Polyphyllin D-induced Monocytic Differentiation Of HL-60Cells And Its Mechanism

Objective: Paris polyphylla is a liliaceous medicine herb widely dustributed inAsia, especially Yunnan province in China. Its roots can be used in traditional medicinefor antalgic, antipyretic, anti-inflammatory, antibacterial, and antitumor properties andso on. Recently, Polyphyllin I, Polyphyllin II, Polyphyllin D are extracted from the rootof P.polyphylla. Acute promyelocytic leukemia (APL) is a relatively common anddangerous acute leukemia. APL is common in young,90%of which patients is easy tobleed caused by disseminated intravascular coagulation (DIC). Differentiation therapy iswidely adopted in leukemia in clinical. In our study, HL-60was used to study the role ofPolyphyllin D extracted from Parispolyphylla in the differentiation of HL-60cells andfurther to explore its possible molecular mechanism of differentiation.Methods:1. HL-60was chosen as the subject of research.2. Groups of experiment:(1) Control: HL-60cells were treated with0.05%DMSO.(2) Experimental groups:1)0.05μM PD group: HL-60cells were treated with different concentrations ofPolyphyllin D0.05μM for72h,2)0.1μM PD group: HL-60cells were treated withdifferent concentrations of Polyphyllin D0.1μM for72h,3)0.2μM PD group: HL-60cells were treated with different concentrations of Polyphyllin D0.2μM for72h.(3)Inhibitor groups:1) P38inhibitor groups: SB203580: HL-60cells were treated with10μmol/l P38inhibitor SB203580for30min, SB203580+PD: HL-60cells weretreated with0.1μM Polyphyllin D for72h after10μmol/l P38inhibitor SB203580for30min pretreatment,2) ERK1/2inhibitor groups: U0126: HL-60cells were treatedwith20μmol/l ERK1/2inhibitor U0126for30min, U0126+PD: HL-60cells weretreated with0.1μM Polyphyllin D for72h after20μmol/l ERK1/2inhibitor U0126for 30min pretreatment.3. HL-60cells were treated with different concentrations (0、0.025、0.05、0.025、0.05、0.4、0.8、1μM) of Polyphyllin D at different time points (24h,48h,72h), meanwhile, the cell viability was tested by MTT test.3. The mononuclear cellsurface CD14was detected by flow cytometry in control, experimental and inhibitorgroups.4. The morphology of HL-60cell treated with0.1μM PD and control wasobserved by Wright-Giemsa staining.5. The protein changes in HL60were detected byWestern blot for P-ERK1/2, ERK1/2, P-P38, P38.Results:1. The proliferation of HL-60cell was obviously inhibited at dose andtime dependence by MTT test after treated with different concentrations of PolyphyllinD.2. Compared with control group, the morphology of HL-60cell treated with0.1μMPolyphyllin D changed: nuclear of one side became flat or depressed andcytoplasm staining became shallow in several cells.3. With the increase ofconcentrations of PD (0,0.05,0.1,0.2μM), the levels of CD14significantly increased,and the results of CD14increased from2.93%to8.22%,23.33%,42.65%; treated withPD+U0126, CD14increased to11.3%; treated with SB203580and PD+SB203580,levels of CD14were6.96%and33.8%, respectively.4. Western blot results showed thatPolyphyllin D could upregulate the expression of ERK1/2and downregulate theexpression of P-P38; ERK1/2inhibitor U0126decreased P-ERK1/2; P38inhibitorSB203580upregulated P-ERK1/2. ERK1/2was referred as the total.(p<0.05)Conclusion:Polyphyllin D could activate ERK to induce the monocytic differentiation ofHL-60cells, the study might provide the basis for the oncotherapy of Polyphyllin D anda potential position in treatment of APL.