Publications by authors named "Hwan-Mook Kim"

198 Publications

Remodeling of Cancer-Specific Metabolism under Hypoxia with Lactate Calcium Salt in Human Colorectal Cancer Cells.

Cancers (Basel) 2021 Mar 25;13(7). Epub 2021 Mar 25.

Gachon Institute of Pharmaceutical Sciences, Gachon University 191 Hambangmoe-Ro, Yeonsu-Gu, Incheon 21936, Korea.

Hypoxic cancer cells meet their growing energy requirements by upregulating glycolysis, resulting in increased glucose consumption and lactate production. Herein, we used a unique approach to change in anaerobic glycolysis of cancer cells by lactate calcium salt (CaLac). Human colorectal cancer (CRC) cells were used for the study. Intracellular calcium and lactate influx was confirmed following 2.5 mM CaLac treatment. The enzymatic activation of lactate dehydrogenase B (LDHB) and pyruvate dehydrogenase (PDH) through substrate reaction of CaLac was investigated. Changes in the intermediates of the tricarboxylic acid (TCA) cycle were confirmed. The cell viability assay, tube formation, and wound-healing assay were performed as well as the confirmation of the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). In vivo antitumor effects were evaluated using heterotopic and metastatic xenograft animal models with 20 mg/kg CaLac administration. Intracellular calcium and lactate levels were increased following CaLac treatment in CRC cells under hypoxia. Then, enzymatic activation of LDHB and PDH were increased. Upon PDH knockdown, α-ketoglutarate levels were similar between CaLac-treated and untreated cells, indicating that TCA cycle restoration was dependent on CaLac-mediated LDHB and PDH reactivation. CaLac-mediated remodeling of cancer-specific anaerobic glycolysis induced destabilization of HIF-1α and a decrease in VEGF expression, leading to the inhibition of the migration of CRC cells. The significant inhibition of CRC growth and liver metastasis by CaLac administration was confirmed. Our study highlights the potential utility of CaLac supplementation in CRC patients who display reduced therapeutic responses to conventional modes owing to the hypoxic tumor microenvironment.
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http://dx.doi.org/10.3390/cancers13071518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037473PMC
March 2021

Sesquiterpene Alcohol Cedrol Chemosensitizes Human Cancer Cells and Suppresses Cell Proliferation by Destabilizing Plasma Membrane Lipid Rafts.

Front Cell Dev Biol 2020 21;8:571676. Epub 2021 Jan 21.

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, South Korea.

Chemosensitization of cancer cells with small molecules may improve the therapeutic index of antitumoral agents by making tumor cells sensitive to the drug regimen and thus overcome the treatment resistance and side effects of single therapy. Cell membrane lipid rafts are known to transduce various signaling events in cell proliferation. Sensitizing cancer cells may cause modulation of membrane lipid rafts which may potentially be used in improving anticancer drug response. Cedrol, a natural sesquiterpene alcohol, was used to treat human leukemia K562 and colon cancer HT-29 cell lines, and effects were observed. Cedrol decreased the cell viability by inducing apoptosis in both cell lines by activation of pro-apoptosis protein BID and inhibition of anti-apoptosis proteins Bcl-X , Bcl-2, and XIAP. Cedrol activated the caspase-9-dependent mitochondrial intrinsic pathway of apoptosis. Furthermore, cedrol inhibited the levels of pAKT, pERK, and pmTOR proteins as well as nuclear and cytoplasmic levels of the p65 subunit of NF-κB. Cedrol caused redistribution of cholesterol and sphingomyelin contents from membrane lipid raft, which was confirmed by a combined additive effect with methyl-β-cyclodextrin (lipid raft-disrupting agent). Lipid raft destabilization by cedrol led to the increased production of ceramides and inhibition of membrane-bound NADPH oxidase 2 enzyme activity. Cholesterol/sphingomyelin-redistributing abilities of cedrol appear as a novel mechanism of growth inhibition of cancer cells. Cedrol can be classified as a natural lipid raft-disrupting agent with possibilities to be used in general studies involving membrane lipid raft modifications.
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http://dx.doi.org/10.3389/fcell.2020.571676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874189PMC
January 2021

Combination Antitumor Effect of Sorafenib via Calcium-Dependent Deactivation of Focal Adhesion Kinase Targeting Colorectal Cancer Cells.

Molecules 2020 Nov 13;25(22). Epub 2020 Nov 13.

Gachon Institute of Pharmaceutical Science, Gachon University, 191, Hambangmoe-ro, Yeonsu-gu, Incheon 21936, Korea.

Sorafenib has been recently used for the treatment of patients with advanced colorectal cancer (CRC) and is recognized for its therapeutic value. However, the continuous use of sorafenib may cause resistance in the treatment of cancer patients. In this study, we investigated whether sorafenib exerts an enhanced anticancer effect on CRC cells via the calcium-mediated deactivation of the focal adhesion kinase (FAK) signaling pathways. The appropriate dose of sorafenib and lactate calcium salt (CaLa) for a combination treatment were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Then, cell cycle analysis was performed following treatment with 2.5 μM sorafenib and/or 2.5 mM CaLa. CRC cells were found to be in the G1 phase by sorafenib treatment, and they accumulated in the sub-G1 phase with CaLa treatment. Western blots and enzyme-linked immunosorbent assays were performed to analyze the elements of the recombinant activated factor (RAF) and focal adhesion kinase (FAK) signaling cascades. Sorafenib-inhibited RAF-dependent signaling in CRC cells, however, either did not affect the expression of Akt or increased it. As the upstream signaling of FAK was suppressed by CaLa, we observed that the expression of the sub-signaling phospho (p) AKT and p-mammalian target of rapamycin was also suppressed. Treatment with a combination of sorafenib and CaLa enhanced the antitumor activity of CRC cells. The % viability of CRC cells was significantly decreased compared to the single treatment with sorafenib or CaLa, and the accumulation of Sub G1 of CRC cells was clearly confirmed. The migration ability of CRC cells was significantly reduced. The findings of this study indicate that sorafenib will show further improved antitumor efficacy against CRC due to overcoming resistance through the use of CaLa.
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http://dx.doi.org/10.3390/molecules25225299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697278PMC
November 2020

Potential Anticancer Effect of Calcium-mediated Src Degradation on Hormone-dependent Breast Cancer.

