Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells

    Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells ´ Guilherme Alvaro Ferreira-Silva, Carla Carolina Lopes Lages, Patricia Sartorelli, Fl´avia Rie Hasegawa, Marisi Gomes Soares, Marisa Ionta PII: DOI: Reference:

S0887-2333(16)30219-3 doi:10.1016/j.tiv.2016.10.011 TIV 3869

To appear in: Received date: Revised date: Accepted date:

29 June 2016 27 October 2016 28 October 2016

´ Please cite this article as: Ferreira-Silva, Guilherme Alvaro, Lages, Carla Carolina Lopes, Sartorelli, Patricia, Hasegawa, Fl´avia Rie, Soares, Marisi Gomes, Ionta, Marisa, Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells, (2016), doi:10.1016/j.tiv.2016.10.011

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Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells

Institute of Biomedical Sciences, Federal University of Alfenas, Rua Gabriel Monteiro da

Silva, 700, zip code 37130-000, Alfenas, MG, Brazil

Institute of Chemistry, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, zip

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code 37130-000, Alfenas, MG, Brazil

Institute of Environmental Sciences, Chemical and Pharmaceutical, Federal University of Sao

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Paulo, Diadema, SP, Brazil

The authors contributed equally to this study

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*

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a

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Hasegawac, Marisi Gomes Soaresb**, and Marisa Iontaa**

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Guilherme Álvaro Ferreira-Silvaa*, Carla Carolina Lopes Lagesb*, Patricia Sartorellic, Flávia Rie

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**

Corresponding authors: Federal University of Alfenas

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700, Gabriel Monteiro da Silva Street, Zip code: 37130-000, Alfenas, MG, Brazil Institute of Biomedical Sciences, Federal University of Alfenas.

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E-mail address: [email protected]

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ABSTRACT Cancer is a public health problem which represents the second cause of death in the world. In

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this framework, it is necessary to identify novel compounds with antineoplastic potential. Plants

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are an important source for discovering novel compounds with pharmacological potential. In

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this study, we aimed to investigate the antiproliferative potential of isolated compounds from Casearia sylvestris on tumor cell lines. Crude extract effectively reduced cell viability of 4 tumor cell lines (HepG2, A549, U251-MG, and HT-144) after 48h treatment. HepG2 and HT-

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144 were the most responsive cells. Three fractions (aqueous ethanol, n-hexane and ethyl acetate) were tested against HepG2 and HT-144 cells and we observed that compounds with

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antiproliferative activity were concentrated in n-hexane and ethyl acetate fractions. The casearins A, G and J were isolated from n-hexane fraction, while casearin D was obtained from

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ethyl acetate fraction. We demonstrated that casearin D significantly inhibited the clonogenic

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capacity of HepG2 cells after 24h exposure indicating its antiproliferative activity. In addition, G1/S transition cell cycle arrest in HepG2 cells was also observed. These effects are related, at

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least in part, to ability of the casearin D in reducing ERK phosphorylation and cyclin D1 expression levels.

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Keywords: Hepatocellular carcinoma, cell cycle arrest, Cyclin D1 expression, casearins, Casearia sylvestris Highlights

Casearin D inhibits cell cycle progression of HepG2 cells. Casearin D treatment reduces ERK phosphorylation levels in HepG2 cells. Cyclin D1 expression is reduced in HepG2 cells after treatment with Casearin D.

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1. Introduction

Cancer is a public health problem in the world which represents a second cause of

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death. According to GLOBOCAN estimates, about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide. Although incidence rates for all cancers combined

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are nearly twice as high in more developed than in less developed countries in both males and females, mortality rates are only 8% to 15% higher in more developed countries (Torre et al.,

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2015).

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Hepatocellular carcinoma (HCC) represents the most frequent primary liver cancer. This disease usually arises as a result of a chronic liver disease but may appear without any

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underlying disease (Díaz-González et al., 2016). Unfortunately, many cases are still diagnosed

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at advanced stages when treatment options are only palliative (Lorenzi, 1998). Excessive alcohol consumption and Hepatitis B and C viruses are important risk factors for HCC

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(Nsonde-Ntandou et al., 2005). Currently, therapeutic approaches for HCC at the advanced stage include the use of sorafenib, a kinase inhibitor (Bruix et al., 2016); however, clinical trials have proven that sorafenib to yield a modest survival benefit. Therefore, it is appropriate to

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identify new compounds with promising antitumor activity against HCC.

