Juwon Park

Curcumin in combination with bortezomib synergistically induced apoptosis in human multiple myeloma U266 cells

Growth of multiple myeloma cells is controlled by various factors derived from host bone marrow microenvironments. Interaction between multiple myeloma cells and bone marrow stromal cells (BMSCs) plays an important role in the expression of adhesive molecules and secretion of growth factors involved in multiple myeloma (MM) cell growth, survival, and resistance to anticancer drugs. Recently, the possibility of developing novel anti‐cancer therapeutic strategies targeting both MM cells and MM cell–BMSC interactions has been discussed. Here we present data showing that curcumin, a major constituent of turmeric compounds extracted from the rhizomes of the plant Curcuma longa, effectively reduced the growth of MM cells and BMSCs. Upon treatment with curcumin, IL‐6/sIL‐6R‐induced STAT3 and Erk phosphorylation was dramatically reduced in the co‐cultured cells. In addition, curcumin inhibited the production of pro‐inflammatory cytokines and VEGF, factors that are associated with the progression of multiple myeloma, from both MM cells and BMSCs. In a combination treatment with curcumin and bortezomib, IL‐6/sIL‐6R‐induced STAT3 and Erk phosphorylation was effectively inhibited. Moreover, this combination treatment synergistically inhibited the growth of MM cells co‐cultured with BMSCs as compared to controls. Taken together, these results indicate that curcumin potentiates the therapeutic efficacy of bortezomib in MM suggesting this combination therapy to be of value in the clinical management of MM.

Correlation between clinical diagnosis based on RDC/TMD and MRI findings of TMJ internal derangement

This study was conducted to investigate the value of magnetic resonance imaging (MRI) in the diagnostic process based on the Research Diagnostic Criteria for Temporomandibular Disorder (RDC/TMD) by evaluating agreement between RDC/TMD and MRI diagnosis of disc displacement (DD) and correlation amongst MRI findings such as DD, joint effusion (JE), degenerative change and superior lateral pterygoid muscle (SLPM) attachment. Randomly selected MRIs of 200 joints from 100 TMD patients differentiated into RDC/TMD group II representing DD by clinical examination were reviewed retrospectively. The results show that Cohens kappa value was 0.336 showing overall disagreement between RDC/TMD group II and MRI diagnoses (P<0.001). The Cohens kappa value for group IIa, DD with reduction (DDWR), was -0.223 (P<0.01) showing disagreement, whilst the value was 0.546 for group IIb, DD without reduction (DDWOR) with limited opening, and 0.490 for group IIc, DDWOR without limited opening, showing moderate agreement (P<0.001). JE was detected with a higher probability as the state of DD advanced (P<0.001) and when degenerative joint changes were present (P<0.05). The difference of DD according to SLPM attachment was insignificant. MRI could be used when clinical examination cannot predict the true position of the disc.

RNA interference-directed caveolin-1 knockdown sensitizes SN12CPM6 cells to doxorubicin-induced apoptosis and reduces lung metastasis

Human renal cell carcinoma (HRCC) is characterized by a high level of resistance to all treatment modalities. Therefore, the investigation of global gene expression in HRCC might help understand its biologic behavior and develop treatment strategies. Using cDNA microarray analysis, we initially compared gene expression profiles between HRCCs and adjacent normal tissues, and found that 87 were up-regulated and 127 genes were down-regulated. Next, a subset of genes, twofold differentially expressed, were validated by Northern blotting. Unexpectedly, caveolin-1, a gene reported to be a tumor suppressor gene, was found to be up-regulated in HRCC tissues. Expression level of caveolin-1 in SN12CPM6 (high metastatic clone) was higher than in SN12C (low metastatic clone), and SN12CPM6 was more resistant to doxorubicin (DXR) than SN12C. Caveolin-1 gene was slightly induced in surviving SN12C cells after DXR treatment. Furthermore, SN12CPM6-siCav1 cells, which were transfected with siRNA of cavelon-1 gene, were more sensitive to DXR, compared to SN12CPM6, but reduction of caveolin-1 gene expression did not affect tumor formation in subcapsule of kidney and lung metastasis. On the other hand, induction of caveolin-1 gene affected the production of lung metastasis under anti-cancer drug treatment: the incidence of pulmonary metastasis was significantly lower in SCID mice injected with SN12CPM6-siCav1 cells, and the number of pulmonary nodules decreased significantly (p = 0.0004). The above results together suggest that caveolin-1 may confer a growth advantage to cancer cells during DXR chemotherapy and surviving HRCC cells eventually might develop lung metastasis.

