[Study on signal transduction pathway in differentiation and apoptosis of leukemia cells induced by heat shock protein inhibitor].
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To explore the signal transduction pathway in the differentiation and apoptosis of leukemia cells induced by heat shock protein 90 (HSP90) inhibitor 17-Allyl amide-17-demethoxygeldanamycin (17AAG).Kasumi-1 cells were treated with increasing concentrations or exposure time of 17AAG. The total kit protein (CD117), phosphorylated kit protein and its downstream signaling molecules were measured by Western blot analysis. Mutated kit protein from control and 17AAG-treated Kasumi-1 cells was immunoprecipitated and immunoblotted for associated chaperones.Total kit protein and kit activity were decreased in 17AAG treated cells, but c-kit mRNA level was not. Total AKT protein and phospho-AKT, as well as phospho-STAT3 were rapidly down-regulated in Kasumi-1 cell after treatment with 17AAG. There was no change in total STAT3 protein. Immunoprecipitation showed that 1 microM 17AAG treatment for 1 hour caused kit associated HSP90 decrease and HSP70 increase.17AAG-induced apoptosis of Kasumi-1 cells is associated with a decline in Asn822Lys mutated kit protein level and phosphorylated kit, and with a downregulation in its downstream activated signaling molecules involved in proliferation. AKT is a client protein of HSP90. The changes of kit associated HSP90 and HSP70 satisfy the circulation mode of molecular chaperone complex.Keywords:
Hsp90 inhibitor
Hsp27
GRB2
Immunoprecipitation
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Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 µM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.
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The present study aimed to explore the regulatory effects of endoplasmic reticulum stress (ERS) on the phosphoinositide 3‑kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, and its subsequent effects on autophagy and apoptosis of human leukemia cells. Human leukemia cells were cultured and treated with various concentrations of tunicamycin for 0, 24, 48, 72 and 90 h. Subsequently, human leukemia cells were assigned into the ER activation group, which was treated with 100 ng/ml tunicamycin, the ER activation + TO901317 (PI3K inhibitor) group, and the control group. An MTT assay was conducted to detect cell proliferation. In addition, a monodansylcadaverine (MDC) assay was used to detect the formation of autophagosomes and Annexin V‑fluorescein isothiocyanate/propidium iodide double staining was used to examine cell apoptosis. Western blotting was performed to detect the expression levels of 78‑kDa glucose‑regulated protein (GRP78), phosphorylated (p)‑protein kinase R‑like endoplasmic reticulum kinase (PERK), p‑α subunit of eukaryotic initiation factor 2 (eIF2α), microtubule‑associated protein 1A/1B‑light chain 3 (LC3), caspase‑3, CCAAT‑enhancer‑binding protein homologous protein (CHOP), PI3K, AKT and mTOR. Treatment with 100 ng/ml tunicamycin for 72 h was considered the optimal condition for further experiments. Compared with in cells prior to treatment, human leukemia cells treated with tunicamycin exhibited increased expression of p‑PERK, p‑eIF2α and GRP78 after 72 h (P<0.05). In addition, the expression levels of mTOR, AKT and PI3K were decreased in the ER activation group compared with in the control and ER activation + TO901317 groups (P<0.05). Compared with in the control group, cell proliferation was inhibited and MDC fluorescence intensity was increased in the ER activation group (P<0.05). Furthermore, compared with in the control and ER activation + TO901317 groups, western blotting indicated that the expression levels of LC3‑II were increased in the ER activation group (P<0.05). The apoptotic rate was also higher in the ER activation group compared with in the control group (P<0.05), and caspase‑3 and CHOP expression was elevated in the ER activation group (P<0.05). These findings indicated that ERS may induce autophagy and apoptosis of human leukemia cells via inhibiting the PI3K/AKT/mTOR signaling pathway.
