Abstract
Prostate cancer is second major problem and cause of death in developed countries especially in USA. Androgen therapy was useful in past but now resistance has been developed against it. Now 1, 25-hydroxyvitamin D3 (biologically active form of vitamin D) has been shown beneficial effects in treatment of prostate cancer. It causes apoptosis of cells and results into peaceful elimination of cell from cancer tissue. It is involved in extrinsic or intrinsic pathway of apoptosis. In this experiment we try to find out its pathway of apoptosis. LNCaP call line was grown and treated with calcitriol (10-7M). After 24hours, 48hours, 72hours and 96 hours of treatment samples were collected. These samples were analyzed for live dead assay for confirmation of apoptotic induction. Furthermore Caspase8 expression analysis was made for determination of extrinsic pathway and cytochrome c expression analysis was done for analysis of intrinsic pathway of apoptosis. Results were measured by flurometery method. Finally statistical analysis was made by using Graph Pad Prism 5.03 statistical software.
Introduction
In America, prostate cancer is second most prevalent and reason of death in males (Carter and Coffey., 1990). During 2003, in America 220,900 new cancer cases were diagnosed (Stewart and Weigel et al., 2004). Androgen therapy has beneficial effects in reducing prostate cancer but now resistance has developed against androgens. So there is need of differentiation agents. 1, 25-dihydroxyvitamin D3 (calcitriol) is basically a steroid hormone which regulate calcium homeostasis along with formation of bone in body. But in addition to all these functions it has ability to control growth and differentiation in prostate cancer cells (Pols., et al 1990: Gross et al., 1997). Calcitriol action is mainly mediated by vitamin D receptors. Calcitriol inhibit growth of many cell lines including prostate cancer cell line LNCaP (Skowronski et al., 1993). Effect of vitamin D3 provokes through its interaction with particular nuclear vitamin D receptors (VDR). VDR is involved in regulation of ligand- dependant transcription. VDR basically have contact with specific DNA sequences and this contact is especially with RXR by binding through either homodimer or as heterodimer. VDR directly target to specific genes including cell cycle inhibitor p21Waf1 and p27Kip1 (Darwish and DeLuca., 1996)
It is very clear that vitamin D receptors are very important for inhibition of growth in prostate cancer cell lines. Vitamin D receptor expression level and transcriptional level are not only responsible growth inhibition. There are certain other factors involved in this complete process (Zhuang et al., 1997). Calcitriol hold the proliferating cancer cells at G1 phase during the cell cycle (Blutt et al., 1997), or collect in G2 phase (Eisman et al., 1989), or undergo apoptosis. This will ultimately results into alteration in growth patterns. In prostate cancer cell line some conflicts have been reported on induction of apoptosis by calcitriol (Zhuang., 1998).
Extrinsic pathway of apoptosis is also present which includes pathway of apoptosis activated by tumor necrosis factor family death receptors. It involves ligand binding, these death receptors form clusters at the plasma membrane. Furthermore it take on caspase-binding adapter proteins to related cytosolic domains and initiating protease activation via induced proximity mechanism The apical protease in this pathway is caspase-8, which cleaves and triggers directly or indirectly a variety of downstream effectors proteases, caspase-3, caspase-6, and caspase-7. This cleavage in apoptosis encourages the degradation of basic structural proteins, comprising PARP and endonuclease regulator ICAD which resulting into chromatin condensation, DNA fragmentation, and other actions involved in apoptosis (Deveraux et al., 2001).
Intrinsic pathway of apoptosis mainly involves loss of transmembrane potential along with discharge of cytochrome c in cytosole. This cytochrome c then forms apoptosome with help of APAF-1 and procaspase-9. It will then activate procaspase-9 which cleave and activates caspase-3 and caspase-7 resulting into cell elimination (Hongbo et al., 2005). It is reported that cytochrome c is released from mitochondria. It is enough for induction of "apoptosomes" multi proteins complex which have zymogen i.e. caspase 9, Apaf-1 and caspase 3 (Jonathan et al., 2002).
Caspase-8 and caspase-9 are responsible for activation of caspase pathways. IAPs is the family of anti apoptotic proteins which control mitochondrial ("intrinsic") and death receptor ("extrinsic") pathways for apoptosis. Its action mainly depends on its IAP repeat domain, present in all of its family members. In humans, IAPs consists of XIAPs, c-IAP1, and c-IAP2 which have direct binding with procaspase-9 and shielding its start in reply to Cyt-c and resulting into suppression of the protease activity in effectors proteases, caspase-3 and caspase-7. IAPs act as endogenous antagonists of certain caspases. It includes downstream proteases that operate at the junction of the intrinsic and extrinsic pathways for apoptosis. Some time, it's over expression is also reported which results into adverse effects (Guzey et al., 2002).
