Introduction

To reduce the occurrence of cancer, one promising approach is its prevention, specifically by chemical intervention through minor non-nutrient dietary constituents. Important to chemoprevention is the fact that carcinogenesis is a long-term process of cellular growth, division and subsequent clonal expansion of initiated cells exemplified by steps known as initiation, promotion and progression (Gupta and Mukhtar, 2002). One advantage of chemoprevention is that agents can be targeted against each stage of tumorigenesis. Thus, inhibition or slowing of any stage of carcinogenesis can potentially prevent cancers from becoming clinically significant.

The intervention of cancer at the promotion stage, however, seems to be most appropriate and practical. The major reason for this is the fact that tumor promotion is a reversible event at least in early stages and requires repeated and prolonged exposure of a promoting agent (Bickers and Athar, 2000). For this reason, it is important to identify antitumor-promoting agents. A number of compounds have been evaluated by our laboratory and others, for their potential chemopreventive activity, and many of them are of plant origin (Gupta and Mukhtar, 2001). Therefore, considerable attention has been focused on identifying edible and medicinal phytochemicals that possess the ability to interfere with carcinogenic or mutagenic processes (Conney et al., 1997; Surh, 2003).

Lupeol (Lup-20(29)-en-3-ol) is a naturally occurring triterpene found in various fruits, vegetables and in many medicinal plants (Figure 1). Of particular note is that a significant quantity of this compound is present in olive, mango, strawberry and fig plants (Sosa, 1963; Anjaneyulu et al., 1982; Saeed and Sabir, 2002). Lupeol is found as an active constituent of various medicinal plants used by native people in the treatment of various skin aliments in North America, Japan, China, Latin America and Caribbean islands (Fournet et al., 1992; Lin et al., 2001; Miura et al., 2001; Beveridge et al., 2002; Kakuda et al., 2002; Badria et al., 2003; Dos Santos Pereira and De Aquino Neto, 2003). Lupeol also has been shown to possess various pharmacological properties (Ulubelen et al., 1997; Geetha and Varalakshmi, 1999; Nagaraj et al., 2000; Sunitha et al., 2001; Vidya et al., 2002; Saleem et al., 2003). Lupeol has been shown to possess strong anti-inflammatory, antiarthritic, antimutagenic and antimalarial activity in vitro and in vivo systems (Guevara et al., 1996; Geetha and Varalakshmi, 1999; Geetha and Varalakshmi, 2001; Ziegler et al., 2002). Lupeol has been shown to act as a potent inhibitor of protein kinases and serine proteases (Hasmeda et al., 1999; Rajic et al., 2000; Hodges et al., 2003) and inhibit the activity of DNA topoisomerase II, a target for anticancer chemotherapy (Moriarity et al., 1998; Wada et al., 2001). Lupeol has also been shown to improve the epidermal tissue reconstitution (Nikiema et al., 2001). Recent studies have shown that Lupeol induce differentiation and inhibit the cell growth of melanoma cells (Hata et al., 2002, 2003).

Figure 1.

Structure of Lupeol

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In view of the anti-inflammatory, antimutagenic and antioxidative activities of Lupeol, as well as its inhibitory potential against prostaglandin (PGE₂) and cytokine production (Fernandez et al., 2001), we considered that Lupeol may possess a significant antitumor-promoting potential. In this study, we assessed the antitumor-promoting effect of Lupeol on CD-1 mouse skin and delineated the mechanism of its action.

Results

Inhibitory effect of Lupeol on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced cutaneous edema

Studies from our laboratory and by other have shown that TPA application to mouse skin results in cutaneous edema (Katiyar et al., 1996; Liang et al., 2002). In the present study, we evaluated the protective effects of topical application of Lupeol in TPA-mediated cutaneous edema in CD-1 mouse. We tested four doses 0.25, 0.5, 1 and 2 mg of Lupeol per animal in our preliminary studies. Since 0.25 and 0.5 mg of Lupeol did not exhibit any significant effect on primary biomarkers of tumor promotion (data not shown), therefore we selected dose of 1 and 2 mg of Lupeol for further studies. The CD-1 mice were topically treated with Lupeol (1 and 2 mg/mouse) and 30 min later were topically treated with TPA (3.2 nmol/mouse). As determined by the weight of 1 cm diameter punch of the dorsal skin, application of TPA to CD-1 mouse skin resulted in a significant development of skin edema at 24 and 48 h post-TPA treatment compared to control and Lupeol-treated groups (Table 1). The skin application of Lupeol 30 min prior to that of TPA application showed a significant protection against TPA-induced skin edema measured at 24 (48%; P<0.01) and 48 (43%; P<0.01) h post-treatment. We found that topical application of Lupeol alone to mice did not result in an increase in skin edema at 24 and 48 h post-treatment (Table 1).

Table 1 - Inhibitory effect of topically applied Lupeol on TPA-induced cutaneous edema.

Full table

Inhibitory effect of Lupeol on TPA-induced epidermal hyperplasia

The effect of topical application of Lupeol on TPA-mediated induction of epidermal hyperplasia was then assessed. As shown in Figure 2, topical application of TPA resulted in an increase in epidermal hyperplasia at 24 and 48 h after treatment when compared to control treated animals. The topical application of Lupeol, however, prior to that of TPA application to mouse skin resulted in inhibition in the induction of epidermal hyperplasia (Figure 2). Lupeol alone did not induce any epidermal hyperplasia as the histology of these animals was comparable to that of control mice (Figure 2).