Anticancer Res 2020 Apr;40(4):1989-1996

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea

Background/aim: The antitumor effect of sustained calcium supply on Src degradation was investigated in the context of hormone-dependent breast cancer, followed by elucidation of the underlying mechanisms.

Materials And Methods: Hormone-dependent T-47D breast cancer cells were used. Lactate calcium salt (LCS) was used as the source of sustained calcium supply, and the applicable concentration of LCS was determined by the colorimetric MTT assay. LCS-mediated deactivation of downstream signaling via Src degradation was identified by western blot and immunocytochemistry.

Results: Calcium-mediated degradation of Src decreased survival signaling via phosphoinositide 3-kinase and protein kinase B and resulted in significant inhibition of the clonogenic ability of hormone-dependent breast cancer cells. Tumor volume was significantly decreased in response to LCS injection in a heterotopic xenograft model, and immuno histochemistry revealed tumor necrosis.

Conclusion: Sustained supply of calcium inhibited survival signaling via degradation of Src in hormone-dependent breast cancer cells.
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http://dx.doi.org/10.21873/anticanres.14154DOI Listing
April 2020

Investigation of the preventive effect of calcium on inflammation-mediated choroidal neovascularization.

Life Sci 2019 Sep 2;233:116727. Epub 2019 Aug 2.

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea. Electronic address:

Aims: Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in elderly people. The pathogenesis of neovascular AMD is known but is closely related to inflammation and choroidal neovascularization (CNV). The aim of this study was to investigate the anti-inflammatory and anti-angiogenic effects of calcium on neovascular AMD.

Main Methods: Human retinal pigment epithelial cells (ARPE-19) were used to identify protein markers of inflammation induced by differentiated macrophages. Choroidal neovascularization (CNV) mouse model was established by rupturing the Bruch's membrane using laser photocoagulation in C57BL/6 mice. Mice were divided into the following groups: untreated control and calcium supplemented. The expression levels of toll-like receptor isotype (TLR) 4, nuclear factor kappa B (NF-κB), hypoxia-inducible factor-1α (Hif-1α), and vascular endothelial growth factor (VEGF) were investigated to check whether calcium supplementation results in suppression of inflammation and has an anti-angiogenic effect. CNV was evaluated by immunofluorescence staining on choroidal flat mounts.

Key Finding: The inflammation-induced expression of TLR4, NF-κB, and Hif-1α was decreased in ARPE-19 cells after calcium supplementation. Inhibition of the transcriptional activation of ARPE-19 cells by Hif-1α suppression resulted in decreased VEGF expression. In the laser-induced CNV mouse model, calcium supplementation inhibited inflammatory mediators and neovascularization in the retinal tissue.

Significance: Supplementation with calcium seems to constrain inveterate symptoms of neovascular AMD by inhibiting inflammation and angiogenesis in the laser-induced CNV mouse model.
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http://dx.doi.org/10.1016/j.lfs.2019.116727DOI Listing
September 2019

Isolation of MLL1 Inhibitory RNA Aptamers.

Biomol Ther (Seoul) 2019 Mar;27(2):201-209

Gachon Institute of Pharmaceutical Sciences, College of Pharmacy, Gachon University, Incheon 21936, Republic of Korea.

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (ystemic volution of igands by ponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers-APT1 and APT2-represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5΄-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3΄. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.
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http://dx.doi.org/10.4062/biomolther.2018.157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430220PMC
March 2019

Investigation into Enhancing Capecitabine Efficacy in Colorectal Cancer by Inhibiting Focal Adhesion Kinase Signaling.

Anticancer Res 2018 Aug;38(8):4667-4676

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea

Background/aim: Capecitabine is a pro-drug of 5-fluorouracil (5-FU), and is an orally available chemotherapeutic used to treat colorectal cancer (CRC). Recently, research has focused on improving its efficacy at lower doses in order to minimize its well-known toxicities. In this study, we investigated the possibility of improving the antitumor effect of capecitabine against CRC by destabilizing focal adhesion kinase (FAK) signaling.

Materials And Methods: Optimal dosages for capecitabine and lactate calcium salt (LCS) were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide MTT assay. The viability of CRC cells was investigated by MTT and clonogenic assays after single or combination treatment with capecitabine and LCS. Western blot analyses were used to determine changes in the expression of components of the FAK and AKT signaling cascade, and this information was used to elucidate the underlying mechanism. A xenograft model was established to evaluate the antitumor efficacy of the combination treatment, as well as its necrotic effect and organ toxicity.

Results: The addition of LCS to capecitabine treatment led to an increase in the proteolysis of the FAK signaling cascade components, including SRC proto-oncogene, non-receptor tyrosine kinase; AKT serine/threonine kinase 1; and nuclear factor-kappa B, resulting in a decrease in the viability and clonogenic ability of CRC cells. In vivo antitumor efficacy, including tumor necrosis, was significantly increased with the combination treatment relative to both single treatments, and no organ toxicity was found in any experimental group.