The aim of this experiment is to determine apoptotic pathway of vitamin D3 on LNCaP cell line. In this laboration caspase-8 and cytochrome c activity is analyzed along with live dead count analysis after treatment of vitamin D3 to LNCaP cell line. Finally results will be analyzed statistically.
Materials and Methods
Materials
LNCaP cell line, RPMI 1640 (Invetrogen, USA), cell culture plates (TPP, Switzerland), centrifuge machine (Hettich universal 32, USA), 1α,25-dihydroxyvitamin D3 (calcitriol), Caspase-8 Colorimetric Assay Catalog Number: BF4100 (R&D Systems, USA), Quantikine Human Cytochrome c Immunoassay kit Catalog Number DCTC0 (R&D Systems, USA), LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen Detection Technology, USA), Incubator (Sanyo, Japan),
Cell culture
LNCaP cells were grown on cell culture flask 75 cm (TPP, Switzerland) in RPMI 1640 supplemented with 0.1 mM nonessential amino acids, 4 mM L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin (sigma-Aldrich), supplemented with 10% FBS. Cells were incubated at 37° C in 95 % humidity and 5% CO2 in incubator (Sanyo, Japan). After 72 hours monolayer was formed in cell culture flask.
Live Dead assay
Cell culture flask was put in bio safety hood class II. Trypsin (5ml) was added in cell culture flask and incubated it for 15mints. After that 5ml of culture medium was added in the flask. Then all medium along with cells were transferred in falcon tube (50ml) followed by centrifugation at 1000 rpm for 15 mints. Supernatant was removed and pellet was re suspended in 4 ml of media. 50µl of Cells were mixed with 50µl of trypan blue dye. Cells were counted by using cell counting chamber.
Cells were seeded in 6 replicates on 96 well micro titer flat bottom plates at the rate of 5000cells per well in 100 µl of culture media. Plate was incubated for 72 hours at 37° C in 95 % humidity and 5% CO2 in incubator (Sanyo, Japan). After 72 hours cells in micro titer plate well A1-6 were treated with 10µl vitamin D3 in dilution of 10-7. Cells in well B1-6 were treated with 10µl of ethanol as control. After treatment plate was again incubated for 72 hours in same conditions.
Solution of calcein AM and EthD was prepared in 2x concentration by adding 2 µl of stock solution of 2mM EthD-1 in 4 ml PBS and 1 µl of 4mM calcein AM in same falcon tube as per protocol given by LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen Detection Technology, USA). Finally after 72 hours of incubation 100µl working solution of EthD and calcein was added in each well of micro titer plate. Plate was incubated for 25 mints and readings were taken by flurostar plate reader (BMG LABTECH, Germany) at emission EM520p and excitation EX485P for calcein and for ethidium at emission 630-10 and excitation 530-10. Total four readings were taken after 24hours, 48hours, 72hours and 96 hours of treatment with calcitriol.
Cytochrome C assay
Cells were counted and 500,000 cells per well were seeded on six well cell culture plate (TPP, Switzerland) using 3ml media. Cells were seeded in three replicates. Plate was incubated in same conditions as described above. After 72 hours three wells are treated with 3µl of Vitamin D3 in final dilution of 10-7 and three wells with 3µl of ethanol as control. Plate was incubated. Protein concentration was estimated by use of BCA protein assay kit (Thermo scientific, USA).
After 24 hours, 48hours, 72hours, and 96 hours of treatment cytochrome c immunoassay was performed as per protocol given in manual of Quantikine Human Cytochrome c Immunoassay kit (R&D Systems, USA). Results were measured by flurostar plate reader (BMG LABTECH, Germany).
Caspase 8 colorimetric assay
After cell counting 500,000 cells were seeded per well in 3ml media in six well cell culture plate (TPP, Switzerland). Total three replicates were seeded. Plate was incubated. After incubation of 72 hours three wells were treated with vitamin D3 in final dilution of 10-7 and three wells with 3µl of ethanol as control. Plate was again incubated in same conditions. Protein concentration was estimated by use of BCA protein assay kit (Thermo scientific, USA).
After 24hours, 48hours, 72hours, and 96 hours of incubation with vitamin D3, caspase 8 colorimetric assay was performed according to protocol given in manual of Caspase-8 Colorimetric Assay kit (R&D Systems, USA). Finally reading was taken at weave length 405 on flurostar plate reader (BMG LABTECH, Germany).
Statistical Analysis
Experiment of live dead assay was performed in replicates of six. Cytochrome c assay and caspase 8 assays were performed in replicates of three. ANOVA analysis was used to study the difference between the treated and untreated cells. Statistical software Graph Pad Prism 5.03 was used for statistical analysis of results.
Results and Discussion
LNCaP cells were cultured and counted 8.8x 106/ml. These cells were further cultured treated with vitamin D3. Effects of treatment were analyzed at different time intervals 24hours, 48hours, 72hours, and 96hours after treatment. Basic idea behind the experiment is to determine the extrinsic or intrinsic pathway of apoptosis caused by vitamin D3. For that cytochrome c immunoassay was performed for determination of mitochondrial intrinsic pathway of apoptosis and caspase 8 assay was done for analysis of extrinsic pathway of apoptosis. Furthermore live dead assay was also evaluated for studying apoptotic effect of vitamin D3 on LNCaP cells.
