to groups 2, 4 and 6 (Figure 1). Mice in group 6 were topically treated with 10 µmol curcumin in 0.1 ml acetone 20 min prior to TPA application. Animals of group 1 treated with 0.1 ml acetone alone and group 5 with 10 µmol curcumin in acetone served as the respective controls. To investigate the involvement of PKC in TPA-elicited molecular alterations in mouse skin, animals in group 4 were topically treated with 1 nmol of PKC inhibitor, Ro-31-8220 (30) in acetone, 20 min prior to TPA application and those in group 3 with 1 nmol of Ro-31-8220 in acetone alone. Mice were killed at various time intervals after TPA treatment (0, 1, 2, 4 and 6 h) for the determination of optimal duration of TPA exposure to elicit early response markers. Thereafter in subsequent experiments, mice were killed 4 h after TPA application and their dorsal skin was excised. A part of the skin was fixed in 10% buffered formalin for histological analysis, whereas rest of the tissue was stored at –80°C. Tissues of animals belonging to the various treatment groups were washed with acetone since this facilitates the removal of maximal colour of curcumin from the skin before separation of the epidermis. This was done to avoid colour interference in protein determination and subsequent analysis.


Figure 1
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  		Fig. 1. Experimental design for studying the effect of curcumin pre-treatment on TPA-induced PKC translocation and downstream targets in mouse skin.

 
Determination of hyperplasia
To ascertain the inhibitory effect of curcumin or Ro-31-8220 pre-treatment on TPA-induced hyperplasia, histopathological analysis was performed on formalin-fixed, paraffin-embedded 5 µm tissue sections and stained with haematoxylin and eosin.

Protein immunoblotting
Epidermis was gently separated from skin using Watson's skin grafting knife with suitably adjusted cutting angle. Total cell, cytosolic or nuclear extracts were prepared from the epidermis by previously described cell fractionation protocol (16). The lysates were aliquotized and stored at –80°C until further use. Protein concentrations were determined using bovine serum albumin as a standard (31). Cytosolic (50 µg), nuclear (100 µg) and total cell proteins (50 µg) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (8–12%) and transferred to polyvinylidene difluoride membranes. Blots were then blocked with 5% milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and probed with respective primary antibodies at a dilution of 1:1000 overnight at 4°C. The membranes were washed thrice with Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 for 20 min and incubated with anti-rabbit or anti-goat or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilutions). Immunoreactive bands were detected with enhanced chemiluminescence reagent from Amersham Biosciences (Buckinghamshire, UK), followed by autoradiography (32). Expressions of COX-2, ODC, JNK, ERK, p38, Bcl2 and Bax were assayed in epidermal lysates, wherein β-actin was used as a loading control. Alternatively, proteins such as c-jun, c-fos, NF-{kappa}B p65 and cyclin D1 were studied in nuclear extracts and I{kappa}B{alpha} and p-I{kappa}B{alpha} in cytosolic fraction where histone H1 and β-tubulin were used as the respective loading controls. Quantitation was done by densitometric analysis software, UV-P lab ware for windows.

PKC activity and protein levels
For measuring PKC activity and expression of its isozymes known to be expressed in mouse skin ({alpha}, β, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}) (13,33), cytosolic and particulate fractions were prepared as described (15). Briefly, extracted epidermis was homogenized in tissue extraction buffer A [20 mM Tris–HCl (pH 7.5), 0.5 mM ethylene glycol-bis(β-aminoethylether)-N, N, N', N'-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, 300 mM sucrose, 1 mM dithiothreitol with freshly added protease inhibitors] and centrifuged at 100 000 x g for 1 h at 4°C, and supernatant or the cytosolic fractions were collected. Pellet was re-suspended in tissue extraction buffer A supplemented with 0.5% Triton X-100 and centrifuged again; this supernatant constitutes the particulate fraction. Both cytosolic and particulate lysates were aliquotized and protein content determined and stored at –80°C. PKC activity in cytosolic and particulate fractions was measured using protein kinase C biotrak enzyme assay kit (Amersham Biosciences) as per the manufacturer's instructions, wherein PKC present in the samples catalyzed the transfer of {gamma}-phosphate group of adenosine-5'-triphosphate to a peptide which is specific for PKC. The extent of phosphorylation, thus, represents the PKC activity and results were expressed as pmol/min/mg protein. To study the cellular distribution of PKC isozymes ({alpha}, β, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}), 50 µg of cytosolic and particulate proteins were separated on an 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted as described above using isozyme-specific antibodies (1:1000 dilution).

