The 4th IEEE International Conference on E-Health and Bioengineering - EHB 2013

Grigore T. Popa University of Medicine and Pharmacy, Iaşi, Romania, November 21-23, 2013

978-1-4799-2373-1/13/$31.00 ©2013 IEEE

Antiangiogenic and antiproliferative effects of halotolerant Penicillium chrysogenum var. chrysogenum on the human umbilical vein

endothelial cells (HUVECs)

Miriş Dikmen, Zerrin Cantürk, Selin Engür, Buket Demirtaş, Semra İlhan

Anadolu University, Pharmacy Faculty, Department of Pharmacology, Eskisehir, Turkey mirisd@anadolu.edu.tr; sengur@anadolu.edu.tr; bktdmrts@gmail.com

Anadolu University, Pharmacy Faculty, Deparment of Pharmaceutical Microbiology, Eskisehir, Turkey, zkcanturk@anadolu.edu.tr

Osmangazi University, Faculty of Science and Letters, Departments of Biology, Turkey silhan@gmail.com

Abstract—Inhibitors of angiogenesis have been currently developed which offer the hope of new treatment options such as diabetic retinopathy, psoriasis, rheumatoid arthritis and cancer. In this study was to investigate the antiproliferative and antiangiogenic effects of ethylacetate extract of Penicillium chrysogenum var. chrysogenum on the HUVECs. Real-time cell analysis system was used to analyze antiproliferative and antiangiogenic effects according to change of cell index values. In our study different concentrations of extract for cell prolifetation analysis were investigated. Antiangiogenic effect was evaluated according to invasion and migration activity. Antiproliferative effects of extract were increased on HUVECs according to concentration and time depending. IC50 concentration was analyzed as 78.97µg/mL on 72h. In the invasion and migration assays, 50µg/mL extract concentration significantly suppressed angiogenic responses. This finding suggests that extract be potentially more pharmacologically active. According to these results natural antioxidants gained from fungi are very essential resources in terms of biotechnology. Keywords—antiproliferative; antiangiogenic; HUVEC; Penicillium chrysogenum

I. INTRODUCTION Angiogenesis is a physiological process including of proliferation, migration and differentiation of endothelial cells. It is distinctly regulated by specific receptors, and intracellular signaling pathways [1]. Cell invasion is the irruption in and extinction of adjacent tissues, specifically on cancer cells. Cell migration is the movement of cells from one region to another, commonly in response to chemical signal, and is significant in diverse physiological and pathological processes including embryonic development, cell differentiation, wound healing, immune response, inflammation and, especially, cancer metastasis[2,3].

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and many human diseases. Fumagillin and ovalicin create a class of structurally concerned natural products that effectively inhibit angiogenesis by blocking endothelial cell proliferation [4]. Recently, fungi have appeared as an substantial source of antioxidant compounds. Phenolic compounds are considered to be the main secondary metabolites in plants, mushrooms, and fungi liable for their antioxidant activity. Penicillium chrysogenum is a commonly used in industry for produce important antibiotics which are a variety of economically important secondary metabolites, like penicillins [5-7].

II. MATERIALS AND METHODS A. Preparation of Extraction

In this study Penicillium chrsogenum var. chrysogenum was isolated from Tuz Lake which is one of the biggest lake in Turkey and is located in the Central Anatolia Region. The stock culture of Penicillium chrysogenum var. chrysogenum was grown on the slant of malt extract agar (MEA) and maintained at 25 °C. Ethylacetate extract was prepared from isolate of Penicillium chrsogenum var. chrysogenum culture medium which included secondary metabolites. Also we determined classical and molecular identification of Penicillium chrsogenum var. chrysogenum.

B. Cell Culture Human umbilical vein endothelial cells (HUVECs) were cultured in F12K containing 2 mM Lglutamine, 0.1 mg/ml heparin, 1.5 g/l NaHCO3 (sodium bicarbonate), 0.03 mg/ml endothelial cell growth complement and 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence in growing medium. The cells were harvested at

confluence with 0.05 % trypsin – 0.02 % EDTA and plated in tissue culture dishes and grew with fresh medium. Fungal ethylacetate extract was dissolved in DMSO solution.

