Vitamin D and prevention of prostate cancer

Pharmacology I

from 2013-09-10 to 2013-09-18

Master program of biomedicine

Karolina Minta ()

Supervisor: Karin Lilja ()

Abstract

The main aims for the laboration were to improve the knowledge of culture technology and to study the antiproliferate action of vitamin D3 (1,25(OH)2D3) treated prostate cancer cells (LNCaP). Vitamin D is a secosteroid, prohormone, which has shown to have antitumor effect on prostate cancer cells. In order to confirm the relation between vitamin D and prostate cancer cells, the cells were grown in vitamin D and ethanol and then the DNA were exctracted from the cells in PCR to create copies of the DNA. The concentration of the hormon amounted to 10-9 and the duration of the treatment averaged 48 hours. After PCR an electrophoresis was made to confirm the relationship between vitamin D and prostate cancer. The electrophoresis did not give any clear results, but since there were less cancer cells in the wells with the vitamin D than in the wells with the ethanol, vitamin D must have had an antiproliferate effect on prostate cancer cells.

Introduction

Vitamin D is a group of secosteroids that are fat-soluble and they are responsible for revising the body absorption of phosphate and calcium. Vitamin D is also a prohormone which is inactive biologically. (Holick, 2006) Thanks to ultraviolet light from the sun we can receive previtamin D3 from 7-dehydrocholesterol. This process occurs in the plasma membrane of skin cells. Previtamin D3 isomerizes to vitamin D3 called cholecalciferol, which is transported to the liver and is there converted to calcidiol (major circulating form of vitamin D3 with only one OH group attached to the 25th carbon). The calcidiol is converted by the kidneys to the form of calcitriol (with one OH group attached to the 1st and one OH group attached to the 25th carbon). The calcitriol is a biologically active form with central role in calcium homeostasis and in intestinal absorption of calcium. Vitamin D becomes hormonally active when it binds to VDR (vitamin D receptor, nucleic of target cell) and it is involved in cell proliferation and differentiation. (Laing and Cooke, 2004), (Lilja, 2013)

The prostate is an uneven exocrine gland of the human male reproductive system. (Romer et al, 1977). The prostate is divided into different zones: central zone, transition zone, pheripheral zone, periethral gland region and fibromuscular zone. It is interesting to add that 70-80% of prostate cancer can be found in the fibromuscular zone. The main way to cure patients from prostate cancer is surgery, where the prostate is removed or through radiation therapy. The prostate gland becomes bigger by the increase of testosterone which is produced in the testis. The testosterone converts to DHT (dihydrotestosterone), which is a more potent androgen. DHT can bind and active AR (androgen receptor), which is related to the progesterone receptor, and higher amount of progestins block the AR. One of the most important functions of this receptor is a DNA-binding transcription factor, which regulate gene expression. (Lilja, 2013)

Research has shown that vitamin D acting in the prostate cancer has an anti tumour effect by inhibition of cell differentiation and growth. After treatment with 1,25(OH)2D3 in the LNCaP (which is a cell line from an injuried prostate adenocarcinoma human cells), they appear to be androgen dependent because AR antagonist can block this process (G0-G1 phase in the cell cycle). Cholecalciferol can also cause apoptosis in LNCaP cells. (Lilja, 2013)

During the laboration different methods were used, namely trypzination, cell counting, RNA isolation, RNA quantification, RT-PCR (reverse transcription), PCR and elecrophoresis. (Lilja, 2013)

The main aims for the laboratory process were to get refained skills in cell culture technology and to research action of vitamin D3 on LNCaP cells and if the secosteroid has impact on the transcription of the androgen and nuclear vitamin D receptor. (Lilja, 2013)

Materials and methods

The laboration was performed according to the laboration manual except instead of ethidium bromide 20 ul gel red was used.

The biological material used in the laboration was: prostate cancer cells from homo sapiens.

The methods used in the laboration were:

cell counting, electrophoresis, PCR, RNA isolation, RNA quantification, RT-PCR, trypzination.

VITAMIN D TREATMENT

Vitamin D was performed on treated cells and ethanol was added into controlled cells. Then they were checked through a microscope.

CELL COUNTING

Cell counting is a method for the quantification of cells using haemocytometer.

TRYPZINATION

Trypzination is a process in which the cells are trypsinized, meaning separation between the cells and the walls of cell monolayer. That was needed in order to count the cells by haemocytometer.

RNA ISOLATION

RNA isolation was used to separate and isolate RNA strands.

RNA QUANTIFICATION

RNA quantification is a way to estimate the RNA purity by measuring the absorbance.

RT-PCR

RT-PCR is a synthesis of cDNA from RNA using the enzyme reverse transcriptase.

PCR

PCR is polymerase chain reaction needed to create many copies of a particular DNA sequence.

ELECTROPHORESIS

Electrophoresis is a process which separates chemical compounds for example in genetic science. In this case this method was used to show the amount of androgen receptors and nucleic vitamin D receptors in both the treated LNCaP and the untreated (control).

The key chemical materials used in the laboration were:

1,25(OH)2vitamin D3 solubilized in 99% ethanol, GAPDH-FW primer, GAPDH-RV primer, AR-FW primer, AR-RV primer, nVDR-FW primer, nVDR-RV primer.

The key material used in the laboration was:

Qiagen RNeasy Mini Kit (Qiagen), the 100bp ladder.

The temperatures of the PCR used in the laboration were:

Initial denaturation (95 C), denaturation (95 C), annealing (58.9 C), extension (72 C), final extension (72 C). (Lilja, 2013)

Results and discussion

The results from the electrophoresis are shown in figure 1.

Figure 1. shows the results from the electrophoresis where CC1 is the control sample with nVDR primer, CT2 is the treated sample with nVDR primer, CC3 is the control sample with AR primer and CT4 is the treated sample with AR primer using the 100bp ladder.

In tube one (CC1) the electrophoresis only showed the presence of GAPDH.

There weren’t any results shown in the electrophoresis from tube two (CT2). This indicate that something apparently went wrong since there should be GAPDH in every tube. This could be caused by a lack of RNA since the amount of cells from the second trypzination only was 2.7 * 10 4 of the treated cells and 4.7 *10 4 of the control cells. After the RNA isolation the RNA concentration shown from the nanodrop only were 7.9ng/ul for the treated and 5.2ng/ul for the control, when they should have been at least over 10ng/ul.

In tube three (CC3) and four (CT4) the electrophoresis show the presence of both GAPDH and AR.

According to another study on the effect of vitamin D in prostate cancer cells (Ahmad HK et al.) 10-9 gave the best results. Since this laboration was executed with the concentration 10-9 the results from this lab should have been fairly good. Eventhough the electrophoresis does not show al the results from the lab there were still more cells in the control (ethanol) than in the treatment with vitamin D, this proves that vitamin D must have an antiproliferate effect on prostate cancer cells.

References:

Ahmad HK, Ferdhos K, Mamunur RS (2013) Role of Vitamin D as an Anti‐Proliferative Compound on Prostate Cancer

Holick MF (2006) High prevalence of vitamin D inadequacy and implications for health. Mayo Clin. (p. 353–73)

Laing CJ, Cooke NE (2004) Vitamin D Binding Protein. Vitamin D 1 (2nd edition) (p.117–134)

Lilja K (2013) Vitamin D and prevention of prostate cancer, Pharmacology

Romer, Sherwood A. , Thomas S (1977). The Vertebrate Body. (p.395)