Conclusion: The addition of LCS increased the anticancer efficacy of capecitabine at a lower dose than is currently used in human patients.
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http://dx.doi.org/10.21873/anticanres.12772DOI Listing
August 2018

Perspective on Cancer Therapeutics Utilizing Analysis of Circulating Tumor Cells.

Diagnostics (Basel) 2018 Apr 11;8(2). Epub 2018 Apr 11.

Gachon Institute of Pharmaceutical Science, Gachon University, 191, Hambangmoe-ro, Yeonsu-gu, Incheon 21936, Korea.

Various methods are available for cancer screening, and the methods are performed depending on the origin site of cancer. Among these methods, biopsy followed by medical imaging is the most common. After cancer progression is determined, an optimal treatment-such as surgery, chemotherapy, and/or radiation therapy-is selected. A new assay has been developed that detects circulating tumor cells (CTCs). Tracking changes in CTCs may reveal important tumoral sensitivity information or resistance patterns to specific regimens and prompt changes in therapy on a personalized basis. Characterization of CTCs at the DNA, RNA, and protein levels is important for gaining insight for clinical applications. A small number of CTCs can be analyzed to obtain genome information such as the progression of cancer including metastasis, even in a single cluster. Although many clinical studies, particularly CTC enumeration and detection of specific oncogene expression, have increased the success rate of diagnosis and predicting prognosis, there is no consensus regarding the technical approaches and various aspects of the methodology, making it difficult to standardize optimal methods for CTC analysis. However, ongoing technological advances are currently being achieved and large-scale clinical studies are being conducted. Applying CTC analysis in the clinic would be very useful for advancing diagnosis, prognosis prediction, and therapeutics.
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http://dx.doi.org/10.3390/diagnostics8020023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023425PMC
April 2018

Antitumor Effect of Calcium-Mediated Destabilization of Epithelial Growth Factor Receptor on Non-Small Cell Lung Carcinoma.

Int J Mol Sci 2018 Apr 11;19(4). Epub 2018 Apr 11.

Gachon Institute of Pharmaceutical Science, Gachon University, 191, Hambangmoe-ro, Yeonsu-gu, Incheon 21936, Korea.

Despite the development of numerous therapeutics targeting the epithelial growth factor receptor (EGFR) for non-small cell lung carcinoma (NSCLC), the application of these drugs is limited because of drug resistance. Here, we investigated the antitumor effect of calcium-mediated degradation of EGFR pathway-associated proteins on NSCLC. First, lactate calcium salt (LCS) was utilized for calcium supplementation. Src, α-tubulin and EGFR levels were measured after LSC treatment, and the proteins were visualized by immunocytochemistry. Calpeptin was used to confirm the calcium-mediated effect of LCS on NSCLC. Nuclear expression of c-Myc and cyclin D1 was determined to understand the underlying mechanism of signal inhibition following EGFR and Src destabilization. The colony formation assay and a xenograft animal model were used to confirm the in vitro and in vivo antitumor effects, respectively. LCS supplementation reduced Src and α-tubulin expression in NSCLC cells. EGFR was destabilized because of proteolysis of Src and α-tubulin. c-Myc and cyclin D1 expression levels were also reduced following the decrease in the transcriptional co-activation of EGFR and Src. Clonogenic ability and tumor growth were significantly inhibited by LSC treatment-induced EGFR destabilization. These results suggest that other than specifically targeting EGFR, proteolysis of associated molecules such as Src or α-tubulin may effectively exert an antitumor effect on NSCLC via EGFR destabilization. Therefore, LCS is expected to be a good candidate for developing novel anti-NSCLC therapeutics overcoming chemoresistance.
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http://dx.doi.org/10.3390/ijms19041158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979318PMC
April 2018

Polyunsaturated fatty acid-based targeted nanotherapeutics to enhance the therapeutic efficacy of docetaxel.

Drug Deliv 2017 Nov;24(1):1262-1272

a College of Pharmacy , Yeungnam University , Gyeongsan , Republic of Korea.

Since breast cancer is one of the most lethal malignancies, targeted strategies are urgently needed. In this study, we report the enhanced therapeutic efficacy of docetaxel (DTX) when combined with polyunsaturated fatty acids (PUFA) for effective treatment of multi-resistant breast cancers. Folic acid (FA)-conjugated PUFA-based lipid nanoparticles (FA-PLN/DTX) was developed. The physicochemical properties, in vitro uptake, in vitro cytotoxicity, and in vivo anticancer activity of FA-PLN/DTX were evaluated. FA-PLN/DTX could efficiently target and treat human breast tumor xenografts in vivo. They showed high payload carrying capacity with controlled release characteristics and selective endocytic uptake in folate receptor-overexpressing MCF-7 and MDA-MB-231 cells. PUFA synergistically improved the anticancer efficacy of DTX in both tested cancer cell lines by inducing a G2/M phase arrest and cell apoptosis. Combination of PUFA and DTX remarkably downregulated the expression levels of pro-apoptotic and anti-apoptotic markers, and blocked the phosphorylation of AKT signaling pathways. Compared to DTX alone, FA-PLN/DTX showed superior antitumor efficacy, with no signs of toxic effects in cancer xenograft animal models. We propose that PUFA could improve the therapeutic efficacy of anticancer agents in cancer therapy. Further studies are necessary to fully understand these findings and achieve clinical translation.
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http://dx.doi.org/10.1080/10717544.2017.1373163DOI Listing
November 2017

The novel hypoxia-inducible factor-1α inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth.

Cell Death Dis 2017 06 1;8(6):e2843. Epub 2017 Jun 1.

Personalized Genomic Medicine Research Center, KRIBB, Daejeon 305-806, Korea.

HIF-1 is associated with poor prognoses and therapeutic resistance in cancer patients. We previously developed a novel hypoxia-inducible factor (HIF)-1 inhibitor, IDF-11774, a clinical candidate for cancer therapy. We also reported that IDF-1174 inhibited HSP70 chaperone activity and suppressed accumulation of HIF-1α. In this study, IDF-11774 inhibited the accumulation of HIF-1α in vitro and in vivo in colorectal carcinoma HCT116 cells under hypoxic conditions. Moreover, IDF-11774 treatment suppressed angiogenesis of cancer cells by reducing the expression of HIF-1 target genes, reduced glucose uptake, thereby sensitizing cells to growth under low glucose conditions, and decreased the extracellular acidification rate (ECAR) and oxygen consumption rate of cancer cells. Metabolic profiling of IDF-11774-treated cells revealed low levels of NAD, NADP, and lactate, as well as of intermediates in glycolysis and the tricarboxylic acid cycle. In addition, we observed elevated AMP and diminished ATP levels, resulting in a high AMP/ATP ratio. The level of AMP-activated protein kinase phosphorylation also increased, leading to inhibition of mTOR signaling in treated cells. In vivo xenograft assays demonstrated that IDF-11774 exhibited substantial anticancer efficacy in mouse models containing KRAS, PTEN, or VHL mutations, which often occur in malignant cancers. Collectively, our data indicate that IDF-11774 suppressed hypoxia-induced HIF-1α accumulation and repressed tumor growth by targeting energy production-related cancer metabolism.
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http://dx.doi.org/10.1038/cddis.2017.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520894PMC
June 2017

Enhancing 5-Fluorouracil Efficacy in a Primary Colorectal Cancer by Long-lasting Calcium Supplementation.

Anticancer Res 2017 06;37(6):2959-2964

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea

Background/aim: 5-Fluorouracil (5-FU) over-use has led to an urgent need for alternative treatment regimens, such as a lower concentration of the drug because of its toxic effects. The aim of this study was to investigate the possibility of improving the antitumor effect of 5-FU without toxicity by targeting primary colorectal cancer (CRC) with sustained calcium supplementation.

Materials And Methods: The viability of CRC cells was determined after treatment of 5-FU, lactate calcium salt (CaLac), or the combination of te two. Western blot analysis for the focal adhesion kinase (FAK) signaling cascade was performed to investigate the underlying mechanism. A xenograft model was established to evaluate antitumor efficacy of each treatment, and the necrotic effect was also observed in tumor tissues.

Results: By the combined treatment, proteolysis of FAK signaling cascade, was mediated by sustained calcium supplementation resulting in further decrease in the clonogenicity of CRC cells. The in vivo anticancer efficacy including tumor necrosis was significantly increased by the combination treatment compared to single treatment of with 5-FU.

Conclusion: Sustained calcium supplementation was able to enhance the potency of 5-FU targeting the primary CRC.
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http://dx.doi.org/10.21873/anticanres.11649DOI Listing
June 2017

Enhancement of the Antitumor Effect of Methotrexate on Colorectal Cancer Cells via Lactate Calcium Salt Targeting Methionine Metabolism.

Nutr Cancer 2017 May-Jun;69(4):663-673. Epub 2017 Mar 29.

a Gachon Institute of Pharmaceutical Science , Gachon University , Incheon , Republic of Korea.

Methionine (Met) is involved in one-carbon de novo nucleotide synthesis and is an essential amino acid for cell survival. The impact of lactate calcium salt (CaLa) on the Met metabolism was investigated to evaluate the enhanced antitumor effect of methotrexate (MTX) on colorectal cancer (CRC) cells. Met dependency relating to homocysteine (Hcy) and betaine was investigated in human CRC cells (HCT-116 and HT-29) using a viability assay and liquid chromatography-mass spectrometry. Expression of betaine transporter-1 (BGT-1) following treatment with MTX alone or with CaLa was determined by Western blot. Enhanced antitumor effect due to malfunction of Met synthesis was confirmed. CRC cell viability decreased in Met-restricted medium, but was maintained after Hcy and betaine treatment while overcoming Met restriction. BGT-1 expression was downregulated following the treatment of dose-increased CaLa, whereas there was no effect on BGT-1 expression after MTX treatment. CaLa in combination with MTX induced reduced Met synthesis when CRC cell viability was reduced. The results indicated that CaLa-mediated BGT-1 downregulation inhibits Met synthesis by disrupting betaine homeostasis. CaLa raised the antitumor effect of MTX via secondary role in the inhibition of the de novo nucleotide synthesis. Combination therapy of MTX and CaLa could maximize the effectiveness of CRC treatment.
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http://dx.doi.org/10.1080/01635581.2017.1299879DOI Listing
June 2017

Synthetic strategy for increasing solubility of potential FLT3 inhibitor thieno[2,3-d]pyrimidine derivatives through structural modifications at the C and C positions.

Bioorg Med Chem Lett 2017 02 23;27(3):496-500. Epub 2016 Dec 23.

Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul 03722, Republic of Korea; Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seodaemun-gu, Seoul 03722, Republic of Korea. Electronic address:

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cell. In AML, a mutation in FLT3 is commonly occurs and is associated with poor prognosis. We have previously reported that thieno[2,3-d]pyrimidine derivative compound 1 exhibited better antiproliferative activity against MV4-11 cells which harbor mutant FLT3 than AC220, which is a well-known FLT3 inhibitor, and has good microsomal stability. However, compound 1 had poor solubility. We then carried out further structural modification at the C and the C positions of thieno[2,3-d]pyrimidine scaffold. Compound 13b, which possesses a thiazole moiety at the C position, exhibited better antiproliferative activity than compound 1 and showed increased solubility and moderate microsomal stability. These results indicate that compound 13b could be a promising potential FLT inhibitor for AML chemotherapy.
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http://dx.doi.org/10.1016/j.bmcl.2016.12.034DOI Listing
February 2017

Synergistically Anti-metastatic Effect of 5-Flourouracil on Colorectal Cancer Cells via Calcium-mediated Focal Adhesion Kinase Proteolysis.

Anticancer Res 2017 01;37(1):103-114

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea

Aim: To investigate the possibility of enhancing an anti-metastatic effect of 5-fluorouracil (5-FU) on colorectal cancer (CRC) cells by combining it with continuous calcium supplementation.

Materials And Methods: Optimal doses of 5-FU with/without lactate salt (CaLa) were determined via clonogenicity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays using human CRC cells cultured on normal or low-attachment plates. Invasion and migration assays confirmed the enhanced anti-metastatic effect of combining 5-FU and CaLa. Western blot analysis for elements of the focal adhesion kinase (FAK) signaling cascade and epithelial-mesenchymal transition (EMT) markers was used to investigate the underlying mechanism.

Results: 5-FU (2.5 μM) had no antitumor activity against unanchored CRC cells, while it significantly suppressed anchorage-dependent cell proliferation. In contrast, treatment with CaLa (2.5 mM), alone and in combination with 5-FU, exerted antitumor activity against both anchored and unanchored CRC cells via calcium-mediated FAK proteolysis and inhibition of EMT markers, such as vimentin and SNAIL.

Conclusion: Calcium supplementation represents a method of enhancing the potency of existing antitumor agents such as 5-FU, augmenting their clinical effectiveness.
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http://dx.doi.org/10.21873/anticanres.11295DOI Listing
January 2017

Engineering of caveolae-specific self-micellizing anticancer lipid nanoparticles to enhance the chemotherapeutic efficacy of oxaliplatin in colorectal cancer cells.

Acta Biomater 2016 09 6;42:220-231. Epub 2016 Jul 6.

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of Korea. Electronic address:

Unlabelled: Novel nanomaterials for the intracellular transport of therapeutic cargos have been actively sought to effectively breach cell-membrane barriers. In this study we developed novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells (CRCs). We demonstrated that NPs with special affinity to caveolae could be designed and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. We demonstrated a remarkable uptake of OL-loaded SMAL NPs (SMAL-OL) in HCT116 and HT-29 cells via the caveolae-mediated endocytosis (CvME) pathway. The higher accumulation of SMAL-OL in the intracellular environment resulted in a significantly elevated anticancer effect compared to that of free OL. Cell cycle analysis proved G2/M phase arrest, along with substantial presence of cells in the sub-G1 phase. An immunoblot analysis indicated an upregulation of pro-apoptotic markers (Bax; caspase-3; caspase-9; and PARP1) and downregulation of Bcl-xl and the PI3K/AKT/mTOR complex, indicating a possible intrinsic apoptotic signaling pathway. Overall, the ability of SMAL NPs to confer preferential specificity towards the cell surface domain could offer an exciting means of targeted delivery without the need for receptor-ligand-type strategies.

Statement Of Significance: In this work, we developed a novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells. We demonstrated that NPs with special affinity to caveolae could be realized and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. In addition, oxaliplatin-loaded SMAL were efficiently endocytosed by the cancer cells and represent a significant breakthrough as an effective drug delivery system with promising potential in cancer therapy. We believe this work holds promising potential for the development of next generation of multifunctional nanocarriers for an exciting means of targeted delivery without the need for receptor-ligand-type strategies.
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http://dx.doi.org/10.1016/j.actbio.2016.07.006DOI Listing
September 2016

Identification of Targets of the HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes.

Bioconjug Chem 2016 08 18;27(8):1911-20. Epub 2016 Jul 18.

College of Pharmacy, Dongguk University-Seoul , Goyang 410-820, Korea.

We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774's target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00305DOI Listing
August 2016

Purification and characterization of chitinase showing antifungal and biodegradation properties obtained from Streptomyces anulatus CS242.

Arch Pharm Res 2016 Jul 23;39(7):878-86. Epub 2016 May 23.

Department of Pharmacy, Chosun University, Gwangju, 501-759, Korea.

In an effort to identify a microbial enzyme that can be useful as a fungicide and biodegradation agent of chitinous wastes, a chitinase (Chi242) was purified from the culture supernatant of Streptomyces anulatus CS242 utilizing powder of shrimp shell wastes as a sole carbon source. It was purified employing ammonium sulfate precipitation and gel permeation chromatography techniques. The molecular weight of the purified chitinase was ~38 kDa by SDS-PAGE. The N-terminal amino acid sequence (A-P-G-A-P-G-T-G-A-L) showed close similarity to those of other Streptomyes chitinases. The purified enzyme displayed optimal activity at pH 6.0 and 50 °C respectively. It showed substantial thermal stability for 2 h at 30-60 °C, and exhibited broad pH stability in the range 5.0-13.0 for 48 h at 4 °C. Scanning electron microscopy confirmed the ability of this enzyme to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. Chi242 could proficiently convert colloidal chitin to N-acetyl glucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)2 signifying that this enzyme is suitable for bioconversion of chitin waste. In addition, it exerted an effective antifungal activity towards fungal pathogen signifying its role as a biocontrol agent. Thus, a single microbial cell of Streptomyces anulatus CS242 justified its dual role.
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http://dx.doi.org/10.1007/s12272-016-0747-3DOI Listing
July 2016

Combination of lactate calcium salt with 5-indanesulfonamide and α-cyano-4-hydroxycinnamic acid to enhance the antitumor effect on HCT116 cells via intracellular acidification.

Oncol Lett 2016 Mar 20;11(3):1866-1872. Epub 2016 Jan 20.

College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-840, Republic of Korea.

Maintenance of a neutral intracellular pH (pHi) is favorable for the survival of tumors, and maintenance of highly acidic extracellular pH (pH) facilitates tumor invasiveness. The aim of the present study was to investigate the antitumor effects of lactate calcium salt (CaLa), 5-indanesulfonamide (IS) and α-cyano-4-hydroxycinnamic acid (CA) via pH regulation in colon cancer cells. HCT116 cells were treated with CaLa, IS, CA and combinations of the three. Subsequently, the concentration of intracellular lactate was determined. pHi and pH were measured using cell lysates and culture media. Colony formation assay, cell viability assay and western blot analysis were additionally performed to analyze the consequences of the pH changes. CaLa, IS, CA and combination treatments induced an increase in the concentration of intracellular lactate. Lactate influx into the tumor microenvironment produced an acidic pHi in colon cancer cells. Consequently, colony formation and cell viability were significantly decreased, as well as poly(adenosine diphosphate-ribose) polymerase degradation. The tumor microenvironment may be exploited therapeutically by disrupting the mechanism that regulates pHi, leading to cell apoptosis. The present study indicated that treatment with CaLa, IS and CA induced intracellular acidification via lactate influx, causing apoptosis of colon cancer cells. Additionally, the findings suggested that the combination of CaLa with IS and CA may enhance antitumor activity, and may provide a potential therapeutic approach for the treatment of colon cancer.
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http://dx.doi.org/10.3892/ol.2016.4137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774552PMC
March 2016

Ninjurin1 suppresses metastatic property of lung cancer cells through inhibition of interleukin 6 signaling pathway.

Int J Cancer 2016 07 16;139(2):383-95. Epub 2016 Feb 16.

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, Republic of Korea.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell surface molecule that can mediate homophilic adhesion and promote neurite outgrowth from cultured dorsal root ganglion (DRG) neurons. Interestingly, Ninj1 overexpressed in human cancer; however, its role in metastasis is not clear. This study showed that inhibition of Ninj1 promotes lung cancer metastasis through interleukin 6 (IL-6)/STAT3 signaling. Ninj1 levels were relatively low in highly motile lung cancer cells. While inhibition of Ninj1 enhanced cell migration in lung cancer cells, overexpression of Ninj1 significantly suppressed it. We found that inhibition of Ninj1 significantly increased expression and secretion of IL-6 in A549 cells. We also found that inhibition of IL-6 decreased intercellular adhesion molecule 1 (ICAM-1) expression. In addition, inhibition of Ninj1 significantly increased cell motility and invasiveness of lung cancer cells. In an in vivo model, we found that Ninj1 suppression did not affect tumor growth but induced significant increase in incidence of lung metastasis, and sizes and number of tumor nodules. Taken together, our data clearly demonstrate that Ninj1 suppresses migration, invasion and metastasis of lung cancer via inhibition of the IL-6 signaling pathway in vitro and in vivo.
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http://dx.doi.org/10.1002/ijc.30021DOI Listing
July 2016

Lactate calcium salt affects the viability of colorectal cancer cells via betaine homeostasis.

Life Sci 2016 Feb 20;147:71-6. Epub 2016 Jan 20.

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea. Electronic address:

Aims: Betaine plays an important role in cellular homeostasis. However, the physiological roles of betaine-γ-aminobutyric acid (GABA) transporter (BGT-1) are still being disputed in cancer. In this study, we tried to find the possibility of the antitumor effect on colorectal cancer (CRC) cell via lactate calcium salt (CaLa)-induced BGT-1 downregulation.

Main Methods: The CRC cell viability and clonogenic assay was performed using different doses of BGT-1 inhibitor. The expression level of BGT-1 was measured following the treatment of 2.5mM CaLa. Betaine was treated to confirm the resistance of the antitumor activity by CaLa. Tumor growth was also measured using a xenograft animal model.

Key Findings: Long-term exposure of 2.5mM CaLa clearly decreased the expression of BGT-1 in the CRC cells. As a result of the downregulation of BGT-1 expression, the clonogenic ability of CRC cells was also decreased in the 2.5mM CaLa-treated group. Reversely, the number of colonies and cell viability was increased by combination treatment with betaine and 2.5mM CaLa, as compared with a single treatment of 2.5mM CaLa. Tumor growth was significantly inhibited in the xenograft model depending on BGT-1 downregulation by 2.5mM CaLa treatment.

Significance: These results support the idea that long-lasting calcium supplementation via CaLa contributes to disruption of betaine homeostasis in the CRC cells and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC.
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http://dx.doi.org/10.1016/j.lfs.2016.01.030DOI Listing
February 2016

Sulfiredoxin inhibitor induces preferential death of cancer cells through reactive oxygen species-mediated mitochondrial damage.

