Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling

Background

The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML) cell line U937.

Methodology/Principal Findings

Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x_L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2), the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP) SH2 domain-containing phosphatase (SHP)-1, and the addition of sodium pervanadate (a PTP inhibitor) prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells.

Conclusions/Significance

Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.     

Inactivation of HDAC3 and STAT3 is Critically Involved in 1-Stearoyl-sn-Glycero-3-Phosphocholine-Induced Apoptosis in Chronic Myelogenous Leukemia K562 Cells

We here investigated the anticancer mechanism of 1-stearoyl-sn-glycero-3-phosphocholine (LPC), one of the lysophosphatidylcholines, in chronic myelogenous leukemia (CML) K562 cells. LPC significantly showed cytotoxicity at 80 μM and induced apoptosis by sub-G1 accumulation, increase in Annexin V positive and caspase activation. LPC enhanced histone H3 acetylation but decreased histone deacetylase (HDAC) activity and HDAC3 expression. LPC also inhibited phosphorylation of STAT3, its DNA binding activity and nuclear co-localization of HDAC3 and STAT3. In addition, LPC effectively attenuated the expression of survival genes such as Cyclin D1, Cyclin E, Bcl-x_L, Bcl-2 and survivin but did not affect COX-2 expression in K562 cells. Furthermore, LPC suppressed phosphorylation of Src and Janus activated kinase 2 while promoted the expression of tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1). Consistently, silencing SHP-1 and pervanadate, an inhibitor of protein tyrosine phosphatase, reversed inactivation of HDAC and STAT3, cleavages of caspase 3 and poly (ADP-ribose) polymerase in LPC-induced apoptosis. Of note, chromatin immunoprecipitation assay revealed that LPC suppressed the binding of HDAC3 and STAT3 to Bcl-x_L, Bcl-2 and survivin promoter. Overall, our findings indicate that inactivation of STAT3 and HDAC mediates LPC-induced apoptosis in CML K562 cells.     

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.     

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.     

Src homology region 2 domain-containing phosphatase 1 positively regulates B cell receptor-induced apoptosis by modulating association of B cell linker protein with Nck and activation of c-Jun NH2-terminal kinase

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.     

γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

Although γ-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In this study, we investigated whether γ-T3 can modulate the STAT3 cell signaling pathway, closely linked to inflammation and tumorigenesis. We found that γ-T3 but not γ-tocopherol, the most common saturated form of vitamin E, inhibited constitutive activation of STAT3 in a dose- and time-dependent manner, and this inhibition was not cell type-specific. γ-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the γ-T3-induced down-regulation of STAT3 activation, suggesting the involvement of a protein-tyrosine phosphatase. When examined further, we found that γ-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of γ-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of γ-T3. Also γ-T3 down-modulated activation of STAT3 and induced SHP-1 in vivo. Eventually, γ-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that γ-T3 is a novel blocker of STAT3 activation pathway both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.     

miR-451a targeting IL-6R activates JAK2/STAT3 pathway,thus regulates proliferation and apoptosis of multiple myeloma cells

Objectives:To investigate the effects of miR-451a targeting interleukin-6 (IL-6) on the proliferation and apoptosis of multiple myeloma (MM) cells and its potential mechanism via JAK2/STAT3 pathway.Methods:mRNA expression of miR-451a and IL-6R in the plasma of patients with MM and normal controls were determined by RT-qPCR. U266 cells were cultured, transfected with miR-451a mimics, the proliferative ability of U266 cells was determined by CCK-8. Potential targets of miR-451a were predicted with the biological software TargetScan, and the direct relationship between miR-451a and the target IL-6R was analyzed by a dual-luciferase reporter assay. U266 cells were stimulated with IL-6R (100 ng/ml), and the proliferative ability and apoptosis rate were determined by CCK-8 and flow cytometry after 48h.Results:In the plasma of patients with MM, miR-451a expression was low and IL-6R expression was high. miR-451a targeted and negatively regulated IL-6R. Overexpressing miR-451a inhibited the proliferation and promoted the apoptosis of U266 cells. IL-6R acting on U266 cells promoted the proliferation and inhibited the apoptosis of U266 cells. Overexpressing miR-451a inhibited the activation of JAK2/STAT3 pathway and down-regulating miR-451a promoted the activation of JAK2/STAT3 pathway.Conclusions:miR-451a targeting IL-6R activates JAK2/STAT3 pathway, thus regulates the proliferation and apoptosis in MM cells.     

