Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Khuda-Bukhsh AR. J Chin Integr Med. 2010; 8(7): 645-654. Received January 25, 2010; accepted April 22, 2010; published online July 15, 2010. Indexed/abstracted in and full-text link-out at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao. Free full text (HTML and PDF) is available at http://www.jcimjournal.com. Forward linking and reference linking via CrossRef. DOI: 10.3736/jcim20100708

Correspondence: Anisur Rahman Khuda-Bukhsh, PhD, Professor; Tel: +91-33-25828768, +91-33-25828750-315; E-mail: prof_arkb@yahoo.co.in, khudabukhsh_48@rediffmail.com

     The screening of plant extracts and natural products have shown that higher plants represent a potential source of new therapeutic agents, as well as of new drugs from natural products for primary lead compounds. Direct anti-tumor effects have been reported for herbal extracts containing various alkaloids and coumarins, demonstrating an inhibitory growth capacity for a number of malignant cell lines in vitro^［1,2］. Coumarins comprise a very large class of natural compounds found throughout the plant kingdom. However, till date, most of the pharmacological and biochemical studies have been carried out on coumarin itself and on monohydroxy- and dihydroxy-coumarins or methoxy-coumarins^［2,3］, but not on any hydroxy-methoxy coumarin. 7-hydroxy-6-methoxy coumarin (scopoletin), is present as one of the natural coumarins in the plant Gelsemium sempervirens. This plant is commonly known as American yellow jasmine and is indigenous to Southern United States. Ethanolic extract of the dried rhizomes and roots is reported to contain some 45 alkaloids, many of which are toxic^［4］. There are several alkaloids that are very toxic, as for example, gelsemine, gelsemicine, gelsedine and sempervirine. However, scopoletin, a relatively less toxic plant ingredient, has been chemically characterized as C10H8O4 with a molecular weight of 192.17^［2］, and is reported to have anti-cancer activity^［5］. This coumarin compound fluoresces when exposed to ultraviolet (UV) light and its anti-cancer potentials were verified earlier on an in vitro system, using human HeLa cell line. Although several studies were carried out earlier with the induced skin cancer^［6-9］, to our knowledge the anti-cancer effect of this compound had not been tested so far on in vivo system of any mammalian model, for which we undertook this study.
     Therefore in the present investigation the hypotheses to be tested were: i) if scopoletin can also demonstrate its anti-cancer potentials against 7,12-dimethylbenz［a］antracene (DMBA)-induced skin cancer in mice (Mus musculus); for this we considered to ascertain cell signaling mechanism through immunoblot analysis of several downstream signal proteins like cytochrome P450 1A1 (CYP1A1), signal transducer and activator of transcription-3 (Stat-3) (anti-apoptotic), proliferating cell nuclear antigen (PCNA), and reverse transcription-polymerase chain reaction (RT-PCR) analysis of aryl hydrocarbon receptor (AhR), Stat-3, caspase-3, p53 (tumor-suppressor), PCNA, cyclin D1, c-myc and survivin to understand molecular mechanisms for chemoprevention, if any; and ii) to examine if action of scopoletin is mediated through competitive inhibition/suppression of DMBA-induced up-regulation of AhR protein, deploying additionally immunofluorescence localization of PCNA proteins. Histological study was also provided along with the incidence and rate of growth of papilloma on periodic rubbing of DMBA (initiator) and croton oil (promoter) on skin surface of the back of mice.