Free Radic Biol Med 2016 Feb 23;91:264-74. Epub 2015 Dec 23.

Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 120-750, South Korea. Electronic address:

Recent studies have shown that many types of cancer cells have increased levels of reactive oxygen species (ROS) and enhance antioxidant capacity as an adaptation to intrinsic oxidative stress, suggesting that cancer cells are more vulnerable to oxidative insults and are more dependent on antioxidant systems compared with normal cells. Thus, disruption of redox homeostasis caused by a decline in antioxidant capacity may provide a method for the selective death of cancer cells. Here we show that ROS-mediated selective death of tumor cells can be caused by inhibiting sulfiredoxin (Srx), which reduces hyperoxidized peroxiredoxins, leading to their reactivation. Srx inhibitor increased the accumulation of sulfinic peroxiredoxins and ROS, which led to oxidative mitochondrial damage and caspase activation, resulting in the death of A549 human lung adenocarcinoma cells. Srx depletion also inhibited the growth of A549 cells like Srx inhibition, and the cytotoxic effects of Srx inhibitor were considerably reversed by Srx overexpression or antioxidants such as N-acetyl cysteine and butylated hydroxyanisol. Moreover, Srx inhibitor rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells and significantly suppressed the growth of A549 xenografts without acute toxicity. Our results suggest that Srx might serve as a novel therapeutic target for cancer treatment based on ROS-mediated cell death.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.023DOI Listing
February 2016

Neonatal capsaicin treatment in rats induces chronic hyperthermia resulting in infectious disease.

Exp Ther Med 2015 Dec 23;10(6):2417-2423. Epub 2015 Oct 23.

College of Pharmacy, Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of Korea.

Treatment of neonatal animals with capsaicin has previously been associated with long-lasting hyperthermia and severe cutaneous lesions. The present study analyzed the effects of capsaicin-induced hyperthermia on the occurrence of infectious disease and pruritic dermatitis in a rat model. Pregnant Sprague-Dawley (SD) rats were obtained 1 week prior to parturition. Pups from each litter were randomly assigned to the following experimental groups: Capsaicin-treated (cap-treated; n=10) or vehicle-treated (n=5). Capsaicin (50 mg/kg) or vehicle were systemically administered to the SD rat pups (age, 48 h), after which body temperature was measured using a biotelemetry system, and the effects of hyperthermia on the ability of the rat pups to resist bacterial infection were analyzed. Furthermore, pruritus-induced scratching behavior and dermatitis were assessed, and changes in interleukin (IL)-4- and IL-13-induced immunoglobulin E expression were measured. Treatment of neonatal rats with capsaicin resulted in chronic hyperthermia, which had negative effects on the host immune defense response. The expression levels of T-helper type 2 cell-associated cytokines were significantly increased (P<0.01) in the cap-treated rats following bacterial infection with or . Furthermore, cap-treated rats exhibited pruritus-induced scratching behavior and dermatitis. The results of the present study suggested that treatment of neonatal rats with capsaicin induces chronic hyperthermia and decreases the effectiveness of the host defense system. Therefore, a cap-treated neonatal rat model may be considered useful when investigating the association between hyperthermia and infectious disease.
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http://dx.doi.org/10.3892/etm.2015.2829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665930PMC
December 2015

Investigation of the functional difference between the pathological itching and neuropathic pain-induced rat brain using manganese-enhanced MRI.

Acta Radiol 2016 Jul 17;57(7):861-8. Epub 2015 Sep 17.

Department of Biomedical Laboratory Science, College of Health and Medical Science, Daejeon University, Daejeon, Republic of Korea

Background: There is a remarkable similarity in the central sensitization of itch and pain. However, the interactions between itch and pain are only partially understood.

Purpose: To investigate the functional activity of cerebral regions to provide clear information on the neuronal pathways related to both pathological itching (PI) and neuropathic pain (NP).

Material And Methods: Sprague-Dawley rats were used in this study. PI was induced via neonatal capsaicin treatment, and scratching behavior was counted. NP was induced via lumbar spinal nerve 5 (L5) ligation, and mechanical allodynia was measured. The activated cerebral regions in the control, PI, and NP rats were measured using a 4.7 T magnetic resonance imaging (MRI) system and manganese-enhanced MRI (MEMRI). Subsequently, the cerebral activation regions were identified, and the signal intensity was compared.

Results: Cerebral activities of the PI-induced rats were found in three regions -7.10 and -4.20 mm, and two regions -2.45 mm from the bregma. In the NP-induced rats, cerebral activities were found in two regions 7.10 and -2.45 mm, and one region -4.20 mm from the bregma. Comparing the PI and NP rats, the cerebral activities were different in one region -7.10 mm and -2.45 mm, and two regions -4.20 mm from the bregma. The different regions were the midbrain area, the geniculate complex, the hypothalamic area, and the amygdala area.

Conclusion: Our MEMRI investigation indicates functionally different activity of cerebral regions due to the effect of PI or NP. These findings provide clear information of the signal transduction in the brain regarding PI or NP that share a similar neuronal pathway.
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http://dx.doi.org/10.1177/0284185115604514DOI Listing
July 2016

Novel self-micellizing anticancer lipid nanoparticles induce cell death of colorectal cancer cells.

Colloids Surf B Biointerfaces 2015 Nov 28;135:793-801. Epub 2015 Aug 28.

