The Molecular Mechanism Of PRF-induced NB4 Cell Apoptosis In Vitro

[Background and objective]Puerariae radix flavones (PRF) is the main effective consituent of Chinese medicine Puerariae radix. In recent years, our research group found that the PRF in the concertration of 0-50μg/mL could induce the NB4 cells to apoptosis in a time and concentration-dependent manner and retard the cell in S-phase. It has also been proved that when PRF induces NB4 cell to apoptosis, the JNK protein is activated via up-regulation of phosphorylation level of JNK, the expression of ERK is up regulated while p38 down-regulated, and the expression of the cleaved caspase3 is up-regulated. To further investigate the molecular mechanism of PRF-induced NB4 cell apoptosis, this research detect the cellular proliferation inhibition influence of the certain concentration of 0-50μg/ml PRF on NB4 cell and the expression of the relative proteins of apoptosis in the case of blocking the JNK, p38, ERK, caspase signal pathway by JNK inhibitor SP600125, p38 inhibitor SB203580, ERK inhibitor U0126 and caspase inhibitor Z-VAD-FMK.[Method]1.0,10,30,50μg/ml PRF±10μmol/L sp600125, 10μmol/L SB203580, 10μmol/L U0126,20μmol/L Z-VAD-FMK interfere the NB4 cell for 24,48 and 72 hours, detect the NB4 cells cellular proliferation inhibition rate by MTT assay.2. 10μg/ml PRF±10μmol/L SP600125 interfere the NB4 cell for 24,48,72 hours, detect the relative proteins of apoptosis.3.0,10,30,50 μg/ml PRF±10μmol/L SP600125 interfere the NB4 cell for 48 hours, detect the relative proteins of apoptosis by Western Blot.4.0,10,30,50μg/ml PRF±20μmol/L Z-VAD-FMK interfere the NB4 cell for 48 hours, detect the relative proteins of apoptosis by Western Blot.5.30μg/mlPRF±SP600125、SB203580、U0126、Z-VAD-FMK interfere the NB4 cell for 48 hours, detect the relative proteins of apoptosis by Western Blot.6.30μg/mlPRF interfere the NB4 cell for 0,12,24,36,48 hours, detect the relative proteins of apoptosis by Western blot.[Results]1. SP600125 could obviously increase the proliferation inhibition rate of PRF in the concertration of 10μg/mL, and so as in the concertration of 30μg/mL, but as action time of PRF prolongs, the difference diminish. By 72 hours, there was no differrence. SP600125 doesn’t affect the proliferation inhibition rate of PRF in the concertration of 50μg/mL, Z-VAD-FMK could supress the proliferation inhibition rate of PRF. With the duration of PRF extending, the difference become more notable; SB203580, U0126 doesn’t affect the proliferation inhibition rate of PRF on NB4 cell. There is no differrence.2. NB4 cells exposed to 10μg/mL PRF for 24,48,72 hours, the JNK protein were activated in a time-dependent manner, while the expression of caspase-3, bcl-2, p-bcl-2 were up-regulated and reach the peak level on 48 hours; when pretreated by SP600126 for 2 hours, compared to single PRF group, the expression of p-bcl-2 were up-regulated; and the expression of JNK, caspase3、bcl2 were up-regulated by 24 hours but down-regulated by 48 and 72 hours.3. Interfered by PRF the NB4 cell for 48 hours, the expression of JNK was up-regulated on a concentration-dependent manner. The expression of caspase3, CDK2-cyclinA were up-regulated while bcl-2 down-regulated. The expression of p53 had no obvious change. Compared to single PRF group, the expression level of JNK was increased and caspase3, p53 was inhibited when pretreated by Z-VAD-FMK for 2 hours. Meanwhile the expressions of bcl-2, CDK2-cyclinA were mildly increased. When pretreated by Z-VAD-FMK for 2 hours, the expression level of JNK, caspase3, ERK were inhibited while bcl-2 was increased.4. As 30μg/ml PRF interfered the NB4 cell for 48 hours, the expression of caspase3, JNK, ERK, p53 were up-regulated while the expression of bax was down-regulated. When pretreated with SB203580 for 2 hours, compared to single PRF group, the expression level of caspase3, JNK, ERK, p53, bax were increased. When pretreated with SP600125, U0126, Z-VAD-FMK, the expression level of JNK, ERK, p53, caspase3 were inhibited while bax were increased.5. As the time of NB4 cell incubated by 30μg/mlPRF prolonged, the expression of JNK was up-regulated, midly decreased on 24 hours. The expression of p38 was down-regulated while caspase 3 were up-regulated in a time-dependent manner. The expression of ERK, cyclinA increased on 12 hours, then gradully decreased. The expression of CDK2 increased and reached the peak le vel on 12h. The expression of p53 didn’t vary with time.[Conclusion]The molecular mechanism of PRF-induced NB4 cell apoptosis varies with the concentration of PRF. The activted JNK signal pathway of NB4 cell induced by PRF probably could not only contribute to apoptotic response, but also mediate survival signaling. Therefore, PRF of low concentration would lead to transient JNK activation causing the survival signal takes dominant, which turns to promote the proliferation of NB4 cell. But whether PRF achieved the effect through induction of cell differentiation still needto be confirmed. When the higher concentration of PRF induced JNK to sustained activation, the apoptotic signals were dominant, which leaded NB4 cells to apoptosis, mainly in the mitochondrial apoptosis pathway. ERK and p38 pathway both participated the apoptosis induced by PRF, in contact with JNK pathway, neither of which is the indispensible signal molecule. Furthermore, we also proved that the molecule mechanism of PRF retarding NB4 cell in S-phrase, which is related with the abnormal expression of cyclinA and CDK2. The up regulation of cyclinA-CDK2 accelerated the NB4 cell to S phase, leading to the disfunction of DNA duplication, which further promoted the cell apoptosis.