For live dead analysis of cells, calcein florescent dye was used for live cells and EthD florescent dye was used to observe dead cells. Mean and standard deviation SD for live cell count are as given in table 1. A graph is plotted as in fig.1 between time interval of treatment and finally measured value of green light at wavelength 485 and 520nm. It shows the kinetic properties of cell viability. The P value observed is statistical analysis regarding importance of time in interaction of vitamin D3 to cell viability is 0.0003. This p value shows that time has great significance in vitamin D effect on cell viability. Cell survival increases as time increase. So with passage of time vitamin D3 losses its effect. Best effect seen at 24hours to 48 hours for live cells viability. P value is <0.0001 for over all time effect which shows matter of great significance.
For dead assay EThD florescent dye was used. When it was observed in flurostar plate reader it shows very high values to be measured. This shows that we have used high concentration of EThD florescent dye. So its lower concentration is needed for proper analysis of results.
Table 1 shows mean and SD values for ethanol treated control and calcitriol conc. 10-7 treated cells after different time intervals. Samples were run in replicates of 6.
Fig 1 shows relation between time interval and live cell growth after treatment of cells with vitamin D3.
Meral Guzey also reported similar results in his studies for cell viability. Meral also use 1α, 25-Dihydroxyvitamin D3 on LNCaP and found that it arrest growth of cells at G1phase (Meral et al., 2002).
Cytochrome c is involved in mechanism of apoptosis. It is involved in intrinsic mitochondrial pathway of apoptosis and in this experiment we try to find out effect of vitamin D3 on cytochrome c release. Experiment was run in replicates of three. SD and mean of observed values is shown in table 2.
Table 2 shows mean and SD values for cytochrome c assay after treatment of cells with vitamin D3 and treat of ethanol as control at different time intervals.
A graph is plotted between time (hours) after treatment and relative absorbance units of expressed cytochrome c protein as a response of treatment as in fig.2.
Fig.2 shows cytochrome c expression after treatment of vitamin D3 (10-7M) in different time intervals.
All these results measurement shows that cytochrome c is released at best after 72hours of treatment. P value for time is calculated as 0.0327. As size of experiment is small it shows 3.3% of random observations which has significant effect in protein expression. If we increase size of experiments means replicates of experiment and got more readings at more time intervals probability of protein expression may be increase with time. It is also reported that cytochrome c independently cause mitochondrial apoptosis without involvement of caspase8 or caspase9 activation. It is also reported that cytochrome c induce apoptosis by altering expression between apoptotic and anti apoptotic proteins (Mathiasen et al., 1999).
Caspase8 is involved in extrinsic pathway of apoptosis. SD and mean values obtained from the Caspase 8 colorimetric assay, readings of results are shown in table 3.
Table 3 includes mean and SD values obtained from caspase8 colorimetric assay results
For further statistical analysis of results a graph is plotted between time intervals and relative absorbance units obtained from flurostar plate reader as given in fig.3.
Fig.3 indicates effect of calcitriol on expression of caspase8 at different time intervals
Results shows at 96 hours calcitriol induce apoptosis having p value 0.0004 which shows its great significance. But over all statistical analysis of results shows that calcitriol is not involve in extrinsic pathway of apoptosis. Many scientists reported that calcitriol is involved in intrinsic pathway of apoptosis.
All three experiments show that calcitriol is involved in apoptosis of LNCaP cancer cell line. Live dead assay indicate that calcitriol arrest cell growth at G1 or G0 phase of cell growth. So it is involved in alteration of cell growth patterns. Hence inhibition of growth by calcitriol is confirmed by experiment of live cell viability. But unfortunately unable to analyze dead cells analysis because we used higher concentration of EThD which cannot be measured by flurostar plate reader. So it can be recommended for future that lower concentration of EThD should be use for proper analysis of dead cells. For further analysis of apoptotic induction pathway of calcitriol flurometry was done for measurement of cytochrome c and caspase8 expression. Cytochrome c assay indicates involvement of calcitriol in mitochondrial apoptotic pathway. Furthermore Caspase 8 colorimetric assay results show that calcitriol is not involved in extrinsic pathway of apoptosis. Although at 96hours calcitriol shows some positive relationship in apoptosis with help of caspase8 but it may be due to procedural pippeting error. It is concluded from this experiment that calcitriol is not involved in death receptor pathway (extrinsic pathway) So based on the above mentioned evidences it can be concluded that cytochrome c release is involved significantly in inducing apoptosis in LNCaP call line. After 72 hours of treatment with calcitriol 10-7M to LNCaP cells exhibits most significant apoptotic behavior.
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