Physapubescin B Induces Apoptosis Of Human Gastric Cancer Cells Through Inhibiting JAK2/STAT3 Signaling Pathway

Objective:We aimed to demonstrate that physapubescin B suppresses cell proliferation and increases apoptosis of human gastric cancer cells through inhibition of JAK2/STAT3 signaling pathway and down-regulation the expression of STAT3 target genes such as XIAP and C-myc.Physapubescin B may serve as a potential therapeutic agent for human gastric cancer.Methods:1.MGC803 and SGC7901 cells were treated with various concentrations of physapubescin B for 24h and 48h.CCK8 Assay was performed to detect the cell viability.2.Flow cytometry was used to investigate the apoptosis rate and cells lysates were used to detect the expression of apoptosis-associated proteins including Caspase3?Caspase9 and PARP after human gastric cancer cells were treated with different concentrations of physapubescin B for 24h.3.MGC803 and SGC7901 cells were treated with physapubescin B and the phosphorylation of JAK2/STAT3 was detected by Western blot.4.Western blot was performed to detect the phosphorylation of STAT3 which was induced by IL-6.5.Immuno-fluoresence staining was used to confirm the localization of pSTAT3(Tyr705)in the cells.6.To confirm the inhibition of STAT3 transcriptional activity of physapubescin B,we used Dual-luciferase reporter assays.7.MGC803 cells were treated with 15?M physapubescin B for indicated time,RT-PCR was used to detect the mRNA level of downstream target genes(XIAP?CyclinD1and C-myc)of STAT3 and western blot was used to measured the equivalent protein expression levels.8.MGC803 xenograft models was used to evaluate the antitumor activivity of physapubescin B in vivo.Results:1.Physapubescin B suppressed the proliferation of MGC803 and SGC7901 cells obviously in a dose and time dependent manner.And the inhibitory rate of 15 ?M physapubescin B on MGC803 and SGC7901 cells were 49.80 ±5.68%,56.76± 3.78%,respectively.2.Flow cytometry results exhibited that physapubescin B also induced apoptosis of cancer cells and the apoptosis rate of 15 ?M physapubescin B on MGC803 and SGC7901 cells were 44.2 ± 13.29%,48.13 ±10.38%,respectively.Also physapubescin B could increase the cleavage of caspase-3,caspase-9 and PARP.3.Physapubescin B inhibited phosphorylation of JAK2 and STAT3 in a does-and time-dependent manner.4.Physapubescin B could surppress constitutive and IL-6 induced phosphorylation of STAT3 in Tyr705.5.Physapubescin B could surppress the nuclear translocation of p-STAT3(Tyr705)in MGC803 cells.6.Physapubescin B could inhibit the transcription activity of STAT3 in a does-dependent manner.7.Physapubescin B inhibited the transcription and expression level of XIAP?CyclinD 1 and C-myc in MGC803 cells.8.Physapubescin B had significantly anticancer activity in vivo and the anti-tumor rate of 60mg/kg physapubescin B was 47.11 ± 12.85%.Conclusion:Our study showed that physapubescin B could induce apoptosis in human gastric cancer cells through inhibition of JAK2/STAT3 signaling pathway as a JAK2 inhibitor in vitro and vivo.These result indicated that physapubescin B had the potential to as a anticancer drug that targets the JAK2/STAT3 signaling pathway in human gastric cancer.