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1.
Int J Oncol ; 53(2): 877-885, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29901185

ABSTRACT

The major hallmarks of tumor progression are angiogenesis, migration and metastasis. Among the components of Rhodiola rosea, salidroside (p­hydroxyphenethyl-ß­d-glucoside) is one of the most potent, and is present in all Rhodiola species. Recent data have revealed the anticancer effects of salidroside; however, the mechanism underlying its ability to inhibit tumor angiogenesis remains unknown. The present study aimed to analyze how salidroside affects major factors involved in breast cancer, and to elucidate its ability to inhibit angiogenesis and invasion. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis and migration, which interacts with matrix metalloproteinases (MMPs). Specifically, MMPs act as a downstream target for STAT3. Using western blotting and reverse transcription-quantitative polymerase chain reaction analysis, the present study demonstrated that treatment of MDA­MB 231 triple-negative breast cancer (TNBC) cells with salidroside led to inhibition of invasion and migration markers, and of STAT3 signaling. Furthermore, in vitro angiogenesis analyses in human umbilical vein endothelial cells confirmed the anti-angiogenic activity of salidroside. An electrophoretic mobility shift assay also demonstrated that salidroside may inhibit the DNA-binding activity of STAT3, preventing STAT3 from binding to a novel binding site of the MMP2 gene promoter. In conclusion, the present results demonstrated that salidroside may downregulate the STAT3 signaling pathway, and inhibit cell viability, migration and invasion through MMPs in breast cancer cells.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Glucosides/pharmacology , Matrix Metalloproteinase 2/metabolism , Phenols/pharmacology , Signal Transduction/drug effects , Triple Negative Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Matrix Metalloproteinase 2/genetics , Neoplasm Invasiveness , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics
2.
Int J Mol Sci ; 18(5)2017 Apr 30.
Article in English | MEDLINE | ID: mdl-28468300

ABSTRACT

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER⁺) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER⁺ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER⁺ breast cancer cells.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Flavones/pharmacology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Paxillin/metabolism , Receptors, Estrogen/metabolism , STAT3 Transcription Factor/metabolism , src-Family Kinases/metabolism , Cell Proliferation , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , MCF-7 Cells , Signal Transduction
3.
Oncol Rep ; 37(6): 3270-3278, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28440514

ABSTRACT

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.


Subject(s)
Janus Kinase 2/genetics , Matrix Metalloproteinase 2/genetics , STAT3 Transcription Factor/genetics , Silymarin/administration & dosage , Triple Negative Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Female , Humans , Neoplasm Invasiveness/genetics , Phosphorylation , Signal Transduction/drug effects , Silybin , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology
4.
Anticancer Res ; 37(4): 1637-1646, 2017 04.
Article in English | MEDLINE | ID: mdl-28373424

ABSTRACT

Gingival squamous cell carcinoma is a rare form of cancer that accounts for less than 10% of all head and neck cancers. Targeted therapies with natural compounds are of interest because they possess high efficacy with fewer side-effects. Methylsulfonylmethane (MSM) is an organic sulfur-containing compound with anticancer activities. The main goal of this study was to induce proliferation inhibition and apoptosis in the metastatic YD-38 cell line. MSM up-regulated expression of P21Waf1/Cip1 and P27Kip1 genes and down-regulated expression of cyclin D1 (CCND1) and CDK4. Moreover, treatment with MSM induced apoptosis and up-regulation of BAX in YD-38 cells. In accordance, the expression of the BCL-2 and BCL-XL, were inhibited, indicating the role of mitochondria in MSM-induced apoptosis. Analysis of mitochondrial integrity showed a loss of mitochondrial potential with an increased level of cytochrome c in the cytosol compared to mitochondria. Active CASPASE-3 (CASP3) was also observed, confirming that MSM-induced apoptosis is caspase-mediated.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Cycle Checkpoints/drug effects , Dimethyl Sulfoxide/pharmacology , G1 Phase/drug effects , Gingival Neoplasms/pathology , Mitochondria/pathology , Sulfones/pharmacology , Apoptosis/drug effects , Blotting, Western , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytochromes c/metabolism , Gingival Neoplasms/drug therapy , Gingival Neoplasms/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Tumor Cells, Cultured
5.
J Cell Mol Med ; 21(4): 720-734, 2017 04.
Article in English | MEDLINE | ID: mdl-27862996

