Bortezomib induces apoptosis by interacting with JAK/STAT pathway in K562 leukemic cells.

In the current study, we aimed to identify the cytotoxic and apoptotic effects of bortezomib (BOR) on human K562 chronic myelogenous leukemia cells and to evaluate the potential roles of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway members STAT3, STAT5, and JAK2 on BOR-induced cell death of leukemic cells. Cell viability was assessed via trypan blue dye exclusion test, and cytotoxicity of the BOR-treated cells was conducted by 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay. The relative messenger RNA (mRNA) expression levels of STAT3, STAT5A, STAT5B, and JAK2 were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). On the other hand, their protein expression levels were detected by western blot method. The obtained results indicated that BOR treatment reduced cell viability and induced leukemic cell apoptosis in a dose- and time-dependent manner as compared to untreated control cells. While mRNA expression levels of STAT5A, STAT5B, and STAT3 were significantly reduced following BOR treatment when compared to untreated controls, it had no effect upon JAK2 mRNA expression. As for protein levels, STAT expressions were downregulated after BOR treatment especially at 72nd and 96th hours. Our results pointed out that BOR treatment had a significant potential of being an anticancer agent for chronic myelogenous leukemia therapy, and this effect could be due to the expressional downregulations of JAK/STAT pathway members.

Repression of STAT3, STAT5A, and STAT5B expressions in chronic myelogenous leukemia cell line K-562 with unmodified or chemically modified siRNAs and induction of apoptosis.

Signal transducers and activators of transcription (STAT) proteins are latent cytoplasmic transcription factors that affect several cellular processes including cell growth, proliferation, differentiation, and survival. Following phosphorylation, STATs are activated, and their upregulated expressions increase in malignancies with playing a role in the development of leukemia. In this study, transfection of K-562 cells with either unmodified or chemically modified anti-STAT3, -STAT5A, -STAT5B siRNAs for duration of 12 days, determining gene silencing at mRNA and protein levels, evaluating apoptosis rate, and detecting JAK/STAT pathway members' gene expression profiles via array method were aimed. Quantitative RT-PCR and Western blot assays indicated that STAT expressions were downregulated both at mRNA and protein levels, and TUNEL assay showed that leukemic cell apoptosis was induced due to inhibition of STATs. Array analysis resulted with decreases in signal transducer, phosphorylation inducer, and oncogene expressions, whereas increased expressions in STAT inhibitor and apoptosis inducer genes were observed. These results point out that siRNA application could constitute a new and alternative curative method for supporting therapy of CML-diagnosed patients in the future.

Two cases with hypereosinophilic syndrome shown with real-time PCR and responding well to imatinib treatment.

The aim of this work was to report two cases of hypereosinophilic syndrome (HES). FIP1L1-PDGFRA fusion was assessed with two protocols at RNA level. The fusion transcript was found positive at the RNA level with both PCR methods in two cases. In this study, the efficiency of imatinib treatment and a dramatic response in two HES cases with multisystemic involvement showing the characteristics of a chronic myeloproliferative disease were presented. Both cases showed complete responses confirming that imatinib mesylate treatment could be successful even in patients with advanced HES having myeloproliferative disease.

Cisplatin resistance induced by decreased apoptotic activity in non-small-cell lung cancer cell lines.

We have investigated defective steps in apoptosis that might account for the development of resistance. For this purpose, A549 and Calu1 NSCLC (non-small-cell lung cancer) cell lines were treated with cisplatin to obtain resistant sub-lines. Gene expression profiles and the phosphorylation status of the BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were determined for each cell line. Cell death and cytochrome c release were analysed after treating cell lines with their appropriate cisplatin doses. Gene expression of BAD, Bid, caspases 4 and 6 were clearly decreased in the resistant cell lines, and the differential phosphorylation status of BAD also seemed to play a role in the development of cisplatin resistance. Since this is a new cisplatin-resistant Calu1 cell line, it is noteworthy that DNA fragmentation, apoptotic cell ratio and cytochrome c levels were most decreased in the CR-Calu1 cell line.

Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18)(q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based onthe detection of mbr or mcr in paraffin-embedded tissue samples. MATERIAL AND METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2(B-cell leukemia-lymphoma 2) (spectrum orange) probes. RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12)was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all,24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(14;18) positive via the FISH method. CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells.

Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC(50) of IFN-alpha-2b on K562 cells was found to be 600IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.

Differential effects of doxorubicin and docetaxel on nitric oxide production and inducible nitric oxide synthase expression in MCF-7 human breast cancer cells.

Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was detected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin.

