Study On The Effects Of Arsenic Trioxide On The Expression Of PML、p53and Survivin In Hut-78Cell Line

Objective:To investigate the possible mechanisms in apoptosis of T cell lymphoma after treated with Arsenic Trioxide(ATO),by detecting the apoptosis rate,effects of cell cycles expression of the genes,such as PML、PML-Ⅰ、PML-Ⅳ、PML-Ⅴ、p53and survivin and the correlation between them in cutaneous T-cell lymphoma (CTCL)cell line Hut-78.Methods:Hut-78cells were treated with the concentration of2,5,10umol/L of ATO for24h,48h and72h respectively.The apoptosis rate and cell cycle distribution proportion of experimental group and control group were detected by Flow cytometry with PI staining and terminal deoxynucleotidy transferase-mediated dUTP nick end-labeling (TUNEL) assay; At the same time,the expressions of PML,PML-Ⅰ,PML-Ⅳ,PML-Ⅴ,p53and survivin genes were detected by the SYBR green I reverse transcriptase-polymerase chain reaction(RT-PCR),and then analysis the correlation between them.Results:1. The effect of ATO on celll cycles showed by Flow cytometry with PI staining: In the range of0-10umol/L, with the concentration increase of ATO, the cell proportion of S phase gradually reduced, while the G0/G1phase increased after the first decreased,and the G2/M phase decreased after the first increased; When the concentration of ATO was2umol/L, the G0/G1ratio reached to minimum,and the G2/M ratio reached to the highest; Later, when the concentration of ATO was higher than2umol/L,the increase extent of G0/G1phase cells was equivalent to the sum of the decrease of the proportion of G2/M and S phase. When the cells were treated with2-5umol/L ATO, the most obvious changes of apoptosis rate and the proportion of each phase in cell cycle were detected.2. The rate of apoptosis and necrosis showed by Flow cytometry with PI staining: With the increase of concentration and extending of action time, the rate of apoptosis and necrosis of Hut-78cells was gradually increased, and the most obvious change appeared between2-5umol/Lof ATO. Hut-78cells were treated with different concentration of ATO for24hours,the differences of apoptosis and necrosis rate between control group and5umol/L,10umol/L group respectively was statistically significant (p=0.035;p=0.014) After treated for48hours, the differences of apoptosis and necrosis rate between control group and5umol/L,10umol/L group respectively had statistically significant(p<0.05),2umol/L group and5umol/L,10umol/L group respectively also had statistically significant(p<0.05); After treated for72hours, the difference of apoptosis and necrosis rate between each group had statistically significant (p<0.05)except between5umol/L and10umol/L group(p=0.513).3. The rate of apoptosis and necrosis showed by TUNEL:With the increasing of concentration and extending of action time, the rate of apoptosis and necrosis of Hut-78cells was gradually increased. Hut-78cells were treated with different concentration of ATO for48hours, the differences of apoptosis rate between control group and10umol/L group had statistically significant(p=0.031). After treated for72hours, the differences of apoptosis rate between control group and2,5,10umol/L group respectively had statistically significant(p<0.05). The difference of necrosis rate between control group and5,10umol/L group,2umol/L group and5umol/L,10umol/L group had statistically significant (p=0.000)4. The expression of genes showed by RT-PCR:When the Hut-78cells was treated with a certain concentration of ATO,the expression of PML、PML-Ⅰ mRNA gradually decreased with reaction time (24-72h)extending.In control group and2umol/L group, expression of PML-Ⅳ and PML-V mRNA also decreased gradually,while in5umol/L group and10umol/L group, the expression decreased after the first increased. Expression of p53and survivin mRNA gradually decreased with acting time extending in control group and2umol/L group while increased in5umol/L group and10umol/L group. After treated with ATO for24h, PML, PML-Ⅰ, PML-Ⅳ, p53, survivin gene mRNA decreased gradually with increasing of the concentration of ATO while PML-Ⅵ gene mRNA first increased and then decreased. After treated with ATO for48h,PML-Ⅳ and PML-V gene mRNA increased gradually with increasing of the concentration of ATO while PML-Ⅰ and survivin gene mRNA first increased and then decreased. The highest expression of PML-Ⅰ gene mRNA was detected when the concentration of ATO was5umol/L. But there was no significant change in PML and p53gene mRNA.After treated with ATO for72h, PML and its isoforms,survivin mRNA expression increased first and then decreased,with the increase of ATO concentration, and the highest expression of these genes mRNA were detected when the concentration of ATO was5umol/L. There was no significant change in p53gene mRNA with increasing of the concentration of ATO.5.The correlation between the genes:There was no correlation between the expression of PML and p53,survivin mRNA(p>0.05),while highly correlated between p53and survivin mRNA. The correlation between PML and PML-Ⅰ、PML-Ⅴ mRNA may be related to the content of its own.Conclusions:1. Low concentration of ATO can significantly inhibit the proliferation of Hut-78cells,while high concentrations can induce apoptosis and necrosis,which is dependent on time and dose. And the rate of apoptosis is always greater than necrosis.2. ATO plays a role in inhibiting cell proliferation and inducing apoptosis in Hut-78cells by regulating the cell cycle maybe,especially arresting cell cycle in the G2/M phase.3.ATO can inhibit cell proliferation and induce apoptosis and necrosis of Hut-78cells, which may be related to the expression of PML, PML-Ⅰ、PML-Ⅳ、PML-Ⅴ.And the apoptotic pathway may not be dependent on p53and survivin gene expression.