Eunkyung Bae

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.

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.

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.

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.

Abstract 1527: CD44v9 involving in multiple myeloma cells adhesion to bone marrow stromal cell promotes bone erosion by augmenting the activation of HGF-receptor/cMet

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of multiple myeloma cells. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support multiple myeloma cell survival. In this study, we found that CD44s and CD44 variants were differentially expressed between fraction of CD138+ fraction and CD138- fraction. Expression levels of CD44v6, CD44v9, and CD44v10, respectively, correlated with bone erosion (p=0.029, p=0.013, p=0.032), suggesting that CD44 variant molecules are involved in multiple myeloma progression. Binding studies using CD44 isoform specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM. In 3D culture, CD44v6 and CD44v9-mediated plasma cell binding resulted in a significant induction of HGF secretion by bone marrow stromal cells. CD44v6 and CD44v9-mediated plasma cell binding significantly induces PI3K/Akt via activation the Src-kinase Lyn. In bone marrow serum of MM patients, the expression levels of IL-6, OPN, and hepatocyte growth factor (HGF), respectively, statistically correlated with bone erosion of MM patients (p=0.021, p=0.001, p=0.036). HGF derived from bone marrow stromal cells with multiple myeloma cells stimulates CD44 signaling via activation of HGF-receptor/cMet. Specific CD44 shRNA suppresses HGF-mediated CD44 signaling. Taken together, the role of CD44 variants in adhesion induced HGF- secretion may explain the previously observed correlation between CD44 variants expression and adverse prognosis in multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1527. doi:1538-7445.AM2012-1527

Abstract 1518: Multiple myeloma cells cross talk with bone marrow stroma lead to induction of DKK1 expression and produce bortezomib resistance

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The importance of the micro-environment in tumor progression is now well established. Dynamic interaction between multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) plays critical role in the progression of MM including drug response and bone erosion. Firstly, we found that DKK1 expression was statistically increased in MM cells (MOLP8, KMS12BM, KMS12PE, NCIH929, LP1, MOLP2, EJM, U266, RPMI8226, and 536MM, respectively) co-cultured with BMSCs obtained from MM patients when compared to MM cells alone (p<0.05). Also, MM cells co-cultured with BMSCs were significantly more resistant against 25 nM bortezomib than MM cells alone (p<0.05). We, therefore, established a bortezomib-resistant cell line using U266 cells (U266/velR), and explored the characteristics of U266/velR cells. Cytotoxic effect of bortezomib in U266/velR was 1.5 folds lower than U266 cells, and the cross-resistance against thalidomide was observed. DKK1 expression in U266/velR was higher than that in parental cells. We found that elevated levels of p-p65 were detected in U266/velR, and the degree of p65 and I-κB expression levels reduced by bortezomib was different between U266/velR and U266. Elevated levels of p-p65/p65 were effectively suppressed by treating with NF-κB activator inhibitor (6-aminoquinazoline). Also, combined treatment of bortezomib and 6-aminoquinazoline reduced the expression of DKK1 in co-culture of U266 cells and BMSCs. The expression of HGF in CD138− fractions of MM bone marrow cells was higher than that in its CD138+ fractions. Whereas, IL-6 and OPN in CD138+ fraction of MM bone marrow cells was higher compared to its CD138− fractions. In co-culture of U266 and BMSCs, IL-6, OPN and HGF was dramatically increased compared to U266 alone and BMSC alone (p<0.01; p<0.05). Taken together, cytokines and growth factor regulated by dynamic interplay between MM cells and BMSCs contributes the bortezomib response and bone erosion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1518. doi:1538-7445.AM2012-1518