Alterations in the activity and expression of serine/threonine protein phosphatases during all trans retinoic acid-induced apoptosis in breast cancer cells.

Retinoids exert different effects on malignant cells with various phenomena. They can induce differentiation and apoptosis in various cancer cells. However, the underlying mechanism of these effects is not clear. There are data related to the role of protein phosphatases during retinoid-induced leukemic cell differentiation. The aim of this study was to evaluate effects of the All trans retinoic acid (ATRA) on protein/serine phosphatases during ATRA induced apoptosis in the breast cancer cells. The MTT assay was used to determine drug-mediated cytotoxicity. A cell death detection ELISA kit was used for detection of the DNA fragments. The activity of serine/threonine protein phosphatases was assayed by the serine/threonine phosphatase system. The expression of serine/threonine protein phosphatases was evaluated by Western blot. During ATRA treatment, a significant decrease in the activity of serine/threonine phosphatases 2A, B and C occurred. The decreased activity of PP2A correlated with the up-regulation of PP2A catalytic and PP2A/B gamma, PP2A/B alpha regulatory subunits. The decrease in activity of the PP2B correlated with down-regulation of PP2B catalytic and up-regulation of PP2B regulatory subunit expression. In addition, there was an up-regulation in PP4C and down regulation in PP2C alpha/beta subunits protein expression. We demonstrated clear alteration in the activity and expression of serine/threonine protein phosphatases in breast cancer cells during ATRA treatment, and we suggest that the ATRA-induced apoptosis of the MCF-7 cells is significantly related to the phosphorylation dynamics.

Potential Involvement of Calcineurin in Regulating the State of Differentiation and Apoptosis of HL-60 Cells During Methylprednisolone-Treatment.

To evaluate the role of calcineurin (protein phosphatase type 2B, PP2B) in methylprednisolone-induced differentiation and apoptosis of leukaemic cells, we have investigated the induction of apoptosis, calcineurin specific protein phosphatase activity and expression of regulatory and catalytic subunits of calcineurin and calmodulin after induction of HL-60 leukaemic cells with methylprednisolone. The cells underwent differentiation and apoptosis within 72 hours time period after methylprednisolone added to cell culture media. Before apoptosis occurred, the specific calcineurin enzyme activity revealed gradual increase during the differentiation process. However, immunoblots of catalytic and regulatory subunits of calcineurin showed no amplification in the amount of these cellular signaling mediators during methylprednisolone-induced differentiation and apoptosis but calmodulin expression gradually increased during the process. Significant increase in the specific calcineurin enzyme activity during differentiation and apoptosis might be crucial to the posttranslational modifications of calcineurin during methylprednisolone-induced differentiation.

Suppression of STAT5A and STAT5B chronic myeloid leukemia cells via siRNA and antisense-oligonucleotide applications with the induction of apoptosis.

Signal transducers and activators of transcription (STAT) proteins function in the JAK/STAT signaling pathway and are activated by phosphorylation. As a result of this signaling event, they affect many cellular processes including cell growth, proliferation, differentiation, and survival. Increases in the expressions of STAT5A and STAT5B play a remarkable role in the development of leukemia in which leukemic cells gain uncontrolled proliferation and angiogenesis ability. At the same time, these cells acquire ability to escape from apoptosis and host immune system. In this study, we aimed to suppress STAT-5A and -5B genes in K562 CML cells by siRNA transfection and antisense oligonucleotides (ODN) targeting and then to evaluate apoptosis rate. Finally, we compared the transfection efficiencies of these approaches. Quantitative RT-PCR and Western blot results indicated that STAT expressions were downregulated at both mRNA and protein levels following siRNA transfection. However, electroporation mediated ODN transfection could only provide limited suppression rates at mRNA and protein levels. Moreover, it was displayed that apoptosis were significantly induced in siRNA treated leukemic cells as compared to ODN treated cells. As a conclusion, siRNA applications were found to be more effective in terms of gene silencing when compared to ODN treatment based on the higher apoptosis and mRNA suppression rates. siRNA application could be a new and alternative curative method as a supporting therapy in CML patients.

STAT pathway in the regulation of zoledronic acid-induced apoptosis in chronic myeloid leukemia cells.

In this study, we aimed to evaluate the cytotoxic and apoptotic effects of zoledronic acid on K562 chronic myeloid leukemia (CML) cells and to examine the roles of STAT genes on zoledronic acid-induced apoptosis. The results showed that zoledronic acid decreased proliferation, and induced apoptosis in K562 cells in a dose- and time-dependent manner. mRNA and protein levels of STAT3, -5A and -5B genes were significantly reduced in zoledronic acid-treated K562 cells. These data indicated that STAT inhibition by zoledronic acid may be therapeutic in CML patients following the confirmation with clinical studies.

