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Vitamin D is a fat soluble vitamin involved in maintaining calcium haemostasis in the body and causing absorption of calcium from the gut. It also has some antiproliferative effects on prostate cancer cells. In this experiment we have made an effort to prove this antiproliferative effect of vitamin D on prostate cancer cells. Thus cell culture, RNA synthesis and polymerase chain reaction (PCR) amplification was done. Knowing that Nuclear vitamin-D receptors (nVDR) receptor have crucial role in cell growth and androgen recptors (AR) receptor have proliferative role in prostate cancer. From our PCR amplification, it is seen that AR gene is more expressed as compare to nVDR gene in both treated and control sample. Thus proving that vitamin D has up regulated AR more than nVDR.
It is hydroxylated in the liver into 25(OH) vitamin D and in kidney into 1, 25(OH) 2 vitamin D (Weisman 2010). This is active form or metabolite of vitamin D that enters the cells and binds to its respective receptor, that is vitamin D receptor (VDR) and then subsequently to its gene which is a calcium binding protein (Weisman 2010). After processing the protein such as osteocalcin or calcium binding protein is formed (Weisman 2010). This protein is responsible for causing calcium absorption from gut (Weisman 2010). Vitamin D is important for musculoskeletal system. It also has role in extra skeletal tissues (Wang et al. 2010). Parathyroid hormone stimulate the production of 1, 25(OH) 2 VD and calcium decreases its production (Weisman 2010).
The deficiency of vitamin D is seen in premature infants, elderly people, obese people and those suffering from malabsorption(Weisman 2010). RICKETS and OSTEOMALACIA caused by vitamin D deficiency in children and adults respectively (Weisman 2010). People with deficiency of vitamin D are more prone to viral respiratory infections (CANNELL et al. 2006).
Vitamin D deficiency lead multiple disorders like increased the blood pressure, fatigue, cancer, fragile bones (R. Zhang et al. 2010). Taking vitamin D supplement by those who are suffering from its deficiency lead to decreased parathyroid hormone level, thus increasing bone density and becoming less prone to bone fracture (Van Den Bergh et al. 2011). The active metabolite that is 1, 25 (OH) 2 vitamin D has antiproliferative effects and thus it down regulates inflammatory marker (Weisman 2010).
Studies about role of vitamin D3 in prostate cancer have revealed that vitamin D3 inhibits growth of cancer cell in prostate gland and this role is androgen dependent. (Murthy et al. 2005).
Prostate is a gland of male reproductive system and prostate cancer is a form of cancer that occurs in prostate gland. Age, genetic factor, diet and hormonal factor contribute to risks for developing prostate cancer (Ramis et al. 2011). Prostate cancer can be diagnose by physical examination like digital examination, raised level of prostate specific antigen(PSA) in the blood and ultimately by doing a biopsy and examining it under the microscope(Kristal et al. 2010). Studies of vitamin D on prostate cancer have shown its anti proliferative, antimetastatic effect (Schwartz 2009). Moreover normal prostate cell are also capable of synthesizing 1,25(OH)2 D. While treating prostate cancer with vitamin D we should have check of Serum calcium level, considering hypercelcemia to be a major risk factor (Schwartz 2009).
Method and Material
On day one trypzinisation was done by removing spent cell culture medium and washed with monolayer cells using phosphate buffer solution (PBS). After adding try psin to PBS solution, flask was rotated and incubated at 37 degree centigrade for 5 minutes and then growing cells were observed under microscope. Then cell culture medium was added slowly to inactivated tryp sin. The whole solution put into falcon tube and centrifuged for 5 minutes. The supernatant was removed and the pellet was resuspended with fresh cell culture. Then 40 micro litter from this solution was taken into eppendrof tube and the remaining solution was placed on ice for seeding cell after cell counting.
Into eppendorf 40 micro litter suspension and 40 micro litter of trypan blue (to colour the dead cell) was added and mixed by gentle pipetting. Heamocytometer was prepared by moistening cover slip with our breath. Two drop of cell suspension were added by capillary force on each side and seen under microscope.
3'A'square were counted
Total cells in3A sq=176
= 58.6 cell/Asq
Number of cell * dilution factor /0.001
=11.7*105 *4ml (initial media concentration)
Cell concentration=1.17*106 cell/ml
=42 micro litter
Thus 2958 micro litter of medium + 42micro litter cells.
Since we have to prepare 7wells instead of 6 wells to keep some excess so, 2958*7= 20.7 ml media
=294 micro litter of cell
Therefore we take 3ml/well.
