Effects And Mechanism Of MTOR Inhibitor Rapamycin On Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia (CML) is a type of common chronicleukemia in China, which can occur in various age groups. Traditional drugssuch as hydroxycarbamide and IFN can relieve some of the patients'symptoms, but they can not cure the disease or block its process. Most of thepatients entered accelerated or blastic phases after a period of chronic phase,and died in hematopoietic failure or complications. bcr/abl fusion gene is amain molecular biological characteristic of CML. Its encoding productBcr/Abl kinase is a critical factor in pathogenesis of CML. Inhibitors ofBcr/Abl kinase (such as imatinib) have achieved satisfactory effects in thetreatment of patients with CP CML and become the first line therapeutic drug.However, there are always some patients that resistance to Bcr/Abl kinaseinhibitors. Studies have shown that Bcr/Abl kinase had intensive tyrosinekinase activity. It could activate many important signaling pathways includingRas/MAPK, Jun/STAT5and PI3K/Akt/mTOR, in sequence affect cell cycledistribution and cell apoptosis. Some studies have proved that mTORcontributed to tyrosine kinase inhibitors (TKIs) resistance of CML, andinhibition of mTOR signaling could enhance the sensitivity ofBCR/ABL-positive cells to TKIs.Mammalian target of rapamycin (mTOR) is a kind of serine/threoninekinase and plays a critical role in many physiological functions such as proteintranslation, cell cycle distribution, cell proliferation and apoptosis.4E-BP1and p70S6K are two major molecules in downstream of mTOR. Theyundertake most of the mTOR signaling's functions. When mTOR signaling isactivated, the phosphorylations of4E-BP1and p70S6K are increased. Asresults, the synthesis of various proteins is dysregulated both at transcriptionand translation level. Abnormal activation of mTOR signaling could be observed in many kinds of tumor cells, such as gastric cancer, small cell lungcancer, cervical cancer, ovarian cancer and hepatocellular carcinoma.Activation of mTOR signaling might also be involved in the tumorigenesisand progress of some hematological malignants, for example acute myeloidleukemia and lymphoma. Furthermore, PI3K/Akt/mTOR signaling wasrequired in the malignant transformation of Bcr/Abl-positive cells and initialcellulars survival. Studies showed that mTOR inhibitors (rapamycin andRD001) significantly inhibited the growth of various tumor cells in vitro. Theprecise mechanism of mTOR in pathogenesis of CML needs to be clarified,and mTOR may become a therapeutic target of CML in the furtuer.In the present study, to study the role of mTOR signaling in pathogenesisof CML and the mechanism of potential therapeutic effects of rapamycin onCML, we will detect four parts in our study:1. Expression of mTOR signalingin CML bone marrow cells;2. Effects of rapamycin on cell cycle distributionand apoptosis in K562cells;3. Mechanism of cell cycle arrest and apoptosisinduced by rapamycin in K562cells;4. Effcets of rapamycin combined withcelecoxib treatment on K562cells. The aim of this study is to explore thefunction of mTOR signaling in pathogenesis and progression of CML, and toprovide theoretical evidence for targeting mTOR signaling in CML therapy.This study includes four parts:Part ⅠExpression of mTOR signaling in CML bone marrow cellsObjective: To explore the possible role of mTOR signaling inpathogenesis of CML by detecting the expression of mTOR and itsdownstream effectors,4E-BP1and p70S6K, in CML bone marrow cells.Methods: Thirty-four patients with chronic phase CML included in thisstudy were collected from the Second Hospital, Hebei Medical University andHebei General Hospital between June2009and December2010. Theexpression of mTOR,4E-BP1and p70S6K and their phosphorylation in CMLbone marrow cells was detected by Western Blot.Results:1.1Expression of mTOR and p-mTOR in CML bone marrow cells Western Blot analysis showed that the positive expression rate of mTORin bone marrow cells with CML showed no statistically significant differenceas compared with that in normal bone marrow cells (P>0.05). However, theexpression of p-mTOR in bone marrow cells with CML was obviouslyincreased, compared with that in normal bone marrow cells (70.6%vs30.0%,P<0.05). There was no relationship between the expression of mTOR and itsphosphorylation and patient's age and gender (P>0.05).1.2Expression of4E-BP1and p-4E-BP1in CML bone marrow cellsThe positive expression rate of4E-BP1in CML bone marrow cellsshowed no statistically significant difference as compared with that in normalbone marrow cells (P>0.05). The expression of p-4E-BP1in CML bonemarrow cells was significant increased as compared with that in normal bonemarrow cells (76.5%vs40.0%,P<0.05). The expression and phosphorylationof4E-BP1showed no relationship with patient's age and gender (P>0.05).1.3Expression of p70S6K and p-p70S6K in CML bone marrow cellsWestern Blot analysis showed that the positive expression rate of p70S6Kin CML bone marrow cells showed no statistically significant difference ascompared with that in normal bone marrow cells (P>0.05). The higherexpression of p-p70S6K was detected in in bone marrow cells with CML,compared with that in normal bone marrow cells (73.5%vs20.0%,P<0.05).However, there was no relationship between expression of p70S6K, p-p70S6Kand patient's age and gender (P>0.05).Conclusion:The phosphorylation level of mTOR,4E-BP1and p70S6Kin CML bone marrow cells was significantly increased, indicating that mTORsignaling was activated and played an important role in pathogenesis of CML.Part Ⅱ Effects of rapamycin on cell cycle, proliferation and apoptosis inK562cellsObjective: To explore Effects of rapamycin on cell cycle, proliferationand apoptosis in K562cells.Methods: The proliferation, cell cycle distribution and apoptosis of K562cell was detected by Flow cytometry (FCM) assay, Western Blot and RT-PCR after treated with different concentrations of rapamycin.Results:2.1Effects of rapamycin on the survival rates in K562cellsMTT result showed obvious inhibition of rapamycin on the proliferationof K562cells. The cell survival rates in groups treated with rapamycindisplayed a significant decrease in a dose-dependent manner ranging from20to1000nmol/L (P<0.05). The cell survival rates in1nmol/L,5nmol/L and10nmol/L rapamycin treatment groups showed no statistically significantdifference as compared with that in control group (P>0.05).2.2Rapamycin arrests K562cells at G0/G1phaseExponentially growing K562cells were treated with rapamycin for24hand48h, and detected by flow cytometry. After treated with rapamycin for24h the proportion of cells in G0/G1phase in treated groups was significantlyincreased, compared with that in solvent control group (29.56±2.89, P<0.05).With regard to S phase cells, the decreased population was accompanied withthe increase of G0/G1phase cells. No difference was found in G2/M cellpopulations after treatment with rapamycin, as compared with the controlgroup. Furthermore, the significant increase of G0/G1phase cells and decreaseof S phase cells were found in groups with rapamycin treatment for48h, aswell as the treated groups for24h. These results indicated that rapamycincould arrest K562cells at G0/G1phase.The FCM analysis also showed that the proportion of cells at G0/G1phasein groups treated with20nmol/L,40nmol/L,80nmol/L, and160nmol/Lrapamycin for48h was significantly increased, compared with that for24h(P<0.05). The proportion of cells at G0/G1phase in10nmol/L rapamycintreatment group showed no obvious change no matter how long the treatmentperiod was (P>0.05). However, the proportion of cells at S phase in treatedgroups with10nmol/L~80nmol/L rapamycin for48h was obviouslydecreased, compared with that treated for24h (P<0.05). As regard to theproportion of cells at S phase in160nmol/L rapamycin treatment groups,there was no difference between treatment for24h and48h (P>0.05). 2.3Effects of rapamycin on CyclinD1, p21, and CyclinB1in K562cellsRT-PCR analysis showed the lower expression of CyclinD1mRNA ingroups with20nmol/L,40nmol/L and80nmol/L rapamycin treatment than incontrol group (P<0.05). On the contrary, higher expression of p21at mRNAlevel was detected in cells with40nmol/L and80nmol/L rapamycin treatment(P<0.05). Expression of CyclinB1show no variety in K562cells afterrapamycin incubation.2.4Rapamycin induced apoptosis in K562cellsTo determine if rapamycin resulted in increased apoptosis of K562cells,we studied apoptosis after treatment of rapamycin with increasingconcentrations for24h. The percentage of apoptotic cells in treated groupswith20,40and80nmol/L was significantly higher than in control group(P<0.05). The result indicated that treatment with20nmol/L or higherconcentrations of rapamycin increased apoptotic cells, including early and lateapoptotic cells, in a concentration dependent manner.Furthermore, the percentage of early apoptotic cells of K562cells ingroups treated with20nmol/L,40nmol/L and80nmol/L rapamycin was9.53±1.10%,11.47±0.90%and15.90±1.67%respectively, significantly higherthan in solvent control group (P<0.05). Meanwhile, the higher percentage oflate apoptotic cells of K562cells was detected in groups with40nmol/L and80nmol/L rapamycin treatment (P<0.05), and no significant difference wasfound in20nmol/L rapamycin treatment group (P>0.05).2.5Effects of rapamycin on Caspase-3and Bcl-2in K562cellsTo study the underlying apoptotic mechanisms, expression of apoptosisregulators, caspase-3and bcl-2, were assessed after treatment with20,40and80nmol/L rapamycin. Caspase-3(32kD) was detected in all groups includingrapamycin treatment groups and solvent control group. However, thecaspase-3activity (17kD) was only detected in K562cells in treated groups.It indicated that rapamycin could activate Caspase-3in K562cells, and mightinduce apoptosis through Caspasse-3activation.The relative expression quantity of Bcl-2protein in treated groups with 40nmol/L and80nmol/L rapamycin was0.733±0.123and0.603±0.095,respectively, significantly lower compared with that in solvent control group(1.080±0.137, P<0.05).Conclusion:Rapamycin, as the inhibitor of mTOR, could arrest K562cells at G0/G1phase by regulationg key factors of the cell cycle and induceapoptosis by decreasing Bcl-2expression and activating Caspase-3.Part Ⅲ Effects of rapamycin on expression of mTOR,4E-BP1and p0S6Kin K562cellsObjective: To explore the possible mechanism of cell growth inhibitionand apoptosis induced by rapamycin in K562cells, the expression of mTORand its downstream molecules was studied.Methods: The expression of mTOR,4E-BP1and p70S6K at mRNA andprotein level was detected by RT-PCR and Western Blot, respectively.Results:3.1Effects of rapamycin on mTOR expression in K562cellsThe expression of mTOR at protein level in rapamycin treatment groupsshowed no statistically significant difference as compared with that in controlgroup (P>0.05). However, the lower expression of p-mTOR was found inrapamycin treatment groups (P<0.05).The expression of mTOR at mRNA level detected by RT-PCR showedthe coincident result with at protein level investigated by Western Blot.3.2Effects of rapamycin on4E-BP1expression in K562cellsWestern Blot showed that the expression of4E-BP1and p-4E-BP1ingroups with rapamycin treatment was significantly decreased, compared withthat in control group (P<0.05). Moreover, RT-PCR also displayed thesignificantly lower expression of4E-BP1mRNA in rapamycin treatmentgroups (P<0.05). It indicated that rapamycin could decrease the expression of4E-BP1in K562cells.3.3Expression of p70S6K and p-p70S6K in K562cells after rapamycintreatmentThe lower expression of p70S6K and p-p70S6K was investigated in rapamycin treatment groups (P<0.05). Accordingly, the mRNA expression ofp70S6K in groups with20nmol/L,40nmol/L and80nmol/L rapamycintreatment was significantly decreased detected by RT-PCR (P<0.05). Theresults indicated that rapamycin could play its role by inhibiting p70S6K.Conclusion:Rapamycin might have an effect on cell cycle and apoptosisby inhibiting mTOR and its downstream substrates (4E-BP1and p70S6K),and then inhibit the growth of tumor cells.PART ⅣEffects of rapamycin combined with celecoxib on mTORsignaling in K562cellsObjective: To explore effects of rapamycin combined with celecoxib onmTOR signaling by detecting proliferation and apoptosis of K562cells.Methods: The treated concentration was determined by MTT, and thenK562cells were incubated with rapamycin (20nmol/L), celecoxib (10μmol/L)and rapamycin combined with celecoxib. The apoptosis of K562cells wasmeasured by flow cytometry, and the expression of mTOR,4E-BP1andp70S6K was detected by RT-PCR and Western Blot as before.Results:4.1Effects of celecoxib on the survival rates in K562cellsMTT assay showed that celecoxib had a potent inhibitory effect on theproliferation of K562cells (P<0.05). The cell survival rates in celecoxibtreated groups displayed a significant decrease in a dose-dependent mannerranging from10to160μmol/L (P<0.05). The cell survival rate in5μmol/Lcelecoxib treatment group showed no statistically significant difference,compared with that in control group (P>0.05).4.2Effects of rapamycin combined with celecoxib on the survival rates inK562cellsMTT study showed the lower survival rate of K562cells was investigatedin the group with two drugs, compared with rapamycin, celecoxib or no drug(P<0.05), indicating that rapamycin combined with celecoxib could inhibitK562cells growth.4.3Rapamycin combined with celecoxib induced K562cells apoptosis Cells Apoptosis induced by rapamycin combined with celecoxib wasdetermined by annexin V/PI apoptosis detection kit. The result showed that theapoptosis rate in groups with rapamycin treatment, celecoxib treatment andrapamycin combined with celecoxib treatment were significantly higher thanthat in solvent control group (P<0.05). And, more apoptosis cells weredetected in the group treated with two drugs than treated with one drug(P<0.05).Furthermore, the early apoptosis of K562cells incubated with rapamycinwas obviously increased as compared with solvent control (P<0.05). On thecontrary, the late apoptosis cells was more in the group with celecoxibtreatment than in solvent control group (P<0.05). The rate of the earlyapoptosis and the late apoptosis in rapamycin combined with celecoxibtreatment group was10.10±1.37%and5.77±0.83%, respectively, significantlyhigher as compared with that in solvent control group (P<0.05). These datasshowed that rapamycin or celecoxib could induce apoptosis in K562cells, andthe rapamycin combined with celecoxib was able to enhance the role of onedrug. It indicated that Rapamycin and celecoxib might have synergistic effectsin inducing apoptosis of K562cells.4.4Effects of rapamycin combined with celecoxib on major molecules ofmTOR signaling in K562cellsWestern Blot showed the expression of mTOR and its phosphorylation inthe group treated with rapamycin and celecoxib was significantly decreased ascompared with that in control group (P<0.05). With regard to expression ofmTOR, there was no significant difference between the K562cells incubatedwith one drug and solvent (P>0.05), however, the lower phosphorylation levelof mTOR was found in two groups treated with one drug (P<0.05). RT-PCRshowed lower mRNA expression of mTOR in K562cells treated by bothrapamycin and celecoxib, as well as the data detected by Western blot. Theresults indicated that rapamycin combined with celecoxib could affect theexpression of mTOR and its phosphorylation level in K562cells.Expression of4E-BP1and its phosphorylation in K562cells incubated with one or both drugs was significantly decreased as compared with that incontrol group (P<0.05). RT-PCR showed the lower expression of4E-BP1mRNA in K562cells, accordance with protein expression.With regard to p70S6K, the lower expression of both at mRNA andprotein level was investigated in the groups treated with rapamycin, celecoxiband rapamycin combined with celecoxib (P<0.05). Interestedly, the expressionof p70S6K in K562cells incubated with both drugs was significantlydecreased as compared with only one drug (P<0.05). Moreover, thephosphorylation level of p70S6K was obviously decreased in the group withrapamycin combined with celecoxib treatment, as well as the expression ofp70S6K studied by Western blot.Conclusion: Rapamycin combined with celecoxib could inhibit cellproliferation and induce apoptosis,and increased the inhibitory effects inK562cells than single agent.Conclusions:1The phosphorylation level of mTOR,4E-BP1and p70S6K in CML bonemarrow cells was significantly increased, indicating that mTOR signalingwas activated and played an important role in pathogenesis of CML.2Rapamycin, as the inhibitor of mTOR, could arrest K562cells at G0/G1phase by regulationg key factors of the cell cycle and induce apoptosis bydecreasing Bcl-2expression and activating Caspase-3.3Rapamycin might have an effect on cell cycle and apoptosis by inhibitingmTOR and its downstream substrates (4E-BP1and p70S6K), and theninhibit the growth of tumor cells.4Rapamycin combined with celecoxib could inhibit cell proliferation andinduce apoptosis,and increased the inhibitory effects in K562cells thansingle agent.