Celastrol Inhibits Differentiation Syndrome Induced By All-trans Retinoic Acid And The Underlying Mechanisms

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia.The main therapeutic regimen for early treatment of APL was Anthracyclines combinedwith cytarabine, but the complete remission rate is very low. And concurrency diffuseintravascular coagulation or primary fibrinolysis often occurred during the process oftreatment, which can cause serious bleeding and eventually leading to early death.All-trans retinoic acid (ATRA) is a landmark drug, which improved the treatment ofAPL. ATRA can induce cell differentiation and apoptosis of APL, and after treatment withATRA, the rate of4-5years disease-free survival rate of APL patients may reach70-80%.Currently, ATRA has become the top choice for treatment of APL. While during theprocess of ATRA treatment, differentiation syndrome (DS) may occurred in about a quarterof the patients. DS is a potentially fatal complication of ATRA treatment, which ischaracterized by respiratory distress, unexplained fever, weight loss, interstitial pulmonaryinfiltrates, pleural or pericardial effusion, hypertension and acute renal failure, among thempulmonary infiltration is the most common symptom. Early diagnosis of DS and treatmentwith hormone such as dexamethasone can reduce the mortality of DS, but early diagnosisof DS is very difficult and hormone drugs are not recommended for long-term use becauseof its toxicity. To reveal the mechanism of DS development, and to find effective measuresto prevent or treat DS become an important subject, which is urgently to be solved and hasgained extensive attention from researchers.The pathogenesis of DS is microvascular injury, and its typical Symptoms compriseedema, hemorrhage, fibrin effusion, leukocyte infiltration, etc., which is similar toinflammation manifestations. Dexamethasone treatment is effective further supports theevidence that DS has inflammatory characteristics. However, the mechanisms underlyingthis pathological changes is remain unclear, the present research generally believed that theoccurrence of the syndrome includs at least three aspects: Firstly, the increase of cellaggregation in vascular. In the process of ATRA-induced cell differentiation andmaturation, the expression of many important adhesion molecules relate with cell adhesionand aggregation such as intercellular adhesion molecular1(ICAM-1) are increased on thesurface of APL cells, which increase adhesion ability of APL cells (leukemia cells/leukemia cells), leading to cell aggregation and small blood flow slows down or evenblocked. Secondly, the increase of cell migration. After APL cells were induced by ATRA into differentiation and maturation, the expression of adhesion molecules on cell surfaceand the ability of adhere to vascular endothelial cell increase, which contribute to cellmigration through the blood vessel. Finally, the increased ability of the migrated cells toresponse to inflammation. The mature APL cells induced by ATRA secrete factors andenzymes relate with inflammation, trigger reactions like inflammation and cause tissuedamage. Therefore, to stop the pathophysiology process, such as blocking the expressionof adhesion molecules or inhibiting the release of inflammatory factors, was believed to beefficient for the treatment of DS.Chinese herb Tripterygium wilfordii, a celastraceae plants has been used in thetreatment of autoimmune diseases such as rheumatoid arthritis. Celastrol is a triterpenemonomer derived from Tripterygium wilfordii, which has a strong anti-inflammatory effect.Researchs in a variety of animal disease models showed that celastrol has potential roles intreatment of collagen-induced arthritis, asthma, systemic lupus and Alzheimer’s disease.Further studies have revealed that celastrol might exert the anti-inflammatory role throughinhibiting the synthesis and release of inflammatory cytokines such as TNF-alpha andIL-1beta, or blockinging the effects induced by the interaction between inflammatory factorsand their targets, such as, inflammatory factors can cause endothelial cell activation andinduce the expression of adhesion molecules including ICAM-1, VCAM-1and E-selectin,while which can be significantly reversed by celastrol.In the previous study, we established an inflammatory response cell model throughstimulating primary cultured human umbilical vein endothelial cells (HUVEC) withinflammatory cytokine, and found that treatment with celastrol could inhibit the expressionof adhesion molecules induced by inflammatory factors and adhesion, including ICAM-1,VCAM-1and E-selectin. Compared to dexamethasone, ibuprofen and other drugs commonlyused for anti-inflammatory, celastrol showed far stronger ability to inhibit the adhension betweenactivated endothelial cells and leukocyte. Given the molecular basis of pulmonaryinfiltration of DS is the expression of a variety of adhesion molecules on leukemia cellsurface and the secretion of inflammatory cytokines induced by ATRA, in the present studywe used acute promyelocytic leukemia cell line NB4cell as cell model, and found thatcelastrol could inhibit ATRA-induced increase of adhesion of leukemia cells to endothelialcells. Based on these results, we hypothesized that celastrol might be useful in thetreatment of DS.1. Celastrol inhibits differentiation syndrome caused by all-trans retinoic acid The previous study found that celastrol could inhibit the adhesion between leukemiacells and endothelial cells induced bu ATRA, suggesting celastrol may be used in thetreatment of differentiation of syndrome (DS). But whether celastrol will interfere with theeffect of ATRA on inducing APL cells differentiation? To answer the question we first toinvestigate the effects of celastrol on cell differentiation and maturation of ATRA-inducedNB4cells. The results showed that, after treatment with ATRA NB4cells appearing themorphological characteristics of granulocyte differentiation such as increase of the amountof cytoplasm, deviation of cell nucleus, and some nucleus changed to kidney-shaped,rod-shaped or lobulated nucleus. NB4cells showed no obvious changes after treatmentwith celstrol together with ATRA compared to treatment with ATRA alone. The results ofnitro blue tetrazolium (NBT) test revealed that celastrol showed synergy effect with ATRAon inducing NBT reduction reaction capability of NB4cells. For regulating the expressionof differentiation antigens, in addition to CD33celastrol showed no effect on the change ofother differentiation antigen induced by ATRA, such as CD11b, CD13and CD15. Theresults above demonstrated that celastrol do not affect the ability of ATRA to induce APLcell differentiation and maturation.Celastrol potent to inhibit the adhesion between leukemia cells and endothelial cellsinduced by ATRA, without interfering with the ability of ATRA in inducing differentiationand maturation of leukemia cells. Therefore, it is worth to further study whether celastrolcan be used as a new drug to treat DS. According to the literature, severeimmunodeficiency (NOD/SCID) mice were introduced to build DS mouse model throughinjection differentiated NB4cells, which were induced in vitro, via the tail vein, aftertreatment with ATRA through oral administration or with celastrol through intraperitonealinjection, the mice were sacrificed, and lungs were collected. The results ofimmunohistochemistry showed that after treatment with ATRA, cell infiltration wasinduced, while which can be suppressed by celastrol.In conclusion, the results showed above suggest that celastrol can inhibitATRA-induced NB4cells pulmonary infiltrates without interfere with the ability of ATRAto induce NB4cells differentiation and maturation.2. Celastrol inhibits the expression of ICAM-1on NB4cells induced by ATRAA variety of factors are involved in the ATRA-induced tissue infiltration, but theincrease of adhesion between cells is the most important for cell transmigration and tissue infiltration in the primary stage of DS develop. During the differentiation of APL cellsinduced by ATRA, a large number of molecules associated with cell adhesion expressed onthe cells surfaces, making APL cells easier to traverse the blood vessels and infiltrate thelungs, eventually resulting in ultra-inflammation reactions. So, we hypothesized that thesupression effect of celasreol on DS may be related with the inhibition of the expression ofcell adhesion molecules. To verify this hypothesis, we investigated the effects of celastrolon the expression of many adhesion molecules in ATRA-induced leukemia cell.Firstly, in vivo experiment was carried out in animal model of DS. By using flowcytometry analysis we found that the expression of ICAM-1was increased in leukemiacells of peripheral blood of DS mice, which were reversed after treatment with celastrol.The result indicated that, celastrol inhibits leukocyte lung infiltration induced by ATRAmight be due to its inhibition role in ATRA-induced cell adhesion. To further reveal themechanism underlying celastrol inhibit leukocyte pulmonary infiltrate induced by ATRA,NB4cells were intrduced as cell model. The results showed that after ATRA induction, theexpressions levels of CD11a, CD11b, CD11c, CD49d are elevated with or without celastrol.VCAM-1and E-selectin are important in white blood cells traversing blood vessels, andthey are not been induced by ATRA or celastrol. Interestingly, ICAM-1, an importantmolecular in the early DS, which can be significantly induced by ATRA, and this inductioncan be obviously inhibited by celastrol.Inflammatory factors and chemokines which are secreted from leukemia cells are alsoimportant for DS development. In the following study, we found that inflammatory factorIL-1βand TNF-α, chemokines MCP-1and IL-8in NB4cells were increased significantlyafter been treated with ATRA. Co-treated with celastrol do not change the secreation ofMCP-1and