Prostaglandin E2 measurement
Epidermis was gently separated from skin as described above and TPA-induced inflammatory response was assessed by measuring prostaglandin E2 (PGE2) levels in epidermis employing PGE2-EIA Kit from Cayman Chemical Company (Ann Arbor, MI) as per the manufacturer's instructions. Results were expressed as PGE2 pg/ml of homogenate.

8-Hydroxy-2'-deoxyguanosine measurement
Levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) were measured in DNA isolated (34) from mouse skin epidermis. DNA samples were digested with nuclease P1 and alkaline phosphatase and purified before they were quantitatively measured for the oxidative DNA adduct, 8-OH-dG, employing ELISA kit (JAICA, Fakuroi City, Japan) as per the manufacturer's instructions. Results were expressed as 8-OH-dG ng/ml of homogenate.

Electrophoretic mobility shift assay
Electrophoretic mobility shift assay was performed as described previously (32). Briefly, NF-{kappa}B (5'-agcttGAGGGGATTCCCTTA-3') and AP-1 (5'-agcttCGCTTGATGACTCAGCCGGAA-3') oligonucleotide probes were separately labelled with [{alpha}-32P] deoxyadenosine triphosphate using Klenow enzyme and purified on a Sephadex G-25 column. The binding reaction was carried out at 25°C for 10 min with or without 5 µg nuclear protein, 1 µg poly(dIdC), 106 c.p.m. of labelled probe in a final volume of 20 µl binding buffer [10X buffer: 100 mM Tris–HCl (pH 7.5), 500 mM NaCl, 50 mM MgCl2, 100 mM ethylenediaminetetraacetic acid, 10 mM dithiothreitol, 1% Triton-X 100, 50% glycerol]. Binding specificity was confirmed by cold competition analysis wherein 50-fold molar excess of unlabelled (cold) AP-1/NF-{kappa}B probe was added to the binding reaction. DNA–protein complexes were separated on a 6% non-denaturing polyacrylamide gel at 200 V. The gel was fixed, dried and DNA–protein complexes were visualized by autoradiography.

Statistical analysis
The statistical analysis was done with the SPSS 14.0 software for Windows. Data are presented as mean ± SE. Means of all data were compared by analysis of variance with post hoc testing. P ≤ 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
TPA induces COX-2 and ODC protein expression maximally at 4 h in mouse epidermis
To select the optimal time duration for TPA response, expressions of COX-2 and ODC (early response markers) were analysed at 0, 1, 2, 4 and 6 h in mouse skin following application of TPA. As shown in Figure 2, topical application of 5 nmol TPA onto the dorsal skin of Swiss bare mice resulted in increased COX-2 and ODC protein expressions as compared with acetone-treated animals. However, the increase in COX-2 and ODC expressions were maximal after 4 h of TPA application and then their protein levels gradually subsides but were still higher than the control animals treated with acetone (Figure 2). Therefore, killing after 4 h of TPA application was selected as the time point for all further evaluations in the study.


Figure 2
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  		Fig. 2. Effect of TPA treatment on early response markers in mouse skin. Dorsal skin of female Swiss bare mice was topically treated with 5 nmol TPA or acetone. Animals were killed at various time points after TPA application. Total cell extract (50 µg) prepared from skin epidermis was analyzed for COX-2 and ODC protein expression by western blotting. Quantification was done by normalizing the band density to that of β-actin. Data represent mean ± SE of six observations. ‘a’ significantly different from animals killed at 0 h of TPA application; ‘b’ significantly different from animals killed at 4 h of TPA application (P ≤ 0.05).

