Triptolide Induces Apoptosis In Human Leukemia Cells Through Caspase-3-mediated ROCK1Activation And MLC Phosphorylation

Leukemia is a hematopoietic malignancies which is harmful for human health. Leukemiaranks first among all cancers in children and the people who under the age of35. In recentyears, the incidence and mortality of leukemia keep rising, therefore it is important and urgentto looking for a novel therapeutic strategy for treatment of leukemia. The active ingredientsisolated from natural plant are important sources of anticancer drugs. Triptolide is one of themain active ingredients of traditional Chinese medicine Tripterygium. It has been exhibitedthe anti-infection, immune suppression and other effects, and is used commonly to treatmentarthritis and autoimmune diseases. Recent studies have found that triptolide has been shownto exhibit anti-tumor activity for a variety of cancers including leukemia. However, themolecular mechanism by which triptolide exhibits anti-leukemic activity has not beenexplored in depth.As an important downstream effector of Rho GTPase family members, ROCK1proteinplays an important role in regulating cell shape, cell polarity, cytoskeletal reorganization, cellmigration, and so on. Recent studies indicated that ROCK1plays an important role inregulating cell survival, apoptosis, invasion and metastasis in cancer cells. Several evidencerevealed that ROCK1activity can be regulated by several proteins, such as RhoA or caspase-3.ROCK1is activated usually by binding with activated RhoA which coupled to GTP. ROCK1can be also activated through proteolytic cleavage by capsase-3. ROCK is a serine/threoninekinase that can directly phosphorylate the myosin light chain (MLC) or indirectly increaseMLC phosphorylation by inactivating the myosin phosphatase (MYPT). MLCphosphorylation and inactivation of MLC phosphatase by ROCK1plays importantphysiological roles, such as apoptosis.Our previous studies indicate that triptolide selectively induces apoptosis in humanleukemia cells. However, the molecular mechanism of triptolide-mediated apoptosis has notbeen fully understood. In this study, we investigated the roles of triptolide in apoptosis and cell signaling events in human leukemia cell lines and primary human leukemia blasts byusing several molecular techniques including flow cytometry, Western blotting,immunofluorescence, TUNEL assay, immunohistochemistry, and genetic approaches such assiRNA. We also investigated the antileukemic activity of triptolide in vivo using nude miceleukemia cell xenograft model. Triptolide selectively induced apoptosis, which wasaccompanied by the loss of mitochondrial membrane potential, cytochrome c release,apoptosis-inducing factor accumulation in nucleus, and Bax translocation from the cytosol tothe mitochondria. Our results revealed that caspase-3-mediated ROCK1activation and MLCphosphorylation may play important roles in triptolide-induced apoptosis. Our in vivo studyalso showed that both ROCK1activation and MLC phosphorylation were associated with thetumor growth inhibition caused by triptolide in mouse leukemia cell xenograft models.Collectively, these findings suggest that triptolide-mediated ROCK1activation and MLCphosphorylation may be a novel therapeutic strategy for treating hematological malignancies.Firstly, we studied the effects of triptolide on apoptosis and mitochondrial damagein human leukemia cell lines and primary human leukemia cells.We investigated the dose-and time-dependent effects of triptolide on apoptosis in U937leukemia cells by flow cytometry. We found that triptolide potentially induced apoptosis inU937cells in dose and time-dependent manners. To determine whether triptolide-inducedapoptosis was specific for U937cells, parallel studies were performed using other humanleukemia cell types, including Jurkat T-lymphoblasts and HL-60promyelocytic leukemia cells.These cell lines exhibited apoptotic effects similar to those in U937cells. We nextinvestigated the effects of triptolide on apoptosis in primary leukemia cells. Primary humanleukemia blasts and normal mononuclear cells were isolated from the bone marrow orperipheral blood samples of44leukemia patients and6healthy donors. We detected apoptosisby flow cytometry after exposure of cells to triptolide. Exposure of primary human leukemicblasts to triptolide resulted in a significant increase in apoptosis in a time-dependent manner.However, the same triptolide concentrations and exposure lengths had minimal lethality innormal CD34+cells isolated from the bone marrow of six healthy donors. We alsoinvestigated the effects of triptolide on PARP cleavage, caspases cleavage/activation in U937cells using Western