Vitamin D treatment
A 6well culture plate was prepared by reseeding 50000 cell to each well. Thus 3ml of final volume as calculated above was added to each well. Then vitamin D3 solubilised with 99% Ethanol hydroxide (EtOH) to a concentration of 10-7 was added to 3 wells. A prepared stock solution of 10-3 nM was added to three control wells. Also EtOH of same volume was added to three controls well because vitamin D added to prior 3 well was treated with EtOH. So we have to see that any change in the number of cell is ether due to vitamin D or due to EtOH.
Then culture plate was incubated for 42hr.
Again trypzinisation was done like day 1 but now 1ml of trypsin and culture media each instead of 3ml were taken and incubated at 37degree centigrade for 5 minutes. From three treated plates solution was put in 1 tube and labelled as treated. Similarly solution from three control well was taken in another tube and labelled and centrifuged. The supernatant was removed and pellet was resuspended. Then 40 micro litter of this solution was mixed with 40 micro litter of trypan blue in eppendorf tube. Haemocytometer was prepared. Now cell were counted in 9Asq.
Treated cells in 9Asq=4/9
Number of cell * dilation factor/0.0001
=8.8*103 * 2ml
Cell in control slide in 9Asq = 9/9
=1 cell/ A sq
1*2ml/0.0001= 20000 cells/ml
20,000 * initial media concentration
= 4*104 cells
(Here we have counted cells in more squares than day1 because now cell are treated and there count is reduced so we need to see cells in more squares.)
From above calculation we see treated cell count is reduced as compare to cell in control solution.
RNAase free environment was maintained. RNA preparation was done by using lab manual and QIAGEN kit. RNA was prepared by doing cell lysis, cell homogenation in QIA shredder and than bound with RNA filter and then undergoing several washing steps and ultimately elution steps was done and RNA was prepaid.
Finding of RNA QUANTIFICATION
Vitamin D 1.7ng/micro litter
Control 4.7ng/micro litter
Average RNA quantification of all groups for control is 6.5ng and for treated is 3.4ng. Treated RNA is less than control because we have less number of cell in treated wells. Thus RNA quantification result supports our cell count result. We have hypothesized that vitamin D decreases cell growth and induced apoptosis. Thus our hypothesis is supported by cell count and RNA quantification.
Reverse transcriptase -PCR synthesize cDNA. RNAase free environment was maintained 2RT-PCR reaction was run, for control and treated respectively. Material for making master mix to prepare cDNA is as follows.
2*RT Buffer=10 micro litter
2*RT enzyme=1 micro litter
Nuclease free water =6 micro litter
Control= 3 Micro litter
Total = 20 micro litter
2*RT Buffer=10 micro litter
2*RT enzyme=1 micro litter
Nuclease free water =4 micro litter
Vitamin D treated= 5 Micro litter
Total = 20 micro litter
(We have borrowed RNA from other group having concentration in control 7.1 and in treated 10.4 because we have to prepare 50ng /cDNA reaction and our RNA quantity is less. If we use our own RNA then we require more than 9micro litter of RNA and we have to prepare a total of 20 micro litter master mix. Thus we have to use RNA from the above mentioned group)
RT-PCR master mix is prepared mixed thoroughly by slowly pipetting up and down. Then PCR tubes are placed in pre heated thermal cycler at 37° C for 60 minutes and then heated to inactivate reverse transcriptase for 5 minutes at 95° C because we don't want the enzyme to be activated again and then kept the tubes on eyes at 4° C.
Step2 PCR reaction
We require master mix for 4 reactions but prepare master mix for 5 reaction to avoid pipetting variations.
dNTP0 =5micro litter
DNA polymerase=1 micro litter
10*PCR buffer=12.5 micro litter
Nuclease free water=85 micro litter
Total= 85 micro litter
GAPDH-F=1 micro litter size of GAPDH is 600 base pair
AR-F=1micro litter size of AR is 200 base pair
VDR-F=2micro litter size of VDR is 350 base pair
CDNA-control= 2 micro litter
CDNA-treated=2 micro litter
PCR was run according to condition given in manual.
Agarose gel was prepared by using material as fallow
1*TAE Buffer - electrophoresis
Ethimide bromide- stains DNA
We have succeeded with treated but not with control PCR good amplification of GAPDH and AR is seen but VDR shows faint bands. VDR bands are weaker than GAPDH and AR bands which mean that VDR is less expressed.
Conclusion and Discussion
All managed to get PCR product with good result and correct size. All managed to make cDNA. All manage to extract RNA. We did not see any change in gene expression because it might be that we have incubated cells for less time. Regarding VDR, shows weaker bands because we have stopped PCR at 28 cycles if these cycles were allowed to continue than VDR would have expressed as strong band.