 
Ro-31-8220 inhibits PKC activity, whereas curcumin decreases TPA-induced PKC translocation
PKCs are the major intracellular target for activation by tumour-promoting phorbol esters. As compared with controls, topical application of TPA onto the dorsal skin of bare mouse resulted in relative decrease in PKC activity in the epidermal cytosolic fraction (Figure 3A), whereas the activity increased significantly in particulate fraction (Figure 3B). In concordance with the enzyme activity, TPA treatment enhanced the translocation of PKC isozymes ({alpha}, β, {gamma}, {varepsilon}, {eta}) from cytosolic to particulate fraction, as evident from their relative protein levels in the two compartments (Figure 3C). However, TPA treatment did not alter the levels of PKC {delta} and {zeta} in the two compartments. Topical application of 1 nmol Ro-31-8220 (PKC inhibitor) alone attenuated the PKC activity in cytosolic as well as in particulate fraction as compared with controls (Figure 3A and B) but did not alter the protein levels of PKC isozymes (Figure 3C). Interestingly, while, pre-treatment of mouse skin with Ro-31-8220 inhibited PKC activity in both the compartments (Figure 3A and B), TPA-induced translocation of protein remained unaltered, resulting in similar levels of proteins as observed in animals treated with TPA alone (Figure 3C). In contrast, PKC activity as well as the levels in both the compartments of mouse treated with curcumin alone was comparable with acetone-treated animals (Figure 3D–F). However, topical application of 10 µmol curcumin prior to TPA significantly decreased the TPA-induced translocation of PKC isozymes ({alpha}, β, {gamma}, {varepsilon}, {eta}) from cytosol to membranes, measured in terms of protein levels (Figure 3F). This was further correlated with the effects on enzyme activity, wherein curcumin pre-treatment significantly reduced both the TPA-mediated decrease in cytosolic PKC activity (20%, Figure 3D) as well as the TPA-mediated increase in particulate PKC activity (up to 29%, Figure 3E).


Figure 3
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  		Fig. 3. Effect of Ro-31-8220 and curcumin on PKC activity in mouse skin. Swiss bare mice were topically treated with 1 nmol Ro-31-8220 or 10 µmol curcumin 20 min prior to TPA (5 nmol) application; mice were then killed 4 h after TPA application. Cytosolic and particulate cell lysates were prepared from skin epidermis. Effect of Ro-31-8220 treatment, alone or prior to TPA, on PKC activity in (A) cytosolic fraction and (B) particulate fraction of mouse epidermis. (C) Protein levels of PKC isozymes ({alpha}, β, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}) were assayed in cytosolic (50 µg) and particulate (50 µg) fraction of epidermis of mice belonging to the different treatment groups. Ace, acetone + acetone; TPA, acetone + TPA; Ro, Ro-31-8220 + acetone; Ro + TPA, Ro-31-8220 + TPA. Data represent mean ± SE of five observations. Differences among groups were determined by one-way analysis of variance followed by Bonferroni test, P ≤ 0.05. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA; c, significantly different from Ro. Effect of curcumin treatment (applied alone or prior to TPA) on PKC activity in (D) cytosolic fraction and (E) particulate fraction as well as on (F) the protein levels of PKC isozymes ({alpha}, β, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}) in cytosolic (50 µg) and particulate (50 µg) fraction of epidermis of mice belonging to the different treatment groups. Ace, acetone + acetone; TPA, acetone + TPA; Cur, curcumin + acetone; Cur + TPA, curcumin + TPA. Data represent mean ± SE of five observations. Differences among groups were determined by one-way analysis of variance followed by Bonferroni test, P ≤ 0.05. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA; c, significantly different from Cur.

 
Overall, results suggest that Ro-31-8220 inhibits PKC activity with no effects on protein translocation, whereas curcumin decreased the TPA-induced PKC translocation without inhibiting the activity.