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Abstract We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal–regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low-AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high-AKT” myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity. [Mol Cancer Ther 2009;8(1):83–93]
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Our previous in vitro study demonstrated that the rat hyperplasia suppressor gene (rHSG) inhibited the proliferation of C6 cells. In the present study, we investigated further the effects of rHSG overexpression on the apoptosis of C6 cells and the possible pathways involved. Hoechst 33342/PI double staining and comet assay were used to examine the morphological characteristics of apoptosis and to examine the effects of rHSG on the apoptosis of the C6 cells. Western blot analysis was used to determine the effects of rHSG overexpression on the protein expression levels of poly(ADP‑ribose) polymerase (PARP), cleaved caspase-3, phosphorylated extracellular signal-regulated kinase 1/2 (p-Erk1/2), phosphorylated Akt (p-Akt) and phosphoinositide 3-kinase (PI3K)/Akt, as well as on the mitogen-activated protein kinase (MAPK) pathways induced by insulin‑like growth factor (IGF)-1. Our results revealed that the C6 cells transfected with the rHSG adenoviral vector (Adv-rHSG-GFP group) efficiently expressed rHSG protein; Hoechst 33342/PI double staining and comet assay revealed that rHSG increased C6 cell apoptosis and induced DNA damage. Western blot analysis indicated that rHSG overexpression significantly increased the level of full-length PARP at 24 and 72 h (P<0.01), but decreased the level at 48 h following transfection (P<0.01), while the proteins levels of cleaved PARP and cleaved caspase-3 increased significantly (P<0.01). The protein expression of p-Erk1/2 and p-Akt began to decrease at 48 h post-transfection (P<0.01). In addition, the protein levels of Akt and Erk1/2 induced by IGF-1 were significantly inhibited. On the whole, the findings of the present study demonstrate that rHSG overexpression induces the apoptosis of rat glioma cells, and that these effects may involve the PI3K/Akt and MAPK pathways.
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The aim of the present study was to investigate the synergistic effect of phenylhexyl isothiocyanate (PHI) and LY294002 [an inhibitor of phosphoinositide 3-kinase (PI3K)] on the PI3K/protein kinase B (Akt) signaling pathway, modulating histone acetylation, inhibiting cell viability and inducing apoptosis in HL-60 cells. The inhibition of HL-60 cell viability was monitored using an MTT assay. Cell apoptosis was measured using flow cytometry. Expression of acetylated histone H3 and histone H4, and the Akt signaling pathway proteins phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated ribosomal protein S6 kinase (p-p70S6K) was detected using western blotting. The results of the present study identified that PHI and LY294002 were able to inhibit cell viability and induce cell apoptosis in HL-60 cells. The combination exhibited a synergistic effect on cell viability and apoptosis. PHI treatment led to an accumulation of acetylated histone H3 and histone H4, but LY294002 treatment had no effect on histone acetylation. However, LY294002 was identified to enhance the effect of PHI on histone acetylation in HL-60 cells. PHI and/or LY294002 were identified to dephosphorylate proteins in the PI3K/Akt signaling pathway, with a synergistic effect observed when used in combination. The results of the present study indicated that the combination of PHI and LY294002 may offer a novel therapeutic strategy for acute myeloid leukemia.
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Abstract Cell confluence induces resistance to chemotherapeutic agents in solid tumor cells and phosphorylation of heat shock protein 27 (HSP27), a low-weight molecular chaperon and stress response protein. Here, we show that human glutathione S-transferase P1 (GSTP1) inhibits phosphorylation of HSP27 induced by both epidermal growth factor (EGF) stimulation and cell confluence in malignant brain tumor cells, resulting in enhancement of resistance to cisplatin. Using the paired human medulloblastoma cell lines, UW228 (GSTP1-ve) and UW228-GSTP1 (GSTP1+ve), we showed that growth at high cell density (confluence) induced resistance to cisplatin, regardless of GSTP1 status. Western blotting analysis of extracts of GSTP1-ve cells growing at high density or stimulated with EGF stimulation showed significantly higher phosphorylation levels of HSP27, whereas the HSP27 phosphorylation was inhibited in GSTP1+ve cells. Interestingly, the induction of HSP27 phosphorylation in GSTP1-ve cells was independent of HSP27 upstream kinase activation and serum starvation, suggesting that the inhibition in GSTP1+ve cells results from a direct interaction of HSP27 with GSTP1, rather than a blockade of HSP27 upstream signaling. Inhibition of HSP27 phosphorylation by kinase-specific inhibitors and siRNA-mediated knock-down of the MAP kinase-activated protein kinase-2, MK2, both showed significant inhibition of HSP27 phosphorylation and enhancement of cisplatin resistance