ABSTRACT

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti-oxidant with anti-proliferative effects on multiple cancers. However, its ability to modulate gene-specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non-canonical STAT pathways to impose the gene-specific induction of G1-arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1-arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non-canonical STAT pathways, each with a specific role in TA-induced anti-cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA-binding activity of STAT1 and in turn enhanced expression of p21Waf1/Cip1 . However, TA binds to EGF-R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL-2 DNA-binding activity. As a result, the expression and mitochondrial localization of BCl-2 are declined. This altered expression and localization of mitochondrial anti-pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF-R/Jak2/STAT1/3 and P38/STAT1/p21Waf1/Cip1 pathways and induce G1-arrest and intrinsic apoptosis in breast carcinomas.


Subject(s)
Breast Neoplasms/metabolism , ErbB Receptors/metabolism , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Tannins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Drug Synergism , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Gefitinib , Humans , Phosphorylation/drug effects , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Transport/drug effects , Quinazolines/pharmacology , Tamoxifen/pharmacology
6.
Anim Biotechnol ; 28(3): 189-197, 2017 Jul 03.
Article in English | MEDLINE | ID: mdl-27874312

ABSTRACT

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.


Subject(s)
Angiopoietins/metabolism , Diterpenes/pharmacology , Hydroxymethylglutaryl-CoA Synthase/antagonists & inhibitors , Ketosis/metabolism , Lactones/pharmacology , Lipoprotein Lipase/metabolism , Signal Transduction/drug effects , Animals , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Hydroxymethylglutaryl-CoA Synthase/metabolism , Ketone Bodies/metabolism , Mice , Models, Biological , STAT5 Transcription Factor/metabolism
7.
PLoS One ; 11(7): e0159891, 2016.
Article in English | MEDLINE | ID: mdl-27447722

ABSTRACT

Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3's involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity.


Subject(s)
Bone Resorption/metabolism , Dimethyl Sulfoxide/pharmacology , Macrophages/drug effects , Macrophages/metabolism , NF-kappa B/metabolism , RANK Ligand/metabolism , STAT3 Transcription Factor/metabolism , Sulfones/pharmacology , Animals , Biomarkers , Bone Resorption/genetics , Cell Differentiation/drug effects , Gene Expression , Mice , Osteoclasts/drug effects , Osteoclasts/metabolism , RANK Ligand/pharmacology , STAT3 Transcription Factor/genetics , Signal Transduction , Tartrate-Resistant Acid Phosphatase/metabolism
8.
Mol Med Rep ; 14(1): 460-6, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27175741

ABSTRACT

As human lifespans have increased, the incidence of osteoporosis has also increased. Methylsulfonylmethane (MSM) affects the process of mesenchymal stem cell (MSC) differentiation into osteoblasts via the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (STAT)5b signaling pathway, and bone morphogenetic protein 2 (BMP­2) is also known to significantly affect bone health. In addition, the phosphorylation of small mothers against decapentaplegic (Smad)1/5/8 regulates the Runt­related transcription factor 2 (Runx2) gene, which encodes a transcription factor for osteoblast differentiation markers. In the present study, the differentiation of MSCs treated with MSM, BMP­2, and their combination were examined. The differentiation of osteoblasts was demonstrated through observation of morphological changes and mineralization, using alizarin red and Von Kossa staining. Western blotting analysis demonstrated that the combination of MSM and BMP-2 increased the phosphorylation of the BMP signaling-associated protein, Smad1/5/8. Combination of MSM and BMP-2 significantly increased osteogenic differentiation and mineralization of the MSCs compared with either MSM or BMP-2 alone. Additionally, reverse transcription-polymerase chain reaction analysis demonstrated that combination of MSM and BMP-2 increased the expression level of the Runx2 gene and the osteoblast differentiation marker genes, alkaline phosphatase, bone sialoprotein and osteocalcin, in MSCs compared with controls. Thus, the combination of MSM and BMP-2 may promote the differentiation of MSCs into osteoblasts.


Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation/drug effects , Dimethyl Sulfoxide/pharmacology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Osteoblasts/cytology , Osteogenesis/drug effects , Sulfones/pharmacology , Animals , Biomarkers , Bone Morphogenetic Protein 2/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Gene Expression Regulation , Immunohistochemistry , Male , Mesenchymal Stem Cells/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects
9.
Int J Oncol ; 48(2): 836-42, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26648017

ABSTRACT

Breast cancer is the most common cancer in women globally. The factors that increase risk include: late age at first birth, alcohol, radiation exposure, family history of breast cancer, and postmenopausal hormone therapy. Numerous drugs are being developed to treat breast cancer. Among them, Herceptin is used for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cases and targets HER2 effectively and efficiently, but it is very expensive. Methylsulfonylmethane (MSM) is an organic sulfur-containing natural compound having no reported toxicity. We examined MSM in breast cancer cell lines and found it inhibited the proliferation of estrogen receptor-positive and HER2-positive breast cancer cells in a dose-dependent manner. It also suppressed the activation of STAT5b and expression of HER2 in breast cancer cells. We determined the STAT5b binding site (GAS element) in the HER2 gene. Detailed analysis showed that MSM decreased the ability of STAT5b to bind the promoter of the HER2 gene and a luciferase assay demonstrated reduced activity. We confirmed that MSM can effectively regulate STAT5b, and thereby decrease HER2 expression. Therefore, we recommend the use of MSM as an inhibitor for the management of HER2-positive breast cancers.


Subject(s)
Breast Neoplasms/drug therapy , Dimethyl Sulfoxide/pharmacology , Receptor, ErbB-2/metabolism , STAT5 Transcription Factor/metabolism , Sulfones/pharmacology , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Promoter Regions, Genetic/drug effects , Signal Transduction/drug effects
10.
Int J Oncol ; 47(3): 1111-20, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26202061

ABSTRACT

Tannic acid (TA), is a potent anti-oxidant, showing anti-proliferative effects on numerous cancers. The ability of TA to induce proliferation inhibition on the rare tumor, gingival squamous cell carcinoma (GSCC), comprising <10% of all head and neck squamous cell carcinomas was studied in the YD-38 cell line. The main goal was to modulate the Jak2/STAT3 pathway using TA and to induce cell cycle arrest and apoptosis in GSCC. TA treatment induced G1 arrest and apoptosis in YD-38 cells. Molecular analysis revealed that TA inhibits Jak2/STAT3 pathway by preventing their expression as well as phosphorylation. This inhibition of STAT3 phosphorylation prevented the nuclear translocation and DNA binding capability of STAT3. Together with the inhibition of transcriptional regulatory function of STAT3, TA inhibited the expression of G1 phase modulators CDK-4, CDK-6, cyclin D1 and cyclin E. It is also evidenced that TA exerted an intense activation of p21Waf1/Cip1, p27Kip1 and p53 genes confirming its role in G1 phase inhibition. Additionally, upon treatment with TA, the expression of mitochondrial pore factors Bax, Bcl-2 and Bcl-XL were changed. We observed inhibition of Bcl-2 and an increase in mitochondrial localization of Bax leading to the loss of mitochondrial membrane potential, resulting in the release of cytochrome c to the cytosol. In addition, we perceived the activation of caspases upon TA treatment. Specific inhibition of caspase protected the cells from TA induced apoptosis. Taken together, this study reveals that TA significantly inhibits the Jak2/STAT3 signaling pathway and induces G1 arrest and mitochondrial apoptosis in YD-38 cells.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/metabolism , Gingival Neoplasms/metabolism , Mitochondria/drug effects , Signal Transduction/drug effects , Tannins/pharmacology , Apoptosis , Carcinoma, Squamous Cell/drug therapy , Cell Cycle/drug effects , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Gingival Neoplasms/drug therapy , Humans , Janus Kinase 2/metabolism , Phosphorylation/drug effects , STAT3 Transcription Factor/metabolism
11.
BMC Cancer ; 15: 474, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-26084564

ABSTRACT

BACKGROUND: Combination therapy, which reduces the dosage intensity of the individual drugs while increasing their efficacy, is not a novel approach for the treatment of cancer. Methylsulfonylmethane (MSM) is an organic sulfur compound shown to act against tumor cells. Tamoxifen is a commercially available therapeutic agent for breast malignancies. METHODS: In the current study, we analyzed the combinatorial effect of MSM and tamoxifen on the suppression of ER-positive breast cancer xenograft growth and metastasis. Additionally, we also validated the molecular targets by which the drug combination regulated tumor growth and metastasis. RESULTS: We observed that the combination of MSM and tamoxifen regulated cell viability and migration in vitro. The intragastric administration of MSM and subcutaneous implantation of tamoxifen tablets led to tumor growth suppression and inhibition of the Janus kinase 2 (Jak2)/signal transducer and activator of transcription 5b (STAT5b) pathway. Our study also assessed the regulation of signaling molecules implicated in the growth, progression, differentiation, and migration of cancer cells, such as Jak2, STAT5b, insulin-like growth factor-1Rß, and their phosphorylation status. CONCLUSIONS: Study results indicated that this combination therapy inhibited tumor growth and metastasis. Therefore, this drug combination may have a synergistic and powerful anticancer effect against breast cancer.