IL-8, while IL-1βand TNF-αsecretion was significantly inhibited.These results suggest that, in vivo, celastrol can inhibit the expression of leukemiacell adhesion molecule; In vitro, celastrol can significantly inhibit the expression ofICAM-1and the secretion of IL-1βand TNF-αinduced by ATRA, but show no effect on theATRA-induced secretion of MCP-1and IL-8. These findings suggest that celastrolinhibited ATRA-induced cell lung infiltration is related with its inhibition ofATRA-induced ICAM-1expression.3. Celastrol inhibits ATRA-induced expression of ICAM-1through impairingMEK-ERK pathway Many studies have shown that the elevated expression level of ICAM-1is one of theimportant molecular factors for DS. Our previous results have demostrated that celastrolinhibited ATRA-induced cell lung infiltration is related with its inhibition ofATRA-induced ICAM-1expression. However, how celastrol exert the role in inhibiting theexpression of ICAM-1induced by ATRA is still unknown. In the following study, we usedmany small molecule inhibitors combined with Western blot to screen the signalingpathways which are related with the regulation of ICAM-1and try to identify themechanism underlying celastrol inhibits ICAM-1expression in ATRA-induced NB4cells.We and other research groups found celastrol is a novel HSP90inhibitor. HSP90regulates the expression of ICAM-1by binding to IKK and inhibiting the NF-κB activity.Therefore, we firstly used17-AAG, an HSP90inhibitor, to treat NB4cells. To our surprise,instead of inhibitory effect,17-AAG has synergy effect with ATRA to stimulate theexpression of ICAM-1in NB4cells. NF-κB is an important transcription factor in theregulation of ICAM-1-induced expression, and celastrol shows a strong inhibitory effect onNF-κB activation in a variety of cell models. Then we next to observe the impact of ATRAon NF-κB activation and found that ATRA can activate NF-κB of NB4cells (P65).Addition of celastrol can significantly inhibited NF-κB activation caused by ATRA.However, NF-κB inhibitor BAY11-7802and PDTC do not inhibit the ATRA-inducedICAM-1expression. The result indicated that celastrol inhibited ATRA-induced ICAM-1expression was not directly through inhibition of NF-κB pathway. STAT1is also involvedin regulating the induced expression of ICAM-1, and it was reported that ATRA canactivate STAT1of NB4cells. In our research, we also found that ATRA treatmentincreased the expression of p-STAT1, which is in accordance with the previous study, andcelastrol treatment can definitly inhibit the activated ATAT1induced by ATRA. While treatNB4cells with STAT1inhibitor AG490also can not inhibit ATRA-induced ICAM-1expression.MAPK signaling pathway is also important in regulating the ICAM-1-inducedexpression. It was reported that ATRA can activate p38and ERK, which are key membersof MAPK signaling pathway. In our study, we found that p-P38and p-ERK expressionwere significantly increased after been induced by ATRA, while p-JNK show nosignificant changes. Addition of celastrol can not inhibit p38activation but suppress ERKactivation which induced by ATRA. Whether celastrol play the inhibitory effect on theATRA-induced ICAM-1expression through inhibiting the activation of ERK? We next stimulate NB4cells with ATRA after pretreatment with ERK inhibitor PD98059. Theresults showed that the increased ICAM-1expression induced by ATRA can be suppressedby PD98059, which means that the role of celastrol in inhibiting ATRA-induced ICAM-1expression is related with the inhibition of ERK phosphorylation. Research hasdemostrated that celastrol directs binding to ERK. In the present study, we used computerprediction to investigat the combination between celastrol and ERK, and found that the C6of celastrol and C164-SH of ERK may form a chemical bond. Given that the activation ofERK is also regulated by the upstream protein MEK, we tried to verify that whethercelastrol interfer with ICAM-1expression induced by ATRA through inhibiting ERKactivation. The results showed that, after stimulating NB4cells with ATRA for15min,MEK phosphorylation level is elevated, and the amount of p-MEK reached the peak at60min, while celastrol may obviously inhibit the phosphorylation of MEK induced byATRA. Similarly, after treating NB4with MEK inhibitor U0126, the expression ofICAM-1induced by ATRA was also inhibited.In summary, in this study we found that celastrol has potential to inhibitdifferentiation syndrome, the fatal side effects of ATRA treatment, without interfering withthe ability of ATRA-induced cell differentiation. We also found that celastrol inhibits theexpression of ICAM-1and the release of inflammatory factor IL-1beta and TNA-alphawhich are induced by ATRA. The further study revealed that celastrol inhibits ICAM-1induced by ATRA through suppression of MEK-ERK signaling pathway. The results of thisstudy will provide new drugs and useful treatment targets for clinical to treat differentiationsyndrome caused by ATRA.