blotting. Consistent with the results related to apoptosis, exposing cells totriptolide resulted in significant increases in PARP cleavage, caspase-3and caspase-9 cleavage/activation. In addition, Jurkat and HL-60cells had comparable degrees of PARPcleavage, caspase-3and caspase-9cleavage/activation. We determined the expression ofapoptosis related proteins by Western blotting after exposure of primary human leukemiablasts and normal mononuclear cells to triptolide. Treating leukemia blasts from four AMLpatients with triptolide also resulted in increases in PARP cleavage, caspase-3and caspase-9cleavage/activation. Triptolide treatment also had no apparent effect on PARP, caspase-3andcaspase-9cleavage in normal CD34+cells.We investigated the dose-and time-dependent effects of triptolide on mitochondrialinjury (loss of mitochondrial membrane potential, m) in U937leukemia cells by flowcytometry. We found that triptolide potentially induced mitochondrial damage in U937cellsin dose and time-dependent manners. We also investigated the effects of triptolide oncytochrome c release from the mitochondria to the cytoplasm, as well as nuclearapoptosis-inducing factor (AIF) accumulation in nucleus in U937cells using Western blotting.Consistent with the results related to apoptosis, exposing cells to triptolide resulted insignificant increases in cytochrome c release, and AIF accumulation in nucleus. Thetime-dependent nuclear AIF accumulation was also observed by immunofluorescence in cellsexposed to triptolide. In addition, Jurkat and HL-60cells had comparable degrees ofcytochrome c release, and nuclear AIF accumulation. We next examined the intracellularlocalization of Bax in mitochondrial and cytosolic fractions using Western blotting. Bax waspredominantly found in the cytosolic fraction of untreated control cells. Bax was translocatedfrom the cytosolic to the mitochondria upon triptolide treatment. We also investigated thesub-cellular localization of Bax during triptolide-induced apoptosis usingimmunofluorescence microscopy. In untreated cells, Bax was mostly distributed in the cytosol;upon triptolide treatment, however, there was a clear shift in Bax localization from the cytosolto the mitochondria. This Bax translocation was also observed in Jurkat and HL-60cellstreated with triptolide. These findings suggest that Bax translocation may contribute toapoptosis in cells that are exposed to triptolide. Taken together, these findings suggest thattriptolide selectively induced caspase-dependent cell death, which was accompanied by theloss of mitochondrial membrane potential, cytochrome c release, AIF accumulation in nucleus,and Bax translocation from the cytosol to the mitochondria. Secondly, we invesitigated the effects of triptolide on ROCK1/MLC signalingpathway.To explore the molecular mechanisms of triptolide induced apoptosis in leukemia cells,we examined the effects of triptolide on ROCK1/MLC signaling pathways by Westernblotting. Exposure of cells to triptolide resulted in increases in cleavage/activation of ROCK1and phosphorylation of MLC and MYPT in dose-and time-dependent manners. SinceROCK1is a major target of RhoA, we also evaluated the effect of triptolide on RhoAactivation. RhoA activation was measured in U937cells treated with triptolide using aGST-RBD (glutathione S-transferase-RhoA binding domain of Rhotekin) pull-down assay.Unfortunately, triptolide treatment did not affect RhoA activation (RhoA-GTP) and totalRhoA levels. We also investigated the effects of C3exoenzyme, a RhoA inhibitor, on ROCK1cleavage and activation during triptolide-induced apoptosis. Pretreatment of U937cells andAML blasts with C3did not diminish the triptolide-mediated increase in apoptosis, PARPcleavage, and caspase3and caspase9cleavage/activation. In addition, pretreatment with C3did not abrogate the ROCK1activation and MLC and MYPT phosphorylation induced bytriptolide. We also examined the effects of caspase inhibitors (z-VAD-fmk or z-DEVD-fmk)on ROCK1cleavage and MLC phosphorylation during triptolide-induced apoptosis.Pretreatment with z-VAD-fmk or z-DEVD-fmk significantly abrogated the triptolide-inducedincrease in apoptosis in both U937cells and primary AML blasts. Cotreatment of triptolidewith z-VAD-fmk or z-DEVD-fmk also blocked the previously observed PARP and caspase-3cleavage. Interestingly, inhibiting caspase activity by z-VAD-fmk or z-DEVD-fmk alsosignificantly blocked the ROCK1activation and MLC phosphorylation mediated by triptolide.These findings suggest that caspase-3is responsible for activating ROCK1and, therefore,phosphorylation MLC during triptolide-induced apoptosis. Taken together, these findingsindicate that caspase-3, rather than