1  Materials and methods
1.1  Reagents  All the chemicals used were of analytical grade and procured from Sigma, USA.
1.2  Animals  Swiss albino mice (Mus musculus) of both sexes were reared and inbred in the animal house under the supervision of Animal Welfare Committee, Department of Zoology, University of Kalyani, India. It is certified that the experiments were performed with the approval of the Ethical Committee, University of Kalyani (Certificate for Proposal No. KU/IAEC/Z-11/07 dt. 18.5.2007). All mice were given normal diet (standard diet) and water ad libitum. A total number of 30 healthy mice weighing between 22 to 26 g were selected randomly for use in the experiment. The selected mice were divided into 4 groups, each comprising 6 mice in the first 3 groups and 12 in the fourth group. Group 1 — Negative control: mice were fed normal diet and water ad libitum without any treatment. Group 2 — Carcinogen-administered: mice were gently rubbed regularly with 100 μg DMBA (once a week) and with 1% croton oil (twice a week) on their backs and continued till 24 weeks. The continuous use of DMBA was consciously made once a week ^［3］. Group 3 — Carcinogen-administered 2% alcohol (placebo)-fed: DMBA and croton oil-intoxicated mice were fed for 24 weeks 100 μL of 2% alcohol (placebo) (scopoletin was dissolved in 2% ethanol), once daily. Group 4 — Carcinogen-administered drug-fed: this group comprised 12 mice all of which were administered DMBA and croton oil; a subgroup of mice (n=6) from this group 4 were fed scopoletin at a daily dose of 50 and the other 100 mg/kg body weight, once daily for 24 weeks.
1.3  Isolation and characterization of scopoletin  Scopoletin was isolated from the homeopathic mother tincture of Gelsemium by the standard column chromatography method (ratio of petroleum ether to ethyl acetate was 3︰1; silica gel 60 to 120 mesh size). With the help of 1H nuclear magnetic resonance (1H NMR), 13C nuclear magnetic resonance (13C NMR), correlation spectroscopy (COSY) and Fourier transform infrared (FTIR) spectroscopy, mass-spectroscopy and high-performance liquid chromatography analyses, the structure of principal component of Gelsemium was determined to be C10H8O4, as shown in Figure 1. Spectral data of scopoletin are as follows. a) Mass spectra of purified compound (MS): m/z=193 (M+). b) 1H NMR spectra of purified compound: 1H NMR (DMSO, 300 MHz), δH=3.95 (S, 3H, OCH3), 6.15 (S, 1H, OH), 6.25 (d, 1H, J=9.4 Hz, ArH) 6.84 (S, 1H, ArH) 6.92 (S, 1H, ArH) 7.58 (d, 1H, J=9.4 Hz, ArH). c) 13C NMR spectra of purified compound (DMSO, 75 MHz): 56.8, 103.5, 110.3, 111.3, 112.4, 145.4, 146.1, 150.3, 151.9, 161.6 per million.

