The Inhibition Effect Of Deuterium Depleted Water On Non-small Lung Cancer A549 And Initial Studies Of Its Molecular Mechanisms

Objective: To study the effects of deuterium-depleted water(DDW)on human A549 lung cancer cell line and to explore the underlying molecular mechanisms.Methods: A549 cell line was cultured in media prepared with 50 ppm DDW or in media prepared with normal water..Cell growth measured using the colorimetric cell counting kit-8(CCK-8)reagent to compare the growth rate of cells in control and DDW groups.The two groups of cells were synchronized by serum starvation and then stimulated with media containing serum.36 and 48 hours after the stimulation,cells from each group were trypsinized,stained with propidium iodide(PI)and then analyzed using fluorescence-activated cell sorting(FACS)to determine the percentages of cells distributed in G1,S and G2/M phases during cell cycle.Total proteins were extracted from cells cultured in normal media or DDW media,In order to study the molecular mechanism of DDW inhibiting the proliferation of A549 cells,Protein chip experiments screened the signal pathway of DDW affecting A549 cell proliferation,western blot was conducted to detect the expression levels of MEK1 and EGFR;phosphorylation of serine 217 and serine 298 on MEK1;phosphorylation of tyrosine 1016 and tyrosine 1110.The expression levels of several alveolusspecific protein markers in each group of cells were also compared.The expression levels of several alveolus-specific protein markers in each group of cells were also compared.The mice were randomly divided into the experimental group(drinking DDW)and the control group(drinking ordinary water),record the date of death,calculate the average life expectancy.In order to study the molecular mechanism of DDW inhibiting the proliferation of A549 cells,Protein chip experiments screened the signal pathway of DDW affecting A549 cell proliferation.Result: Culturing A549 cells in DDW media for 60 days significantly inhibited cell proliferation,notable morphological transformation from cuboidal,adherent and piled-up growth to spindle,protruded and separated growth were also observed.36 hours after the serum-starved cells were stimulated with serum;the percentage of cells grown in DDW media had a significantly higher percentage of cells(70.51±3.12)than the cells grown normal media(56.69±2.13)in G1 phase,but a lower percentage of cells(22.15±0.95)than cells cultured in normal media(34.28±1.04).The results obtained by western blot showed that DDW treatment did not change the protein levels of MEK1 and EGFR;DDW did not change the phosphorylation of EGFR on tyrosine 1016 and 1110 but significantly reduced the phosphorylation levels of MEK1 on both serine 1016 and serine 1110.The tumor growth of the experimental group was slower than that of the control group.DDW treatment also increased the expression levels of type I alveolus markers: AQ5 and T1 a as well as two type II alveolus markers: SPC1 and SPC2.The results of the mouse's life test showed that the female rats drinking DDW had a longer life expectancy than the normal water.Protein chip results showed that DDW may inhibit the growth of A549 cells by inhibiting the MAPK-Mtor-Akt signaling pathway that associated with cell proliferation.Conclusion: Long-term cultivation of A549 lung cancer cells with DDW media significantly inhibited the cell growth by G1/S retardation;this is correlated with reduction in MEK1 phosphorylation.DDW treatment also causes alteration in the differentiation status of A549 cells.