Methylprednisolone induces apoptosis by interacting with the JAK/STAT pathway in HL-60 and K-562 leukemic cells.

OBJECTIVE: To determine the gene expression profiles of the JAK/STAT pathway members STAT3, STAT5A, STAT5B at both mRNA and protein levels in HL-60 and K-562 leukemia cells that were undergoing apoptosis following high-dose methylprednisolone (MP) treatment. METHODS: HL-60 cells were treated with 0.1 mM MP and K-562 cells were treated with 0.4 mM MP according to their IC(50) values. STAT3, STAT5A, and STAT5B mRNA relative expression levels were determined by qRT-PCR whereas the protein levels were detected via western-blot analysis and apoptosis was evaluated by Annexin V method. RESULTS: A significant decrease was seen in STAT5A mRNA relative expression level at 48 hours of MP treatment (P < 0.05) both in HL-60 and K-562 cells. Other STATs showed a lower downregulation in their relative expressions at 48 hours at mRNA level for both of the cell lines. STAT proteins showed no expression change in K-562 cells in time course experiments but while STAT5A expression was downregulated; STAT5B showed an increase at 96 hours in HL-60 cells. Apoptosis was triggered by high-dose MP treatment that was evaluated by fluorescent microscopy. CONCLUSION: The JAK/STAT pathway components may play an important role in the apoptosis mechanism of leukemic cells under MP treatment in HL-60 and K-562 cells. Other pathways may also be involved with a post-translational modification seen in the HL-60 cell line, with both upregulation and downregulation of protein expression levels of STAT5B and STAT5A, respectively.

Enalapril-induced apoptosis of acute promyelocytic leukaemia cells involves STAT5A.

BACKGROUND: In this study, we aimed at evaluating the cytotoxic and apoptotic effects of enalapril on human HL60 acute promyelocytic leukaemia cells and at clarifying the roles of signal transducers and activator of transcription proteins (STATs) on enalapril-induced cell death. MATERIALS AND METHODS: Cell viability and cytotoxicity tests were conducted by Trypan blue dye exclusion and 2,3-Bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assays, respectively. Apoptotic analyses were performed by the AnnexinV-enhanced green fluorescent protein (EGFP) staining method and by fluorescence microscopy. Expression levels of STAT3, -5A and -5B genes were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The results showed that enalapril reduced viability and proliferation, and induced apoptosis in HL60 cells in a dose- and time-dependent manner as compared to untreated controls. The expression levels of STAT5A gene were significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. CONCLUSION: Taken together, all data showed for the first time that enalapril has significant anticancer potential for the treatment of acute premyelocytic leukaemia.

Suppression of STAT5A increases chemotherapeutic sensitivity in imatinib-resistant and imatinib-sensitive K562 cells.

STAT proteins are cytoplasmic transcription factors that are involved in the regulation of numerous cellular activities such as cell growth, differentiation, and survival. In this study, we aimed to identify the expression pattern of STAT genes in imatinib-sensitive and -resistant K562 cells, and further, to reveal the effects of STAT5A siRNA knockdown on cell growth and apoptosis induction. The XTT cell proliferation assay showed that both sensitive and resistant K562 cells were sensitized to imatinib upon transfection with STAT5A siRNA. Caspase-3 enzyme activity was increased significantly in both cells. These results may open up new opportunities to overcome chemotherapeutic resistance in leukemia.

Immunohistochemical analysis of inducible and endothelial forms of nitric oxide synthase in cyclosporin A-induced gingival overgrowth.

BACKGROUND: The contribution of nitric oxide (NO) to immune response and matrix degradation in the periodontal environment suggests a role for NO and NO-synthase (NOS) activity in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth (GO). However, current knowledge on this topic is limited to experimental animal studies. The present study was undertaken on the basis of a hypothesis whether altered nitrite/nitrate levels in gingival crevicular fluid (GCF) and endothelial NOS (eNOS) and inducible NOS (iNOS) immunoreactivity in gingiva of CsA-treated patients contribute to the pathogenesis of CsA-induced GO. METHODS: Twenty-four CsA-medicated renal transplant patients with GO (GO+; n = 12) or without GO (GO-; n = 12), 10 gingivitis, and 10 healthy subjects were included in the study. GCF samples from two proximal sites facing interdental papilla were collected, and papilla was excised. iNOS and eNOS were determined by immunohistochemistry. GCF nitrite/nitrate levels were analyzed based on the Griess reaction. RESULTS: Weak iNOS immunostaining was observed in the healthy and GO- groups. In the gingivitis and GO+ groups, iNOS immunostaining significantly increased in connective tissue. Epithelial immunostaining of iNOS was localized to basal keratinocytes and the lower layer of stratum (str.) spinosum in the gingivitis group. In the GO+ group, iNOS immunostaining was differentially localized to keratinocytes of str. superficiale but considerably decreased in the str. basale. Weak eNOS immunostaining was found in the healthy and GO- groups, whereas higher immunostaining was observed in the gingivitis and GO+ groups. No intergroup differences were observed regarding nitrite/nitrate levels in GCF. CONCLUSION: CsA differentially upregulated iNOS, but not eNOS, in overgrown gingiva, which may play a pivotal role in the pathogenesis of CsA-induced GO.