Figure 2.

Inhibitory effect of Lupeol on TPA-induced hyperplasia in CD-1 mice: 24 and 48 h after treatment, the animals were killed, skin biopsies were processed for hematoxylin and eosin staining. Representative pictures are shown. Details are given under Materials and methods

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Inhibition of TPA-caused induction of epidermal ornithine decarboxylase (ODC) activity by Lupeol

In order to determine the effect of Lupeol against the TPA-induced ODC activity in CD-1 mice, groups of animals were treated topically with Lupeol (1 or 2 mg/animal, 30 min prior to topical application of TPA (3.2 nmol/animal)). All the test substances were applied in 0.2 ml acetone. As shown in Figure 3, pretreatment of animals with Lupeol resulted in a dose-dependent inhibition of the TPA-caused induction of epidermal ODC activity. At the highest dose of Lupeol (2 mg/animal), used in this study, 75% inhibition (P<0.005) was observed as compared to TPA-treated control (Figure 3). Lupeol at lower dose (1 mg/animal) also caused a substantial inhibition (50%; P<0.005) in the epidermal ODC activity in mice treated with TPA. Topical application of Lupeol alone (2 mg/animal) was without any effect on basal epidermal ODC activity.

Figure 3.

Inhibitory effect of Lupeol on TPA-induced epidermal ODC activity: ODC enzyme activity was determined using 0.4 ml epidermal supernatant by measuring the release of ¹⁴CO₂ from the D, L-[¹⁴C] ornithine as described in Material and methods. Data are represented as means.e. of four individual values (^*P<0.005). The epidermis from two animals was pooled for each determination

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Inhibition of TPA-caused induction of epidermal ODC protein expression by Lupeol

Next, we assessed the effect of skin application of Lupeol on TPA-caused enhanced expression of ODC protein in the epidermis. Western blotting revealed that at 6 h post-treatment of TPA, there was maximum expression of epidermal ODC protein expression and it gradually declined with the passage of time, that is, at 12, 24 and 48 h post-TPA treatment (Figure 4a). Treatment of TPA caused a four-fold increase in epidermal ODC protein level as compared to acetone-treated control, while pretreatment of animals with Lupeol resulted in a significant inhibition against TPA-caused induction of epidermal ODC protein expression in a dose-dependent manner at all time points investigated (Figure 4a). Densitometric analysis of these blots indicated that, under experimental conditions used, the inhibition varied from 50–70% in Lupeol-pretreated animals. Topical application of Lupeol alone up to 2 mg/animal was without any effect on enzyme level and did not cause any induction of epidermal ODC protein expression.

Figure 4.

Inhibitory effect of Lupeol on TPA-induced epidermal ODC, iNOS and COX-2 protein expression in CD-1 mice: at different time after treatment, the animals were killed, epidermal protein lysate was prepared, and ODC and COX-2 protein expression were determined as described under Materials and methods. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for -actin. The immunoblots shown here are representative of three independent experiments with similar results. The values above each lane indicate relative density of the band normalized to -actin

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Inhibitory effect of Lupeol on TPA-induced epidermal cyclo-oxygenase (COX-2) and induced nitric oxide synthase (iNOS) protein expression

COX-2 and iNOS are well-established biomarkers of inflammation and tumor promotion. We next assessed the effect of skin application of Lupeol on TPA-induced epidermal iNOS and COX-2 protein expression. We found that topical application of TPA to CD-1 mice resulted in an increase in epidermal COX-2 protein expression, which was maximum (2.7-fold) at 6 h post-TPA treatment when compared to the acetone-treated control (Figure 4b). The TPA-caused induction in the expression level of epidermal COX-2 gradually declined with the time, that is, at 12, 24 and 48 h post-TPA treatment. However, at all time points, the expression of COX-2 in mouse skin following TPA application remained higher than corresponding acetone-treated control. At the lowest dose of Lupeol (1 mg/animal), there was 40% inhibition in TPA-caused increased epidermal COX-2 protein expression and the highest dose of Lupeol (2 mg/animal) restored the level of TPA-induced COX-2 protein almost to its basal level (Figure 4b).

We observed that topical application of TPA to CD-1 mice resulted in a significant increase in the expression of epidermal iNOS protein (Figure 4c). The expression of iNOS was observed to reach its peak at 6 h post-TPA treatment and it declined to its basal level at 12 h post-TPA treatment. Topical application of TPA alone caused five-fold increase in iNOS protein expression in mouse skin as compared to vehicle-treated controls; however, pretreatment of Lupeol to the skin caused a dose-dependent inhibition against TPA-caused increases of iNOS protein expression. Densitometric analysis of blots revealed that mice pretreated with Lupeol (2 mg/animal) showed 74% inhibition against TPA-induced epidermal iNOS protein expression (Figure 4c). The application of Lupeol alone at the dose of 2 mg did not produce any change in epidermal COX-2 and iNOS protein expression when compared with vehicle-treated control animals.