Subject(s)
Breast Neoplasms/drug therapy , Dimethyl Sulfoxide/administration & dosage , Janus Kinase 2/genetics , STAT5 Transcription Factor/genetics , Sulfones/administration & dosage , Tamoxifen/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Estrogen Receptor alpha/genetics , Female , Humans , Janus Kinase 2/antagonists & inhibitors , Neoplasm Metastasis , Receptors, Somatomedin/antagonists & inhibitors , Receptors, Somatomedin/genetics , STAT5 Transcription Factor/antagonists & inhibitors , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
12.
Int J Oncol ; 44(3): 883-95, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24402583

ABSTRACT

Human urinary bladder cancer is the fifth most common cancer, with a worldwide estimate of about two million patients. Recurrence after complete transurethral prostatic resection is the most important problem in therapy. Combination therapy is a new approach in the treatment of cancers that do not respond to current therapies. These therapies have many advantages over conventional therapies, such as fewer side-effects and greater efficiency. Research efforts using natural compounds for the elimination or growth suppression of the cancer arise from studies on methylsulfonylmethane (MSM). MSM is a natural sulfur compound with no side-effects. AG490 is a tyrosine kinase inhibitor that has been extensively used for inhibiting Jak2 in vitro and in vivo. In our study, the combinatorial effect of these two agents on human bladder cancer cell lines and xenografts was analyzed. We observed that the combination of AG490 and MSM inhibited cancer cell viability and cell migration in vitro. This combination inhibited VEGF mRNA expression in bladder cancer cell lines. In vivo experiments showed that oral administration of AG490 and MSM combination significantly inhibited the growth of tumor xenografts in mice. Our study clearly demonstrates that the predominant effect of this combination is the reduction of signaling molecules including STAT3, STAT5b, IGF-1R, VEGF and VEGF-R2 which are involved in the growth, progression and metastasis of human bladder cancer. The anti-metastatic ability of this drug combination is confirmed using metastatic animal models. Therefore, this combination could have the effect of genesistasis and powerful anticancer effects against bladder cancer.


Subject(s)
Cell Movement/drug effects , Janus Kinase 2/biosynthesis , STAT3 Transcription Factor/biosynthesis , Urinary Bladder Neoplasms/genetics , Animals , Cell Survival/drug effects , Dimethyl Sulfoxide/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Janus Kinase 2/antagonists & inhibitors , Mice , RNA, Messenger/biosynthesis , Receptor, IGF Type 1/biosynthesis , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/biosynthesis , STAT5 Transcription Factor/genetics , Signal Transduction/drug effects , Sulfones/pharmacology , Tyrphostins/pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Xenograft Model Antitumor Assays
13.
Mol Med Rep ; 8(3): 891-6, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23877734

ABSTRACT

Sorghum is a principal cereal food in a number of parts of the world and is critical in folk medicine in Asia and Africa. However, its effects on bone are unknown. Growth hormone (GH) is a regulator of bone growth and bone metabolism. GH activates several signaling pathways, including the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathways, thereby regulating expression of genes, including insulin­like growth factor (IGF)­1. Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage, increasing the pool of IGF­1 target cells, the mature osteoblasts. In the present study, the effects of Hwanggeumchal sorghum extracts (HSE) on GH signaling via the Jak/STAT pathway in osteoblasts were investigated. HSE was not observed to be toxic to osteoblastic cells and increased the expression of BMP7 and GH­related proteins, including STAT5B, p­STAT5B, IGF­1 receptor (IGF-1R), growth receptor hormone (GHR) and Jak2 in MC3T3­E1 cells. In addition, HSE increased BMP7 and GHR mRNA expression in MC3T3­E1 cells. The expression of HSE­induced BMP7 and GHR was inhibited by AG490, a Jak2 kinase inhibitor. The observations indicate that HSE­induced signaling is similar to GH signaling via the GHR­Jak2 signaling axis. Using small interference RNA (siRNA) analysis, STAT5B was found to play an essential role in HSE­induced BMP7 and GH signaling in MC3T3­E1 cells. Results of the current study indicate that HSE promotes bone growth through activation of STAT5B.