PKC acts as a key player in TPA-induced biochemical and molecular alterations in mouse skin
Ro-31-8220 being the inhibitor of PKC activity was employed for delineating the downstream molecular targets of PKC in mouse skin, elicited in response to TPA. It was observed that pre-treatment of mouse skin with Ro-31-8220 significantly decreased the TPA-induced levels of COX-2 and PGE2 (inflammatory markers) (Figure 4A and B), ODC (marker of proliferation and differentiation) (Figure 4A) and 8-OH-dG (marker of oxidative DNA damage) (Figure 4B) in mouse epidermis. As compared with acetone treatment, topical application of TPA significantly increased the hyperplasia and expression of cyclin D1 (proliferation marker) and Bcl2, an anti-apoptotic protein, while it decreased the levels of apoptotic protein, Bax, in mouse epidermis (Figure 4C–E). Importantly, pre-treatment of mouse skin with Ro-31-8220 decreased the TPA-induced hyperplasia (Figure 4D) and expression of cyclin D1 (Figure 4E) and Bcl2 (Figure 4C), whereas it prevented the depletion of Bax (Figure 4C), thereby demonstrating that PKC plays an important role in TPA-induced proliferation and inhibition of apoptosis in mouse skin. It is well documented that these TPA-induced effects, in turn, are regulated by transcription factors such as NF-{kappa}B and AP-1 and upstream MAPKs. Because phosphorylation is indicative of activation of MAPK, anti-phospho-MAPK antibodies were employed to assay their levels by immunoblotting. Importantly, pre-treatment with Ro-31-8220 significantly decreased the TPA-induced phosphorylation of MAPKs (ERK, p38 and JNK) (Figure 4C) as well as it diminished the nuclear protein levels of NF-{kappa}B, c-jun and c-fos in mouse skin (Figure 4E). Besides, pre-treatment of mice with Ro-31-8220 blunted the TPA-mediated increased p-I{kappa}B{alpha} protein expression, which in turn accounts for the reduced TPA-mediated degradation of I{kappa}B{alpha} protein (Figure 4E). This is in agreement with the observed decrease in TPA-induced nuclear translocation of NF-{kappa}B upon pre-treatment with Ro-31-8220. Additionally, Ro-31-8220 pre-treatment attenuated the TPA-induced AP-1 and NF-{kappa}B DNA binding in mouse epidermal nuclear extracts (Figure 4F). Results, thus, suggest PKC to be one of the key players in TPA-induced molecular or biochemical alterations in mouse skin.


Figure 4
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  		Fig. 4. Effect of Ro-31-8220 pre-treatment on various TPA-responsive markers in mouse skin. Swiss bare mice were topically treated with Ro-31-8220 (1 nmol) 20 min prior to TPA (5 nmol) application; mice were then killed 4 h after TPA application. Total cell, cytosolic and nuclear extracts were prepared from skin epidermis. (A) Protein levels of COX-2 and ODC were measured in epidermal total cell extracts (50 µg) by western blotting using specific antibodies. Quantification was done by normalizing the band density to that of β-actin. Effect of Ro-31-8220 pre-treatment on TPA-induced (B) inflammatory response measured as levels of PGE2 in skin epidermis by enzyme immuno assay (left panel) and oxidative DNA damage quantitated as 8-OH-dG levels in epidermal DNA by enzyme-linked immunosorbent assay (right panel). Data represent mean ± SE of five observations. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA. (C) Effects of Ro-31-8220 pre-treatment on TPA-induced responses on cellular kinases (p-ERK, p-p38 and p-JNK) and apoptosis-related markers (Bax and Bcl2) were studied by measuring their protein levels in total cell extract (50 µg) by immunoblotting. β-Actin was used as the loading control. (D) Effects of Ro-31-8220 pre-treatment on TPA-induced hyperplasia analyzed by the histopathological observation of formalin-fixed, haematoxylin and eosin-stained tissue sections, magnification x400. (E) Effects of Ro-31-8220 pre-treatment were studied on TPA-induced levels of c-jun, c-fos, NF-{kappa}B p65, cyclin D1 in epidermal nuclear extracts (100 µg) (left panel), whereas levels of p-I{kappa}B{alpha} and I{kappa}B{alpha} were measured in cytosolic extracts (50 µg) of mice belonging to the various treatment groups (right panel). Band density was normalized to histone H1 and β-tubulin in respective cellular compartments (bottom panel). Ace, acetone + acetone; TPA, acetone + TPA; Ro, Ro-31-8220 + acetone; Ro+ TPA, Ro-31-8220 + TPA. Data represent mean ± SE of five observations. Differences among groups were determined by one-way analysis of variance followed by Bonferroni test, P ≤ 0.05. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA. (F) Binding of NF-{kappa}B and AP-1 to consensus sequence in epidermal nuclear extracts were analyzed by electrophoretic mobility shift assay. The binding specificity was confirmed by cold competition, wherein 50-fold molar excess of unlabelled NF-{kappa}B or AP-1 consensus sequence was added. The position of DNA-bound NF-{kappa}B or AP-1 is indicated by arrow; ‘FP’ indicates the free probe. Data show representative electrophoretic mobility shift assay blot from four independent experiments, showing the similar trend.

