3,4-dihydroxyphenyl acetic acid and (+)-epoxydon isolated from marine algae-derived microorganisms induce down regulation of epidermal growth factor activated mitogenic signaling cascade in Hela cells
- Mi Jeong Jo†1,
- Seong Ja Bae†1,
- Byeng Wha Son2,
- Chi Yeon Kim3Email author and
- Gun Do Kim1Email author
© Jo et al.; licensee BioMed Central Ltd. 2013
Received: 5 December 2012
Accepted: 21 May 2013
Published: 25 May 2013
Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase (RTK) family. Epidermal growth factor induces its dimerization and stimulates phosphorylation of intracellular tyrosine residues. Phosphorylation of EGFR is studied for cancer therapy because EGFR regulates many cellular processes including cell proliferation, differentiation, and survival. Hence, down-regulation of EGFR kinase activity results in inhibition of signaling cascades amenable for proliferation and progression of cell cycle.
In the study, we purified 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon from Aspergillus sp. isolated from marine brown alga Ishige okamurae and Phoma herbarum isolated from marine red alga Hypnea saidana respectively and determined its anti-tumor activities against HeLa human cervical cancer cells.
Two compounds suppressed EGFR activity in vitro with IC50 values for 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon were 2.8 and 0.6 μg/mL respectively and reduced the viable numbers of HeLa cells. Immunoblotting analysis exhibited that the compounds induced inhibition of cell growth by causing downregulation of the mitogenic signaling cascade, inactivation of p90RSK, and release of cytochrome c from mitochondria.
Results suggest that decreased expression of active EGFR and EGFR-related downstream molecules by treatment with the compounds may results in the inhibition of cell growth and inducement of apoptosis.
KeywordsEGFR HeLa Marine alga Ishige okamurae Hypnea saidana Mitogenic
Epidermal growth factor (EGF) receptor (EGFR) is a type 1 receptor tyrosine kinase or member of the ErbB (HER) receptor family . The EGFR receptor is divided into an extracellular ligand-binding domain, which is an anchor domain that spans the membrane, and an intracellular component that activates tyrosine kinase and induces further downstream signaling . After ligand activation, the members of the family bind to each other, forming homodimers or heterodimers . It has been shown that EGFR is involved in signaling pathways regulating cellular growth, cell cycling, and differentiation . EGFR is overexpressed in various solid tumors including breast, colorectal, ovarian and non-small-cell lung cancer, and excessive EGFR signaling is associated with the development of a wide variety of benign and metastatic tumors . Furthermore, it is reported that when EGFR is overexpressed, it activates the signaling transduction system, and therefore cancer cells grow more aggressively, and with the invasiveness increasing, the transition occur more easily, affecting negative effects to the survival rate . Therefore knowledge of the inhibitory mechanism related to epidermal growth factor (EGF) would assist in searching for targets in cancer therapy .
Over the past several decades, many studies on EGFR-targeted therapy in cancer have been performed and numerous targets for anticancer agents have emerged. Especially, monoclonal antibodies (Cetuximab) and tyrosine kinase inhibitors (Gefitinib and Erlotinib) have been developed to inhibit receptor activation [8, 9]. Currently, people prefer natural products from the ocean or soil rather than chemical compounds made in a laboratory. In recent years, seaweed extracts have been found to have anti-tumor activities , and many researchers have identified algae extracts such as fucoidan and carrageenan which demonstrate anti-tumor effects. The results indicate that the extracts from a wide variety of marine algae could suppress tumor activities and restrain the ability of tumors to grow [11, 12]. For this reason, many types of algae extracts have been studied and possible reasons that the extracts inhibit a variety of cancers have been elucidated in the field of cell signaling pathways involving apoptosis, death receptor, and cell cycling [13, 14].
Marine fungi were reported having a rich profile of biologically active metabolites . The ecological pressures of a unique marine environment may drive the production of new secondary metabolites by microorganisms. As an example, salinosporamide A was isolated from Salinispora tropica, and was found to cause significant proteasome inhibition in clinical trials . Chaetomugilins have been isolated from a strain of Chaetomium globosum originally isolated from the marine fish Mugil cephalus, and exhibited significant growth inhibition against human cancer cell lines .
