In Vitro Study Of The Proliferative Inhibitory And Abti-inflammatory Effects Of JAK1 Inhibitor SHR0302 On Myproliterative Neoplasms

Objective To explore the effects and the molecular mechanism of the selective JAK1 inhibitor SHR0302 on myeloproliterative neoplasms patients and cell lines UKE1,SET2 in vitro,and to compare with Ruxolitinib to clarify the inhibitory effect of SHR0302 on JAK1 pathway and the difference with Ruxolitinib.Methods CCK8 assay was used to detect the effect of SHR0302 and Ruxolitinib at different concentrations on the proliferation of UKE1 and SET2 cells at different time;The effect of SHR0302 and Ruxolitinib at different concentrations on the colony formation ability of MPN cell lines and patients’ primary cells were tested by colony formation assay;RT-PCR was used to detect the effect of SHR0302 and Ruxolitinib at different concentrations on the expression levels of TNF-α and IL-1β mRNA of UKE1 and SET2 cells after 3h;Multi-factor assay kit MSD was used to detect the effects of SHR0302 and Ruxolitinib at different concentrations for 24h on the protein levels of ten inflammatory factors in UKE1 and SET2 cells;Flow cytometry Annexin V/PI double staining assay was used to detect the effect of SHR0302 at different concentrations on apoptosis of UKE1 and SET2 cells for 48h;Western blot analysis was performed to detect the effect of SHR0302 and Ruxolitinib at different concentrations on the phosphorylation level of protein in the JAK-STAT signaling pathway after 3h.Results SHR0302 inhibited the proliferation of UKE1 and SET2 cells in a time-dependent and concentration-dependent manner.The IC50 of SET2 and UKE1 cells which were treated with SHR0302 for 72h was 1.611μmol/L and 5.034μmol/L,respectively.The IC50 of Ruxolitinib for 72h was 103.4nmol/L and 3.276μmol/L,respectively.Compared with the control group,SHR0302 inhibited MPN cell lines and patient colony formation in a concentration-dependent manner after 24h.5μmol/LSHR0302 inhibited colony formation in UKE1 and SET2 cells by 32%and 69%,respectively,and inhibited colony formation in primary red line cells(BFU-E)of MPN patients(2PV,2ET)by 33%,48%,34%and 47%,respectively.The inhibition rate of Rxolitinib on BFU-E in MPN patients(2PV,2ET)at 0.4μmol/L was 43%,50%,60%and 73%,respectively.SHR0302 inhibited TNF-α and IL-1β mRNA expression levels in UKE1 and SET2 cells in a concentration dependent manner at 1μmol/L(P<0.05)and Ruxolitinib at 0.1μmol/L(P<0.05)after 3h,so SHR0302 inhibited TNF-α and IL-1βmRNA expression levels less than Ruxolitinib.SHR0302 and Ruxolitinib inhibited the expression of ten inflammatory cytokines in SET2 and UKE1 cells in a concentration-dependent manner at 24h,respectively.SHR0302 reduced SET2 and UKE1 cell inflammatory factor protein expression levels by about 50%at 1.6μmol/L and 2.5μmol/L concentrations,respectively(P<0.05),which was equivalent to that of Ruxolitinib at 1μmol/L.SHR03 02 was also found to be weaker than Ruxolitinib in the expression of inflammatory factor protein.After 48h with SHR0302,the apoptosis rate of MPN cell lines increased with increasing drug concentration compared with the control group(P<0.05).The JAK-STAT signaling pathway was significantly inhibited by SHR0302 in SET2 and UKE1 cells at different concentrations for 3h,p-STAT(Tyr701)and p-STAT3(Tyr705)were down-regulated at 1μmol/L,p-JAK1(tyr1022/1023)and p-STAT5(Tyr694)were down-regulated at 5μmol/L,and the differences were statistically significant(P<0.05).The reference drug Ruxolitinib significantly inhibited the downstream STAT protein at 0.1μmol/L,the activity of p-STAT5(Tyr694)was stronger than that of p-STAT1(Tyr701)and p-STAT3(Tyr705).The inhibitory effect of SHR0302 on the JAK-STAT signaling pathway was weaker than that of Ruxolitinib.Conclusion SHR0302 can effectively inhibit proliferation and promote apoptosis of MPN cell lines and patients’ primary cells and inhibit the expression of inflammatory factors by down-regulating p-JAK1 and its downstream p-STAT1,p-STAT3 and p-STAT5 protein phosphorylation levels,indicating that SHR0302 is a promising new drug for the treatment of myeloproliterative neoplasms.SHR0302 has less antiproliferative and anti-inflammatory effects than Ruxolitinib,but its differences with the targets of Ruxolitinib suggest the possibility of combination therapy.