 
Curcumin decreases TPA-induced activation of transcription factors and upstream MAPKs
Having observed the response of curcumin pre-treatment on TPA-mediated PKC translocation and the importance of PKC in TPA-mediated cellular responses in mouse skin, we next investigated the effects of curcumin on some of the TPA-responsive markers (the downstream targets of PKC) in mouse skin. When applied topically onto the dorsal skin of mice 20 min prior to TPA, 10 µmol curcumin significantly decreased the TPA-induced phosphorylated levels of JNK, ERK and p38 in mouse epidermis, although the extent of decrease varied for the three kinases (Figure 5A). This was similar to that observed with pre-treatment of Ro-31-8220 (Figure 4D). The levels of total form of each kinase remained unaltered among the various treatment groups under the experimental conditions (Figure 5A). Furthermore, similar to the PKC inhibitor Ro-31-8220 (Figure 4E), pre-treatment with curcumin also significantly decreased the TPA-induced nuclear protein expressions of c-jun and c-fos (59–61%) in mouse epidermis (Figure 5B). Pre- treatment with curcumin resulted in decreased TPA-induced phosphorylation of I{kappa}B{alpha} significantly, thus averting the TPA-mediated degradation of I{kappa}B{alpha}, which consequently resulted in decreased TPA-induced nuclear translocation of NF-{kappa}B p65 (Figure 5B). Besides, as observed with Ro-31-8220 (Figure 4F), pre-treatment with curcumin blunted the TPA-induced DNA-binding ability of AP-1 and NF-{kappa}B in epidermal nuclear extracts of mice (Figure 5C). The protein levels of c-jun, c-fos as well as NF-{kappa}B were similar in epidermal nuclear extracts from mice treated with acetone or curcumin alone (Figure 5B).


Figure 5
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  		Fig. 5. Effect of curcumin pre-treatment on TPA-responsive markers in mouse skin. Swiss bare mice were topically treated with curcumin (10 µmol) 20 min prior to TPA (5 nmol) application; mice were then killed 4 h after TPA application. Total cell, cytosolic and nuclear extracts were prepared from skin epidermis. (A) Protein levels of phosphorylated MAPKs were assessed in total cell extract (100 µg) prepared from epidermis of mice belonging to the various treatment groups. Protein levels of p-ERK, p-JNK and p-p38 were normalized to that of total kinase; levels of each of which remained unaltered under same experimental conditions. (B) Effect of curcumin pre-treatment on TPA-induced levels of c-jun, c-fos and NF-{kappa}B p65 was studied in epidermal nuclear extracts (100 µg), whereas levels of p-I{kappa}B{alpha} and I{kappa}B{alpha} were measured in epidermal cytosolic extracts (50 µg) of mice belonging to the various treatment groups. Band density was normalized to histone H1 and β-tubulin in respective cellular compartments. Ace, acetone + acetone; TPA, acetone + TPA; Cur, curcumin + acetone; Cur + TPA, curcumin + TPA. Data represent mean ± SE of five observations. Differences among groups were determined by one-way analysis of variance followed by Bonferroni test, P ≤ 0.05. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA; c, significantly different from Cur. (C) Binding of NF-{kappa}B and AP-1 to consensus sequence in epidermal nuclear extracts was analyzed by electrophoretic mobility shift assay. The binding specificity was confirmed by cold competition, wherein 50-fold molar excess of unlabelled NF-{kappa}B or AP-1 consensus sequence was added. The position of DNA-bound NF-{kappa}B or AP-1 is indicated by arrow; ‘FP’ indicates the free probe. Data show representative electrophoretic mobility shift assay blot from four independent experiments, showing the similar trend.