Absence of apolipoprotein B-3500 mutation in Turkish patients with coronary and cerebrovascular atherosclerosis.

OBJECTIVE: The arginine- to-glutamine change at codon 3500 of the apolipoprotein B-100 (apo B) is a well- known genetic cause of hypercholesterolemia. Since increased cholesterol levels lead to atherosclerosis, identification of the carries of the apo B-3500 mutation is an important step in the risk stratification of individuals and families with hypercholesterolemia. The prevalence of this mutation in Turkish population is not well known. We aimed to investigate the frequency of apo B-100 mutations (codon 3500) C9774T (Arg 3500-->Trp) and G9775A (Arg 3500-->Gln) in patients with atherosclerosis in comparison with healthy subjects. METHODS: This cross-sectional study included 442 unrelated subjects living on the West coast of Turkey. Subgroups consisted of 165 patients with coronary artery disease, 163 patients with ischemic stroke, and 114 healthy control subjects. RESULTS: We did not find any apo B-100 mutation both in the patient and control groups. CONCLUSION: As it is hypothesized that this mutation arose within the Central European region from a common ancestor approximately 7000 years ago and spread across Europe, our result of the absence of the R3500Q mutation in Turkish patients give an important information about the geographical distribution of the apo B-R3500Q, that the mutation has not reached to Anatolia.

Enteral resveratrol supplementation attenuates intestinal epithelial inducible nitric oxide synthase activity and mucosal damage in experimental necrotizing enterocolitis.

BACKGROUND/PURPOSE: The release of various enzymes including inducible nitric oxide synthase (iNOS) leads to enterocyte apoptosis through free nitrogen radicals, which in turn leads to impaired mucosal barrier and bacterial translocation with resultant sepsis in necrotizing enterocolitis (NEC). Resveratrol, a polyphenol compound from phytoalexins with antioxidant and scavenger properties, also play a critical role in modulating key enzymes in cell cycle including iNOS. We therefore hypothesized that resveratrol would prevent mucosal damage in experimental NEC in rats. METHODS: Newborn rats were randomized into 3 groups: group 1 was left to breast-feed (BF), whereas group 2 (NEC) was induced by enteral formula feedings twice daily and by being subjected to hypoxia thrice. The third group (R) received the same treatment as the NEC group but the enteral feeds were supplemented with resveratrol. Rats were killed on day 4, and their terminal ileal samples were harvested for histopathologic analysis. Expression of iNOS was assessed by sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis and immunohistochemistry. Band densities were quantified by using the software NIH image. RESULTS: The epithelial structure in group BF was normal. In the NEC group, there were marked loss of the brush border, vacuolization, and necrosis. The epithelial structure was found to be preserved in group R. Western blot analysis revealed marked elevation in the expression of iNOS protein at 130 kD molecular weight (band densities in groups BF, NEC, and R were 0.3 +/- 3.5, 3.7 +/- 2.9, and 0.6 +/- 5.1, respectively; P < .01). Immunohistochemical analysis revealed that iNOS staining was significantly increased in the NEC group, whereas it remained minimal for the BF and R groups. Ileal tissue nitrate/nitrite levels for groups BF, NEC, and R were 178.3 +/- 7, 191.4 +/- 4.1, and 181 +/- 3.6 micromol/(L x g), respectively (P < .01). CONCLUSIONS: These findings may provide insights for the beneficial effect of enteral resveratrol supplementation on inflammatory conditions of the bowel including NEC through attenuating the release of iNOS and preservation of mucosal integrity.

Protein phosphatase 2A (PP2A) has a potential role in CAPE-induced apoptosis of CCRF-CEM cells via effecting human telomerase reverse transcriptase activity.

Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC(50) of 1 muM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC(20) of inhibitors and IC(50) of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC(50) of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE.