ABSTRACT
Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.
Subject(s)
Alzheimer Disease , Tauopathies , Alzheimer Disease/drug therapy , Animals , Brain/metabolism , Disease Models, Animal , Humans , Immunization, Passive , Mice , Mice, Transgenic , Tauopathies/drug therapy , tau Proteins/metabolismABSTRACT
Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies/chemistry , Asparagine/chemistry , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Amides/chemistry , Animals , Antibody Affinity , Drug Discovery , High-Throughput Screening Assays , Humans , Mutation/genetics , Protein Binding , Protein StabilityABSTRACT
BACKGROUND AND AIM: The preventive effect of intrarectal administration of mouse cathelicidin (mCRAMP) and oral administration of mCRAMP-encoding Lactococcus lactis (N4I) has been shown in murine experimental colitis. It is pivotal to understand the ability of N4I whether it can promote mucosal repair in existing colitis. METHODS: Mice with dextran sulfate sodium-induced ulcerative colitis (UC) were treated orally with L. lactis or its transformed strain with or without nisin induction. The body weight, clinical symptoms, and histological changes of colonic tissues were determined. Sulfasalazine was used as a reference drug. Young adult mouse colon cells were used to further elucidate the direct action and possible mechanisms of mCRAMP to promote colonic wound repair. RESULTS: Results showed that N4I could improve the clinical symptoms, maintain crypt integrity and preserve mucus-secreting layer in colitis animals. The preparation also could prevent cell death and promote cell proliferation. In contrast, effective dose of sulfasalazine only alleviated clinical symptoms but not the mucosal damage and repair in the colon. In vitro study further showed that mCRAMP could directly promote wound repair by accelerating cell migration but not cell proliferation through the GPCR/MAPK pathway. CONCLUSIONS: mCRAMP-encoding L. lactis could be a potential therapeutic preparation better than the traditional anti-inflammatory agent in the treatment of UC.
Subject(s)
Antimicrobial Cationic Peptides/administration & dosage , Antimicrobial Cationic Peptides/pharmacology , Colitis, Ulcerative/drug therapy , Intestinal Mucosa/physiology , Lactococcus lactis , Wound Healing/drug effects , Administration, Oral , Administration, Rectal , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Colitis, Ulcerative/pathology , Colitis, Ulcerative/physiopathology , Colitis, Ulcerative/therapy , Colon/cytology , Epithelial Cells , Intestinal Mucosa/pathology , Male , Mice, Inbred BALB C , CathelicidinsABSTRACT
The spread of tau pathology correlates with cognitive decline in Alzheimer's disease. In vitro, tau antibodies can block cell-to-cell tau spreading. Although mechanisms of anti-tau function in vivo are unknown, effector function might promote microglia-mediated clearance. In this study, we investigated whether antibody effector function is required for targeting tau. We compared efficacy in vivo and in vitro of two versions of the same tau antibody, with and without effector function, measuring tau pathology, neuron health, and microglial function. Both antibodies reduced accumulation of tau pathology in Tau-P301L transgenic mice and protected cultured neurons against extracellular tau-induced toxicity. Only the full-effector antibody enhanced tau uptake in cultured microglia, which promoted release of proinflammatory cytokines. In neuron-microglia co-cultures, only effectorless anti-tau protected neurons, suggesting full-effector tau antibodies can induce indirect toxicity via microglia. We conclude that effector function is not required for efficacy, and effectorless tau antibodies may represent a safer approach to targeting tau.
Subject(s)
Alzheimer Disease/metabolism , Microglia/metabolism , tau Proteins/metabolism , Alzheimer Disease/pathology , Animals , Antibodies/immunology , Brain/metabolism , Brain/pathology , Cells, Cultured , Coculture Techniques/methods , Cytokines/metabolism , Mice, Transgenic , Neurons/metabolismABSTRACT
AIMS: To study the characteristics of miltirone-induced anti-colon cancer effects. MATERIALS AND METHODS: Cell viability was detected using MTT assay. LDH (lactate dehydrogenase) leakage was detected using CytoTox96® non-radioactive cytotoxicity kit. Apoptosis was detected by FCM (flow cytometry). Caspase activation was determined by chemiluminescence or western blotting. AIF (apoptosis-inducing factor) expression in the cell fraction was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by confocal microscope. Intracellular calcium was detected by both FCM and confocal microscope. To determine the roles of ROS and Ca(2+) in the pro-apoptotic activity of miltirone, colon cancer cells were pretreated with kinds of antioxidants, dicoumarol, calpeptin or BAPTA-AM in some cases. KEY FINDINGS: Miltirone exhibited potent cytotoxicity on colon cancer cells with a better selectivity than that of dihydrotanshinone. The pro-apoptotic activity of miltirone was p53- and ROS-dependent. In detail, miltirone induced direct mitochondrial damage, including significant decrease of mitochondrial ROS, MMP, mass and increase of intracellular ROS and Ca(2+). NQO1 (quinone oxidoreductase1) was supposed to be a defender for the cytotoxicity induced by miltirone in colon cancer cells. Furthermore, miltirone induced time- and concentration-dependent translocation of AIF and activation of caspases. SIGNIFICANCE: In this study, ROS- and p53-dependent apoptosis induced by miltirone on colon cancer cells was firstly revealed. Strong positive feedback between mitochondrial dysfunction and accumulation of intracellular Ca(2+) was suggested to be the characteristic of the anti-colon cancer activity of miltirone.