Although a few studies elicited the bioactivities of metabolites from marine microorganisms, there is no report about the marine microorganisms symbiotic with marine algae. The present study was conducted in order to elucidate the mechanism of the anti-tumorigenic effects of algae derived microorganism extracts, we purified 3,4-dihydroxyphenyl acetic acid from Aspergillus sp. on the marine brown alga Ishige okamurae and (+)-epoxydon from Phoma herbarum on the marine red alga Hypnea saidana, respectively. HeLa human cervical epithelial cancer cells were used in the study as highly express EGFR tyrosine kinase on their surface. We studied the inhibitory effects of the compounds on EGF induced phosphorylation of EGFR in HeLa cells. The results of this investigation may provide new insights into the mechanism of tumor suppression and the possibility for applications in tumor prevention and treatment, because control of the activation of EGFR tyrosine kinase has an important role in tumorigenesis .
Results and discussion
Physiochemical data for 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon from marine algae derived microorganisms
The molecular weight (M.W.) of (+)-epoxydon is 156 (C7H8O4) (Figure 1). [α]D + 71.6° (c 0.3, MeOH); IR (neat) νmax 3356, 1680, 1400, 1236, 1027, 903, 867 cm-1; UV (MeOH) λmax (log ϵ) 203 (3.72), 237 (3.68) nm; CD (MeOH) (Δϵ) 338 (+0.95), 245 (-1.76) nm; 1H NMR (400 MHz, DMSO-d6) δ 6.39 (1H, dddd, J = 2.7, 2.6, 2.2, 2.1 Hz, H-3), 4.70 (1H, ddddd, J = 6.2, 2.8, 2.8, 2.6, 2.1 Hz, H-4), 5.79 (1H, d, J = 6.2 Hz, 4-OH), 3.40 (1H, d, J = 4.2 Hz, H-5), 3.76 (1H, ddd, J = 4.2, 2.8, 2.7 Hz, H-6), 3.96 (1H, dddd, J = 15.2, 5.5, 2.8, 2.2 Hz, Ha-7), 4.07 (1H, dddd, J = 15.2, 5.5, 2.6, 2.1 Hz, Hb-7), 5.01 (1H, t, J = 5.5 Hz, 7-OH); 13C NMR (100 MHz, DMSO-d6) δ 193.9 (s, C-1), 133.8 (s, C-2), 141.4 (d, C-3), 63.7 (d, C-4), 52.9 (d, C-5), 54.0 (d, C-6), 57.3 (t, C-7); CIMS m/z (rel.int.) 156 [M]+ (100), 138 [M-H2O]+ (7), 122 [M-H2O-O]+ (2), 110 [M-CO-H2O]+(3).
Inhibition of EGFR tyrosine kinase activities
Inhibitory effects of the compounds on EGF-induced cell growth
Activation of EGFR tyrosine kinase by EGF as a positive control
Down-regulation of the activated EGFR by the compounds
EGF plays an important role in the regulation of cell growth, proliferation, and differentiation by binding to its receptor EGFR  and stimulating the associated protein tyrosine kinase activity . Epidermal growth factor receptor (HER1) tyrosine kinase is a recognized target for tumor therapy and anti-cancer drugs have been developed to inhibit receptor activation . Researchers have shown that the receptor was suppressed by tyrosine kinase inhibitors (Iressa) , monoclonal antibodies such as Cetuximab, and other compounds .
Inhibition of EGFR-mediated mitogenic signaling
One of the most important protein kinase cascades activated by tumor promoters, such as EGF, is the mitogen-activated protein kinase (MAPK), induced by the activation of EGFR. Ras is a small guanine-nucleotide binding proteins (G-proteins) cycle between active (GTP-bound) and inactive (GDP-bound) forms . Receptor tyrosine kinases and G-protein-coupled receptors activate Ras, which then stimulates the Raf-MEK-MAPK pathway . Mitogen-activated protein kinases (MAPKs) constitute a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines [27, 28] and is an important target in the diagnosis and treatment of cancer . Upon stimulation, a sequential 3-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK  and cytochrome c . Cytochrome c is a well conserved electron-transport protein and is part of the respiratory chain localized to the mitochondrial intermembrane space . Upon apoptotic stimulation, cytochrome c is released from mitochondria, and this event eventually leads to apoptosis .
Relative inhibitory effect of the compounds to Tyrphostin AG 1478 on the activities of EGFR and EGF-related downstream molecules
In this study, we demonstrated that 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon from marine algae reduced expression of the mitogenic signaling cascade and EGFR activation, leading to apoptosis in HeLa cells because EGFR has been indicated to be an important target in cancer therapy.
3,4-dihydroxyphenyl acetic acid is a metabolite of the neurotransmitter dopamine, and can be oxidized by hydrogen peroxide, leading to the formation of toxic metabolites which destroy dopamine storage vesicles in the substantia nigra. (+)-epoxydon is a new secondary metabolite from a marine algae derived fungus. In the present study, we purified 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon from Aspergillus sp. isolated from marine brown alga Ishige okamurae and Phoma herbarum isolated from marine red alga Hypnea saidana respectively and determined its anti-tumor activity against HeLa human cervical cancer cells.