C. Cell growth and proliferation assay using Real-Time Cell Analysis System (RTCA DP)

Kinetics of cell adhesion and spreading were experimented using the Real-Time Cell Analyser (RTCA DP). The growth characteristics following seeding were monitored to determine the optimum seeding density and time to beginning of the death. The system measures electrical impedance across interdigitated micro-electrodes incorporated on the bottom of tissue culture E-Plates. The impedance measurement, which is indicated as cell index (CI) value, provides quantitative information about the condition of the cells, involving cell number, viability, and morphology [8-10]. Background of the E-plates was measured in 100 µl medium in the Real-Time Cell Analyzer (RTCA DP) station. Afterwards 100 µl of a HUVEC cell suspension was added (20.000 cells/well). Plates were incubated for 30 min at room temperature and E-plates were placed into the Real-Time Cell Analyzer and impedance was measured every hour. Approximately 20h after seeding, when the cells were in the log growth phase, the cells were treated to 100 µl of medium containing different concentrations of ethylacetate extract (25, 50, 100, 200 and 400 µg/mL) and impedance monitoring continued for another 72 hours. Impedance was demonstrated as Cell Index (CI) values. Dose-response curves at 72 hours were generated to determine IC50 values during the incubation time. Control was received medium+DMSO with a final concentration of 0.1%. The electrical impedance was analysed by the RTCA-connected software of the RTCA DP system as a dimensionless parameter called CI.

D. Cell invasion and migration assay using Real-Time Cell Analysis System (RTCA DP)

The RTCA DP instrument uses the CIM -Plate 16 (cellular invasion/migration), which properties microelectronic sensors connected onto the underside of the microporous polyethylene terephthalate (PET) membrane of a Boyden-like chamber. As cells migrate from the upper chamber through the membrane into the bottom chamber in reply to chemoattractant, they contact and adhere to the electronic sensors on the underside of the membrane, ensuring in a raise in impedance [8-10]. 1) Invasion assay: The invasion assays were created on CIM-16 plates using RTCA DP system. Wells were coated with 20 μL of 20% matrigel and incubated to gel at 37 °C, 5% CO2 for 4 h. After 4 h, the wells of the bottom chamber were

filled with 160 μL of 10% serum containing medium and the top and bottom parts of the CIM-16 plates were assembled together. The assembled CIM-16 plate was allowed to stabilize for 1 h at 37 °C, 5% CO2 after the addition of 20 μL of serum-free medium to the top chamber wells. For seeding, cells were trypsinized and centrifuged at 1000 rpm for 5 min, and washed with serum-free medium before resuspension in serum-free medium. Cells (20.000 cells⁄well) were seeded onto the top chambers of CIM-16 plates in 80 μL of serum-free media, then added to 100 μL of extract concentrations and placed into the RTCA DP system for data collection after a 30 min incubation at room temperature. The RTCA DP software was set to collect impendence data (reported as cell index) at least once every 10 min [2,8]. 2) Migration assay: To evaluate cell migration, we used CIM-16 plates using RTCA System. The wells of the bottom chamber were filled with 160 µl of 10% serum containing medium and the top and bottom parts of the CIM-16 plates were assembled together. Then cells (20.000 HUVECs/well) were added to the top chamber in 100 μL serum free medium and added to 100 μL of extract concentrations. After stabilizing CIM-plates for 30 minutes at room temperature, the chamber was loaded in the RTCA DP machine, and the cell indices were measured continuously for 24 hours. The RTCA DP software was set to collect impendence data (reported as cell index) every 10 minutes [11].

III. RESULT According to DPPH method belonging to Penicillium chrsogenum var. chrysogenum, the percent value of inhibition free radical scavenging activity was found 72% and total phenolic content was found 62 mg/mL. Also, we determined that Penicillium chrsogenum var. chrysogenum extract included Ortho-coumaric and protocatechic acids with HPLC analysis. Penicillium chrsogenum var. chrysogenum inhibited cell growth, invasion and migration in a concentration response dependent manner (Figure 1, 2, 3 and 4). Cell migration and invasion were almost completely abrogated inhibited at a concentration of 200 mg/mL according to control. Also, cell proliferations were gradually reduced between of 100-400 μg/mL concentrations of extract. This study indicated that the increased inhibitory effect displayed by the extract could have been a result of additive effect of ethylacetate extract which includes fungal secondary metabolites, derivatives and/or synergistic effects among them, or could have been a result of other, totally different compounds.

Fig. 1. Real-time monitoring of cell proliferative effects of Penicillium chrysogenum var. chrysogenum on HUVECs using RTCA-DP System. (n =6).

Fig. 2. Effect of Penicillium chrysogenum var. chrysogenum extract during 72 hour exposure on the viability of HUVEC cells were determined based on the concentration–response curves of the cell index and determined IC50 rates by the RTCA DP system. (n =6).

Fig. 3. Invasion kinetics of Penicillium chrsogenum var. chrysogenum on HUVEC cells, as shown by permanent monitoring of live-cell invasion for 24h. Results shown are mean ± s.d. (n =4).

Fig. 4: Migration kinetics of Penicillium chrsogenum var. chrysogenum on HUVEC cells, as shown by permanent monitoring of live-cell migration for 24h. Results shown are mean±s.d. (n=4).

IV. DISCUSSION Angiogenesis is a widely regulated process that includes a complex cascade of events and displays an important role in tumor growth and metastasis. It is a key process in the progression of cancer [12]. Cancer cells cannot grow without angiogenesis, and they remain in immobile phase. Consequently, angiogenesis is a very important target for cancer chemoprevention. It was reported that many natural health products inhibit angiogenesis. Among the known “natural” angiogenesis inhibitors, polyphenols show to play an important role [13-14]. However, the mechanisms of antiangiogenic effect of polyphenols are not well known [15]. Ronen Ben et al. (2009), founded that secreted secondary metabolites of a fumigatus inhibited HUVEC differentiation, migration, and capillary tube formation in vitro and suppressed angiogenesis in neutropenic mice. Moreover, they explained that gliotoxin has a specific role in mediating the antiangiogenic activity of a fumigatus [16]. As a result, we were determined to the antiproliferative and antiangiogenic effects of ethylacetate extract of Penicillium chrysogenum var. chrysogenum culture medium on the human umbilical vein endothelial cells (HUVECs). This study was preliminary investigation for new studies which include the angiogenesis-related signaling pathways of Penicillium chrsogenum var. chrysogenum secondary metabolites on HUVEC and different cancer cells. These findings indicate Penicillium chrsogenum var. chrysogenum secondary metabolites may have potential as preventive and therapeutic agents in diseases involving pathological angiogenesis.

ACKNOWLEDGMENT This study was carried out as a part and support of Anadolu University Scientific Research Projects numbered 1003S117.

REFERENCES [1] F. Bussolino, A. Mantovani, G. Persico, “Molecular mechanisms

of blood vessel formation,” Trends in Biochemical Sciences 22, 251–256,1997.

[2] M. C. Eisenberg, K. Yangjin, R. Li, W. E. Ackerman, D. A. Kniss, A. Friedman, “Mechanistic modeling of the effects of myoferlin on tumor cell invasion,” BMC Complementary and Alternative Medicine, 1998.

[3] X. Yu, Y. Tong, X. Han, H. Kwok, G. Yue, C. Lau, W. Ge, “Anti Angiogenic Activity of Herba Epimedii on Zebrafish Embryos In Vivo and HUVECs In Vitro,” Phytother, Res. 27: 1368–1375 2013.

[4] E. C. Griffith, Z. Su, S. Niwayama, C. A. Ramsay, Y. Chang, J. O. Liu, “Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2,” Proc. Natl. Acad. Sci. USA.Vol. 95, pp. 15183–15188, 199.

[5] T. Jayakumar, P. A. Thomas, P. Geraldine, “In-vitro antioxidant activities of an ethanolic extract of the oyster and Emerging Technologies,” Vol. 10, no. 2, pp. 228–234, 2009.

[6] K.J.A. Davies, “Oxidative stress: the paradox of aerobic life,” Biochem.Soc.Symp. vol.61, pp.1–31, 1994.

[7] J.V. Schloss. “Oxygen toxicity from plants to people,” Planta, vol. 216, pp. 38–43 2002.

[8] C. Bird, S. Kirstein, “Real-time, label-free monitoring of cellular invasion and migration with the xCELLigence system,” Nature methods, August 2009.

[9] K. Solly, X. Wang, X. Xu, B. Strulovici, W. Zheng, “Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays,” Assay Drug Dev. Technol. 2, 363–372 (2004).

[10] E. Urcan, U. Haertel, M. Styllou, R. Hickel, H. Scherthan, F. X. Reichla,” Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts,” Dental materials, 26:51–58, 2010.

[11] E. Lee, J. E. Koskimaki, N. B. Pandey, A. S. Popel, “Inhibition of Lymphangiogenesis and Angiogenesis in Breast Tumor Xenografts and Lymph Nodes by a Peptide Derived from Transmembrane Protein,” Neoplasia 15, 112–124, 2013.

[12] J. Folkman, “Angiogenesis inhibitors: a new class of drugs,” Cancer Biol Ther 2 (Suppl 1): S127-S133, 2003.

[13] T. Fotsis, M. Pepper, E. Aktas, S. Breit, S. Rasku, H. Adlercreutz, K. Wahala, R. Montesano, L. Schweigerer, “Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis,” Cancer Res 57: 2916-2921, 1997.

[14] C. Kanadaswami, L. Lee, P. Lee, J. Hwang, F. Ke, Y. Huang, M. Lee, “The antitumor activities of flavonoids,” In Vivo 19: 895-909, 2005.

[15] J. Mojžiš, M. Šarišský, M. Pilátová, V. Voharová, L. Varinská, G. Mojžišová, A. Ostro, P. Urdzík, R. Dankovčik, L. Mirossay, “In vitro Antiproliferative and Antiangiogenic Effects of Flavin7,” Physiol. Res. 57: 413-420, 2008.

[16] R. Ben-Ami, R. Lewis, K. Levantakos, D. Kontoyiannis, “Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites,” Blood.17;114(26):5393-9,2009.