Impact Of GNMT Expression And Regulation On The Biological Characteristics Of Human Prostate Cancer Cell Lines LNCap And PC3

The incidence of prostate cancer is rising world-widely as well as in China. Numerous molecular biology and genetic mechanisms contribute to the development of the disease. Using metabolomic profiling methods, Sreekumar et al reveal the potential role of sarcosine in prostate cancer progression. They find that sarcosine concentration is obviously high in prostate cancer tissues especially the invasive tumors than that in the benign prostate tissues, and they also find that the addition of sarcosine to benign prostate epithelial cells promotes invasive properties in these cells. The concentration of sarcosine is regulated by the enzyme glycine-N-methyltransferase (GNMT). Latest study discovered that GNMT expressions are more abundant in prostate cancer cells compared with normal prostate epithelial cells, and GNMT knockdown suppresses prostate cancer cell proliferation and induces cellular apoptosis in prostate cancer cells. Thus, the regulation of GNMT expression or function may be a strategy for revealing the development mechanisms and developing novel therapeutics for prostate cancer. As a multi-functional protein, the methyltransferase activity of GNMT can be inhibited by it’s catalysate S-adenosylhomocysteine (SAH), and the effect can be seen both in vitro and in vivo. SAH can also suppress the expression of GNMT at transcriptional level. And another study find retinoid acid (RA) can activate and induce GNMT in rats. So, we can regulate the expression and activity of GNMT by SAH and RA in vitro and in vivo, which may implement our further studies. To our knowledge, till now there are no studies can tell whether the external regulation of GNMT can change the biological characteristics of human prostate cancer cell lines or not. We aim to reveal the impact of GNMT expression and regulation (in vitro and in vivo) on the differentiation, apoptosis, proliferation, invasiveness and tumor generation ability of LNCap and PC3 cell lines.Part ⅠGNMT Expression in Human Prostate Cancer Cell Lines LNCap and PC3OBJECTIVE Investigate the expression difference of GNMT between human prostate cancer cell lines LNCap and PC3, reveal the relationship between GNMT expression and prostate cancer cell invasiveness.METODS Detect the expression in both cell lines LNCap and PC3 of GNMT mRNA by RT-PCR, and GNMT protein by Western Blot.RESULTS The relative expression of GNMT mRNA is (1.000±0.331) in LNCap cell, and (2.579±0.863) in PC3 cell, with statistical difference, p<0.05. The relative expression of GNMT protein is (0.341±0.072) in LNCap cell, and (0.841±0.116) in PC3 cell, with statistical significant difference, p<0.01.CONCLUSION The expression of GNMT correlates with the invasiveness of prostate cancer cell lines, it’s high in PC3 cell with higher invasiveness and low in LNCap cell with lower invasiveness.Part ⅡImpact of GNMT Regulation in vitro On the Biological Characteristics of Human Prostate Cancer Cell Lines LNCap and PC3OBJECTIVE Investigate the impact of GNMT regulation in vitro on the differentiation, apoptosis, proliferation and invasiveness of human prostate cancer cell lines LNCap and PC3.METHODS Up regulation of GNMT activity by addition of GNMT (10μg/ml), and down regulation of that by addition of SAH (10 ng/ml) to the LNCap and PC3 cell lines. Observe the cell differentiation by microscope, detect the cellular apoptosis by TUNEL and flow cytometry, cell proliferation by MTT test, and cell invasiveness by transwell test at four time points (12h,24h,36h and 48h).RESULTS ①Microscope Observation There is no marked change of cell appearance in both cell lines of both groups under 200 X microscope at 48h, and the result is the same by 5d. ②TUNEL In both cell lines, the apoptosis rate is lower in GNMT group and is higher in SAH group than in control group. In LNCap cell, GMNT group vs. control group, there is no statistical difference at 48h (p>0.05), and statistical difference at 12h,24h and 36h (p<0.05); SAH group vs. control group, there is statistical difference at all 4 time points (p<0.05). In PC3 cell, GMNT group vs. control group, there is no statistical difference at 12h and 48h (p>0.05), and statistical difference at 24h and 36h (p<0.05); SAH group vs. control group, there is no statistical difference at 12h (p>0.05), and statistical difference at 24h,36h and 48h (p<0.05). ③ Flow Cytometry In both cell lines, the apoptosis rate is lower in GNMT group and is higher in SAH group than in control group. In LNCap cell, there is statistical significant difference in all the 3 groups at all 4 time points (p<0.01). In PC3 cell, GMNT group vs. control group, there is no statistical difference at 48h (p>0.05), and statistical difference at 12h,24h and 36h (p<0.05); SAH group vs. control group, there is statistical significant difference at all 4 time points (p<0.01).④MTT In both cell lines, the absorbance is higher in GNMT group and is lower in SAH group than in control group. But the statistical difference only appears in PC3 cell, SAH group vs. control group at 48h (p<0.05). ⑤Transwell In both cell lines, the transmembrane cell is more in GNMT group and is less in SAH group than in control group. There is statistical difference in both cell lines at 36h and 48h (p<0.05).CONCLUSION Addition of GNMT and SAH to the LNCap and PC3 cells implements GNMT regulation in vitro, which can impact the cellular biological characteristics except for cell differentiation. Up regulation of GNMT activity results in suppression of cellular apoptosis in both cell lines despite the androgen dependent property, no obvious influence of cell proliferation in both cell lines, and cell invasiveness enhancement in both cell lines especially in PC3 cell. While down regulation of GNMT activity results in promotion of cellular apoptosis in both cell lines despite the androgen dependent property, suppression of cell proliferation in PC3 cell, and attenuation of cell invasiveness in both cell lines despite the androgen dependent property.Part ⅢImpact of GNMT Regulation in vivo On the Tumor Generation Ability of Human Prostate Cancer Cell Line PC3OBJECTIVE Investigate the impact of GNMT regulation in vivo on the tumor generation ability of human prostate cancer cell line PC3 in nude mice.METHODS Inject PC3 cell to the nude mice subcutaneously for prostate cancer tumor generation. Divide the mice into 3 groups randomly,2 experimental groups are fed with cis-Retinoid Acid (CRA,30μmol/kg) and S-adenosylhomocysteine (SAH,10 g/kg) respectively, while control group is fed with normal diet. In 6 weeks, the tumor size is measured weekly for drawing the growth curve. After that, the mice are sacrificed and the expression of GNMT in the tumor is detected by immunohistochemistry.RESULTS After 6 weeks, the tumor size is bigger in CRA group and is smaller in SAH group vs. control group, with statistical difference (p<0.05). The expression of GNMT in the tumor is more abundant in CRA group and lesser in SAH group vs. control group, with statistical difference (Positive Cell Ratio, p<0.01; Integrated Optical Density, p<0.05).CONCLUSION CRA and SAH intake can modulate the growth of human prostate cancer generated by PC3 cell in nude mice. Such impact may due to the regulation of GNMT activity and expression in the tumor, CRA intake can up regulate GNMT and promote tumor growth while SAH intake can down regulate GNMT and suppress tumor growth.