in UW228 cells. Using a cell-free system, recombinant GSTP1 protein inhibited HSP27 phosphorylation via MK2. Immunoprecipitation with anti-HSP27 and anti-GSTP1 antibodies showed formation of a GSTP1-HSP27 complex in starved cells and that the level of this complex increased significantly following EGF treatment. In in vivo xenografts of U87MG, HSP27 formed a complex, not only, with GSTP1, but also with p38, Akt, and MK2. shRNA-mediated GSTP1 knockdown in MGR3 cells increased HSP27 phosphorylation significantly, compared with shRNA negative control. Similarly, GSTP1-knockout mouse embryonic fibroblasts (MEFs) showed higher phosphorylation levels of HSP27 induced by both confluence and EGF than control human GSTP1 knock-in MEF cells. Using 2-D native/SDS-PAGE, the level of large (>200 kD) oligomeric HSP27 species was increased in GSTP1+ve, relative to GSTP1-ve cells. Taken together, these results indicate that GSTP1 binds directly to and inhibits HSP27 phosphorylation resulting in an enhanced HSP27 large oligomer formation and increased chaperon activity. The inhibition of HSP27 occurs directly, rather than through a blockade of HSP27 upstream signaling. The data define HSP27 as a novel downstream effector of GSTP1-mediated cellular signaling that can impact outcome of treatment. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2011-703
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Human telomerase activity is induced by Ag receptor ligation in T and B cells. However, it is unknown whether telomerase activity is increased in association with activation and proliferation of NK cells. We found that telomerase activity in a human NK cell line (NK-92), which requires IL-2 for proliferation, was increased within 24 h after stimulation with IL-2. Levels of human telomerase reverse transcriptase (hTERT) mRNA and protein correlated with telomerase activity. ERK1/2 and Akt kinase (Akt) were activated by IL-2 stimulation. LY294002, an inhibitor of PI3K, abolished expression of hTERT mRNA and protein expression and abolished hTERT activity, whereas PD98059, which inhibits MEK1/2 and thus ERK1/2, had no effect. In addition, radicicol, an inhibitor of heat shock protein 90 (Hsp90), and rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), blocked IL-2-induced hTERT activity and nuclear translocation of hTERT but not hTERT mRNA expression. hTERT was coimmunoprecipitated with Akt, Hsp90, mTOR, and p70 S6 kinase (S6K), suggesting that these molecules form a physical complex. Immunoprecipitates of Akt, Hsp90, mTOR, and S6K from IL-2-stimulated NK-92 cells contained telomerase activity. Furthermore, the findings that Hsp90 and mTOR immunoprecipitates from primary samples contained telomerase activity are consistent with the results from NK-92 cells. These results indicate that IL-2 stimulation induces hTERT activation and that the mechanism of IL-2-induced hTERT activation involves transcriptional or posttranslational regulation through the pathway including PI3K/Akt, Hsp90, mTOR, and S6K in NK cells.
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Hsp90 inhibitor
K562 cells
Histone deacetylase inhibitor
HDAC1
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The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5‑[5‑(2‑nitrophenyl) furfuryl iodine]‑1, 3‑diphenyl‑2‑thiobarbituric acid (UCF‑101) treatment was detected with reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF‑101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF‑101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF‑101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF‑101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF‑101, resulting in the upregulation of the WT1 protein level. However; UCF‑101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF‑101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal‑related kinase (ERK1/2) pathway markedly and persistently, but UCF‑101 exhibited no notable effect on the inhibition of the ERK1/2 pathway. HtrA2 and its regulatory effect on WT1 may affect the sensitivity of BCR/ABL(+) cell lines to target therapy drugs through different mechanisms. Regulation of WT1 by HtrA2 occurs in K562 cells, and the regulation may affect the apoptosis of K562 cells under the stress caused by chemotherapeutic treatment. The p38 MAPK signaling pathway, which serves an important role in cell apoptosis, is a downstream pathway of this regulation.
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The serine/threonine kinases, Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an Akt1 antisense oligonucleotide (AS) to specifically downregulate Akt1 protein in both cancer and normal cells. Our data indicate that Akt1 AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FACS analysis and a caspase activity assay. Conversely, Akt1 AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF), fibroblast from muscle (FBM), and mammary gland epithelial 184B5 cells. In addition, Akt1 AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akt1 is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of Akt1 may provide a powerful sensitization agent for chemotherapy specifically in cancer cells.
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