Plants are an important source for discovering new prototypes with pharmacological potential because they contain a wide spectrum of bioactive secondary metabolites (Zanin et al., 2015; Newman and Cragg, 2016). Casearia sylvestris is a tree found in tropical regions around the world which are commonly widespread in the Americas. In Brazil, the tree is commonly known as “guaçatonga” (Basile et al., 1990) and can be found from the Amazonas (Tapajós river region) to Rio Grande do Sul states (Torres et al., 1986; Hack et al., 2005). It has been used in the Brazilian folk medicine as a diuretic, appetite suppressant, weight loss product, and snakebite agent (Cruz, 1995; Oshima-Franco et al., 2005). Studies show that C. sylvestris has

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different pharmacological properties including antimicrobial, antiulcer, anti-inflammatory, and antileishmanicial activities (Antinarelli et al. 2015, Bou et al., 2014).

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Phytochemical investigations performed from leaf extracts of C. sylvestris showed that clerodane diterpenes (casearins and casearvestrins) are the major constituents (Itokawa et al.,

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1990). Studies have reported these compounds as closely associated with different biological activities attributed to C. sylvestris including cytotoxic activity (Carvalho et al., 1998; Morita et

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al., 1991; Oberlies et al., 2002; Santos et al., 2010).

Different casearins have been isolated (A – X) (Carvalho et al. 1998; Itokawa et al.,

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1989, 1990; Morita et al. 1991; Santos et al. 2010; Wang et al., 2009). Cytotoxic activity of some casearins on tumor cell lines has been evaluated. Itokawa et al. (1990) demonstrated that

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casearins (A – F) had cytotoxic activity against V-79 cells, with casearin C being the most

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active among them. Dupuy et al. (2013) also verified that casearin G was effective in reducing cell viability of MCF-7 and PC-3 cells, which are derived from breast and prostate cancers, with

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PC-3 cells being more responsive to caserin treatment than MCF-7. Ferreira et al. (2014) showed that casearin X had a great cytotoxic effect on HL-60 leukemia cells. Bou et al. (2015)

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described a great cytotoxic activity of a new casearin X (dinor casearin X) on HL-60 cells. Although there is evidence that casearins have cytotoxic activity on cancer cells, the molecular mechanism involved in this process still remains poorly understood. In addition, there has been limited exploration in relation to the antiproliferative activity of the casearins.

Herein, we carried out a bioguided study that allowed us to isolate the casearins A, D, G, and J. Casearin D was isolated from ethyl acetate fraction obtained from Casearia sylvestris crude leaves and we investigated its antiproliferative potential on HepG2 cells, which is derived from human hepatocellular carcinoma.

2. Materials and methods

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2.1. Plant material, preparation of the extracts, and isolation Casearia sylvestris leaves were collected from a single tree in the Atlantic Forest area

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of São Paulo City, SP, Brazil (coordinates 23 53'08.86''S, 46 40'10.45''O), in October 2012.

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Botanical identification was made by Dr. Roseli Buzanelli Torres from Instituto Agronômico de

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Campinas (IAC), in Campinas–SP, Brazil. A voucher specimen (IAC 55272) was deposited in the IAC herbarium. After that, leaves (290 g) were dried, powdered, and extracted with MeOH for three days to obtain 11.1 g of crude extract. The MeOH extract was ressuspended in

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MeOH:H2O (2:1) and partitioned using hexane and EtOAc. The EtOAc phase (3.0 g) was subjected to separation over silica gel (0.063-0.200 mm) CC and eluted with increasing amounts

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of EtOAc in hexane (9:1 to 1:9) to obtain 23 fractions (E1-E23). Fraction E15 (239 mg) was fractionated over Sephadex LH-20, eluted with MeOH, and purified by preparative TLC on

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SiO2 (hexane-EtOAc, 4:1) to provide casearin D (6,4 mg).

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Also the hexane phase (6.4 g) was subjected to SiO2 column chromatography gel eluted with increasing amounts of EtOAc in hexane (9:1 to 1:9) to be separated into 23 fractions (A1 –

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A23). The fractions A11, A12, and A13 were submitted to new chromatographic steps. Thus, fraction A11 (380 mg) was fractionated over Sephadex LH-20, eluted with MeOH, and

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followed by SiO2 prep TLC (hexanes 8: EtOAC 2) to provide caserins G (77 mg) and J (51 mg). Finally, fractionation of A13 (526 mg) over Sephadex LH-20, followed by prep TLC (hexanes 7: EtOAC 3) provided casearin A (43 mg). 2.2. Identification

The chromatographic procedures carried out to fractions H15 and E15 allowed the isolation of two pure substances. The 13C NMR spectra of these substances showed in general from 29 to 30 signals, similar to those reported for casearins in the previous literature. Characteristic signals in 13C NMR spectra were observed at δ 57 (CH3) indicating the presence of a methoxy group and in the structure, and δ 63 and 72 (CH) for the carbinolic carbon (C2) in both isolated compounds. The signals at δ 72 and 75 (CH) were assigned to carbons attached to acetyl and hydroxyl groups (C6). The double

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bond at C-3 and C-4 also characteristic of casearins was observed at δ 123 and 142 respectively. The chemical shifts at δ 53 (C-5) and δ 35 (C-10) were similar to those

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described for casearins and analogous compounds. The signals of methynic carbons at δ

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95 (C-18) and 97 (C-19) indicate the existence of the diacetalic ring system C, while

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those at δ 170 to 174 suggest the presence of acetate and butanoate groups as substituents. Thus the structural identification of casearins A, D, G, and J, isolated in

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this work was performed by comparing the data obtained from the spectral 1H and 13C

2.3 Cell lines and treatment schedule

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NMR with the data previously reported (Itokawa et al., 1990; Morita et al., 1991).

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MCF-7 (breast cancer), HT-144 (melanoma), U251-MG (glioblastoma), A549 (lung cancer),

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and HepG2 (hepatocellular carcinoma) cells were used in this study. The cell cultures were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma, CA, USA) supplemented

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with 10% fetal bovine serum (Vitrocell, Campinas, Brazil). Cells were grown in a 37C humidified incubator containing 5% CO2. Casearia sylvestris extracts or isolated compounds

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(casearins) were solubilized in DMSO for obtaining a stock solution, which was stored at -20o C until use. After attachment (24h), the cells were treated (extracts or isolated compounds) for 24h or 48h depending on the experimental approach.

2.4 Cell viability analysis Cell viability was measured by MTS (dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium) assay using CellTiter 96® Aqueous Non-Radiative Cell Proliferation assay (Promega) according to the manufacturer’s instructions. Cells were seeded (1 x 104 cells/well) into a 96-well plate. Leaf extracts or casearins were used in different concentrations (0 - 40 µg.mL-1) for 48h. Formazan, the reduced form of tetrazolium, absorbs light at 490 nm and

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viable cells rate is directly proportional to the amount of produced formazan by dehydrogenase enzymes. Experiments were conducted in triplicate wells. Data are presented as the mean ±

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standard deviation (SD) of three independent experiments. The IC50 value was determined from

2.5 Cytokinesis block micronucleus assay (CBMN)

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non-linear regression using GraphPad Prism® (GraphPad Software, Inc., San Diego, CA, USA).

The micronucleus assay was performed according to Natarajan and Darroudi (1991) with some

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minor modifications. Briefly, cells were seeded at a density of 2.5 × 105cells per well in 6-well culture plates and treated with crude extract of Casearia sylvestris at 5 µg/mL for 48 h. After

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the exposure period, cells were washed twice with PBS and re-incubated in fresh medium containing 3 μg/mL of cytochalasin B (Sigma) for 28 h. After that, cells were harvested and

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suspended in cold sodium citrate (1%) at room temperature for 5 min. Then, the cells were fixed

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in Carnoy's solution (1:3 mixtures of acetic acid and methanol), and a few drops of formaldehyde were added to preserve the cytoplasm. Immediately after centrifugation at 1000

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rpm for 5 min, the cells were re-suspended in Carnoy's solution and dropped onto clean microscopic slides, air dried, and stained with Giemsa (5%). Analysis was performed using a

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light microscope (Nikon) at 1,000x magnification (immersion oil). Previously to micronuclei (MN) analysis, it was determined the nuclear division index (NDI), which was calculated by the following formula: NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1-M4 represent the number of cells with 1 to 4 nuclei, respectively, and N is the number of cells scored (500) (Eastmond and Tucker, 1989). Micronuclei (MN) were counted in 1000 bi-nucleated cells (BNC) per culture and were scored according to the criteria published by Fenech (2000), in 3 independent experiments. Results are presented as mean ± DP. Doxorubucin was used as a positive control.

2.6 Cell cycle analysis

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Cell cycle analysis was performed according to Pereira et al. (2016). Briefly, cells were treated with casearin D for 24h at 3 or 6 µg.mL-1 for cell cycle analysis. The cells were fixed with 75%

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ethanol at 4 C overnight, rinsed twice with cold phosphate-buffered saline (PBS). Afterwards,

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cells were homogenized in dye solution [PBS containing 30 μg.mL-1 propidium iodide (PI) and

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3 mg. mL-1 RNAase]. DNA was quantified after 1h after staining. The analysis was performed by flow cytometry (Guava easyCyte 8HT, Hayward, CA, USA). Results are presented as mean

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± standard deviations (SD) of 3 independent experiments.

2.7 Clonogenic assay

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Clonogenic assay was performed according to Franken et al. (2006). Briefly, 1 x 104 cells were seeded in 35mm plates. Cells were treated for 24h and recovery in a drug-free medium

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subsequently for 15 days. Aftwards, the colonies were fixed and stained with Cristal Violet.

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Only colonies with >50 cells were counted by direct visual inspection with a stereo microscope at 20X magnification. Assays were performed in triplicate and the data were presented as mean

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2.8 Immunoblot

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± SD of 3 independent experiments.

Cells were homogenized in RIPA lysis buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris pH 8.0) containing both protease and phosphatase inhibitors (Sigma). Lysates were centrifuged (10,000 × g) for 10 min at 4 °C. Supernatants were recovered, total proteins were quantified (BCA kit, Pierce Biotechnology Inc., Rockford, IL, USA) and resuspended in Laemmli sample buffer containing 62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue. An aliquot of 50 μg protein was separated by SDS–PAGE (12%) and transferred (100 V, 250 mA for 2h) onto a PVDF membrane (Amersham Bioscience), which was blocked by incubation for 1h at 4°C with blocking solution [5% non-fat milk in Tris-buffered saline (TBS) + 0.1% (v/v) Tween-20] to prevent nonspecific protein binding. The membrane was probed with primary antibodies: (Tyr

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204) phosphorylated ERK antibody (Santa-Cruz - 1:50), ERK 1 (Santa-Cruz – 1:1000), Cyclin D1 (Sigma – 1: 200) and α-tubulin (Sigma– 1:1000) overnight at 4oC. After washing with TBS-

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tween (0.1%), the membrane was incubated with a secondary antibody (anti-rabbit peroxidase

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conjugated) for 2h at room temperature. Immunoreactive bands were visualized with the ECL

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Western Blotting Detection Kit (Amersham Pharmacia). A reprobing protocol was followed for detecting immunoreactive bands for different antibodies. Results were obtained from three independent experiments. The quantification of immunoreactive bands was performed using a

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public program (Image J).

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2.9 Statistical Analysis

The results were tested for significance using one-way analysis of variance (ANOVA) followed

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by a Tukey’s post-test. The values were expressed as mean ± SD.

3. Results

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Crude extract of Casearia sylvestris at 40 μg.mL-1 was tested against five cancer cell lines (Figure 1). Cell viability was drastically reduced in HepG2, HT-144, A549, and U251-MG.

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Afterwards, dose-response curves were performed to determine IC50 values (concentration which inhibits 50% of the growth). The data are shown in Table 1. IC50 values of HepG2 and HT-144 were comparable to cisplatin, a powerful cytotoxic chemotherapeutic agent.

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Figure 1. Cell viability determined by MTS assay after 48h of treatment with crude extract of C. -1 sylvestris at 40 μg.mL .

HT-144

C. sylvestris

17,78 ± 0,76

10,96 ± 1,04

Cisplatin*

9,54 ± 0,84

9,63 ± 0,66

A549

HepG2

25,63 ± 0,90

9,94 ± 0,35

6,52 ± 0,57

7,50 ± 0,52

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U251-MG

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Table 1. IC50 (μg.mL-1) values determined from MTS data. Cell cultures were treated with crude extract of C.sylvestris for 48h.

*Cisplatin was used as a positive control.

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For the bioguided study, we evaluated the effect of three fractions obtained from crude extract (n-hexanic, ethyl acetate and aqueous ethanol) on cell viability of the most responsive

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cell lines (HT-144 and HepG2). We clearly observed that both ethyl acetate and n-hexanic fractions effectively reduced cell viability in these lineages, while aqueous ethanol fraction was

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compound from these fractions.

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less active. The IC50 values are shown in Table 2. In this framework, we worked in isolating of

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Table 2. IC50 (μg.mL-1) values determined from MTS data. Cell cultures were treated with different fractions of C.sylvestris for 48h.

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Extract hydroalcoholic ethyl acetate n-hexane

Hep G2 > 40.00 6.97±0.48 11.48±0.63

HT 144 > 40.00 3.35±1.05 5.92±0.97

We sought also investigate the possible genotoxic effect of crude extract of C. sylvestris on HepG2 cells through micronucleus assay using a sub-toxic concentration (5 µg.mL-1) for 48 h (Figure 2A). We did not observe a significant increase in micronucleus frequency when compared to control samples (Figure 2B).

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Figure 2. (A) Cell viability of HepG2 cells determined by MTS assay after treatment with crude extract of C. sylvestris for 48 h. (B) Micronucleus analysis performed in HepG2 cell cultures treated with crude extract of C. Sylvestris at 5 µg.mL-1 for 48 h. Three thousand cells were analyzed in each group. Doxorubicin (DXR) was used as a positive control. *Statistically different (p  0.05) from control group according to ANOVA followed by Tukey post-test.

The structural identification of casearins A, D, G, and J, isolated in this work, was

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performed by comparing the data obtained from the spectral 1H and 13C NMR with the findings

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from the literature data (Carvalho et al., 1998; Itokawa et al., 1990; Morita et al., 1991). In the next step, HepG2 cells were selected for evaluating the effect of the casearins A, D, G, and J on cell viablity. All casearins were effective in reducing cell viability of HepG2

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cells, however casearin D drastically reduced cell viability when used at 5 μg.mL-1 (Figure 3 and Table 3). Others casearins (A, G, and J) reduced cell viability only when used in concentrations upward 10 μg.mL-1. These findings motivated us to conduct a deeper investigation of the mechanism involved with the effect that was previously observed of casearin D on HepG2 cells.

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HepG2

casearin A

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casearin D casearin G casearin J

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Concentration (g.mL-1)

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20

10

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Cell viability (%)

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Figure 3. Cell viability determined by MTS assay after 48h of treatment of HepG2 cells with different casearins.

cell line HepG2

23.25 ± 0.65

Compounds

casearin D

casearin G

casearin J

6.04 ± 0.31

22.63 ± 0.56

12.60 ± 0.49

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casearin A

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Table 3. IC50 (μg.mL-1) values determined from MTS data. Cell cultures were treated with different casearins for 48h.

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Afterwards, we evaluated the influence of casearin D on proliferation behavior of HepG2 cells. For this evaluation, we performed cell cycle analysis and clonogenic assay. We

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observed a significant increase in G0/G1 population with concomitant decrease in both S-phase and G2/M populations when casearin D was used at 3 µg.mL-1. A similar profile was observed for cultures treated with 6 µg.mL-1 of casearin D, however in this condition there are significant increase of both SubG1 and G2/M populations (Figure 4A) proving the toxicity of the casearin when used at 6 µg.mL-1. Colony frequencies were significantly reduced in samples treated with casearin D for 24 h. We observed approximately 53% and 97% less colonies in cultures treated at 3 μg.mL-1 and 6 μg.mL-1, respectively, when compared to control cultures (Figure 4B). Thus, when casearin D is used in low concentration, it has antiproliferative activity on HepG2 cells. In next step, we evaluated the expression profile of cyclin D1 and extracellular signalregulated kinase (ERK), a regulator upstream of cyclinD1. According Western blot results, casearin D did not alter the profile of expression of total-ERK. However p-ERK levels were

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significantly reduced. We also observed downregulation of cyclin D1 as a consequence of the

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treatment (Figure 4C).

Figure 4. Cell cultures were treated for 24 h with casearin D at 3 and 6 µg.mL -1. (A) Cell cycle analysis

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was determined by flow cytometry. (B) Clonogenic assay was performed as described in methods. Upper panel of (B) represents an illustrative image of the colony formation assay. (C) Immunoblot showing expression profile of total-ERK, p-ERK and cyclin D1. α-tubulin was used as loading control. Doxorubicin (DXR) was used as a positive control. Immunoreactive bands were quantified using ImageJ. Other quantitative analysis was performed using GraphPad Prism®.*** (p < 0.0001), ** (p < 0.001) and * (p < 0.01) according to ANOVA followed by a Tukey post-test.

4. Discussion Natural products have long been used to prevent and treat diseases including cancers. These products have garnered recent attention as particularly good candidates for the development of anticancer drugs. This current study provides additional insights regarding to antitumor potential of Casearia sylvestris considering antiproliferative potential of the casearins. Cytotoxic activity of these clerodane diterpenes, has been reported (Itokawa et al., 1990; Dupuy et al., 2013; Ferreira et al., 2014), but the molecular mechanism responsible for

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their cytotoxicity remains poorly understood. In addition, only a few studies have been explored antiproliferative activity of the casearins (Ferreira et al. 2010; Felipe et al., 2014).

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We observed that crude extract derived from leaves of C. Sylvestris had a great

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cytotoxic activity against 4 tumor cell lines (HepG2, A549, U251-MG and HT-144). The

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HepG2 and HT-144 were the most responsive. The crude extract was partitioned with different solvents (Itokawa et al., 2000). We tested 3 fractions (ethyl acetate, n-hexane, and hydroalchoolic) against HepG2 and HT-144 cells and verified that cell viability was reduced in

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cultures treated with ethyl acetate and n-hexane extracts, but not in cultures treated with

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hydroalcoholic extract.

We were able to isolate the casearins A, G, and J from n-hexane fraction; while casearin D was obtained from ethyl acetate fraction. These compounds were tested against HepG2 cells.

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Casearin D was the most effective in reducing cell viability of HepG2 cells and therefore it was

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selected for extending our studies concerning to its antiproliferative potential.

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The ability of cancer cells to form colonies is essential for spread of a malignant tumor to distant organs (Eccles and Welch, 2007) and we demonstrated that casearin D significantly reduced the proliferative capacity of HepG2 cells inhibiting their ability in forming colonies

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showing therefore the great antiproliferative activity of casearin D on HepG2 cells. We demonstrated by the first time that caserin D induces cell cycle arrest in G0/G1. This effect is closely associated to its ability in reducing ERK phosphorylation and cyclin D1 expression.

Disturbance of the cancer cell cycle is one of the therapeutic targets for

development of new anticancer drugs (Carnero, 2002). Considering that human cancers commonly show a deregulated control of G1 phase progression, chemical compounds that regulate negatively cell cycle progression are useful for cancer therapy. Although casearin D activity on cell cycle progression had not been reported, Ferreira et al. (2014) evidenced that casearin X, isolated from C. sylvestris caused cell cycle arrest in G0/G1 in HL-60 leukemia cells. Felipe et al. (2014) demonstrated that aqueous ethanol extract

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from C. sylvestris and its chloroform fraction (f-CHCl3) inhibited cell cycle of MCF-7 cells in G0/G1. In the last case, the authors associated the cell cycle arrest with an increase in

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expression of negative regulators of cell cycle in consequence to DNA damage (p53, p16 and -

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H2AX) but they could not establish a correlation between cell cycle arrest with the presence of

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casearin C in these extracts.

Overexpression of cyclin D1 is a common event in HCC (Malumbres and Carnero, 2003; Zhu et al., 2003). Musgrove (2006) demonstrated that deregulation of cyclin D1 in HCC

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is closely associated with altered mitogen-activated protein kinases (MAPKs) signaling pathway including upregulation of extracellular signal-regulated kinase (ERK), a member of MAPKs.

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Therefore, the results obtained in the present study are very important and reveal a possible molecular mechanism involved with antiproliferative activity of casearin D in HepG2 cells.

Conclusion

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inhibited ERK activation.

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According to our results, casearin D effectively induced down-regulation in cyclin D1 and

Casearin D, a clerodane diterpene isolated from C. sylvestris, has an important antiproliferative

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activity on HepG2 cells. This effect is related to its ability in inhibiting ERK phosphorylation and reducing cyclin D1 expression levels. Casearin D therefore represents a promising prototype for further studies in HCC cancer therapy.

Conflicts of Interest The authors declare no conflicts of interest

Acknowledgements The authors are grateful to Brazilian Agencies CNPq, FAPEMIG, CAPES, FAPESP, and FINEP for their financial support and fellowships.

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