 
Curcumin decreases the TPA-induced inflammation, oxidative damage as well as proliferation and it induces apoptosis in mouse epidermis
As has been observed with Ro-31-8220, topical application of curcumin 20 min prior to TPA significantly reduced the TPA-induced protein expressions of COX-2 (63%) and ODC (67%) in mouse epidermis (Figure 6A). It was also observed that pre-treatment with curcumin reduced the TPA-induced levels of PGE2 (49%) in mouse skin significantly, thereby suggesting that curcumin protects against TPA-induced inflammation (Figure 6B). Further, protective effect of curcumin in mouse skin was also observed in abrogation of TPA-induced oxidative damage (up to 51%), measured as levels of 8-OH-dG in mouse epidermal DNA (Figure 6C). The basal levels of COX-2, ODC, PGE2 and 8-OH-dG were detected in skin of animals treated with acetone or curcumin alone. Histopathological observations (Figure 6D) indicated that although the topical application of curcumin alone did not induce any epidermal hyperplasia in mouse skin, it markedly reduced the TPA-induced hyperplasia. This was further complemented with immunoblot analyses of cell proliferation marker, cyclin D1, showing a significant decrease in TPA-induced protein levels of cyclin D1 upon curcumin pre-treatment in skin epidermis (Figure 6D, bottom panel), as was observed with Ro-31-8220 (Figure 4E). Pre-treatment with curcumin also significantly abrogated the TPA-induced expression of anti-apoptotic protein Bcl2, while it increased the expression of Bax, pro-apoptotic marker, in mouse skin (Figure 6E). Curcumin-mediated inhibition of TPA-induced anti-apoptotic response is also reflected in increased Bax to Bcl2 ratio (Figure 6E).


Figure 6
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  		Fig. 6. (A) Protein levels of COX-2 and ODC were measured in epidermal total cell extracts (50 µg) by western blotting using specific antibodies. β-Actin was used as loading control. Effect of curcumin pre-treatment on TPA-induced (B) inflammatory response marker PGE2 in skin epidermis by enzyme immuno assay and (C) oxidative damage marker quantitated as levels of 8-OH-dG in epidermal DNA by enzyme-linked immunosorbent assay. Data represent mean ± SE of five observations. ‘a’ significantly different from Ace; ‘b’ significantly different from TPA. (D) Effect of curcumin pre-treatment on TPA-induced hyperplasia in mouse skin analyzed by the histopathological observation of formalin-fixed, haematoxylin and eosin-stained tissue sections. Magnification x400 (upper panel). Also, the effect of curcumin pre-treatment was studied on TPA-induced cell proliferation marker, cyclin D1 in nuclear lysate (50 µg) of mice belonging to the various treatment groups, levels were normalized to histone H1 (bottom panel). (E) Effect of curcumin pre-treatment on TPA-altered apoptosis-related markers. Protein levels of Bax and Bcl2 were measured in epidermal total cell extracts (50 µg) by western blotting using specific antibodies. β-Actin was used as loading control. Ratio of normalized band density of Bax and Bcl2 was used as the measure of apoptotic index (lower panel). (F) Histogram showing the relative percentage decreases in TPA-induced response markers upon Ro-31-8220 and curcumin pre-treatment.

 
These results, thus, show an analogous response of PKC inhibitor and curcumin on TPA-induced cellular kinases and transcription factors and subsequent downstream effector molecules, mediating cellular responses in mouse skin. However, the extent of decrease in TPA-induced response markers was relatively higher upon Ro-31-8220 pre-treatment as compared with curcumin pre-treatment (Figure 6F).


   Discussion
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 Materials and methods
 Results
 Discussion
Funding
 References
 
Chemopreventive efficacy of curcumin has been established at both the initiation and promotion stages of carcinogenesis (32,35–37). Previously, curcumin was shown to inhibit TPA-induced tumour promotion in mouse skin (23); however, the effects of curcumin on PKC, the major target/receptor protein of TPA, in vivo in mouse skin still remain obscure. In fact, though numerous studies have shown that TPA treatment of mouse skin is associated with inflammatory response (38), oxidative stress (39), proliferation (16) and activation of nuclear oncogenes (40), the precise mechanism of action of TPA in skin is yet not fully understood. PKCs, that play an important role in transducing the signal from mediators across the membranes, have been implicated in regulating numerous cellular events including cell proliferation, cell death and cell differentiation (10,11). Modulation of PKC, therefore, is likely to have therapeutic potential for several diseases including cancer; PKCs could, thus, be one of the promising targets of chemopreventives. The major aim of this study was to understand the role of PKC in TPA-mediated cellular responses and the response of curcumin on TPA-altered PKC in mouse skin and its downstream targets and to obtain an insight into the mechanisms of protective effects of curcumin in skin.