Subject(s)
Apoptosis/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Mitochondria/drug effects , Mitochondria/pathology , Phenanthrenes/pharmacology , Reactive Oxygen Species/metabolism , Apoptosis Inducing Factor/metabolism , Calcium/metabolism , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dicumarol/pharmacology , Humans , L-Lactate Dehydrogenase/metabolism , Membrane Potential, Mitochondrial/drug effects , NAD(P)H Dehydrogenase (Quinone)ABSTRACT
The antimicrobial peptide cathelicidin is critical for protection against different kinds of microbial infection. This study sought to elucidate the protective action of cathelicidin against Helicobacter pylori infection and its associated gastritis. Exogenous cathelicidin was found to inhibit H. pylori growth, destroy the bacteria biofilm, and induce morphological alterations in H. pylori membrane. Additionally, knockdown of endogenous cathelicidin in human gastric epithelial HFE-145 cells markedly increased the intracellular survival of H. pylori. Consistently, cathelicidin knockout mice exhibited stronger H. pylori colonization, higher expression of proinflammatory cytokines IL-6, IL-1ß, and ICAM1, and lower expression of the anti-inflammatory cytokine IL-10 in the gastric mucosa upon H. pylori infection. In wild-type mice, H. pylori infection also stimulated gastric epithelium-derived cathelicidin production. Importantly, pretreatment with bioengineered Lactococcus lactis that actively secretes cathelicidin significantly increased mucosal cathelicidin levels and reduced H. pylori infection and the associated inflammation. Moreover, cathelicidin strengthened the barrier function of gastric mucosa by stimulating mucus synthesis. Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis.
Subject(s)
Cathelicidins/immunology , Gastric Mucosa/immunology , Gastritis/immunology , Helicobacter Infections/immunology , Animals , Antimicrobial Cationic Peptides , Cell Line , Disease Models, Animal , Fluorescent Antibody Technique , Gastric Mucosa/microbiology , Helicobacter pylori/immunology , Humans , Mice , Mice, Knockout , Microscopy, Electron, Scanning , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , TransfectionABSTRACT
FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.
Subject(s)
Fibroblast Growth Factors/metabolism , Gelatinases/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Endopeptidases , Fibroblast Growth Factors/chemistry , Gelatinases/genetics , Gene Deletion , HEK293 Cells , Humans , Macaca fascicularis , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine Endopeptidases/geneticsABSTRACT
BACKGROUND: Dihydrotanshinone I (DHTS) was previously reported to exhibit the most potent anti-cancer activity among several tanshinones in colon cancer cells. Its cytotoxic action was reactive oxygen species (ROS) dependent but p53 independent. PURPOSE: To further study the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer both in vitro and in vivo. METHODS: Caspase activity was detected by fluorescence assay. Apoptosis was detected by flow cytometry and TUNEL assay. Protein levels were analyzed by western blotting. Knockdown of target gene was achieved by siRNA transfection. Formation of LC3B puncta and activation of caspase-3 were detected by confocal fluorescence microscope. In vivo anti-colon cancer activity of DHTS was observed in xenograft tumors in NOD/SCID mice. RESULTS: Anti-colon cancer activity of DHTS by inducing apoptosis and autophagy was observed both in vitro and in vivo. Mitochondria mediated caspase dependent pathway was essential in DHTS-induced cytotoxicity. The apoptosis induced by DHTS was suppressed by knockdown of apoptosis inducing factor (AIF), inhibition of caspase-3/9 but was increased after knockdown of caspase-2. Meantime, knockdown of caspase-2, pretreatment with Z-VAD-fmk or NAC (N-Acety-L-Cysteine) efficiently inhibited the autophagy induced by DHTS. A crosstalk between cytochrome c and AIF was also reported. CONCLUSION: DHTS-induced caspase and ROS dependent apoptosis and autophagy were mediated by mitochondria in colon cancer. DHTS could be a promising leading compound for the development of anti-tumor agent or be developed as an adjuvant drug for colon cancer therapy.
