Abstract:
Reason: The respective experiment was performed at Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India during 2008-2009. Sam Masih, Ramyapriya and Mohd. Zeeshan worked on the project to complete their M.Tech study.
Sam Masih et al. published the initial results of the project in International Journal of Cancer Research Vol. 6 No. 1, 10-18, 2010 without the permission of the concerned authorities.
INTRODUCTION
American cancer society reports show that 7.6 million people died from cancer (a deadly disease in which cells will show uncontrolled growth, metastasis and invasion) worldwide during 2007. Breast cancer is one of the dangerous diseases and is the second common type of cancer worldwide. Breast cancer can be observed in aged women and mainly in developed countries. There are several risk factors for breast cancer which includes stress (Thaker et al., 2006), age, gender, diet and lifestyle (Simonian and Coyle, 1996), ethnicity, family history, inherited susceptibility gene and endocrine factors.
Among all, stress is one of the major cause for breast cancer. Scientists have suggested that the effects of stress on the immune system may in turn affect the growth of some tumor (De Vasconcelos et al., 2001; Schapira, 1999). Age is also a main cause of breast cancer (Kitani et al., 2002; Shelley et al., 2005). Earlier puberty and social isolation were the other major factors that lead to breast cancer (McClintock et al., 2005).
Recent advances in our understanding of the complex signaling pathways involved in immune system regulation have accelerated development of drug-based (agonist and antagonist) strategies for cancer treatment and prevention (Salih and Fentiman, 2001). Breast cancer in women, is a complex process involving the neuroendocrine system and the immune system. There is evidence linking stress, concomitant behavioral response patterns and resultant neurotransmitter changes.
Stress is implicated in immune modulation. Bidirectional talk between immune system and endocrine system occur so that the normal functioning of the body is to be maintained in cancer. Stress leads to reduced state of immune activity. Chronic stress has been shown to suppress different facets of immune function such as antigen presentation, T-cell proliferation, humoral and cell-mediated immunity, mainly through the release of catecholamine and/or glucocorticoid hormone (Salih and Fentiman, 2001).
Deprenyl has been used as a drug for the treatment of Parkinson and Alzheimer disease. A lot of research has been done on rats and they did not showed any body weight change as compare to control. Deprenyl is an irreversible inhibitor of MAO-B (Monoamine Oxidase), which has been used as an adjunct to reduce the oxidation of dopamine. Deprenyl will influence the activation of growth factors synthesis (Kitani et al., 2002; Keri et al., 2003) resulting in regeneration of neurons and neuroprotection of cell bodies. It is a monoamine oxidase inhibitor that leads to improvement of immunological responses (ThyagaRajan et al., 2000). It has been observed that L-deprenyl protects these normal human uroepithelial explants via the expression of Bcl-2 protein (Seymour et al., 2003).
Studies have been conducted in animal models to understand the pathogenesis of disease and therapeutic effect of various drugs. Human breast cancer cell lines have also been used to understand the cellular mechanism of the disease. The MCF-7 is the most commonly used breast cancer cell line that was derived in Michigan Cancer Foundation, 1973. Apart from that there are many other cell lines that are in use viz., MDA-MB 231, T47D and Hs578T (Kabbinejadian et al., 2008). Among them T47D cell line is an Estrogen Receptor (ER) positive cell line and grows faster. It can grow in the absence of estrogen and frequently up regulate signaling molecules such as Raf- intracellular pathways (Hoshino, 1999; Cowley et al., 1994).
Mitogen Activated Protein Kinase (MAPK) pathway links extracellular signals with cellular response and influences to control essential functions such as growth proliferation, differentiation and apoptosis (Lim et al., 2006). There are few factors that will induce intracellular pathways like mitogen, trophin, stress and cytokines. In that mitogen and trophin will bind to tyrosine kinase and induce MAPK (Rozengurt, 2007). In higher animals, RKIP-1 has been reported to inhibit serine proteases such as thrombin and chymotrypsin, despite the fact that it has no apparent homology to any known family of serine protease inhibitors (Sartor et al., 1997). The MAPK can activate nuclear transcription factor (NF-kB) and can influence diverse cellular functions like cell proliferation, differentiation and apoptosis.
Since studies on breast cancer have been limited because of the complication in their pathways and other factors, therefore, present study investigated the effect of deprenyl on viability of T47D cell line by MTT assay and the effect of deprenyl on MAPK pathway.
MATERIALS AND METHODS
Chemicals
RPMI-1640 media was procured from Himedia. L-Glutamine, Penicillin, Streptomycin,
Gentamycin, HEPES, Sodium Pyruvate, Bovine Insulin, Fetal Bovine Serum Deprenyl,
Phosphate Buffer Saline, Trypsin- EDTA and MTT were obtained from Sigma in the
month of April, 2008. Western Blotting Kit was purchased from Genie, Bangalore.
Primary antibody was procured from Invitrogen. T47D human breast cancer cell
lines were purchased from ATCC (American Type Culture Collection) in the second
week of May, 2008.
Cell Culture
In the 3rd week of July, 2008, cell culturing was performed with slight
modifications; T47D cells were maintained in RPMI-1640 in 75 cm2 tissue
culture flasks and incubated in a humidified atmosphere containing 5% CO2
at 37°C. Cells were fed on alternate days and passaged upon reaching
70-80% confluency (usually between 2-3 days). Viability of cells were checked
by using Trypan blue method. Trypan blue is a dye that is taken up by dead cells
and viable cells appear clear under the microscope.
Cryopreserving Cells
Numbers of cells were calculated by haemocytometer and 2x106 cells
per cryovials were stored. Each cryovial contained 1 ml freezing medium (90%
FBS and 10% DMSO).
MTT Assay
MTT assay was performed to check the percentage cell viability which was
done in the month of September, 2008. Three different 96 well plates (24, 48
and 72 h incubation period) were seeded with 5000-7,000 cells/well (90 μL)
in all the wells. 100 μL of medium was added in control. Each plate was
incubated with three different concentrations (10-3, 10-6
and 10-9 M) of deprenyl. No drug was added in the control. After
particular incubation periods, 20 μL MTT solution was added in all the
wells and plates were placed in CO2 incubator for 3-4 h to get the
purple precipitate. After getting purple precipitate, 100 μL of DMSO with
10% SDS was added to all wells, including controls and then were wrapped with
aluminum foil. Plates were incubated for 2 h in dark at 37°C and the readings
were taken at 450 nm and calculations were done by using SPSS 10.0 software
for one way ANOVA with p = 0.5 and graph were plotted on excel sheet with statistical
error.
Western Blotting
Western Blotting was performed to check the effect of deprenyl on MAPK signaling
pathway, done in the month of December, 2008 and January, 2009. Specific antibodies
were purchased from invitrogen and all other chemicals were purchased from Genie.
Three different plates were prepared like MTT assay with different concentration
of deprenyl (10-3, 10-6 and 10-9 M) for three
different incubation periods (24, 48 and 72 h, respectively). After particular
incubation periods protein were lysed with lysis buffer (20 mM Tris, 100 mM
NaCl, 1 mM EDTA and 0.5% Triton X 100) and protein sample was quantified by
Bradford method.
Western blotting was done to check the activation of Mitogen Activated Protein Kinase (MAPK) pathway by different concentrations of deprenyl at different incubation periods. The 30-50 μg protein sample was taken to perform the SDS Page then bands were obtained that were transferred on nitrocellulose membrane and western blotting analysis was done by antibodies specific for the phosphorylation of MAPK. Cells alone were used for the control to identify the effects of different concentrations of deprenyl on T47D human breast cancer cell lines.
RESULTS
Cell Culturing
The 1.68 million cells mL-1 were counted by haemocytometer and
fed with 20 mL RPMI-1640 complete growth media in 75 cm2 tissue culture
flask to start the cell culturing. Cells reached almost 70-80% confluency in
36 h (Fig. 1).
MTT Assay
From MTT assay it was identified that deprenyl at 10-3 M concentration
was toxic and cells died as compared to the control but it was noticed that
as concentration was decreased to 10-6 M there was an increased in
cell viability which decreased again slightly at 10-9 M. While, MTT
assay was done at different time incubation it was noticed that deprenyl showed
good percentage cell viability at same range and that was increased with increase
in incubation period.
Results of 24, 48 and 72 h Incubation Period
Readings were taken by ELISA reader at 450 nm and one way ANOVA test was
performed by SPSS 10.0 software (Table 1).
| Table 1: | ANOVA readings of cells treated with deprenyl for 24, 48 and 72 h incubation period |
| Data is expressed as Mean±SD | |
| Fig. 1: | Cells after 36 h of inoculation shows 70-80% confluency |
| Fig. 2: | Comparative graph of percent cell viability and different Deprenyl concentration over varying incubation period |
24 h Incubation
Plates treated with 10-6 M concentration of deprenyl showed more
viability as compared to cells treated with 10-3 and 10-9
M concentration of deprenyl. 10-3 M concentration showed least viability.
48 h Incubation
From Table 1 it can be concluded that 10-6
M deprenyl showed highest viability followed by 10-9 and 10-3
M concentration of deprenyl. Results indicate that overall viability was increased
after 48 h of incubation as compared to 24 h of incubation.
72 h Incubation
From the table it was proved that 10-3 M concentration of deprenyl
was toxic for cells because at this concentration cells died as compare to the
control. 10-6 M concentration was noticed as best suited for cell
viability as compare to other concentrations of the deprenyl.
Significance of Different Concentrations and Incubation Periods
From Fig. 2 it can be clearly observed that 10-6
M deprenyl treated cells showed maximum viability at all incubation period.
Cells treated with 10-3 M concentration of drug showed maximum viability
in 72 h incubation period. It can be seen that there was an increase in the
percent viability of cells for 10-6 M concentration. From the graph
it can be concluded that for all incubation period the viability of 10-9
M cells decreases as compared to 10-6 M deprenyl treated drug and
for 10-3 M the viability is less. It was also observed that percent
viability of cells is maximum for all the treated concentration of drug in 72
h incubation.
Western Blotting
In Fig. 3, cells were treated with deprenyl for 24 h incubation
period for western blotting analysis to check the effect on MAPK. From the bands
it was noticed that MAPK pathway was phosphorylated at 10-3 M concentration.
From the bands (Fig. 4, 5) it was noticed
that MAPK pathway was phosphorylated at 10-3 M concentration and
it was increased with 48 and 72 h incubation period, respectively. The 10-6
and 10-9 M concentrations did not show appreciate level of activated
MAPK. These results showed that MAPK was phosphorylated at 10-3 M
concentration of deprenyl and its activation was increased with the increase
in incubation period (48 and 72 h, respectively).
| Fig. 3: | Western Blotting analysis after 24 h of incubation at varying concentration of drug |
| Fig. 4: | Western Blotting analysis after 48 h of incubation at varying concentration of drug |
| Fig. 5: | Western Blotting analysis after 72 h of incubation at varying concentration of drug |
DISCUSSION
Stress and age are the major causes for breast cancer. Researchers have revealed that treatment with deprenyl leads to enhance central and peripheral catecholaminergic activity and a readjustment of immunological response (Harman, 1956; Yu, 1996). Deprenyl was mainly used in aging and age-associated disorders (Seniuk et al., 1994). Deprenyl is a monoamine oxidase inhibitor and at proper dose it protects the cells from oxidative stress and apoptosis (Knoll, 1988; Runden et al., 1998). As it has been noticed that deprenyl did not show any body weight change as compared to control when given to rats and researchers have shown that deprenyl enhances the cells survivability and its effect increases mainly during ageing as well as age related disorder (Kitani et al., 1999). Because of this quality, deprenyl has been proved as a drug without any cheese effect, if given to a longer period and studies on Parkinson’s and Alzheimer’s patient have shown that it is a suitable drug for treating these diseases (Knoll, 1988). Studies on rodents suggested that the activity of deprenyl significantly increases with age (Kitani et al., 1994). Results proved that deprenyl at 10-6 M concentration showed more cell viability with increase in incubation periods (48 and 72 h, respectively). Our results were in line with the previous findings that long incubation of deprenyl with proper dose increases the lifespan.
Western blotting was done to check the activation of Mitogen Activated Protein Kinase pathway by deprenyl. SDS Page was started with 50 μg protein to run the gel. Cells alone were run as a control to compare the effect of deprenyl on MAPK pathway. From the results it was observed that at 10-3 M concentration deprenyl showed dark band as compared to other selective concentrations that showed the activation of MAPK pathway at this particular concentration of deprenyl. The activation of MAPK showed that at 10-3 M concentration deprenyl must have cross the cellular membrane to elicit its stimulatory effect. As research suggested that specific cell lines were responsible for the activation of specific intracellular signaling pathways (Heiser et al., 2009). Results suggested that 10-3 M concentration of deprenyl was best suited for the activation of MAPK that increased with longer incubation period (48 and 72 h, respectively).
CONCLUSIONS
The aim of present investigation was to check the effects of deprenyl on T47D human breast cancer cell lines and MAPK signaling pathway. To study this MTT and Western blotting assays have been done to check the viability of T47D human breast cancer cell lines in the presence of different doses of deprenyl and to check the activation of MAPK by different doses of deprenyl respectively. Both the assays have been performed at three different incubation periods (24, 48 and 72 h, respectively).
MTT results showed that 10-3 M concentration of deprenyl was toxic for cells and because at this concentration cells died as compare to the control. Deprenyl at 10-6 M concentration was noticed as best suited for cell viability as compare to other concentrations of the deprenyl. It was observed that at 10-6 M concentration cell viability increased with increase in incubation periods (48 and 72 h, espectively).
Activation of MAPK pathway was analyzed by western blotting. Activation of MAPK pathway was observed at 10-3 M concentration of deprenyl and as incubation period was increased, there was an increase in activation level. These findings suggested that at selective dose (10-6 M), deprenyl increased the cell viability which increased further with longer incubation period and the selective dose of deprenyl (10-3 M) activated MAPK signaling pathway that showed cellular proliferation in T47D human breast cancer cell lines.
ACKNOWLEDGMENT
We thank to Prof. Dr. S. Thyagarajan, School of Bioengineering, SRM University, Chennai, for his valuable suggestion and guidance throughout this work and to provide all the necessary facilities to do this study.