Younghyoun Yoon

Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1

Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.

CXC Chemokine Receptor-4 Antagonist Blocks Both Growth of Primary Tumor and Metastasis of Head and Neck Cancer in Xenograft Mouse Models

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.

Development of a unique small molecule modulator of CXCR4.

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties

Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogs with enhanced activity on NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analog whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1-100 μM) or vehicle (DMSO 1%) for 1h. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1 μg/mL). EF31 (IC(50)~5 μM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC(50)~35 μM) and curcumin (IC(50) >50 μM). In addition, EF31 exhibited greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB, EF31 (IC(50)~1.92 μM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC(50)~131 μM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analog for further therapeutic development.

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

Background A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. Methodology/Principal Findings The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. Conclusions/Significance CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

Monocarbonyl curcumin analogues: Heterocyclic pleiotropic kinase inhibitors that mediate anticancer properties

Curcumin is a biologically active component of curry powder. A structurally related class of mimetics possesses similar anti-inflammatory and anticancer properties. Mechanism has been examined by exploring kinase inhibition trends. In a screen of 50 kinases relevant to many forms of cancer, one member of the series (4, EF31) showed ≥85% inhibition for 10 of the enzymes at 5 μM, while 22 of the proteins were blocked at ≥40%. IC50 values for an expanded set of curcumin analogues established a rank order of potencies, and analyses of IKKβ and AKT2 enzyme kinetics for 4 revealed a mixed inhibition model, ATP competition dominating. Our curcumin mimetics are generally selective for Ser/Thr kinases. Both selectivity and potency trends are compatible with protein sequence comparisons, while modeled kinase binding site geometries deliver a reasonable correlation with mixed inhibition. Overall, these analogues are shown to be pleiotropic inhibitors that operate at multiple points along cell signaling pathways.

Early prediction of response to Vorinostat in an orthotopic rat glioma model.

Glioblastoma is the most common primary brain tumor and is uniformly fatal despite aggressive surgical and adjuvant therapy. As survival is short, it is critical to determine the value of therapy early on in treatment. Improved early predictive assessment would allow neuro‐oncologists to personalize and adjust or change treatment sooner to maximize the use of efficacious therapy. During carcinogenesis, tumor suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. Suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) is an orally active, potent inhibitor of histone deacetylase (HDAC) activity. A major shortcoming of the use of HDAC inhibitors in the treatment of patients with brain tumors is the lack of reliable biomarkers to predict and determine response. Histological evaluation may reflect tumor viability following treatment, but is an invasive procedure and impractical for glioblastoma. Another problem is that response to SAHA therapy is associated with tumor redifferentiation and cytostasis rather than tumor size reduction, thus limiting the use of traditional imaging methods. A noninvasive method to assess drug delivery and efficacy is needed. Here, we investigated whether changes in 1H MRS metabolites could render reliable biomarkers for an early response to SAHA treatment in an orthotopic animal model for glioma. Untreated tumors exhibited significantly elevated alanine and lactate levels and reduced inositol, N‐acetylaspartate and creatine levels, typical changes reported in glioblastoma relative to normal brain tissues. The 1H MRS‐detectable metabolites of SAHA‐treated tumors were restored to those of normal‐like brain tissues. In addition, reduced inositol and N‐acetylaspartate were found to be potential biomarkers for mood alteration and depression, which may also be alleviated with SAHA treatment. Our study suggests that 1H MRS can provide reliable metabolic biomarkers at the earliest stage of SAHA treatment to predict the therapeutic response. Copyright

Benzenesulfonamides: A Unique Class of Chemokine Receptor Type 4 Inhibitors

The interaction of CXCR4 with CXCL12 (SDF‐1) plays a critical role in cancer metastasis by facilitating the homing of tumor cells to metastatic sites. Based on our previously published work on CXCR4 antagonists, we have synthesized a series of aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction. Analogue bioactivities were assessed with binding affinity and Matrigel invasion assays. Computer modeling was employed to evaluate a selection of the new analogues docked into the CXCR4 X‐ray structure and to rationalize discrepancies between the affinity and Matrigel in vitro assays. A lead compound displays nanomolar potency in the binding affinity assay (IC50=8.0 nM) and the Matrigel invasion assay (100 % blockade of invasion at 10 nM). These data demonstrate that benzenesulfonamides are a unique class of CXCR4 inhibitors with high potency.

Synthesis and evaluation of 2,5 and 2,6 pyridine-based CXCR4 inhibitors

Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.

Early prediction of response to Vorinostat in an orthotopic glioma rat model

Glioblastoma is the most common primary brain tumor and is uniformly fatal despite aggressive surgical and adjuvant therapy. As survival is short, it is critical to determine the value of therapy early on in treatment. Improved early predictive assessment would allow neuro‐oncologists to personalize and adjust or change treatment sooner to maximize the use of efficacious therapy. During carcinogenesis, tumor suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. Suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) is an orally active, potent inhibitor of histone deacetylase (HDAC) activity. A major shortcoming of the use of HDAC inhibitors in the treatment of patients with brain tumors is the lack of reliable biomarkers to predict and determine response. Histological evaluation may reflect tumor viability following treatment, but is an invasive procedure and impractical for glioblastoma. Another problem is that response to SAHA therapy is associated with tumor redifferentiation and cytostasis rather than tumor size reduction, thus limiting the use of traditional imaging methods. A noninvasive method to assess drug delivery and efficacy is needed. Here, we investigated whether changes in 1H MRS metabolites could render reliable biomarkers for an early response to SAHA treatment in an orthotopic animal model for glioma. Untreated tumors exhibited significantly elevated alanine and lactate levels and reduced inositol, N‐acetylaspartate and creatine levels, typical changes reported in glioblastoma relative to normal brain tissues. The 1H MRS‐detectable metabolites of SAHA‐treated tumors were restored to those of normal‐like brain tissues. In addition, reduced inositol and N‐acetylaspartate were found to be potential biomarkers for mood alteration and depression, which may also be alleviated with SAHA treatment. Our study suggests that 1H MRS can provide reliable metabolic biomarkers at the earliest stage of SAHA treatment to predict the therapeutic response. Copyright