Mapping of elemental distribution of Cu, Fe and Zn in human prostatic carcinoma cell spheroid culture using synchrotron radiation based on Microbeam X-ray Fluorescence

Full text: Prostate cancer continues to be one of the most common fatal cancers in men. The main function of the prostate is the high production and secretion of citrate which is possible due to the unique accumulation of zinc by prostatic epithelial cells. Iron plays a vital function in oxygen metabolism, oxygen uptake, and electron transport in mitochondria, energy metabolism, muscle function, and hematopoiesis. Copper plays an important role in the aetiology and growth of tumours however, whether intratumoral copper is actually elevated in prostate cancer patients has not been established. Zinc is an essential trace element, critical for diverse biological functions in the human body. The X-ray micro fluorescence technique (μXRF) is a rapid and non-destructive method of elemental analysis that provides useful elemental information about samples without causing damage or requiring extra sample preparations. This study investigated the behavior of cells in spheroids of human prostate cells, tumour cell line (DU145) and normal cell line (RWPE- 1), after supplementation with zinc chloride by 24 hours using synchrotron Xray micro fluorescence (μSRXRF). Besides that, it too was analysed cell viability through colorimetric cytotoxicity assay (MTT) and evaluated the organization of cytoskeleton cell, in order to better characterize morphologically the established cellular spheroids, by fluorescence microscopy. The measurements were performed with a standard geometry of 45º of incidence, excited by a white beam using a pixel of 25 μm and a time of 300 ms/pixel at the XRF beamline at the Synchrotron Light National Laboratory (XRF - 18905 proposal). It was observed that the iron intensity in the normal cells spheroids did not present a standard with respect to the treatment of zinc chloride, whereas in the tumor cell increased from the treatment of 100 μM, whereas in the treatment of 50 μM the intensity remained constant. There was a decline in the intensity of copper in the tumor cells in the treatment of 50, in the treatments of 100, 150, 200 and 250 μM there was an increase when compared to the control. It was observed that the highest intensities were observed in the treatments of 150 and 200 μM. In the normal cells, the intensity of copper did not present a standard with respect to the treatment of zinc chloride, however the intensity remained lower in all the treatment when compared to the control. In the case of zinc, it has been observed that despite the treatment the tumor cell suffers a decrease in the ability to accumulate zinc when compared to normal cells. MTT assay's results showed no significant difference, which indicates that the cell viability was not changed, regardless of treatment in both cell types tested, so the cells are viable. Finally, immunofluorescence assay allowed to evaluate the organization of the cytoskeleton and cellular phenotype, through the identification of cytokeratin and vimentin filaments. (author)