ABSTRACT
Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Fabaceae/chemistry , L-Lactate Dehydrogenase/antagonists & inhibitors , Plant Extracts/pharmacology , Signal Transduction/drug effects , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Female , G2 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HSP90 Heat-Shock Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , L-Lactate Dehydrogenase/genetics , Lactate Dehydrogenase 5 , M Phase Cell Cycle Checkpoints/drug effects , Mice , Plant Extracts/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Anti-angiogenesis targeting VEGFR-2 has been considered as an important strategy for cancer therapy. Ellagic acid is a naturally existing polyphenol widely found in fruits and vegetables. It was reported that ellagic acid interfered with some angiogenesis-dependent pathologies. Yet the mechanisms involved were not fully understood. Thus, we analyzed its anti-angiogenesis effects and mechanisms on human breast cancer utilizing in-vitro and in-vivo methodologies. The in-silico analysis was also carried out to further analyze the structure-based interaction between ellagic acid and VEGFR-2. We found that ellagic acid significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways including MAPK and PI3K/Akt in endothelial cells. Ellagic acid also obviously inhibited neo-vessel formation in chick chorioallantoic membrane and sprouts formation of chicken aorta. Breast cancer xenografts study also revealed that ellagic acid significantly inhibited MDA-MB-231 cancer growth and P-VEGFR2 expression. Molecular docking simulation indicated that ellagic acid could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR-2 kinase unit. Taken together, ellagic acid could exert anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Breast Neoplasms/metabolism , Ellagic Acid/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor Receptor-2/metabolism , Adenosine Triphosphate/chemistry , Angiogenesis Inhibitors/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Binding Sites , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Ellagic Acid/chemistry , Enzyme Activation/drug effects , Female , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Mice , Molecular Docking Simulation , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic/drug effects , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/chemistry , Xenograft Model Antitumor AssaysABSTRACT
LDH-A, as the critical enzyme accounting for the transformation from pyruvate into lactate, has been demonstrated to be highly expressed in various cancer cells and its silencing has also been approved relating to increased apoptosis in lymphoma cells. In this study, we intend to investigate the correlation between LDH-A and other clinicopathological factors of breast cancer and whether LDH-A silencing could suppress breast cancer growth, and if so the potential mechanisms. 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation, and pathological subtypes classified by ER, PR, and Her-2. shRNAs were designed and applied to silence LDH-A expression in breast cancer cell lines MCF-7 and MDA-MB-231. The effects of LDH-A reduction on cancer cells were studied by a series of in vitro and in vivo experiments, including cell growth assay, apoptosis evaluation, oxidative stress detection, transmission electron microscopy observation, and tumor formation assay on nude mice. LDH-A expression was found to correlate significantly with tumor size and to be independent for other clinicopathological factors. LDH-A reduction resulted in an inhibited cancer cell proliferation, elevated intracellular oxidative stress, and induction of mitochondrial pathway apoptosis. Meanwhile, the tumorigenic ability of LDH-A deficient cancer cells was significantly limited in both breast cancer xenografts. The Ki67 positive cancer cells were significantly reduced in LDH-A deficiency tumor samples, while the apoptosis ratio was enhanced. Our results suggested that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer.
Subject(s)
Apoptosis/genetics , Breast Neoplasms/enzymology , Cell Transformation, Neoplastic/genetics , Gene Silencing , L-Lactate Dehydrogenase/genetics , Mitochondria/metabolism , Oxidative Stress , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Female , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , L-Lactate Dehydrogenase/metabolism , Lactate Dehydrogenase 5 , Mice , Signal TransductionABSTRACT
AIM OF THE STUDY: Although herbs have long been alternatively applied for cancer treatment in China, its treatment effects and their potential mechanisms have not been sufficiently investigated. The chinese herb Spatholobus suberectus (SS) is commonly prescribed to cancer patients. In this study, the anti-cancer effect of SS and its molecular mechanisms have been investigated. MATERIALS AND METHODS: The effect of SS on cell proliferation was studied by cell growth assay and flow cytometry on breast cancer cell lines MCF-7 and colon cancer cell line HT-29. The role of SS in apoptosis was studied by flow cytometry, DNA fragmentation assay and mitochondrial membrane potential assay. Expression of proteins associated with cell cycle and apoptosis was determined by Western blot analysis. The in vivo effect of SS was tested in nude mouse cancer xenografts. RESULTS: Cell growth assay showed that SS effectively inhibits tumor cell growth in a dose-dependent manner. Flow cytometry analysis showed that SS could arrest the cell cycle at G2/M checkpoint, which is associated with DNA damage and activation of phosphor-Chk1/Chk2. The pro-apoptotic effect of SS was demonstrated by Annexin V-PI staining and mitochondrial membrane potential assay. In vivo experiments show that the efficiency of SS alone group was superior to docetaxel or to docetaxel and SS combined. No obvious body weight loss or blood toxicity was observed in SS tested animals. CONCLUSIONS: Our data demonstrates that SS is a potential herb for cancer treatment by inhibiting tumor growth via induction of apoptosis and arrest of the cell cycle at G2/M phase.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Drugs, Chinese Herbal/pharmacology , Fabaceae , Neoplasms/drug therapy , Phytotherapy , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , China , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Ethnopharmacology , Fabaceae/chemistry , Female , G2 Phase/drug effects , HT29 Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Nude , Neoplasms/metabolism , Neoplasms/pathology , Plants, Medicinal/chemistry , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
AIM: Compounds containing Bupleurum possess immunomodulating effects. KY88 LIVER LIVO (KY88) is a blend of such compound. The aim of this study is to investigate the effects of KY88 on the production of cytokines by lymphocytes in vitro. METHODS: Seventy Sprague Dawley rats were used of which 40 were orally fed with 4 mg purified KY88 for 35 d. Normal human lymphocytes were isolated and cultured in standard conditions. The culture medium was collected at zero and 72 h after the KY88 treatment. The cytokines, including interleukin-1beta (IL-1beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma, were measured by ELISA kits. RESULTS: TNF-alpha levels in the supernatant of cultured human lymphocytes significantly increased after the treatment of PHA and KY88. The mean levels were (855+/-251), (399+/-145), and (176+/-49) ng/L after the treatment with KY88 at the concentrations of 10, 1 and 0.1 g/mL respectively. However, the level in the control group without specific treatment was only (68+/-4) ng/L. The difference between KY88 10 g/mL and control groups was significant (P<0.05). All other cytokines did not show significant variations between KY88 and the control groups. KY88 may regulate the immune function through the induction of TNF-alpha expression.