Inhibitory effect of Lupeol on TPA-induced epidermal phosphatidyl inositol kinase (PI3K) and phosphorylation of Akt

Studies have shown that PI3K plays an important role in carcinogenesis (Luo et al., 2003; Mills et al., 2003; Osaki et al., 2004). We next investigated whether TPA can induce PI3K protein expression in mouse skin. Western blot analysis revealed that topical application of TPA caused a significant (two- to threefold) increase in the expression of both catalytic (p110) as well as regulatory (p85) subunit of PI3K in mouse skin (Figure 5a and b). A threefold induction in the expression of catalytic subunit p110 was found at 6 h post-TPA treatment only. There was a sustained induction up to 24 h post-TPA treatment in the expression of regulatory subunit p85, with 6 h post-TPA treatment showing the maximum expression. Topical application of Lupeol 30 min prior to TPA application resulted in a significant inhibition of TPA-induced increased expression of both catalytic and regulatory subunits of PI3K (Figure 5a and b). However, there was no significant difference between control and treatments at later time points in the expression of catalytic p110 subunit. Skin application of animals with 2 mg Lupeol prior to TPA application resulted in the recovery of the expression of both p110 and p85 almost to their basal levels.

Figure 5.

Inhibitory effect of Lupeol on TPA-induced activation of PI3K and phosphorylation of Akt in CD-1 mice: at different time after treatment, the animals were killed, epidermal protein lysate was prepared, and PI3K and phosphorylated (Thr³⁰⁸) Akt and total Akt protein expression were determined as described under Materials and methods. The immunoblots shown here are representative of three independent experiments with similar results. The values above each lane indicate relative density of the band normalized to -actin.

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Several biological effects of PI3K are mediated through the activation (phosphorylation) of downstream target Akt. Akt, also known as protein kinase B, is a serine (Ser)/threonine (Thr) kinase, which has been identified as an important component of prosurvival signaling pathway (Downward, 1998). As Akt is a downstream substrate for PI3K, we next assessed whether Akt is involved in cellular responses to TPA by performing Western blot analysis with antibody to phosphorylated form of Akt at Thr³⁰⁸, which is a prerequisite for the catalytic activity of Akt. Densitometric analysis of blots revealed a significant increase in the phosphorylation of Akt at Thr³⁰⁸ in mouse skin treated with a single topical application of TPA (Figure 5c). We observed a maximum phosphorylation of Akt (3.8-fold) at 6 h post-TPA treatment and this induction gradually declined with time after 12 h post-TPA treatment. We observed a dose-dependent inhibition of TPA-induced phosphorylation of Akt at Thr³⁰⁸ with the pre-application of Lupeol prior to TPA application in CD-1 mice skin (Figure 5c). However, no significant phosphorylation of Akt at Thr³⁰⁸ was observed at 24 and 48 h time points in both control as well as treatment groups. Importantly, no change was observed in the total epidermal Akt content in mice treated with TPA as well as Lupeol as compared to vehicle-treated control (Figure 5d).

Inhibitory effect of Lupeol on TPA-induced activation of NF-B and IKK and phosphorylation and degradation of IB protein expression

Studies have shown that Akt can promote survival by activating NF-B signaling pathway (Romashkova and Makarov, 1999). Activation and nuclear translocation of NF-B is preceded by the phosphorylation and proteolytic degradation of IB (Israel, 1995). To determine whether the inhibitory effect of Lupeol was attributable to an effect on IB degradation, we examined the cytoplasmic level of IB protein expression by Western blot analysis. We found that TPA application to mouse skin resulted in the degradation of IB protein expression at 12 and 24 h after treatment (Figure 6a). There was no significant difference between control and treatments at 6 and 48 h in the expression of IB protein. Topical application of Lupeol 30 min prior to TPA application resulted in a significant inhibition of TPA-induced degradation of IB protein (Figure 6a). We next assessed whether TPA application affects the phosphorylation of IB protein. As shown by Western blot, TPA induced a marked increase in the phosphorylation level of IB protein at Ser³² at 12 and 24 h post-TPA treatment. Topical application of Lupeol prior to TPA application exhibited a dose-dependent inhibition in TPA-induced phosphorylation of IB protein (Figure 6b). Studies have shown that IKK activity is necessary for IB protein phosphorylation/degradation (Baldwin, 1996; Maniatis, 1997). To determine whether inhibition of TPA-induced IKK activation by Lupeol is attributable to suppression of IB phosphorylation/degradation, we also measured IKK protein level. Densitometric analysis of the blots revealed that TPA application resulted in a 5.5-fold increase in the expression of IKK protein that in turn phosphorylates and degrades IB protein (Figure 6c). At 12 h post-TPA treatment, IKK protein expression was at its peak followed by a gradual decline in the induction of IKK protein expression with time and no significant difference between control and treatments at 48 h in the expression of IKK protein was observed. Topical application of Lupeol prior to TPA application dose dependently inhibited TPA-induced activation of IKK and at the highest dose of Lupeol, there was almost 95% inhibition in TPA induced activation of IKK protein at 12 h time point (Figure 6c).

Figure 6.