Figure 1  Structure of scopoletin

1.4  Incidence of skin tumors  The onset of tumors (>2 mm) usually surfaced at the 8th week in the DMBA plus croton oil-treated mice, and the administration continued till 24 weeks when 100% of mice showed extensive skin papillomas. The numbers of tumor formation in the DMBA plus croton oil-treated groups and also the carcinogen plus drug-fed groups were recorded weekly. The data expressed as number of papilloma per mouse were plotted as a function of weeks on test.
1.5  Evaluation of antioxidant activity
1.5.1  Scavenging of 2, 2-diphenyl-1-picrylhydrazyl radicals  The test extracts were prepared in different concentrations and added to reaction mixture containing 0.1 mmol 2, 2-diphenyl-1-picrylhydrazyl (DPPH) in ethanol, 0.95 mL of 0.05 mol/L Tris-HCl buffer pH 7.4. The mixture generated 2, 2-diphenyl-1-picrylhydrazyl radicals. The absorbance of the mixture was measured at 517 nm exactly 30 seconds after adding drug, which would give precise amount of DPPH radicals scavenged. Percentage of scavenging activity was calculated according to the procedure of Yokozawa et al^［10］.
1.5.2  Scavenging of nitric oxide  Nitric oxide was generated by chemical reaction of sodium nitroprusside 5 μmol/L in standard phosphate buffer solution (PBS). When the mixture was incubated with different concentrations of drug dissolved in standard phosphate buffer (0.025 mol/L, pH 7.4), and the tubes were incubated at 25 ℃ for 5 hours, part of the generated nitric oxide was scavenged. The amount of the left over nitric oxide was calculated. For this, 0.5 mL of incubation solution was removed and diluted with 0.5 mL Griss reagent. The absorbance of the chromophore was then read at 546 nm^［11］.
1.5.3  Hydroxyl radical scavenging activity  Hydroxyl radicals are known to be generated as a result of reactions in the Fe3+-ascorbate-ethylene diamine tetraacetic acid (EDTA)-H2O2 system. The hydroxyl radical scavenging activity was measured by studying the competition between the hydroxyl radicals generated from this system and scavenging activities of the drug, according to the procedure of Mary et al^［12］.
1.6  Preparation of nuclear and cytosolic extract  For analysis of expressions of nuclear factor-κB (NF-κB) and p53 (Santa Cruz Biotechnology) and immunoprecipitation studies, nuclear extract of skin sample was used. For analysis of all other signal proteins AhR, CYP1A1, PCNA, Stat-3, survivin, matrix metalloproteinase-2 (MMP-2), cyclin D1, c-myc, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), caspase-3, and p38 mitogen-activated protein kinase (p38MAPK) and biochemical studies, cytosolic extract^［13］ of skin sample was used. The homogenized material was centrifuged at 13 000 g for 15 min at 4 ℃.
1.7  Biochemical analysis  Lipid peroxidation (LPO) activity was estimated from the supernatant of skin sample by the method of Ohakawa et al^［14］. Superoxide dismutase (SOD) activity was analyzed by the method of Kakkar et al^［15］. Catalase (CAT) activity was measured by the method of Maehly et al^［16］. Glutathione peroxidase (GPx) was assayed by the method of Paglia et al^［17］. Glutathione S-transferase (GST) was assayed by the method of Habig et al^［18］. All chemicals were purchased from Sigma, USA.
1.8  Histology and immunofluorescence method  The back skin samples of experimental groups were dissected from the lesion and/or tumor regions of both the drug-fed and positive controls (and also of normal back skin of the negative control) and fixed in the normal buffer formalin, followed by dehydration treatment, as per the standard practice^［3］. For the immunofluorescence study, the technique of Arabzadeh et al^［19］ was adopted with a little modification. Briefly, the cut tissue sections were de-paraffinized and incubated separately for 12 hours with mouse monoclonal antibody against PCNA, procured from Abcam International, USA. Then, after being blocked with 3% bovine serum albumin, the tissue sections were incubated for 2 hours with fluorescence isothiocyanite (FITC)-conjugated secondary antibody purchased from Sigma, USA. The fluorescence intensity of FITC was measured under a ZEISS fluorescence microscope.
1.9  Western blot analysis  It was performed as per protocol described previously^［20］ from skin sample. For quantitative analysis of each band, density was determined by using Gel Doc System, Ultra Lum, USA.
1.10  Immunoprecipitation  For immunoprecipitation, cleared lysate was prepared and about 100 μg of protein were immunoprecipitated by using 10 μL of anti-AhR antibody (Abcam, USA) for overnight at 4 ℃ with gentle rotation. Twenty-five microliter protein G CL-Agarose (Bangalore Genei, India, Cat#LIA 43S) was added to the previous mixture, depending on the experiment and mixed for 4 hours at 4 ℃ with gentle rotation. It was then centrifuged at 3 500×g for 2 min. The immunoprecipitates were washed extensively with sterile PBS and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blot analyses with anti-Aryl hydrocarbon receptor translocator (ARNT) antibody (Santa Cruz Biotechonology, USA) as described above and developed by alkaline phosphatase-conjugated secondary antibody (Sigma, USA).
1.11  RNA extraction and quantitative RT-PCR analysis  The total RNA was extracted from the skin tissue of experimental groups by using TRIzol reagent according to the manufacturer’s instructions. The synthetic oligonucleotide primers used for RT-PCR were procured from Bangalore Genei, Bangalore, India and the primer sequences are shown in Table 1. Single-stranded cDNA was used as template for PCR amplification by using Taq polymerase. After 2 min at 94 ℃, amplification was performed for all the samples under the following conditions: 94 ℃ for 30 s, 52 ℃ for 30 s and 72 ℃ for 30 s for 35 cycles, with a final incubation at 72 ℃ for 8 min. Following PCR, 3 μL sample aliquots were subjected to electrophoresis on 1% (W/V) agarose gel for 20 to 30 min and then stained with ethidium bromide and photographed. The densitometric analysis was performed by using Total Lab software (Ultra Lum USA).
 

Table 1  Primer sequences of cancer-related genes (mouse origin) used in this study

 

1.12  Statistical analysis  The significant differences between data of carcinogen plus 2% alcohol-administered mice and carcinogen-administered scopoletin-fed mice were analyzed by the Student’s t test. Additionally, the data were also analyzed by using one way ANOVA (Tukey method; SPSS version 11).

2  Results
2.1  Tumor incidence  The incidence of papilloma growth in different weeks in control and treated mice had been provided^［3］. The papillomas observed in the DMBA plus croton oil-treated group were significantly bigger in size (70% of papillomas≥2.5 mm in diameter) than those of drug-fed group (8% to 10%).
2.2  Histopathological examination  As compared to the skin section with normal histological features (Figure 2A), irregular distribution with finger-like projections (papilloma) indicative of cancerous growth were found in the skin sections of DMBA-administered mice (Figure 2B). The tumors of rats in the DMBA plus croton oil-treated group were composed of focal proliferation of squamous cells, and characterized by presence of some necrotic cells, keratinization and epithelial pearls; these features were indicative of the malignant nature of tumors, particularly for the presence of the well-differentiated squamous cell carcinoma. On the other hand, in the scopoletin-fed mice, these features were visible to a much lesser extent (Figure 2C).
2.3  Immunofluorescence of PCNA  The immunofluorescence analysis for PCNA was also used to assess the proliferation activity in normal (Figure 2D), carcinogen-treated (Figure 2E) and scopoletin-fed (Figure 2F) groups during tumor promotion. The PCNA is associated with the S phase of DNA replication. It was observed through immunofluorescence localization that the PCNA expression was higher in DMBA-administered group, and a characteristic intense staining, as compared to control, was observed (Figure 2E) and also there was a significant modulation of this expression in the scopoletin-fed groups. The data on proportion of fluorescence intensity represented in Figure 2G prepared from three replicates would also validate the above observation.
 

Figure 2  Histology of skin tested by haematoxylin and eosin staining and PCNA protein localization

Histology of skin: A, B, and C; PCNA localization: D, E, and F. A and D: Normal group; B and E: DMBA plus croton oil group; C and F: Scopoletin-fed group. G: Proportion of average fluorescence intensity of PCNA protein in different groups. **P<0.01, vs DMBA plus alcohol group.

2.4  In vitro radical scavenging activity of scopoletin  Table 2 depicts the free radical scavenging activity of scopoletin in different concentrations from 50 to 200 μg/mL. The treatment with 200 μg/mL of drug shows 68.48% inhibition of DPPH radical, 45.17% inhibition in hydroxyl scavenging activity and 28.22% inhibition of nitric oxide scavenging activity.

Table 2  Evaluation of anti-oxidant activity of different doses of scopoletin

2.5  LPO and anti-oxidant biomarkers  A significant increase in basal level of LPO was observed in the carcinogenic control and also the alcoholic control (Table 3), but after administration of scopoletin due to free radical quenching activity of this coumarin, peroxidation of lipids significantly decreased. Marked decrease in the activities of different anti-oxidant enzymes like SOD, CAT, GPx and GST were observed in the mice treated with the carcinogen and in the alcoholic control group (Table 3), which significantly reverted after administration of scopoletin at the higher dose.
 

Table 3  Effects of drugs on LPO and the activities of anti-oxidant enzymes SOD, catalase, GPx and GST in skin of control and experimental mice

2.6  Expressions of AhR and CYP1A1  AhR is the key regulator for polyaryl hydrocarbon intake process in cells. Scopoletin significantly reduced mRNA expression level of AhR (Figure 3). CYP1A1 protein is involved in the metabolic process of DMBA and turns the pro-carcinogen into active carcinogenic form. Our Western blot analysis and mRNA quantification by RT-PCR revealed that scopoletin significantly reduced the CYP1A1 over-expression (Figure 3, Table 4-6).
2.7  Expression of Stat-3  The Stat-3 is a transcription factor that is involved in skin cancer development. The Stat-3 prevents apoptosis in the initiation phase of skin tumorigenesis. The Stat-3 expression from the Western blotting and the mRNA expression of Stat-3 were found to be significantly reduced at both the doses of scopoletin as compared with their respective control (Table 4, Figure 3).
2.8  Expressions of p53 and PCNA  The p53 expression was significantly elevated in the scopoletin-administered groups, as compared with the carcinogen-administered group through mRNA expression (Table 4, Figure 3).
     From the Western blot and RT-PCR analyses, it revealed that the expression level of PCNA was significantly elevated after prolonged administration of DMBA on skin. On the other hand, the elevation was significantly reduced (Table 4, 5 and Figure 3) after the scopoletin treatment. This result was also supported by the result of immunofluroscence in our study.
 

Table 4  Expressions of AhR, CYP1A1, Stat-3, p53 and PCNA mRNAs detected by RT-PCR

Table 5  Expressions of caspase-3, survivin, cyclin-D1, c-myc, MMP-2 and TIMP-2 mRNAs detected by RT-PCR

Table 6  Expressions of CYP1A1, Stat-3, PCNA, caspase-3, NF-κB, p38MAPK proteins detected by Western blotting and immunoprecipitation of ARNT

Figure 3  Immunoblots and mRNA expressions of β-actin, AhR, CYP1A1, Stat-3, p53, PCNA and caspase-3

A: Normal group; B: DMBA plus croton oil group; C: DMBA, croton oil plus alcohol group; D: DMBA, croton oil plus scopoletin (50 mg/kg) group; E: DMBA, croton oil plus scopoletin (100 mg/kg) group.

2.9  Expression of caspase-3  The data on the Western blot and mRNA expressions revealed that caspase-3 was significantly elevated after scopoletin treatment, as compared with that of carcinogenic control (Table 4, 5 and Figure 3). Thus, our data clearly indicate the involvement of caspase-3 in death signaling pathway of the skin cells after administration of scopoletin.
2.10  Expression of survivin  The statistical analysis showed that the mRNA expression of survivin was down-regulated significantly (Table 5, Figure 4) by scopoletin, when compared with that of carcinogen control.
2.11  Expressions of cyclin D1 and c-myc  Two cell cycle regulators that are important for the progression of tumorigenesis are the cyclin D1 and c-myc oncogenes. Thus, in our studies, quantitative PCR was performed to compare the mRNA levels of these genes in the normal, carcinogen-administered, and drug-fed groups. The cyclin D1 and c-myc mRNA levels were significantly increased in carcinogen-administered group, as compared with normal control (Table 5 and Figure 4). After the drug treatment, we observed positive modulation of both the oncogenes.
2.12  Expressions of MMP-2 and TIMP-2  The maintenance of the extracellular matrix architecture is achieved by the balance of the action of MMPs with simultaneously elaborated counter regulatory inhibitors like TIMPs. The MMP-2 mRNA expression was significantly blocked by the scopoletin treatment (Table 5, Figure 4); on the other hand, it elevated the TIMP-2 mRNA expression. Scopoletin may act on the MMP-2 by regulating the activity of TIMP-2 mRNA expression.
 

Figure 4  mRNA expressions and densitometric representation of survivin, cyclin D1, c-myc, MMP-2 and TIMP-2

A: Normal group; B: DMBA plus croton oil group; C: DMBA, croton oil plus alcohol group; D: DMBA, croton oil plus scopoletin (50 mg/kg) group; E: DMBA, croton oil plus scopoletin (100 mg/kg) group.

2.13  Expression of NF-κB  In general most carcinogenic process is associated with inflammation. Therefore, we examined if the expressions of NF-κB was significantly down-regulated after scopoletin administration, as compared with DMBA, croton oil plus alcohol-administered mice. Indeed, the expressions of NF-κB were significantly down-regulated (Table 6, Figure 5).
2.14  Expression of p38MAPK  As illustrated in Figure 5 and Table 5 from Western blotting, scopoletin reduced the expression of p38MAPK in a dose-dependent manner.
 

Figure 5  Immunoblots of expressions of NF-κB and p38MAPK

A: Normal group; B: DMBA plus croton oil group; C: DMBA, croton oil plus alcohol group; D: DMBA, croton oil plus scopoletin (50 mg/kg) group; E: DMBA, croton oil plus scopoletin (100 mg/kg) group.

2.15  Immunoprecipitation of AhR-ARNT complex  From our immunoprecipitation study it is clear that AhR formed complex with ARNT in the presence of DMBA and thus co-precipitated. But with the treatment of scopoletin, this precipitation was significantly reduced for both the doses (Figure 6 and Table 6). Therefore it appeared that scopoletin competitively interacted with ARNT complex and antagonized the hetero-dimer formation, considerably reducing the subsequent activation of CYP1A1.
 

Figure 6  Immunoprecipitation of ARNT blots

A: Normal group; B: DMBA plus croton oil group; C: DMBA, croton oil plus alcohol group; D: DMBA, croton oil plus scopoletin (50 mg/kg) group; E: DMBA, croton oil plus scopoletin (100 mg/kg) group.