A number of agents (sphingosine, staurosporine, tamoxifen, palmitoylcarnitine, cyclosporine, chlorpromazine, gossypol, cremophorEL, clomiphene, amiloride, bromophenacyl bromide, polymyxin sulphate, berberine sulphate, glycyrrhetic acid, H-7, mellitine, trifluoroerzine and quercetin) with diverse structure and pharmacological properties have been shown to inhibit PKC activity (41,42). Because many of these PKC inhibitors are not specific, their inhibitory effects on TPA-induced tumour promotion in mouse skin could not be ascribed with certainty to PKC action (41,43). Ro-31-8220 (a staurosporine analogue) has been shown to be a selective inhibitor of PKC, both over PKA and Ca2+/calmodulin-dependent kinase dependent kinase, in membrane-free systems, competing with adenosine triphosphate for co-substrate site (28). Our results demonstrate for the first time the inhibitory effect of Ro-31-8220, whether applied alone or prior to TPA, on PKC activity in both the soluble and particulate fraction of mouse skin. Although Ro-31-8220, with or without TPA, inhibited PKC activity in both the compartments, it did not alter the TPA-induced PKC protein translocation. Ro-31-8220 has been shown to abrogate the effect of TPA on platelet-activating factor-induced (Ca2+)i elevation in platelets (30) and to block the TPA-induced activation of MAPK phosphatase-1 in Rat-1 fibroblasts (44). However, the in vivo anti-promoting effects of Ro-31-8220 in tissues have not been reported. Our results demonstrate that pre-treatment of mouse skin with Ro-31-8220 decreased the TPA-induced levels of phospho-MAPKs and transcription factors (c-jun, c-fos and NF-{kappa}B) in mouse skin. These effects of Ro-31-8220 pre-treatment eventually led to the attenuation of TPA-induced levels of cyclin D1, Bcl2, ODC, COX-2, PGE2 and 8-OH-dG in mouse skin. These observations thus demonstrate the key role of PKC in signalling cascades elicited in response to TPA in mouse skin and its potential role in regulation of TPA-induced proliferation, inflammation and oxidative damage.

Curcumin and its analogue have been shown to inhibit TPA-induced tumour promotion in the classical two-stage skin carcinogenesis model (17). As mentioned above, our results suggest the crucial involvement of PKC in TPA-induced tumour promotion events; we therefore investigated whether anti-promoting effects of curcumin in mouse skin are mediated via PKC. So far, the evidences for the effects of curcumin on PKC are not consistent. Reddy et al. (45) have shown that in membrane-free systems curcumin (100 µM) acts as an inhibitor of PKC, as against this another group showed that curcumin (6–48 µM) inhibited PKC activity in the absence of membranes, whereas stimulation was observed in the presence of membranes (46). In cultured NIH3T3 fibroblasts, Liu et al. (27) showed that curcumin alone did not affect the PKC activity; it inhibited the TPA-induced PKC activity only in particulate fraction. They also showed that curcumin had no effect on TPA-induced translocation of PKC protein from cytosol to particulate fraction. But, it is surprising that though curcumin inhibited the TPA-induced particulate PKC activity without altering the protein levels, inhibitory effect of curcumin employed at similar concentrations was not observed in particulate activity of control cells. Our observations present initial report in mouse skin demonstrating that curcumin at concentrations employed and have been shown to inhibit TPA-induced tumour promotion (17,23) decreased the TPA-induced translocation of protein of PKC isozymes ({alpha}, β, {gamma}, {varepsilon}, {eta}) from cytosol to membrane. This decrease in protein translocation was also reflected in the observed decrease in TPA-altered activity in the two compartments. Of interest, curcumin alone did not influence both the PKC activity as well as the protein levels in either compartment. Hence, the results suggest that unlike Ro-31-8220, curcumin acts as a modulator of PKC and not as inhibitor of PKC. Our results are in agreement with a recent in vivo study on PKC in mouse skin, though with different polyphenols (15). Accumulating data suggest that various PKC isoforms participate in the regulation of cutaneous inflammation, cell proliferation, differentiation, survival, cell death, epidermal tumour formation and malignant progression (13,33,47,48). Available evidence also shows that the activation of some TPA-dependent PKCs, especially of PKC {alpha} and {varepsilon}, is crucial for induction of tumorigenesis and tumour progression (33). Therefore, the modulatory effects of curcumin on TPA-induced PKC activity and protein levels of isoforms {alpha} and {varepsilon} would be of functional relevance in its chemopreventive actions.