TNF α mediated IL-6 secretion is regulated by JAK/STAT pathway but not by MEK phosphorylation and AKT phosphorylation in U266 multiple myeloma cells.

IL-6 and TNFα were significantly increased in the bone marrow aspirate samples of patients with active multiple myeloma (MM) compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFα than those with MM in plateau phase. TNFα increased interleukin-6 (IL-6) production from MM cells. However, the detailed mechanisms involved in signaling pathways by which TNFα promotes IL-6 secretion from MM cells are largely unknown. In our study, we found that TNFα treatments induce MEK and AKT phosphorylation. TNFα-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKβ or by small interfering RNA (siRNA) targeting TNF receptors (TNFR) but not by MEK, p38, and PI3K inhibitors. Also, TNFα increased phosphorylation of STAT3 (ser727) including c-Myc and cyclin D1. Three different types of JAK inhibitors decreased the activation of the previously mentioned pathways. In conclusion, blockage of JAK/STAT-mediated NF-κB activation was highly effective in controlling the growth of MM cells and, consequently, an inhibitor of TNFα-mediated IL-6 secretion would be a potential new therapeutic agent for patients with multiple myeloma.

Non-A type nucleophosmin 1 gene mutation predicts poor clinical outcome in de novo adult acute myeloid leukemia: differential clinical importance of NPM1 mutation according to subtype

Mutations of nucleophosmin gene (NPM1) are known to be related to good prognosis in AML patients lacking FLT3 internal tandem duplication (FLT3-ITD). Recently, NPM1 mutations other than type A were reported, but their clinical significance is not well known. Retrospective medical record review of 106 de novo AML patients lacking FLT3-ITD, who received induction chemotherapy from three centers in Korea between 1997 and 2007, was performed. Direct sequencing of NPM1 and RT-PCR for FLT3-ITD was performed on genomic DNA derived from blood samples of patients before induction chemotherapy for detection of mutations. NPM1 mutation was detected in 18 patients, where 13 were type A mutants and 5 were non-type A mutants. CR, CR1-D and OS was not different according to NPM1 mutational status overall. But, non-type A NPM1 mutation was related to shorter CR1-D when compared with NPM1 wild types and NPM1 type A mutation (p = 0.004). OS was shorter in non-type A mutants when compared with NPM1 wild-type patients and NPM1 type A mutants (p = 0.001). The type of mutation of NPM1 is important for prognosis in de novo AML lacking FLT3-ITD. Non-A type NPM1 mutation is a poor prognostic factor.

Blockage of interleukin-6 signaling with 6-amino-4-quinazoline synergistically induces the inhibitory effect of bortezomib in human U266 cells.

The transcription factor nuclear factor-kappa B (NF-κB) regulates the transcription of a number of genes involved in a variety of cellular responses, including cell survival, inflammation, and differentiation. NF-κB is activated by a variety of stimuli, proinflammatory cytokines, mitogens, growth factors, and stress-inducing agents. Aberrant NF-κB expression is considered to be one of the oncogenic factors of cancer and the constitutive activation of NF-κB is observed in several hematologic disorders [classic Hodgkins lymphoma, diffuse large B cell lymphoma, and multiple myeloma (MM)], and the modulation of NF-κB activation is emerging as a promising novel anticancer therapeutic strategy.Therefore, we focused on the regulation of NF-κB activation in MM. When U266 cells were treated with 6-amino-4-quinazoline, an NF-κB activation inhibitor, we determined that it most effectively blocked the interleukin (IL)-6-induced activation of MAPK and JAK/STAT pathways among different signaling inhibitors. The results of the luciferase assay indicated that 6-amino-4-quinazoline inhibited NF-κB activation with diminished NF-κB protein bound to NF-κB DNA binding sites. In addition, 6-amino-4-quinazoline suppressed the production of IL-6, which affected MM cell proliferation. Furthermore, combined treatment with bortezomib and 6-amino-4-quinazoline effectively inhibited the IL-6 and soluble IL-6R-induced activation of STAT3 and extracellular signal-regulated kinase phosphorylation. Our data showed that the inhibition of NF-κB activation abrogated MM cell proliferation induced by the IL-6 pathway, and might represent a promising therapeutic strategy for the treatment of MM.