Subject(s)
Bone Morphogenetic Protein 7/metabolism , Growth Hormone/metabolism , Janus Kinase 2/metabolism , Plant Extracts/toxicity , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Sorghum/metabolism , Animals , Bone Morphogenetic Protein 7/genetics , Cell Differentiation , Cell Line , Cell Lineage , Gene Expression/drug effects , Janus Kinase 2/antagonists & inhibitors , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Plant Extracts/chemistry , RNA Interference , RNA, Small Interfering/metabolism , Receptor, IGF Type 1/metabolism , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , STAT5 Transcription Factor/antagonists & inhibitors , STAT5 Transcription Factor/genetics , Sorghum/chemistry , Tyrphostins/pharmacology
14.
JAKSTAT ; 2(4): e24931, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24470975

ABSTRACT

Osteoblast differentiation is a critical step in the maintenance of bone homeostasis. Osteoblast differentiation is generally maintained by growth hormone (GH) and various other endocrine and autocrine/paracrine factors. JAK2-STAT5B pathway is a central axis in the mechanism of GH signaling. Similarly, the autocrine/paracrine signaling factor IGF-1 also mediates its effects through this pathway. Analysis on JAK2-STAT5B pathway showed its importance in the IGF-1/IGF-1R mediated regulation of gene expression and osteoblast differentiation. Persistent activation of STAT5B and inhibition of STAT5B degradation showed increased osteoblastic differentiation and STAT5B/Runx-2 activities. Conditional gene silencing studies showed the importance of the JAK2-STAT5B pathway in stimulation of other transcription factors and expression of various differentiation markers.

15.
PLoS One ; 7(10): e47477, 2012.
Article in English | MEDLINE | ID: mdl-23071812

ABSTRACT

Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.


Subject(s)
Dimethyl Sulfoxide/pharmacology , Growth Hormone/metabolism , Janus Kinase 2/metabolism , Osteogenesis/drug effects , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Sulfones/pharmacology , Analysis of Variance , Animals , Blotting, Western , Cell Differentiation/physiology , Cell Line, Tumor , DNA Primers/genetics , Electrophoretic Mobility Shift Assay , Immunoprecipitation , Luciferases , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , Osteoblasts/physiology , RNA, Small Interfering/genetics , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tetrazolium Salts , Thiazoles
16.
PLoS One ; 7(7): e40531, 2012.
Article in English | MEDLINE | ID: mdl-22792362

ABSTRACT

BACKGROUND: Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model. CONCLUSIONS/SIGNIFICANCE: Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/metabolism , Janus Kinase 2/metabolism , Plant Extracts/pharmacology , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Sorghum/chemistry , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , MCF-7 Cells , Mice , Mice, Nude , Neoplasm Metastasis/drug therapy , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Plant Extracts/administration & dosage , Promoter Regions, Genetic , Protein Binding/drug effects , Receptor, IGF Type 1/metabolism , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
17.
Int J Oncol ; 41(1): 161-8, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22552610

ABSTRACT

Hsp90α is a molecular chaperone protein involved in the structural maturation of oncogenic signaling proteins. Hsp90 was recently identified as an anticancer target; various studies are ongoing to find ways for managing cancer through Hsp90α. However, this approach is limited by reported side-effects. Hypoxia is a hallmark of solid tumors, including those of breast cancer and the extent of tumor hypoxia is associated with resistance to treatment and poor prognosis. One of the major signaling pathways in cancer cells, the Jak2/STAT5b pathway, has been found to be closely correlated with hypoxia. The objective of this study was to investigate the role of Jak2/STAT5b in the regulation of Hsp90α expression so that Hsp90α targeting can be achieved indirectly by modulating the Jak2/STAT5b pathway. We examined the role of the Jak2/STAT5b pathway in the expression of Hsp90α under hypoxic conditions by immunoblotting, reporter gene assays, EMSA and RNA interference analysis. With the help of in vivo models, we also analyzed the expression of Hsp90α in different parts of solid tumor tissues. We found a close association between hypoxic stress and Hsp90α expression. We also determined that STAT5b regulates the expression of Hsp90α during hypoxic stimulation. Under hypoxic conditions the expression of Hsp90α and STAT5b were proportional. siRNA analysis and nucleotide analysis showed that the promoter of Hsp90α has a STAT5b binding domain. Our work confirmed that STAT5b is one of the transcription factors that regulate Hsp90α. We, therefore, concluded that under hypoxic conditions, the Jak2/STAT5b pathway regulates Hsp90α expression and it could serve as a promising target for the treatment of solid tumors.


