Effect of Digitoxin on p53 and p21 genes expression at primary and metastatic melanoma cells Experimental Design, 7.5 hp Spring Term, 2008

Report (2009/02/04 – 2009/02/25)

Author Umer Naveed Chaudhary [email protected] Supervisor Hong Zhang [email protected]

School of Life Sciences Skövde University, Box 408 541 28, Skövde, Sweden

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Abstract Rising incidence rates of cutaneous melanoma have been seen during the last four decades in white populations in several parts of the world, particularly among European countries, cutaneous melanoma is becoming more common. About 8,100 people in the UK are diagnosed with melanoma every year and number of cases are increasing in Sweden. The increase may be due to the skin is exposed to more sunlight than in the past, both by solresor and solarium tanning. To understand melanoma, it is helpful to know about the skin and about melanocytes. Digitoxin is extracted from the plant Digitalis (Digitalis purpurea) and used for the treatment of various heart diseases. Different reports specify that this medicine can also be effective against cancer. Various reports indicate that the patients, who have long-term use of digitoxin, simultaneously have low-rate infect to melanoma. Thus this medicine have been recognized that can be effective against cancer. This experiment was to determine whether digitoxin has function to inhibit melanoma. The results demonstrated that the digitoxin have effect to melanoma cells on protein level. Step by step, researchers will enhance relevant information about melanoma, and the ultimate goal is to cure melanoma cancer.

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Table of contents

Introduction…………………………………………………………4 Methods……………………………………………………….…….5 Results………………………………………………………….……5 Discussion………………… ……………………………………......7 References………………….………………………………....……..7

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Introduction Malignant melanoma is one of the serious type of cancer in human being, which usually starts in the skin. Although the number of people who develop melanoma is rising now a days. In several parts of the world, particularly among European countries, cutaneous melanoma is becoming more common. About 8,100 people in the UK are diagnosed with melanoma every year and number of cases are increasing in Sweden. The increase may be due to the skin is exposed to more sunlight than in the past, both by solresor and solarium tanning (Lundh, 2006). Melanoma expand from melanoma cells of skin known as melanocytes. Melanocytes provide color to the skin. In case melanoma, the melanocytes begin to develop and separate more rapidly than natural and begin to expend into the neighboring surface layers of skin, that occur very slowly within some months. If the melanoma is diagnosis at early stages, it can be cure with surgery. Melanoma having less than 1mm in depth are cured easly . If the melanoma is not treated in early stages, the cells begin to grow down into the deeper layers of the skin that contain tiny blood and lymph vessels and finally move toward the other part of the body in the blood stream and lymph system. The skin is divided into two main layers. The layer nearest the surface is called epidermis, that is made up of squamous cells. Basal cells located under the squamous cells in the epidermis. Melanocytes are present in the lower part of the epidermis. The layer beneath the epidermis is called dermis include blood vessels, hair follicles, lymph vessels and glands. Few of these glands make sweat, that help to regulate the body temperature. Other glands produce sebum, an oily substance that helps to keep the skin dry. p53 (tumor protein) is encoded by TP53 gene, that regulate the cell cycle and functions as a suppression of tumor/cancer. P53 trigger the DNA repair proteins, when DNA become damage and grip the cell cycle at the G1/S regulation point on DNA damage identification can also initiate apoptosis (programme cell death). Activation of p53 responsible for the expression of several genes including p21. G1-S/CDK and S/CDK complexes can inhibit their activities, when attach to p21 and cannot pass through to the next stage of cell division. p53 cannot bind to DNA longer and as a result p21 protein is not more existing to act as a stop signal for cell division. Consequently the cells divide out of control and to develop tumor. As a development of new therapies against cancer, examines the effect of digitoxin on cancer cells. Digitoxin is extracted from the plant Digitalis (Digitalis purpurea) and used for the treatment of various heart diseases. Different reports specify that this medicine can also be effective against cancer. In 1988, Repke review the role of the Na+/K+ ATPase in normal and cancer cell proliferation (Repke et. al., 1998). The increase demand for the action of Na+/K+ ATPase in mitosis was previously established at that time. Switch down of Na+/K+ ATPase by digitalis disrupt the flow of adenosine diphosphate (ADP) and inorganic phosphate (Pi) and increase turnover of glycolysis required for the proliferation cannot take place and there is lack of macromolecule biosynthesis required for proliferation. Nevertheless, the difference in the susceptibility between normal and malignant cells still has to be clarifying (Repke et al., 1995). Ca2+ play essential role in the apoptotic process. Increased intracellular Ca2+ concentration may start apoptosis by itself, and increased intracellular Ca2+ concentration is a step in several cascades leading to apoptosis after receptor interaction. Even in the cascade of events triggered by the ligation of the Fas

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receptor, Ca2+ plays a essential role in several steps of the apoptotic pathway (Sen et al., 1999 & Chien et al., 1999). The aim of this project is to use FM55P and FM55M human melanoma cell lines to examine protein level in terms of genes involved in apoptosis (p53, p21) and analyze either digitoxin has toxic effect on them or not. Both cell lines were established from the primary and second metastasis melanoma from the same patient, respectively and providing precious cell models (Bartkova, Lukas et al. 1996).

Methods Cell culture and treatment FM55P (primary) and FM55M (metastatic) cell lines were used. The cells were cultured with DMEM containing 10% FBS (fetal bovine serum), 1% L-glutamine 2mM and penicillin 100 IU/ml and streptomycin 100 µg/ml. The cells were grown at 37° C in 95 % humidity and 5% CO2. Finally the cells were treated with 60ng/ml Digitoxin for 24 hr. Treated and untreated cells were harvested after detached by Trypsin/EDTA and cell pellets were kept at -80° C for later analysis. Every pellet contained approximately 3-4 x 106 cells. Protein extraction/purification for western blotting Untreated and treated pellets were thawed. Proteins were extracted using AllPrep Protein Mini Kit (Qiagen) according to the lab protocol. The lysate was homogenised with QIAshredder centrifuge column (Qiagen). 5% (w/v) SDS were used as protein buffer due to the protein concentration determination by BCA method (bicinchoninic acid). Protein concentration After the protein purification/extraction, the protein concentration was measured using Bradford Coomassie assay kit (PIERCE). Standards were arranged manually and protein sample, standards and controls were added in 96 well plate. 200µl (mixture of BCA and 4% Cupric Sulfate) were added to each well and incubated in 37° C for half an hour. Cool the plate at room temperature and concentration was measured by FluoStar Galaxy spectrophotometer (BMG Labtechnologies, Tyskland) at 550 nm. Western blotting For western blotting, proteins were analyzed by polyacrylamide gel (SDS-PAGE) along with protein ladder and then transferred in to nitrocellulose membrane according to the standard procedure of Bio- RAD (USA). The membrane was treated with monoclonal antibodies according to the protocol of Santa Cruz Biotechnology kit. LumiGLO Substrates was used for the detection of the western blot.

Results In this experiment, we have used two melanoma cell lines, FM55P (primary) and FM55M (metastatic). The cells were treated with 60ng/ml of Digitoxin and divided in 4 groups: FM55P untreated, FM55P treated, FM55M untreated and FM55M treated. Protein concentration of FM55P untreated and treated cell (PU & MT) is shown in table 1.

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Table 1 FM55P Untreated & Treated cell Protein Concentration PU (p21) 0.833 ug/ul PT (p21) 0.384 ug/ul PU (p53) 1.936 ug/ul PT (p53) 1.264 ug/ul PU: Primary untreated, PT: Primary treated

Amount of protein 20ug 20ug 20ug 20ug

Protein concentration of FM55M untreated and treated cell (PU & MT) is shown in table 2. Table 2 FM55M Untreated & Treated cell Protein Concentration MU (p21) 0.416 ug/ul MT (p21) 0.592 ug/ul MU (p53) 0.592 ug/ul MT (p53) 0.928 ug/ul MU: Metastasis untreated, MT: Metastasis treated

Amount of protein 20ug 20ug 20ug 20ug

p21 and p53 proteins were running on SDS-PAGE and then transfer in to the Western blot (as shown in fig. 1). Finally p53 protein show no bands and p21 show band on FM55P (PU & PT), FM55M (PU).

P53 PU PT MU MT

p21 PU PT MU MT

75kDa

25kDa

21kDa

Figure1. Protein Expression of p53 and p21. p53 has a molecular weight of 53kDa and p21 has molecular weight of 21kDa. The concentration of the primary antibody is 1:500 and secondary antibody is 1:2000. • Marker • PU (FM55P untreated cell) • PT (FM55P treated cell) • MU (FM55M untreated cell) • MT (FM55M treated cells)

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Discussion The aim of this study was to observe if digitoxin had some sort of toxic effect on melanoma cells. If the toxic effect was confirmed, what would the mechanisms behind this product and may be difference between treated and untreated cells seen? The final results show that at the protein level there are some different between treated and untreated cells have been seen. At the Western Blot with concentrations 1:500 demonstrated a low degree of non-specific banding (Fig 1). According to the Fig 1, we can find some different between the bands of both FM55P and FM55M. p21 protein shows band on FM55P (untreated& treated), FM55M (untreated), that demonstrate the digitoxin have effect to melanoma cells. p53 protein shows no bands, these findings are in concomitant with the results of Ping Ren (2008). Absent of band in p53 protein may be due to the expression induce by Digitoxin was not in the late growth phase. Another reason was that, may be Digitoxin straightforwardly affected the viability of melanoma cell.

References Bartkova, J., J. Lukas, et al. (1996). "The p16-cyclin D/Cdk4-pRb pathway as a functional unit frequently altered in melanoma pathogenesis." Cancer Res 56(23): 5475-83. Chien M. M., Zahradka K. E., Newell M. K., Freed J. H. Fasinduced B cell apoptosis requires an increase in free cytosolic magnesium as an early event. J Biol Chem 1999; 274: 7059–7066. Hughes F. M. Jr, Evans-Storms R. B., Cidlowski J. A. Evidence that non-caspase proteases are required for chromatin degradation during apoptosis. Cell Death Differ 1998; 5: 1017–1027. Lundh C.I. (2006) Diagnostik och behandling av malignt melanom – alltid spännande! Tillgänglig på nätet http://www.svenskkirurgi.se/skf/svkir/06-3/melanom.htm [Hämtad 08.01.31] Ping Ren, Hong Zhang Effect of Digitoxin on p53, Bax and Bcl-2 genes expression at primary and metastatic melanoma cells (2008) School of Life Sciences. Skövde University Skövde, Sweden. Repke K. R. H., Benga Gh., Tager J. M. (eds) Biomembranes, Basic and Medical Research. Berlin: Springer Verlag, 1988: 161–173. Repke K. R., Schon R., Megges R., Weiland J., Nissen E., Matthes E. Potential suitability of Na+/K(+)transporting ATPase in prescreens for anti-cancer agents. Anticancer Drug Des 1995; 10: 177–187. Sen C. K., Sashwati R., Packer L. Fas mediated apoptosis of human jurkat T-cells: intracellular events and potentiation by redox-active alpha-lipoic acid. Cell Death Differ 1999; 6: 481–491.