Molecular characterization and prognostic significance of FLT3 in CML progression

To characterize the molecular mechanisms involved in the transition from the chronic phase to blast crisis in chronic myelogenous leukemia (CML), gene expression profiles of leukemic cells from patients in the chronic and blast crisis phases were analyzed using an 8.7K cDNA chip and real-time PCR. A transient transfection analysis was conducted to evaluate the role of FLT3, which was significantly upregulated in the blast crisis patients. Abl and c-Kit induction was detected in K562 cells transfected with FLT3 cDNA (K562/FLT3), and Abl and c-Kit levels were reduced in K562/FLT3 cells transfected with FLT3-siRNA (K562/FLT3-siRNA). The induction of FLT3 in CML cells attenuated imatinib-induced apoptosis. The opposite effect was observed in K562/FLT3-siRNA cells. An increased level of cleaved PARP and decreased level of pro-caspase 3 were noted when K562/FLT3-siRNA cells were treated with imatinib. These findings indicate that FLT3 is associated with disease progression, despite imatinib therapy. These results may help in the prediction of disease progression in CML patients and the development of more appropriate therapeutic modalities.

Combination of SK-7041, one of novel histone deacetylase inhibitors, and STI571-induced synergistic apoptosis in chronic myeloid leukemia.

Although STI571 still plays a key role in the treatment of chronic myeloid leukemia, emergence of resistance to STI571 is a major obstacle to successful outcome. Therefore, new agents that increase the sensitivity of chronic myeloid leukemia cells to STI571 are urgently required. SK-7041 is a novel hybrid synthetic histone deacetylase inhibitor derived from the hydroxamic acid of trichostatin A and pyridyl ring of MS-275. Its cytotoxic effects were examined both as a single agent and in combination with STI571 in acute and chronic myeloid leukemia. SK-7041 exhibited growth inhibition of leukemia cells by downregulation of CDK4, cyclin E and cyclin B1 expression, and by upregulation of p21CIP1=WAF1 expression with subsequent activation of the mitochondria-mediated caspase pathway. SK-7041 showed synergism on growth inhibition, cell cycle arrest and induction of apoptosis in chronic myeloid leukemia (K562) when combined with STI571. The synergistic effect was mediated through the same mechanism as in SK-7041 alone, involving reduction of cyclin D1 and induction of p21CIP1=WAF1. Taken together, our findings suggest that SK-7041 is active against leukemia and offers new prospects for overcoming STI571 resistance in chronic myeloid leukemia.

Establishment of a new Glivec-resistant chronic myeloid leukemia cell line, SNUCML-02, using an in vivo model

OBJECTIVE In this study, we report a newly established chronic myeloid leukemia (CML) cell line, SNUCML-02, which is resistant to imatinib and describe its biological characteristics. MATERIALS AND METHODS Mononuclear cells were obtained from the bone marrow of a CML patient in blast crisis and were cultured in Dulbeccos modified Eagles medium/F12 containing 20% fetal bovine serum. After 2 months of primary culture, these cells were injected into nonobese diabetic/severe combined immune-deficient mice via tail vein. Eight weeks after injection, mice were sacrificed and ex vivo culture was performed from the bone marrow cells isolated from the mice. The established cell line was named as SNUCML-02 and the biological features were characterized by cytogenetic analysis, fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, sequencing analysis, cell proliferation assay, and Western blot analysis. RESULTS Cytogenetic studies using conventional G-banding and fluorescent in situ hybridization of SNUCML-02 demonstrated classical Philadelphia chromosome, (9;22)(q34;q11.2), and other abnormalities, such as add(11)(q23), +19 and +der(9;22). SNUCML-02 has the same BCR-ABL fusion transcript as was seen in K562 cells, but has no mutations in the ABL kinase domain. SNUCML-02 was more resistant to imatinib (STI571, Gleevec, Glivec) than other CML cell lines (K562, Kcl22, and BV173). SNUCML-02 has constitutive activation of extracellular signal-regulated kinase phosphorylation. In addition, interleukin-3 induced c-ABL phosphorylation and constitutively enhanced extracellular signal-regulated kinase phosphorylation was not inhibited by imatinib in SNUCML-02. CONCLUSION SNUCML-02 is a new established cell line with a relatively high level of resistance to imatinib, which is useful for investigating the pathogenesis of CML progression, and will be useful in developing optimal therapeutic strategies for this ailment.

Modified Johnson-Cook model incorporated with electroplasticity for uniaxial tension under a pulsed electric current

An empirical expression describing the electroplastic deformation behavior is suggested based on the Johnson-Cook (JC) model by adding several functions to consider both thermal and athermal electric current effects. Tensile deformation behaviors are carried out for an AZ31 magnesium alloy and an Al-Mg-Si alloy under pulsed electric current at various current densities with a fixed duration of electric current. To describe the flow curves under electric current, a modified JC model is proposed to take the electric current effect into account. Phenomenological descriptions of the adopted parameters in the equation are made. The modified JC model suggested in the present study is capable of describing the tensile deformation behaviors under pulsed electric current reasonably well.