In conclusion, this study demonstrated that the compounds effectively inhibited proliferation and invasion of HeLa cells and suggests that EGFR may be a potential therapeutic agent for cervical cancer.
Isolation for 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon from marine algae derived microorganisms
The compound, 3,4-dihydroxyphenyl acetic acid (DOPAC), was isolated from the surface fungus of the marine brown alga Ishige okamurae collected at Uljin, Gyeongbuk province and Geomoon Island, JeonNam province in South Korea. The fungus was then identified as Aspergillus sp. on the basis of morphological evaluation and 18S rRNA analysis (SolGent, Daejeon, South Korea) (similarity 99%). The fungus was cultured (10 L for 4 weeks (static) at 29°C in a SWS medium consisting of soytone (0.1%), soluble starch (1.0%), and seawater (100%). The resulting broth and mycelia were extracted separately with EtOAc (ethyl acetate) and CH2Cl2-MeOH (methanol) (1:1) to afford the broth extract (430 mg) and the mycelium extract (1.1 g), respectively. The broth extract showed a radical (DPPH) scavenging activity with an IC50 value of 1.1 μg/mL, however, the mycelium extract was inactive. Therefore, broth extracts were subjected to column chromatography on silica gel (n-hexane/EtOAc), and then octadesyl silica (ODS) gel (H2O/MeOH) to provide 5 fractions. Further purification of fraction-4 containing 3,4-dihydroxyphenyl acetic acid by recycling HPLC, followed by HPLC (C18 Apollo, MeOH-H2O = 3:2), yielded 3,4-dihydroxyphenyl acetic acid (3.3 mg).
The other compound, (+)-epoxydon, was isolated from the surface fungus of the marine red alga Hypnea saidana collected in Tongnyeong and Yokjee Island, GyeongNam province in South Korea, and then identified as Phoma herbarum on the basis of morphological evaluation and 18S rRNA analysis (SolGent, Daejeon, South Korea) (similarity, 99%). The culture broth and mycelia were separated, and the broth (10 L) was extracted with ethyl acetate to provide a crude extract (640 mg) which was subjected to silica gel flash chromatography and eluted with n-hexane/EtOAc (5:1), n-hexane/EtOAc (1:1), n-hexane/EtOAc (1:5), n-hexane/EtOAc (1:10), and finally with EtOAc. The collections (30 mL each) were combined on the basis of their TLC profiles to yield 5 major fractions. Medium pressure liquid chromatography (MPLC) of fraction-3 on ODS by elution with MeOH yielded crude (+)-epoxydon (9.0 mg). The isolated crude (+)-epoxydon was further purified by HPLC (YMC ODS-A, MeOH) utilizing a 30 min gradient program of 50% to 100% MeOH in H2O to yield (+)-epoxydon (5.0 mg).
In vitro assay for EGF receptor tyrosine kinase
The substrate (poly [Glu:Tyr] (4:1)) and the AlphaScreen® P-Tyr-100 assay kit (PerkinElmer Inc., Waltham, MA, USA) composed of Donor-streptavidin and Acceptor-P-Tyr-100 beads were used for the EGF receptor tyrosine kinase assay. EGFR enzyme purified from human carcinoma A431 cells was purchased from Sigma-Aldrich. The kinase reactions were performed in a mixture of EGFR enzyme, ATP, and biotinylated poly [Glu:Tyr] (4:1) in a kinase reaction buffer (50 mM Tris (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 2 mM DTT, and 0.01% Tween-20). The mixture was incubated for 1 h at room temperature (RT) and then quenched by addition of detection buffer containing EDTA, Donor-Streptavidin, and Acceptor-P-tyr-100 beads. After further incubation for 1 h at RT, the intrinsic kinetic activities were detected as an AlphaScreen® signal using a Fusion-Alpha microplate analyzer (PerkinElmer Inc.).
Cell culture and treatment
Human cervical cancer HeLa cells and human embryonic kidney HEK293 (American Type Culture Collection, Manassas, VA, USA) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with high-glucose (HyClone Laboratories, Logan, UT, USA), supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories) and penicillin-streptomycin (100 μg/mL penicillin, and 100 units/mL streptomycin) (PAA Laboratories GmbH, PA, Austria) at 37°C with 5% CO2. Human epidermal growth factor (Sigma-Aldrich, St Louis, MO, USA) was dissolved in media and treated at a final concentration of 10 ng/mL. Each compound was reconstituted with DMSO to 10 mg/mL and added to the culture media to the final concentration specified in the test. The same concentration of DMSO was added to the control dishes. HeLa cells were treated with 50 μg/mL of each compound for 40 min and then treated with 10 ng/mL of EGF for 10 min, except for control cells.
Cell viability assay
To estimate the effects of the compounds on cell viability, HeLa and HEK293 cells were seeded (1 × 104 cells/mL) in 96-well plates in 100 μL of DMEM-10% FBS and cultured for 24 h at 37°C in a 5% CO2 incubator. After incubation, the HeLa and HEK293 cells were treated with two compounds (0, 1, 5, 10, 25, 50, 100 and 200 μg/mL) for 40 min, and then exposed to EGF (10 ng/mL) for 10 min. After treatment, 10 μL of EZ-Cytox Cell Viability Assay solution WST-1® (Daeil Lab Service, Jong-No, Korea) was added to each well, and the cells were then incubated for 3 h at 37°C in a 5% CO2. Absorbance was measured at 460 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).
Protein extraction and western blotting
HeLa cells were cultured with each compound for 40 min and then treated with 100 ng/mL of EGF for 10 min. The cells were then washed twice with cold-PBS, harvested and lysed with lysis buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1% Triton X-100, 1% deoxycholate, 0.1% SDS containing proteinase inhibitors (PMSF, EDTA, Aprotinin, Leupeptin, Prostatin A, Intron Biotechnology, Gyeonggi, Korea)]. The lysates were shaken on ice 6 times every 5 min and centrifuged at 14,000 rpm for 20 min at 4°C. Using bovine serum albumin (BSA) as a standard, the Protein Quantification Kit (CBB solution®) (Dojindo Molecular Technologies, Rockville, MD, USA) was used for determining the concentration of whole cell lysates. Each protein was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto nitrocellulose membranes (PALL Life Sciences, Pensacola, MI, USA). The membranes were blocked with phosphate buffered saline-Tween-20 (PBST: 135 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM sodium phosphate, 1.4 mM potassium dihydrogen phosphate, 0.5% Tween-20) containing 5% skim milk for 2 h at RT and hybridized with the appropriate primary antibody (anti-EGFR (pY1068), anti-EGFR, anti-Ras, anti-c-Raf (pS338), anti-MEK 1/2 (pS217/221), anti-MAPK p44/42 (pT202/204), anti-p90RSK (pS380), anti-cytochrome c, anti-GAPDH (Cell Signaling Technology Inc., Danvers, MA, USA)) for overnight at 4°C. Protein bands were visualized by enhanced chemiluminescent (ECL) detection solution (Pierce, Rockford, IL, USA) after hybridization for 1 h with the horseradish peroxidase (HRP)-conjugated secondary antibody from rabbit or mouse (Cell Signaling Technology Inc.).
Immunofluorescence of the phosphorylated EGFR
HeLa cells were incubated on cover glass-bottom dishes (SPL Lifesciences, Gyeonggi, Korea) in DMEM with high-glucose containing FBS (10%) and penicillin-streptomycin. The cells were fixed with 4% formaldehyde (Junsei Chemical Ltd., Japan) for 15 min at RT and then blocked for 1 h in 5% normal serum based on the host primary antibody. After removing the blocking buffer, cells were incubated with 0.1 μg/mL of anti-EGFR (pY1068) overnight at 4°C and then washed 3 times in cold PBS followed by incubation for 1 h with 0.1 μg/mL of anti-rabbit IgG (H + L), and F(ab') fragment (Alexa Fluor 488 conjugate) (Cell Signaling Technology Inc.). After washing, the stained cells were mounted with Prolong Gold Antifade Reagent (Invitrogen, Eugene, OR, USA) and then observed under a Nikon ECLIPS 50i microscope equipped with a charged-coupled device (CDD) camera (Nikon, Tokyo, Japan). Images were captured and processed with High-Content Analysis Software (Cambridge Healthtech Institute, Needham, MA, USA).
The statistical significance of the differences between the values of compound-treated and non-treated groups was determined by GraphPad Prism 5.0. The results are expressed as mean values ± standard deviations of the mean (SDM). Every untreated control group and treated group was measured in differences by t-tests. (p < 0.05 was considered significant). The experiments were performed in triplicates and at least three times each. In case of no error bar in the graph, the variation of values is infinitesimal and thus, the bars are hidden behind.
This research was supported by the National Research Foundation of Korea Grand funded by the Korean Government (KRF-2008-314-F00048).
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