Determination of metallothioneins and alpha-methylacyl-CoA racemase in tumor prostate diseases

Introduction: Metallothioneins (MT) belong to the group of intracellular, low-molecular cysteine-rich proteins with molecular weight from 6 to10 kDa. Due to their high affinity to heavy metal ions (Zn, Cd, As, etc.), their main and crucial function is homeostasis maintenance and detoxification of heavy metals. The role of MT in tumour tissue remains still unclear, but MT can be considered as new promising tumour marker. Alpha-methylacyl-CoA racemase (AMACR) is a -oxidation of branchedâperoxisomal and mitochondrial enzyme involved in the fatty acids and was recently reported that AMACR has very high sensitivity and specificity to prostate adenocarcinoma. Therefore is very important to find new approaches in AMACR detection.

Aim: The main aim of this paper is to study expression of MT and AMACR in tumor prostate cell lines and in patients serum on protein and mRNA level. Moreover, we compare the differences in MT expression between cells influnced with zinc ions.

Material and methods: Biological samples. Prostatic cell lines derived from prostate adenocarcinoma (LNCaP-FGC, PC-3, and 22RVL) and prostatic cell line derived from normal epithelium (PNT1A – human immortalized prostatic cell line) were used. Protein detection. SDS-PAGE electrophoresis and Western-blot analysis with subsequent immunodetection were used. Immunohistochemical detection: Immunohistochemical detection of AMACR was performed by R.T.U Vectastain universal ABC kit (Vector Laboratories, Burlingame, CA, USA).

Results: We investigated influence of zinc ions on MT expression in prostatic cell lines derived from prostate carcinoma and MT level in patients serum samples. For the MT determination we used SDS-PAGE electrophoresis and western blot analysis with subsequent immunodetection. Moreover we used modern electrochemical methods (Brdicka reaction) for detection of MT. We demonstrated up-regulation of prostatic specific antigen expression in prostate tumour cells (LNCaP, PC-3, 22RVL) and contrariwise down-regulation of MT expression. This fact can be explain by decreased concentration of zinc ions in prostatic tumor cells and therefor lower concentration of MT in tumor cell lines in comparing with non-tumor cells. Besides immunodetection we measured MT by adsorptive transfer stripping technique coupled with differential pulsed voltammetry Brdicka reaction and we obtained similar results as in immunodetection. In case of AMACR, results show relatively big diferences between tumor and non-tumor cell lines in AMACR content in mRNA and protein level as well. In case of tumor cells the expression of AMACR was up-regulated in comparison with non tumour cells. Then we cultivated cells on glass slides for immunodetection of AMACR in situ and we obtained similar results.

Conclusions: This study provides important new information about expresion of MT and AMACR in prostate tumor cell lines. Evidently MT expression is significantly down regulated in 22RVL, and PC-3 cells while AMACR was up regulated. We observed significant (á=0.05) difference in MT content between cell lines treated/nontreated with zinc ions by using immunohistochemical methods and electrochemical methods as well.

Acknowledgements: This work was supported by the grants GACR 301/09/P436 and IGA MZ NS10200-3/2009.