Effects Of Tetramethylpyrazine On The Proliferation And Differentiation In Hl-60Cells And In Modeling Scid Mice

ObjectivesHuman acute promyelocytic leukemia HL-60cells and umbilical veinendothelial ECV-304cells were selected in the study. On the basis of thefunction of resisting anoxia and antiangiogenesis in TMP, we detected theeffect of the tetramethylpyrazin(eTMP)alone or in combination of arsenictrioxide(As₂O₃) on proliferation and differentiation in human acutepromyelocytic leukemia HL-60cells, and investigated the possiblemechanism based on the molecular of VEGF and H1F-1a. As well toinvestigate TMP could potentiate As₂O₃activity against leukemia further invivo, we establish the SCID mice model suffering from human AML.Methods1. Effects of TMP on proliferation and differentiation in HL-60cells.The alteration in the cell proliferation of TMP(concentration of100,200,300,400,600,800,1000μg/ml)was detected by MTT experiment; Thefunctional maturity of differentiation in HL-60cells was determined by NBTtest, and filter the suitable concentration in the following experiments; Cell morphology was observed by Wright's staining; Flow cytometry was used todetect the expression of CD11b/CD14and the cell cycle distribution inHL-60cells. The cell cycle related mRNA and protein such as c-myc,p27,CDK2and cyclinE1were detected by RT-PCR and Western blot.2. Effects of TMP alone or in combination of As₂O₃on proliferation inHL-60cells. HL-60cells and ECV-304cells were used in this partialexperiment. There were four groups: control group, TMP treated group,As₂O₃treated group and TMP+As₂O₃treated group. The alteration in theproliferation was detected by MTT; RT-PCR and ELISA were assayed toexamine the expression of VEGF mRNA and protein; Trypan blue stainingwas down to draw the growth curves of ECV-304cells; Flow cytometry wasused to determine the early apoptosis of ECV-304cells; the adhesion degreeof ECV-304cell was observed by inverted microscopy.3. Effects of TMP alone or in combination of As₂O₃on proliferation inHL-60cells. There were four groups: control group, TMP treated group,As₂O₃treated group and TMP+As₂O₃treated group. The alteration in thecell proliferation was detected by MTT experiment; The functional maturityof differentiation in HL-60cells was determined by NBT test, and filters thesuitable concentration in the following experiments; Cell morphology wasobserved by Wright's staining; Flow cytometry was used to detect theexpression of CD11b/CD14and the cell cycle distribution in HL-60cells.The cell cycle related mRNA and protein such as c-m yc, p27,CDK2and cyclinE1were detected by RT-PCR and Western blot assay.4.As well to investigate TMP could potentiate As₂O₃activity againstleukemia further in vivo, we establish the SCID mice model suffering fromhuman AML. HL-60cells (1×10⁶) were injected into SCID mice via a tailvein following irradiated at300cGy (⁶⁰Co source)24h earlier. Theexpression of CD33in bone marrow mononuclear cells detected byimmunohistochemistry and the infiltrated organization observed by HEstaining were used to identify whether the model is successfully established.The SCID mice models were randomly divided into four groups: controlgroup, TMP treated group (200mg/kg, i.p., Qd×21d), As₂O₃treated group(3mg/kg, i.p., Qd×21d), TMP+As₂O₃treated group (200mg/kgTMP+3mg/kg As₂O₃, i.p., Qd×21d). The mice were put to death afteranesthesia at the death hour. The liver and kidney function was detected aftergotten their serum; the expression of the secreted VEGF protein wasexamined by ELISA; Bone Marrow Microvessel density (BMMVD) wasscored using immunofluorescence; the expression of CD33and HIF-1αprotein in bone marrow mononuclear cells detected byimmunohistochemistry.Results1. TMP inhibited the proliferation of HL-60cells in a dose andtime-dependent manner. TMP induced the differentiation of HL-60cells inthe relatively low concentration (200-300μg/ml). After exposed to TMP in the300μg/ml concentration, the cells were blocked in the G0/G1cell cycleprogression. The mRNA and protein expression of c-myc were graduallydecreased, while remarkably up-regulated in the expression of p27. Therewere no obviously changes in the mRAN expression of CDK2and cyclinE1;however the protein expressions of them were extantly declined in atime-dependent manner.2. TMP can obviously potentiate As₂O₃ability of inhibitingproliferation in HL-60and ECV-304cells. The mRNA expression andsecretory protein of VEGF remarkably declined after exposure to thecombination treatment. Both of TMP and As₂O₃can induce the earlyapoptosis and adhesion ability of ECV-304cells, and these effects wereenhanced when treated combined.3. Combination treatments had synergistic effects on the proliferativeinhibition rates. The rates were increased gradually after the combinationtreatment, much higher than those treated by corresponding As₂O₃alone.The cells exhibited characteristics of mature granulocytes and a higherNBT-reducing ability, being a2.6-fold increase in the rate of NBT-positiveratio of HL-60cells within the As₂O₃treatment versus almost a13-foldincrease in the TMP+As₂O₃group. Cells treated with both TMP and As₂O₃expressed far more CD11b antigens, almost2-fold compared with controlgroup. The mRNA and protein expression of c-myc and cyclinE1weregradually decreased, while remarkably up-regulated in the expression of p27. There were no obviously changes in the mRAN expression of CDK2;however the protein expressions of them were extantly declined.4. The high expression of CD33protein in mice bone marrowmononuclear cells and an abundant of leukemic cells appeared in micetissues of liver, kidney, spleen, bone marrow and transplanted tumordemonstrated that SCID mice model suffering from human AML hadalready established. The survival time in each experimental group hadsignificant difference. The prolonged rates of survival time were29.63ï¼…inTMP group,50ï¼…in As₂O₃group and74.07ï¼…in combined treated grouprespectively. The liver and kidney function dramatically got better incombined treated group compared with As₂O₃group. CD33protein inBMMC were strongtly positive expressed in control group, moderatelyexpressed in TMP or As₂O₃group, and low expressed in combined group.HIF-la protein were strongtly positive expressed in control group,moderately expressed in As₂O₃group, low expressed in TMP group, andnegative expressed in combined group. VEGF protein in serum incombined group was dramatically decreased. In control group the lumen inmice bone marrow microvascular got much smaller and the microvascularhad many small branches. The bone MVD in each mice treated group wasdramatically decreased than that in control group, and that of which incombined group was decreased than that in other group. The bone MVD ineach treated alone group had no significant difference. Conclusions1. Small doses of TMP can induce differentiations of HL-60cells, andblock the cells in G0/G1cell cycle progression. The mechanism waspossibly by down-regulating the c-myc mRNA and protein expression,up-regulating the p27mRNA and protein expression and inhibiting thecombination of the protein cyclin E and CDK2.2. TMP potentiates As₂O₃inhibiting proliferation ability in HL-60cellsvia the pathways of VEGF autocrine and paracrine. The study preliminarilydemonstrated that antiangiogenesis is the mechanism of TMP alone or incombination of As₂O₃treated AML.3. Small doses of TMP potentiate As₂O₃-induced differentiation ofHL-60cells, possibly by regulating the expression and activity of G0/G1phase-arresting molecules. Up-regulating the expression of p27caninhibiting the combination of the protein cyclin E and CDK2, conductive tothe DNA synthesis and the down-regulation of relative genes such as c-mycneeded in the transition of the G1→S cell cycle progression. Thecoordination and interaction of the upward and downward genes induceddifferentiation of HL-60cells.4. Injecting HL-60cells into pretreated SCID mice via a tail vein cansuccessfully establish SCID mice-human AML model. TMP can potentiateAs₂O₃anticancer ability in SCID mice suffering from AML, reduce the toxicside effect of As₂O₃in liver and kidney function, possibly by down-regulating the expression of HIF-1a and VEGF, inhibiting the growthof the bone marrow microvacular and increasing the apoptosis rate of HL-60cells.