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of Korea. Electronic address:

In the present study, we developed a novel drug-like self-micellizing anticancer lipid (SMAL), and investigated its anticancer activity and effects on cell death pathways in human colorectal cancer (CRC) cell lines. Three self-assembled nanoparticles were prepared, namely, SMAL102 (lauramide derivative), SMAL104 (palmitamide derivative), and SMAL108 (stearamide derivative) by a thin-film hydration technique, and were characterized for physicochemical and biological parameters. SMAL102 were nanosized (160.23 ± 8.11 nm) with uniform spherical shape, while SMAL104 and SMAL108 did not form spherical shape but formed large size nanoparticles and irregular in shape. Importantly, SMAL102 showed a cytotoxic effect towards CRC cell lines (HCT116 and HT-29), and less toxicity to a normal colon fibroblast cell line (CCD-18Co). Conversely, SMAL104 and SMAL108 did not have an anti-proliferative effect on CRC cell lines. SMAL102 nanoparticles were actively taken up by CRC cell lines, localized in the cell membrane, and exhibited remarkable cytotoxicity in a concentration-dependent manner. The normal colon cell line showed significantly less cellular uptake and non-cytotoxicity as compared with the CRC cell lines. SMAL102 nanoparticles induced caspase-3, caspase-9, and PARP cleavage in HT-29 cells, indicating the induction of apoptosis; whereas LC3B was activated in HCT116 cells, indicating autophagy-induced cell death. Collectively, these results demonstrate that SMAL102 induced cell death via activation of apoptosis and autophagy in CRC cell lines. The present study could be a pioneer for further preclinical and clinical development of such compounds.
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http://dx.doi.org/10.1016/j.colsurfb.2015.08.039DOI Listing
November 2015

Investigation of lactate calcium salt-induced β-catenin destabilization in colorectal cancer cells.

Life Sci 2015 Oct 24;139:160-5. Epub 2015 Aug 24.

Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea. Electronic address:

Aims: Calcium supplements appear to reduce the risk of developing colorectal cancer (CRC), and it is necessary to clarify the mechanisms by which they exert their effects. In the present study, we investigate the supplementation effect of calcium via lactate calcium salt (CaLa) on CRC cells, focusing on β-catenin destabilization.

Main Methods: The clonogenic assay was performed using different doses of CaLa. The expression level of c-Myc and Cyclin D1 was measured in addition to the confirmation of β-catenin expression in the CRC cells. Glycogen synthase kinase (GSK)-3β expression was also confirmed in order to investigate the mechanism of β-catenin degradation. Tumorigenic ability was confirmed using a xenograft animal model.

Key Findings: The number of colonies was significantly decreased after 2.5mM CaLa treatment. CaLa-treated CRC cells showed a decrease in the β-catenin expression. The quantitative level of the β-catenin protein was significantly decreased in the CRC cell lysates, hence the expression level of c-Myc and cyclin D1 was significantly decreased following 2.5mM CaLa treatment. We also confirmed that an increased expression of GSK-3β by CaLa is a key pathway in β-catenin degradation. In the xenograft study, tumorigenicity was significantly inhibited to a maximum of 45% in the CaLa-treated group as compared with the control.

Significance: These results support the idea that calcium supplementation via CaLa contributes to β-catenin degradation and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC.
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http://dx.doi.org/10.1016/j.lfs.2015.08.012DOI Listing
October 2015

Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF.

Oncotarget 2015 Sep;6(27):24047-60

College of Pharmacy, Gachon University, Incheon, Republic of Korea.

The growth factors derived from the microenvironment create an environment conducive to tumor growth and survival. HGF deprivation using neutralizing antibody enhanced chemosensitivity in colorectal cancer cells (CRC). We determined secreted HGF in fibroblast conditioned medium (CM). Combination treatment of anti-HGF antibody and irinotecan (CPT-11) directly enhanced CPT-11 sensitivity in CRC. We generated xenograft in NOD/SCID mice inoculating HCT-116 human colorectal cancer cells subcutaneously with or without fibroblast. We found that the combination of CPT-11 and anti-HGF antibody induced marked suppression of tumor development. These results suggest that HGF produced by fibroblast induce CPT-11 resistance, and that anti-HGF antibody abrogate such resistance in vivo. fibroblast-derived HGF is important determinant of chemoresistance. Anti-HGF monoclonal antibody treatment confirmed the importance of this growth factor for chemoresistance in CRC. These results present new options toward the early diagnosis of chemoresistance and suggest novel combinations of chemotherapy and anti-HGF agents to prevent or significantly delay the onset of therapy resistance.
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http://dx.doi.org/10.18632/oncotarget.4369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695169PMC
September 2015

p53 Modulates Notch Signaling in MCF-7 Breast Cancer Cells by Associating With the Notch Transcriptional Complex Via MAML1.

J Cell Physiol 2015 Dec;230(12):3115-27

Stanley Scott Cancer Center, Louisiana State Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, Louisiana.

p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.
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http://dx.doi.org/10.1002/jcp.25052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549197PMC
December 2015

Evaluation of preventive and therapeutic activity of novel non-steroidal anti-inflammatory drug, CG100649, in colon cancer: Increased expression of TNF-related apoptosis-inducing ligand receptors enhance the apoptotic response to combination treatment with TRAIL.

Oncol Rep 2015 Apr 10;33(4):1947-55. Epub 2015 Feb 10.

College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-840, Republic of Korea.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage‑dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL‑mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.
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http://dx.doi.org/10.3892/or.2015.3793DOI Listing
April 2015

Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain.

PLoS One 2015 28;10(1):e0116984. Epub 2015 Jan 28.

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of Korea.

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116984PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309579PMC
February 2016