Interferon alpha induces cell death through interference with interleukin 6 signaling and inhibition of STAT3 activity

In multiple myeloma, which commonly depends on interleukin 6, IL-6, survival signaling induced by this cytokine is largely mediated by activation of STAT3. Interferon alpha (IFNalpha) treatment of cell lines derived from multiple myeloma or of myeloma tumor cells ex vivo leads to apoptosis. In this study we demonstrate that IFNalpha treatment of the two myeloma cell lines, U266-1984 and U-1958, results in the decrease of STAT3 activity as demonstrated by a diminished STAT3/3 DNA-binding activity and the shift from STAT3/3 towards STAT1/1 and STAT3/1 complexes in EMSA, leading to the down-regulation of known STAT3 target genes such as Bcl-X(L), Mcl-1 and survivin. Ectopic expression of a form of STAT3, STAT3C, rescued U266-1984 cells from IFNalpha-induced apoptosis. IFNalpha promoted sustained accumulation of tyrosine phosphorylated STAT3C in the nucleus and a prolonged DNA binding of the STAT3/3 homodimers in EMSA. The shift towards a sustained STAT3 response in IFNalpha-treated STAT3C-transfected cells led to a hyper-induction of Bcl-2 and Mcl-1 proteins. Thus our data demonstrated that IFNalpha is able to interfere with IL-6 signaling by inhibiting STAT3 activity and that the abrogation of STAT3 activity accounts for the ability of IFNalpha to induce apoptosis in myeloma cells.     

Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells

Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 (PP-1) and 2A (PP-2A). The phosphorylation and dephosphorylation at the serine/threonine residues on proteins play important roles in regulating gene expression, cell cycle progression, and apoptosis. In this study, phosphatase inhibitor okadaic acid induces apoptosis in U937 cells via a mechanism that appears to involve caspase 3 activation, but not modulation of Bcl-2, Bax, and Bcl-X(L) expression levels. Treatment with 20 or 40 nM okadaic acid for 24 h produced DNA fragmentation in U937 cells. This was associated with caspase 3 activation and PLC-gamma1 degradation. Okadaic acid-induced caspase 3 activation and PLC-gamma1 degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 40 nM. Moreover, PMA (phorbol myristate acetate), PKC (protein kinase C) activator, protected U937 cells from okadaic acid-induced apoptosis, abrogated okadaic acid-induced caspase 3 activation, and specifically inhibited downregulation of XIAP (X-linked inhibitor of apoptosis) by okadaic acid. PMA cotreated U937 cells exhibited less cytochrome c release and sustained expression levels of the IAP (inhibitor of apoptosis) proteins during okadaic acid-induced apoptosis. In addition, these findings indicate that PMA inhibits okadaic acid-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase 3 that is involved in the execution of apoptosis.     

Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells

SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells.     

Role of SHP-2 tyrosine phosphatase in the DNA damage-induced cell death response

SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in the regulation of growth factor and cytokine signal transduction. Here we report a novel function of this phosphatase in DNA damage-induced cellular responses. Mutant embryonic fibroblast cells lacking functional SHP-2 showed significantly decreased apoptosis in response to DNA damage. Following cisplatin treatment, induction of p73 and its downstream effector p21(Cip1) was essentially blocked in SHP-2 mutant cells. Further investigation revealed that activation of the nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage induction of p73, was impaired in the mutant cells, suggesting a functional requirement of SHP-2 in c-Abl activation. Consistent with this observation, the effect of overexpression of c-Abl kinase in SHP-2 mutant cells on sensitizing the cells to DNA damage-induced death was abolished. Additionally, we found that in embryonic fibroblast cells 30-40% of SHP-2 was localized in the nuclei, and that a fraction of nuclear SHP-2 was constitutively associated with c-Abl via its SH3 domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was significantly enhanced in response to DNA damage. These results together suggest a novel nuclear function for SHP-2 phosphatase in the regulation of DNA damage-induced apoptotic responses.     

Negative regulation of myeloid cell proliferation and function by the SH2 domain-containing tyrosine phosphatase-1

The SH2 domain containing tyrosine phosphatase SHP-1 has been implicated in the regulation of a multiplicity of signaling pathways involved in hemopoietic cell growth, differentiation, and activation. A pivotal contribution of SHP-1 in the modulation of myeloid cell signaling cascades has been revealed by the demonstration that SHP-1 gene mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten mice. To investigate the role of SHP-1 in regulation of myeloid leukocytes, an HA epitope-tagged dominant negative (interfering) SHP-1 (SHP-1C453S) was expressed in the myelo-monocytic cell line U937 using the pcDNA3 vector. Overexpression of this protein in SHP-1C453S transfectants was demonstrated by Western blot analysis and by detection of decreased specific activity. Growth, proliferation, and IL-3-induced proliferative responses were substantially increased in the SHP-1C453S-overexpressing cells relative to those in control cells. The results of cell cycle analysis also revealed that the proportion of cells overexpressing SHP-1C453S in S phase was greater than that of control cells. The SHP-1C453S-expressing cells also displayed diminished rates of apoptosis as detected by flow cytometric analysis of propidium iodide-stained cells and terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end-labeling assay. While motility and phagocytosis were not affected by SHP-1C453S overexpression, adhesion and the oxidative burst in response to PMA were enhanced in the SHP-1C453S compared with those in the vector alone transfectants. Taken together, these results suggest that SHP-1 exerts an important negative regulatory influence on cell proliferation and activation while promoting spontaneous cell death in myeloid cells.     

The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells

Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.