RhoA, activates ROCK1in triptolide-induced apoptosis.To further characterize the role of ROCK1/MLC signaling pathway intriptolide-mediated apoptosis in leukemia cells, a specific MLC inhibitor ML-7and a ROCK1inhibitor Y-27632were employed. Pretreating both U937cells and primary AML blasts withML-7dramatically attenuated triptolide-mediated apoptosis. Consistently, co-administeringML-7also reduced the PARP cleavage, caspase3and caspase9cleavage/activation, andcytochrome c release induced by triptolide. Western blots also showed that pretreatment withML-7inhibited the triptolide-mediated MLC phosphorylation. Pretreating U937and AML cells with Y27632dramatically prevented triptolide-mediated apoptosis. Consistent with thesefindings, pretreating of U937cells and AML blasts with Y27632also attenuated the PARPcleavage, caspase3and caspase9cleavage/activation, and cytochrome c release induced bytriptolide. Furthermore, pretreating with Y27632dramatically abrogated the triptolide-inducedROCK1activation and MLC and MYPT phosphorylation. Next, we knocked down ROCK1expression in U937cells using a ROCK1-specific siRNA. Knocking down ROCK1expression significantly abrogated triptolide-mediated apoptosis and diminishedtriptolide-induced PARP cleavage, caspase3and caspase9cleavage/activation, andcytochrome c release. Knocking down ROCK1also reduced the triptolide-induced MLC andMYPT phosphorylation. Taken together, these findings suggest that the ROCK1/MLCsignaling pathway has an important functional role in triptolide-mediated apoptosis.Thirdly, we investigated the effect of triptolide on tumor growth in a U937xenograft model, and explored the potential role of ROCK1/MLC signaling pathway intriptolide-mediated antileukemic activity in vivo.To determine whether triptolide exhibits anti-leukemic activity in vivo, nude mice wereinoculated intraperitoneally with U937cells. After inoculation, mice received injections ofeither vehicle control or triptolide (0.15mg/kg) for40days. Tumor volume and body weightwere measured once per5days, and mice status and adverse reactions were evaluated. Ourresults showed that treatment of mice with triptolide significantly inhibited tumor growth. Incontrast, no statistically significant changes in body weight were noted when comparingtriptolide-treated and vehicle control mice. Moreover, mice in the triptolide group did notexhibit any signs of toxicity. These findings indicate that triptolide treatment significantlyinhibited tumor growth of U937xenograft without causing any side effects and/or toxicity inthese mice. We then used hematoxylin and eosin (H&E), the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry toexamine morphological changes and the induction of apoptosis in U937cells in vivo. Tumorsfrom control mice had typical histologic appearances while those of mice treated withtriptolide had a marked decrease in cancer cells and signs of necrosis, inflammatory cellinfiltration, and fibrosis. Administering triptolide also significantly induced apoptosis intumor cells, as measured by TUNEL positivity. Immunohistochemistry analysis furtherrevealed that triptolide-treated mice had increased immunoreactivity for cleaved caspase-3, another indicator of apoptosis.To further evaluate whether ROCK1/MLC signaling pathway could be involved inantileukemic activity mediated by triptolide in vivo, Western blot and immunohistochemistryanalyses were employed. Treatment with triptolide resulted in a clear increase incleavage/activation of ROCK1and the levels of phospho-MLC. Taken together, these findingsindicate that triptolide significantly inhibited the growth of U937xenografts without causingany side effects and that this effect was also associated with ROCK1activation and MLCphosphorylation.In summary, our present findings indicate that triptolide effectively induces apoptosis inhuman leukemia cells and primary leukemia blasts, and inhibits the tumor growth of leukemiaxenografts. These effects are associated with ROCK1activation and MLC and MYPTphosphorylation. Our results also suggest that ROCK1is cleaved by caspase-3, rather thanRhoA, in this process. The activation of ROCK1and caspase-3might represent a positivefeedback loop resulting in triptolide-mediated apoptosis. Collectively, these findings support ahypothetical model of triptolide-induced apoptosis in leukemia cells. In this model, triptolideinduces ROCK1/MLC activation, leading to Bax translocation, and finally in mitochondrialinjury (cytochrome c release), caspase activation, and apoptosis. The ROCK1activation andMLC phosphorylation mediated by triptolide could be a potential therapeutic intervention fortreating leukemia and potentially other hematologic malignancies.