3  Discussion
     In our earlier studies^［2］, we have shown that scopoletin, one of the coumarin compounds present in Gelsemium sempervirens extract, significantly enhanced apoptosis in in vitro culture of human HeLa cells, indicating its anticancer nature. Further, we also observed that this compound had free-radical scavenging activity as revealed from Table 2. Incidentally, several botanicals and/or dietary supplements have been investigated earlier for their possible anti-skin carcinogenic properties, including some flavonoids like silymarin and silibinin showing positive results^［7］. A considerable increase in LPO in the DMBA-treated mice suggests that greater generation of free radicals induced more oxidative stress.
     Apoptosis has been shown to be regulated by tumor suppressing activity of p53 protein^［21,22］. Studies have also indicated that the lack of p53 expression or function is associated with an increased risk of tumor formation^［23,24］. Loss of tumor suppressor gene function is associated with amelioration of apoptosis and increased growth of tumors^［25］. Results also follow the same trend. Survivin, a recently discovered protein and a member of the inhibitors of apoptosis protein family, plays a key role in apoptosis and cell division regulation^［26］. Scopoletin has been shown to markedly reduce the expression of survivin to inhibit the growth and differentiation of skin cells. In the present study, we observed that scopoletin markedly down-regulated the expression of survivin. In one study^［8］, DMBA-induced skin cancer has been claimed to be associated with/mediated through up-regulation of PCNA, but the signaling mechanism was not further investigated beyond that. From the results of the present study it was revealed that signaling of DMBA-induced skin carcinogenesis is possibly mediated through stimulation of AhR, which is ligand-activated. The ligand activation of AhR protein, which has been shown to be up-regulated in 7 out of 11 mammary tumor types^［13］, activates the expression of CYP1A1 and CYP1B1 proteins^［27］, which in turn activates procarcinogenic DMBA into metabolites like 3,4-dihdrodiol-1,2-epoxide^［28］. From the Western blot study and mRNA expression of CYP1A1 we observed that CYP1A1 was over-expressed and acted as active carcinogen, leading to the generation of reactive oxygen species (ROS)^［29］. Incidentally, an increase in ROS generation by DMBA administration has also been observed by us through analysis of the enzymes like SOD, catalase, and LPO. Increase of the ROS then activates PCNA that provokes the cell into faster divisional activity, resulting in down-regulation of p53 as revealed from mRNA expression and a suppression of p53 activity reflects on its inability to function normally to delay the cell cycle giving time for the damaged DNA to be repaired. The result is the uncontrolled proliferation of skin cells showing effective progression of skin cancer (papilloma) observed in the carcinogen-treated mice. Thus, the treatment of the drug in both the doses, 100 mg showing a little more pronounced effects than that of 50 mg, produced some dramatic changes in the expression levels of these signal molecules. First, it shows appreciable suppression in the expression of AhR receptor as revealed from mRNA expression. One explanation could be that either scopoletin itself acts as a ligand to bind on the AhR receptor, thereby competitively blocking the DMBA entry as a stimulator of expression of AhR, or else, restricts the entry of ARNT protein into nucleus, because binding of ligand such as procarcinogen to AhR is followed by the nuclear translocation and heterodimerization with ARNT. The AhR-ARNT heterodimer subsequently binds to the consensus DNA site, present in the promoter region of CYP1A1^［27］. CYP1A1 gene is transactivated by the binding of ligand-activated AhR-ARNT complex. From the immunoprecipitation study, another explanation is possible: scopoletin may block the formation of AhR-ARNT complex formation and thereby reduce the expression of CYP1A1, both at the gene and the cellular levels.
     Caspase-3 is known to be an effector caspase whose early activation triggers apoptotic cells to the “point of no return”^［6］. Therefore, scopoletin might activate caspase-3-mediated apoptotic machinery at the early stage of carcinogenesis process to restrict the growth of preneoplastic cells in the skin from further mutational changes.
     Degradation of basement membranes and extracellular matrix is an essential process in invasion and metastasis in malignant tumors. MMPs, potent proteolytic enzymes, are known to play key roles in this process. Maintenance of extracellular matrix architecture is achieved by the balance of the action of MMPs with simultaneously elaborated counter regulatory inhibitors like TIMPs. The MMP-2 mRNA expression was significantly blocked by scopoletin treatment, and on the other hand, it elevated the TIMP-2 mRNA expression.
     MAPKs play a critical role in the transcriptional activation of NF-κB and AP1 in TPA-treated mouse skin^［30］. In our present experiment we observed that scopoletin inhibited the expression of MAPKs, p38MAPK. In a recent study, we have reported that a synthetic coumarin (4-methyl-7-hydroxy coumarin) attenuated activation of NF-κB^［3］. Our present observation also corroborated the previous observation that substituted coumarin inhibited the transcriptional activation of NF-κB. The inhibitory effects of scopoletin on DMBA and croton oil-induced expression of p38MAPK would suggest a molecular basis of inhibitory activity of skin papilloma. We have also shown evidence that scopoletin can have regulatory roles on some cell cycle regulators like cyclin D1 in normal as well as cancer cells. The cyclin D1 expression was increased in carcinogen-administered mice, and the expression was significantly down-regulated after administration of the drug.
     Thus, we can conclude that scopoletin has considerable anticancer potentials against DMBA-induced skin cancer in mice, and that its major action seemed to be mediated through regulation of AhR.

4  Acknowledgements
      This work was financially supported by a grant sanctioned to Prof. A.R. Khuda-Bukhsh, Department of Zoology, University of Kalyani, Kalyani-741235, India by Boiron Laboratory, Lyon, France. ARKB is grateful to Dr. Philippe Belon, Ex-Director, Boiron Laboratory for his kind encouragements.