Interestingly in the present study, beneficial effects of curcumin were also demonstrated on downstream effector targets of PKC in TPA-treated mouse skin, thereby affecting the TPA-induced proliferation, inflammation or oxidative damage. As reported (25), pre-treatment of mouse skin with curcumin significantly decreased the TPA-induced levels of phospho-MAPKs, nuclear levels of NF-{kappa}B and its DNA-binding ability. The findings in the present study also illustrate significant attenuation of TPA-induced binding ability of AP-1 to consensus sequence and decreased nuclear expressions of c-jun, c-fos, NF-{kappa}B, as was also observed with Ro-31-8220. These in turn accounts for the observed curcumin-mediated abrogation of TPA-induced proliferation, differentiation and inflammation (levels of ODC, COX-2 and PGE2) in murine epidermis as reported (1,17,25). In addition, the present study documents curcumin-mediated decrease in TPA-induced oxidative damage (8-OH-dG) and cyclin D1 expression in murine skin. Moreover, the apoptosis-inducing ability of curcumin in TPA-treated mouse skin, evident from the expressions of apoptotic markers, has not been reported earlier. Together, the results suggest that curcumin decreased the TPA-induced PKC protein translocation to membranes to prevent transmembrane signal transduction and hence biological consequences. Earlier studies with curcumin have reported effects on a few TPA-responsive markers but were not complemented with its effect on PKC, whereas our study is more comprehensive in elucidating its effects on PKC and downstream targets related to cell death, cell proliferation, inflammation, oxidative damage.

In the present study, comparative evaluations of two PKC modulators suggest that relatively lower concentration of Ro-31-8220 (a specific inhibitor, 1 nmol) brought about stronger effects on TPA-induced response markers when compared with curcumin (a natural and non-specific inhibitor, 10 µmol). However, it must also be noted that Ro-31-8220 affected the basal activity, while curcumin decreased only the TPA-induced response without affecting the basal activity. Based on these effects, it may be speculated that Ro-31-8220, although is more efficacious, may affect the physiological functions, and therefore result in toxicity of compound whereas curcumin is not likely to have such effects. Plant-derived polyphenols may prove to be useful modulators of PKC and relevant downstream targets/responses. However, the present study does not provide insight into the exact mechanisms of modulation of PKC by curcumin. It is not clear whether it is the direct/indirect effects on PKC protein. Further studies on the exact mechanism of curcumin-mediated modulation of PKC may be useful in deciding its usefulness.

To surmise, our results suggest that although mode of regulation of PKC in skin by Ro-31-8220 (inhibition of activity) and curcumin (decreasing TPA-induced protein translocation and hence activity) is different, they share commonality in altering TPA-induced cellular responses in skin. This in a way suggests that curcumin modulates TPA-induced molecular and biochemical alterations in mouse skin via a PKC-dependent mechanism. Though in the present study, effect of curcumin on DMBA-initiated and TPA-promoted tumour formation in mouse skin has not been studied, it can be speculated based on the observations here that curcumin might be similarly altering PKC to modulate the signalling cascades, leading to inhibition of TPA-induced tumour promotion in mouse skin.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Indian Council of Medical Research; Council of Scientific and Industrial Research to R.G.


   Acknowledgments
 
The authors thank Dr M.Krishna for critical reading of the manuscript and Mr Prasad Phase and Surendra Gosavi for technical assistance.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received March 14, 2008; revised April 25, 2008; accepted April 29, 2008.


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