Subject(s)
Gene Expression Regulation, Neoplastic , HSP90 Heat-Shock Proteins/genetics , STAT5 Transcription Factor/metabolism , Up-Regulation , Animals , Base Sequence , Cell Hypoxia , Cell Line, Tumor , Consensus Sequence , Genes, Reporter , HSP90 Heat-Shock Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Luciferases, Firefly/biosynthesis , Luciferases, Firefly/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Promoter Regions, Genetic , Protein Binding , STAT5 Transcription Factor/genetics
18.
PLoS One ; 7(4): e33361, 2012.
Article in English | MEDLINE | ID: mdl-22485142

ABSTRACT

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1α, VEGF, BrK, and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90α, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis. Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Dimethyl Sulfoxide/pharmacology , Down-Regulation/drug effects , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Sulfones/pharmacology , Animals , Apoptosis/drug effects , COS Cells , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Chlorocebus aethiops , Female , Gene Expression , Humans , Inhibitory Concentration 50 , Mice , Promoter Regions, Genetic , Protein Binding , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/genetics , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
19.
Int J Oncol ; 36(5): 1243-51, 2010 May.
Article in English | MEDLINE | ID: mdl-20372799

ABSTRACT

Cyclin D1 and insulin-like growth factor 1 receptor (IGF-1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. The purpose of the present study was to investigate the molecular mechanism by which hemin exerts its inhibitory effects on aggressive breast cancer cells. We found that hemin regulates cyclin D1 and IGF-1R proteins and insulin-like growth factor-1 gene expression through STAT5b in breast cancer cells. We confirmed that STAT5b, cyclin D1, and IGF-1R is up-regulated by hypoxia, and the increased STAT5b binds strongly to the STAT5-binding sites contained within the distal 5'-flanking region of IGF-1 gene in breast cancer cells. EMSA studies showed that STAT5 binding activity to the IGF-1 and cyclin D1 promoter was distinctly decreased by hemin in STAT5b-transfected COS-7 or MDA-MB 231 cells. IGF-1 gene expression was also decreased by hemin in mammary epithelial cells. STAT5b expression was inhibited in siRNA experiments and by hemin, leading to decreased levels of IGF-1. These results provide a basis for molecular targets in cancer treatment via the STAT5b/IGF-1 or /cyclin D1 pathway in solid tumor cells. These data indicate that hemin inhibits the cyclin D1 and IGF-1 expression via STAT5b under hypoxia in ERalpha-negative breast cancer cells. These findings are valuable toward understanding the role of hemin-induced inhibition of cyclin D1 and IGF-1 expression under hypoxia in invasive and metastatic breast cancer.


Subject(s)
Cyclin D1/antagonists & inhibitors , Estrogen Receptor alpha/metabolism , Hemin/metabolism , Hypoxia , Insulin-Like Growth Factor I/antagonists & inhibitors , STAT5 Transcription Factor/metabolism , Animals , CHO Cells , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cricetinae , Cricetulus , Gene Expression Regulation, Neoplastic , Hemin/chemistry , Humans , Neoplasm Invasiveness , Neoplasm Metastasis
20.
Int J Oncol ; 33(3): 477-84, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18695876

ABSTRACT

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.


Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Hypoxia/drug effects , Diterpenes/pharmacology , Lactones/pharmacology , STAT5 Transcription Factor/drug effects , Blotting, Western , Breast Neoplasms/metabolism , Cell Hypoxia/physiology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/drug effects , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/drug effects , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p21/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Electrophoretic Mobility Shift Assay , Female , Flow Cytometry , Gene Expression/drug effects , Humans , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Small Interfering , STAT5 Transcription Factor/metabolism , bcl-2-Associated X Protein/drug effects , bcl-2-Associated X Protein/metabolism
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