Inhibitory effect of Lupeol on TPA-induced activation of IKK, and phosphorylation and degradation of IB in CD-1 mice: at different time after treatment, the animals were killed, epidermal cytosolic was prepared and protein expression was determined as described under 'Materials and methods'. Equal loading was confirmed by stripping the immunoblot and reprobing it for -actin. The immunoblots shown here are representative of three independent experiments with similar results. The values above each lane indicate relative density of the band normalized to -actin

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Next, we investigated whether topical application of Lupeol inhibits TPA-induced activation and nuclear translocation of p65, the functionally active subunit of NF-B in mouse skin. As shown by Western blot analysis, we found that TPA application onto mouse skin resulted in the activation and nuclear translocation of NF-B/p65 (Figure 7a). However, topical application of Lupeol prior to TPA application inhibited TPA-induced NF-B/p65 activation and nuclear translocation (Figure 7a).

Figure 7.

Inhibitory effect of Lupeol on TPA-induced NF-B activation and its subsequent translocation to the nucleus in CD-1 mice. (a) At different time after treatment, the animals were killed, nuclear lysates were prepared and protein expression was determined as described under 'Materials and methods'. The values above each lane indicate relative density of the band. (b) At different time after treatment, the animals were killed, nuclear lysates were prepared and DNA binding was determined by EMSA as described under 'Materials and methods'. C1, C2 and C3 refer to inter experimental controls, where C1 represents biotin–EBNA (Epistein–Barr virus nuclear antigen) control DNA, C2 represents biotin–EBNA control DNA and EBNA extract and C3 represents biotin–EBNA control DNA and EBNA extract plus 200-fold molar excess of EBNA DNA. In C1, no protein extract for DNA to bind resulted in an unshifted band. In C2, sufficient target protein resulted in DNA–protein binding, resulting in shift detected in comparison to the band at position C1. C3 demonstrated that the signal shift observed in C2 could be prevented by competition from excess unlabeled DNA

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Inhibitory effect of Lupeol on NF-B–DNA binding using electrophoretic mobility shift assay (EMSA)

We next performed EMSA to investigate the effect of Lupeol treatment on TPA-induced NFB–DNA-binding activity. As shown in Figure 7b, TPA treatment resulted in a marked increase of NF-B–DNA-binding activity in comparison to control and Lupeol groups (Figure 7b). The induction of NF-B–DNA-binding activity coincided with the degradation of IB and activation of IKK (Figure 6c). Prior application of Lupeol to mouse skin significantly inhibited TPA-induced epidermal NF-B–DNA-binding activity (Figure 7b).

Antiskin tumor-promoting effects of Lupeol

Lupeol treatment for 28 weeks did not significantly affect the body weight gain of mice in any group and none of the treated mice exhibited any signs of toxicity (data not shown). Since application of 2 mg Lupeol to mice skin significantly inhibited various molecules that play significant role in the progression of skin tumors, we selected this dose for assessing the antitumor-promoting potential of Lupeol in 7, 12, dimethyl benz(a)anthracene (DMBA)-initiated mouse skin. As shown by the data in Figure 8, topical application of Lupeol prior to that of TPA in DMBA-initiated CD-1 mouse skin resulted in a significant inhibition of tumorigenesis. This inhibition was evident when tumor data were considered as the percentage of mice with tumors (Figure 8a), the number of tumors per mouse (Figure 8b) and the number of tumors per group (Figure 8c). At the termination of the experiment at 28 weeks on test, compared with 100% animals with skin tumors in non-Lupeol-treated group, only 53% of the animals exhibited the appearance of skin tumors. The tumor incidence data revealed that prior application of Lupeol to DMBA-initiated and TPA-treated mouse skin significantly increased the latency period of tumor formation (P<0.05, ² test). At the termination of the experiment at 28 weeks on test, compared with a total of 125 tumors in non-Lupeol-treated group of animals, only 33 tumors in Lupeol-treated group were recorded (Figure 8b). Compared with the non-Lupeol-treated group, such decrease in the total number of tumor in the Lupeol-treated group correspond to 74% inhibition. When these tumor data were considered in terms of the number of tumors per mice, at the termination of the experiment at 28 weeks on test, compared with a 6.25 tumors per mouse in non-Lupeol-treated group of animals, only 1.65 tumors per mouse in Lupeol-treated group were recorded (Figure 8c). Compared with the non-Lupeol-treated group, such decrease in the number of tumor per mouse in the Lupeol-treated group correspond to 74% inhibition.

Figure 8.

Two-stage carcinogenesis tumor data in CD-1 mice: inhibitory effect of Lupeol on DMBA-initiated and TPA-promoted tumor formation in CD-1 mice: in each group, 20 animals were used. Tumorigenesis was initiated in the animals by a single topical application of 200 nmol DMBA in 0.2 ml vehicle on the dorsal shaved skin, and 1 week later, the tumor growth was promoted with twice-weekly applications of 3.2 nmol TPA in 0.2 ml vehicle. To assess its antiskin tumor-promoting effect, Lupeol at a dose of 2 mg/animal was applied topically 30 min prior to each TPA application in different groups. Treatment with TPA alone or Lupeol plus TPA was repeated twice weekly up to the termination of the experiments at 28 weeks. Animals in all the groups were watched for any apparent signs of toxicity, such as weight loss or mortality during the entire period of study. Skin tumor formation was recorded weekly, and tumors larger than 1 mm in diameter were included in the cumulative number only if they persisted for 2 weeks or more. The tumor data are represented as the percentage of mice with tumors (a), the number of tumors per mouse (b), and the number of tumors per group (c). The data were analysed by Wilcoxon's rank-sum test and ² analysis

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Discussion

Cancer chemoprevention is increasingly being realized as an important area for cancer prevention, which, in addition to providing a practical approach of identifying potentially useful inhibitors of cancer development, also affords excellent opportunities to study the mechanisms of carcinogenesis (Conney et al., 1997; Bickers and Athar, 2000; Gupta and Mukhtar, 2002). The mouse skin model of multistage carcinogenesis has been a useful experimental framework to study basic mechanisms associated with the initiation, promotion and progression stages of carcinogenesis and defining newer chemopreventive agents. The intervention of cancer at the promotion stage appears to be most appropriate and practical. The major reason for this relates to the fact that tumor promotion is a reversible event at least in early stages, and requires repeated and prolonged exposure of a promoting agent (DiGiovanni, 1992; Surh, 2003). Further, tumor promotion is an obligatory step in the carcinogenic pathway where clonal expansion of initiated cell population occurs leading to what is referred as march of initiated cells towards malignancy. For this reason, it is important to identify mechanism-based effective novel antitumor-promoting agents. We argue that these agents, which have the ability to intervene at more than one critical pathway in the carcinogenic process, will have greater advantage over other single-target agents. Lupeol, a triterpene, is one such polyphenolic agent found in various edible plants such as olive, fig, mango and strawberry (Sosa, 1963; Anjaneyulu et al., 1982; Saeed and Sabir, 2002). Previously, it has been shown that Lupeol provides strong antioxidant protection against benzoyl peroxide-induced toxicity in Swiss albino mouse skin (Saleem et al., 2001). Lupeol has been shown to significantly reduce the PGE₂ production and inhibit the production of TNF and interlukin-1 in vitro (Fernandez et al., 2001). Current study was designed to establish the chemopreventive potential of Lupeol in CD-1 mice and to provide comprehensive molecular mechanisms involved in this effect.

The topical application of TPA to mouse skin or its treatment in certain epidermal cells is known to result in a number of biochemical alterations, changes in cellular functions and histological changes leading to skin tumor promotion (DiGiovanni, 1991; Katiyar et al., 1997; Katiyar and Mukhtar, 1997; Chun et al., 2002; Seo et al., 2002). Our data clearly demonstrate that preapplication of Lupeol before TPA treatment affords a significant inhibition of TPA-induced skin edema and hyperplasia (Table 1, Figure 2). Previously, Lupeol has been reported to provide protection against croton oil-induced edema in mouse ear and was reported to have more efficacy than indomethacin (Nikiema et al., 2001).

Accumulating information constantly reinforces that ODC, the first and the rate-limiting enzyme in the biosynthesis of polyamines, plays an important role in the regulation of cell proliferation, differentiation and development of cancer (Thomas and Thomas, 2003). The induction of ODC has been suggested to play a significant role in tumor promotion. Studies with the mouse skin model showed an excellent correlation between the induction of ODC activity and the tumor-promoting ability of a variety of substances (Einspahr et al., 2003). It has been shown that overexpression of ODC is a sufficient condition for tumor promotion in mouse skin (Ahmad et al., 2001). Several lines of evidence indicate that aberrations in ODC regulation, and subsequent polyamine accumulation, are intimately associated with neoplastic transformation (Mohan et al., 1999). Elevated levels of ODC gene products are consistently detected in transformed cell lines, virtually all-animal tumors and in certain tissues predisposed to tumorigenesis (Auvinen, 1997). As tumor formation can be prevented by the agents that block the induction of ODC (Verma et al., 1979; Nakadate et al., 1985), ODC inhibition was shown to be a promising tool for screening inhibitors of tumorigenesis. In the present study, topical application of Lupeol prior to that of TPA resulted in a significant inhibition of TPA-mediated induction of epidermal ODC activity (Figure 2). It is reasonable to believe that Lupeol application inhibited the action of the tumor promoter and/or the enzymatic pathway(s) that regulates the ODC induction rather than interacting directly with the enzyme. In addition, our data obtained from Western blot analysis demonstrate that prior application of Lupeol to that of TPA showed an inhibitory effect of Lupeol against TPA-induced increases in the levels of epidermal ODC protein in mouse. The magnitude of the inhibitory effect of topical application of Lupeol on TPA-induced increases in ODC protein expression seem to be similar to that for inhibition of TPA-induced increases in ODC enzyme activity.

Tumor promotion is closely linked to inflammation and oxidative stress, and it is likely that compounds that have anti-inflammatory and antioxidative properties act as antitumor promoters as well (Bhimani et al., 1993). COX-2 isoform and iNOS are important enzymes involved in mediating the inflammatory process (Herschman, 1994; Smith et al., 1996). COX-2 and iNOS have been reported to play an important role in cutaneous inflammation, cell proliferation and skin tumor promotion (Furstenberger and Marks, 1985; Herschman, 1994). There is considerable body of compelling evidence that inhibition of COX-2 and iNOS expression or activity is important for not only alleviating inflammation but also for the prevention of cancer (Kim et al., 2003). In this study, we showed the inhibitory effects of Lupeol against TPA-caused induction of epidermal COX-2 and iNOS protein expression in CD-1 mouse (Figure 3). These inhibitory effects also correlate with the inhibitory effect of Lupeol against TPA-caused induction of skin edema (Table 1) and hyperplasia (Figure 2). These inhibitory effects of Lupeol against TPA-mediated responses in the mouse skin suggest that the primary effect of Lupeol may be against inflammatory responses, which may then result in the inhibition of tumor promotion.

Recent reports indicate that well-known biomarkers of tumor promotion and inflammation, that is, COX-2, iNOS and ODC, are regulated by NF-B transcriptional factor (Callejas et al., 1999). NF-B molecule, in addition to the regulation by IB molecule, is also reported to be regulated by PI3K/Akt signaling pathway (Carpenter and Cantley, 1996). Both NF-B and PI3K/Akt signaling pathways have emerged as promising molecular targets in the prevention of cancer. Since Lupeol significantly inhibited the induction of ODC, COX-2 and iNOS, we investigated if Lupeol exerts effects on these molecules or interferes with the signaling molecules that regulate them. Therefore, we investigated the efficacy of Lupeol in the modulation of molecules involved in NF-B and PI3K/Akt signal pathway. PI3K/Akt are important regulatory molecules that are involved in different signaling pathways and in the control of cell growth, promote cell survival and malignant transformation (Carpenter and Cantley, 1996; Stambolic et al., 1999). Our study clearly demonstrated that topical application of TPA resulted in the activation of both regulatory subunit (p85) and catalytic subunit (p110) of PI3K and phosphorylation of Akt at Thr³⁰⁸protein expression as an early event (Figure 5). Topical application of Lupeol prior to TPA application to mouse skin resulted in the reduction in TPA-induced expression of PI3K and phosphorylation of Akt (Figure 5). The PI3K/Akt promotes cell survival by activating NF-B signaling pathway (Romashkova and Makarov, 1999). Upon phosphorylation and subsequent degradation of IB, NF-B activates and translocates to the nucleus (Bours et al., 2000). Several lines of evidence suggest that proteins from the NF-B and IB families are involved in carcinogenesis. NF-B controls the expression of several growth factors, oncogenes and tumor suppressor genes (c-myc, p53), genes encoding cell adhesion proteins (ICAM-1, ELAM-1, VCAM-1) and proteases of the extracellular matrix (Epinat and Gilmore, 1999). NF-B is activated by various stimuli, including growth factors, carcinogens and tumor promoters including TPA (Ahmad et al., 2000; Afaq et al., 2003). Studies have shown that NF-B activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as ionizing radiation (Epinat and Gilmore, 1999). In the present study, we have demonstrated that topical application of TPA to mouse skin resulted in the activation and nuclear translocation of NF-B (Figure 7).

The IKK complex is believed to be an important site for integrating signals that regulate the NF-B pathway. In the present study, we observed that TPA application to mouse skin resulted in an increased expression of IKK, and phosphorylation and degradation of IB protein (Figure 6). Interestingly, we found that topical application of Lupeol prior to TPA application to mouse skin inhibited TPA-induced NF-B, IKK activation, and phosphorylation and degradation of IB protein (Figures 6 and 7). Phosphorylation of IB, an inhibitory subunit of NF-B, on serine residues 32 and 36 by kinases (IKK), precedes rapid degradation of IB that in turn activates NF-B (Baldwin, 1996; Maniatis, 1997). It is only when IB is degraded that NF-B is transported into the nucleus (Baeuerle and Baltimore, 1996). As Lupeol inhibits IB phosphorylation and degradation, our study suggests that the effect of Lupeol on NF-B/p65 is through the inhibition of phosphorylation and subsequent proteolysis of IB.

The results in Figure 8 show the protective effects of skin application of Lupeol on TPA-caused tumor promotion in DMBA-initiated CD-1 mouse skin. The preapplication of Lupeol to mice skin showed protective effects when tumor data were considered as the total number of tumors or tumors per mouse and the percent mice bearing tumors (Figure 8). These chemopreventive and antitumor promotion effects in murine skin by Lupeol can be explained by the biochemical mechanisms observed in the present study. Based on the outcome of this study, we suggest multiple pathways by which Lupeol results in the inhibition of tumor promotion in mouse skin. This may be explained by the modulation of NF-B mediated by PI3K/Akt that in turn affects COX-2 and iNOS. Lupeol may inhibit ODC activity via modulating NF-B or some other pathway regulating ODC gene. Lupeol seems to act as a modulating agent in multiple signaling pathways, thus proving as an excellent example of being an ideal chemopreventive agent. However, further work is needed to verify this suggestion.

In summary, we have shown that topical application of Lupeol prior to TPA application to CD-1 mice resulted in a significant decrease in skin edema, hyperplasia, epidermal ODC activity and protein expression of ODC, iNOS and COX-2, classical markers of inflammation and tumor promotion. In addition, we have also shown that topical application of Lupeol prior to TPA application also resulted in the inhibition of activation of PI3K and phosphorylation of Akt, activation of NF-B/p65 and IKK, and degradation and phosphorylation of IB. In addition, we showed that preapplication of Lupeol inhibited skin tumorigenesis in CD-1 mice.

Our data clearly demonstrate that Lupeol could be a potent antitumor-promoting agent because it inhibits TPA-induced tumor promotion in an in vivo animal model. One might envision the use of chemopreventive agents such as Lupeol in an emollient or patch for chemoprevention or treatment of skin cancer. In addition, because Lupeol exerts multiple effects on biomarkers associated with carcinogenesis, it could be tested for the cancer chemoprevention of other organs.

Materials and methods

Materials

ODC, iNOS, COX-2, IKK antibodies were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). IB and IB (phospho) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). AKT and PI3K were purchased from Upstate (Lake Placid, NY, USA). NF-B/p65 antibody was procured from Geneka Biotechnology Inc. (Montreal, Canada). Anti-mouse or anti-rabbit secondary antibody horse-radish peroxidase conjugate was obtained from Amersham Life Science Inc. (Arlington Height, IL, USA). Lupeol, DMBA and TPA were purchased from Sigma Chemicals (St Louis, MO, USA). Lightshift™ chemiluminiscent EMSA kit was obtained from Pierce (Rockford, IL, USA). The DC BioRad Protein assay kit was purchased from BioRad Laboratories (Herculus, CA, USA). Novex precast Tris-glycine gels were obtained from Invitrogen (Carlsbad, CA, USA).

Animals

Female CD-1 mice (5–6 weeks old) were obtained from Charles River Laboratories (USA). These mice were housed four per cage under standard animal house conditions with a 12-h light/12-h dark cycle, and housed at 24+2°C and 50+10% relative humidity. Animals were fed a Purnia chow diet and water ad libitum.

Treatment of animals for short-term studies

The animals were shaved on the dorsal side of the skin, divided into four groups and treated topically on the shaved area with either 0.4 ml vehicle (acetone), or TPA (3.2 nmol/0.4 ml acetone/animal, v/v), or Lupeol (1 or 2 mg/0.2 ml acetone/animal, w/v) followed 30 min later with TPA (3.2 nmol/0.2 ml acetone/animal). Animals were killed at different time point, that is, 6, 12, 24 and 48 h post-TPA treatment. The epidermis was separated from the whole skin and homogenized in 0.1 M Tris-HCI buffer (pH 7.2) using a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY, USA) at 100 000 g supernatant, and microsomal fractions were prepared as described earlier (Katiyar and Mukhtar, 1997). The sample of 6 h treatment group was used to assess the ODC activity as this time point has previously been shown for optimal induction of the enzyme (Katiyar et al., 1996).

Edema and hyperplasia

To assess the inhibitory effect of preapplication of Lupeol on TPA-induced edema, 1 cm diameter punches of skin from vehicle-, Lupeol-, TPA- or Lupeol- and TPA-treated mice were removed, made free of fat pads and weighed immediately. After drying for 24 h at 50°C, the skin punches were reweighed and the loss of water content was determined. The difference in the amount of water gain between the control (vehicle treated) and TPA treated represented the extent of edema induced by TPA, whereas that between the control vehicle and Lupeol plus TPA represented the inhibitory effect of Lupeol. For the hyperplasia study, skin was removed, fixed in 10% formalin and embedded in paraffin. Vertical sections (5 m) were cut, mounted on a glass slide and stained with hematoxylin and eosin.

ODC enzyme activity

ODC enzyme activity was determined using 0.4 ml epidermal supernatant by measuring the release of ¹⁴CO₂ from the D, L-[¹⁴C]ornithine as reported earlier (Gupta et al., 1999). The epidermis from the skin was homogenized at 4°C in a glass-to-glass homogenizer in 10 volumes of ODC buffer (50 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM pyridoxal-5-phosphate, 1 mM 2-mercaptoethanol and 0.1% Tween-80). The homogenate was centrifuged at 100 000 g at 4°C and the supernatant was used for enzyme determination. Briefly, 100 l of the supernatant was added to 0.25 ml of the assay mixture (35 mM sodium phosphate (pH 7.2), 0.2 mM pyridoxal phosphate, 4 mM dithiothreitol, 1 mM EDTA, 0.4 mM L-ornithine containing 0.5 Ci of DL-[1-¹⁴C] ornithine hydrochloride) in 15 ml corex centrifuge tube equipped with rubber stoppers and central well assemblies containing 0.2 ml ethanolamine and methoxyethanol in 2 : 1 (v/v) ratio. After incubation at 37°C for 60 min, the reaction was terminated by the addition of 0.5 ml of 2 M citric acid, using a 21-guage needle/syringe. The incubation was continued for 1 h. Finally, the central well containing the ethanolamine:methoxyethanol mixture to which ¹⁴CO₂ has been trapped was transferred to a vial containing 10 ml of toluene-based scintillation fluid and 2 ml of ethanol. The radioactivity was measured in a Beckman LS 6000 SC liquid scintillation counter. Enzyme activity was expressed as pmol CO₂ released/h/mg protein.

Preparation of cytosolic and nuclear lysates

Epidermis from the whole skin was separated as described earlier (Katiyar et al., 1999), and was homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na₃VO₄, 0.5% NP-40, 1% Triton X-100, 1 mM PMSF (pH 7.4)), with freshly added protease inhibitor cocktail, Protease Inhibitor Cocktail Set III (Calbiochem, La Jolla, CA, USA). The homogenate was then centrifuged at 14 000 g for 25 min at 4°C and the supernatant (total cell lysate) was collected, aliquoted and stored at -80°C. For the preparation of nuclear lysate, 0.2 g of the epidermis was homogenized in 1 ml of ice-cold phosphate-buffered saline (pH 7.6) and centrifuged at 12 000 g for 5 min at 4°C. The pellet was resuspended in 1 ml of cold buffer containing 10 mM HEPES (pH 7.9), 2 mM MgCl₂, 10 mM KCI, 1 mM dithiothreitol, 0.1 mM EDTA and 0.1 mM PMSF. After homogenization in a tight-fitting Dounce homogenizer, the homogenates were left on ice for 10 min and centrifuged at 25 000g for 10 min. The nuclear pellet was resuspended in 0.1 ml of the buffer containing 10 mM HEPES (pH 7.9), 300 mM NaCI, 50 mM KCI, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF and 10% glycerol with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem, La Jolla, CA). The suspension was gently shaken for 20 min at 4°C. After centrifugation at 25 000 g for 10 min, the nuclear extracts (supernatants) were collected and quickly frozen at -80°C. The protein content in the lysates was measured by DC BioRad assay (BioRad Laboratories, Hercules, CA) as per the manufacturer's protocol.

Western blot analysis

For Western blot analysis, 40 g of the protein was resolved over 8–12% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot containing the transferred protein was blocked in blocking buffer (5% nonfat dry milk, 1% Tween-20, in 20 mM TBS, pH 7.6) for 1 h at room temperature followed by incubation with appropriate primary antibody in blocking buffer for 1 h to overnight at 4°C. This was followed by incubation with anti-mouse or anti-rabbit secondary antibody horse-radish peroxidase (Amersham Life Sciences, Inc., USA) for 1 h and then washed several times and detected by chemiluminescence ECL kit (Amersham Life Sciences, Inc., USA) and autoradiography using XAR-5 film obtained from Eastman Kodak Co. (Rochester, NY, USA). Densitometric measurements of the band in Western blot analysis were performed using digitalized scientific software program UN-SCAN-IT (Silk Scientific Corporation, Orem, UT, USA).

EMSA

EMSA for NF-B was performed using Lightshift™ chemiluminiscent EMSA kit (Pierce, Rockford, IL, USA) by following the manufacturer's protocol. To start with, DNA was biotin labeled using the Biotin 3'-end labeling kit (Pierce, Rockford, IL, USA). Biefly, in a 50 l reaction buffer, 5 pmol of double-stranded NF-B oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3'; 3'-TCA ACT CCC CTG AAA GGG TCC G-5' was incubated in a microfuge tube with 10 l of 5 TdT (terminal deoxynucleotidyl transferase) buffer, 5 l of 5 M biotin-N4-CTP, 10 U of diluted TdT, 25 l of ultrapure water and incubated at 37°C for 30 min. To extract labeled DNA, 50 l of chloroform : isoamyl alcohol (24 : 1) was added to each tube and centrifuged briefly at 13 000 g. The top aqueous phase containing the labeled DNA was removed and saved for binding reactions. Each binding reaction contained 1 binding buffer (100 mM Tris, 500 mM KCl, 10 mM dithiothretol, pH 7.5), 2.5% glycerol, 5 mM MgCl₂, 50 ng/l poly(dI–dC), 0.05% NP-40, 5 g of nuclear extract and 20–50 fmol of biotin end-labeled target DNA. The content was incubated at room temperature for 20 min. To this reaction mixture was added 5 l of 5 loading buffer, subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. When the transfer was complete, DNA was crosslinked to the membrane at 120 mJ/cm² using a UV crosslinker equipped with 254 nm bulbs. The biotin end-labeled DNA was detected using streptavidin–horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film (XAR-5 Amersham Life Science Inc., Arlington Height, IL, USA) and developed using a Kodak film processor.

Skin tumorigenesis

Female CD-1 mice were used in DMBA- and TPA-induced, two-stage skin tumorigenesis protocol as described earlier (Katiyar et al., 1996). The dorsal side of the skin was shaved using electric clippers, and the mice with hair cycles in the resting phase were used for tumor studies. In each group, 20 animals were used. Tumorigenesis was initiated in the animals by a single topical application of 200 nmol DMBA in 0.2 ml vehicle on the dorsal shaved skin, and 1 week later, the tumor growth was promoted with twice-weekly applications of 3.2 nmol TPA in 0.2 ml vehicle. To assess its antiskin tumor-promoting effect, Lupeol at a dose of 2 mg/animal, which produced a significant inhibition against TPA-caused induction of ODC, was applied topically 30 min prior to each TPA application in different groups. Treatment with TPA alone or Lupeol plus TPA was repeated twice weekly up to the termination of the experiments at 28 weeks. Animals in all the groups were watched for any apparent signs of toxicity, such as weight loss or mortality during the entire period of study. Skin tumor formation was recorded weekly, and tumors larger than 1 mm in diameter were included in the cumulative number only if they persisted for 2 weeks or more.

Statistical analysis

A two-tailed Student's t-test was used to assess the statistical significance between the TPA- and Lupeol plus TPA-treated groups. A P-value <0.05 was considered statistically significant. In tumorigenesis experiments, the statistical significance of difference between TPA and Lupeol plus TPA groups was evaluated by the Wilcoxon's rank-sum test and ² analysis.

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Acknowledgements

This work was supported by United States Public Health Service Grants RO1 CA 78809 and RO3 CA 99909.