Subject(s)
Abietanes/pharmacology , Apoptosis , Autophagy , Caspases/metabolism , Colonic Neoplasms/drug therapy , Animals , Cell Line, Tumor , Colonic Neoplasms/metabolism , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Multidrug resistance (MDR) develops in nearly all patients with colon cancer. The reversal of MDR plays an important role in the success of colon cancer chemotherapy. One of the commonest mechanisms conferring MDR is the suppression of apoptosis in cancer cells. PURPOSE: This study investigated the sensitivity of cryptotanshinone (CTS) and dihydrotanshinone (DTS), two lipophilic tanshinones from a traditional Chinese medicine Salvia miltiorrhiza, in apoptosis-resistant colon cancer cells. METHODS: Cell viability was measured by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein levels were analyzed by western blot analysis. The formation of acidic vesicular organelles was visualized by acridine orange staining. RESULTS: Experimental results showed that multidrug-resistant colon cancer cells SW620 Ad300 were sensitive to both CTS and DTS in terms of cell death, but with less induction of apoptosis when compared with the parental cells SW620, suggesting that other types of cell death such as autophagy could occur. Indeed, the two tanshinones induced more LC3B-II accumulation in SW620 Ad300 cells with increased autophagic flux. More importantly, cell viability was increased after autophagy inhibition, indicating that autophagy induced by the two tanshinones was pro-cell death. Besides, the cytotoxic actions of the two tanshinones were p53-independent, which could be useful in inhibiting the growth of apoptosis-resistant cancer cells with p53 defects. CONCLUSION: The current findings strongly indicate that both CTS and DTS could inhibit the growth of apoptosis-resistant colon cancer cells through induction of autophagic cell death and p53-independent cytotoxicity. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the apoptosis-resistant cancer types.
Subject(s)
Abietanes/pharmacology , Apoptosis , Autophagy , Drugs, Chinese Herbal/pharmacology , Phenanthrenes/pharmacology , Cell Line, Tumor/drug effects , Cell Survival , Colonic Neoplasms/pathology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Humans , Tumor Suppressor Protein p53/metabolismABSTRACT
Vitamin D, beyond its classical roles in the regulation of calcium and phosphorus homeostasis and bone metabolism, has been implicated in multiple pathological processes, including progression from inflammation to cancer development and also involvement in autoimmune diseases as well as cardiovascular disorders. In this review, we shall discuss the different roles of vitamin D and its therapeutic targets in different gastrointestinal diseases, focusing on colorectal cancer (CRC) and inflammatory bowel disease (IBD). To this end, vitamin D deficiency has been identified as a risk factor of CRC. On the other hand the active metabolite of vitamin D, 1, 25- dihydroxyvitamin D3 (1, 25(OH)2D3) has multiple anti-cancerous benefits including inhibition of proliferation, induction of apoptosis, promotion of differentiation and suppression of angiogenesis in tumors. In IBD, vitamin D is involved in the pathogenic process through the normalization of immune responses in the colon. With these experimental findings, well-designed and large-scale clinical trials are warranted to further define the therapeutic action of vitamin D in the prevention and/or treatment of IBD and further on CRC in humans.
Subject(s)
Gastroenteritis/drug therapy , Gastrointestinal Neoplasms/drug therapy , Receptors, Calcitriol/drug effects , Vitamin D/therapeutic use , Vitamins/therapeutic use , Animals , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/physiopathology , Humans , Receptors, Calcitriol/physiology , Vitamin D/pharmacology , Vitamin D/physiology , Vitamins/pharmacology , Vitamins/physiologyABSTRACT
Delivery and penetration of chemotherapeutic drugs into neoplasm through the tumor vasculature are essential mechanisms to enhance the efficiency of chemotherapy. "Vascular targeting" strategy focuses on promoting the infiltration of chemotherapeutic drugs into neoplastic tissues. In this study, we achieved a targeted therapy by coupling tumor necrosis factor α (TNFα) with TCP-1, a novel vascular-targeting peptide, in an orthotopic colorectal cancer model in mice. High dose of TCP-1-conjugated TNFα (TCP-1/TNFα: 5µg/mouse) displayed potent antitumor activity by inducing apoptosis and reducing microvessel number in tumors than unconjugated TNFα, with no evidence of increased toxicity. In the combined therapy, the antitumor action of 5-fluorouracil (5-FU) was potentiated when the mice were pretreated with a low dose of TNFα (1ng/mouse) and to a greater extent by the same concentration of TCP-1/TNFα. In this regard, TCP-1/TNFα combined with 5-FU synergistically inhibited the tumor growth, induced apoptosis and reduced cell proliferation. More importantly, TCP-1/TNFα normalized the tumor vasculature and facilitated the infiltration of immune cells to neoplasm as well as attenuated the immunosuppressing effects of TNFα in bone marrow and spleen. At the same time, TCP-1/TNFα significantly improved 5-FU absorption into the tumor mass. Taken together, these findings underscore the therapeutic potential of TCP-1 as a drug carrier in cancer therapy. TCP-1 is a novel vascular-targeting peptide and appears to be a promising agent for drug delivery. TCP-1 fused with TNFα holds great promise for colorectal cancer therapy.
Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Cell-Penetrating Peptides/administration & dosage , Colorectal Neoplasms/drug therapy , Fluorouracil/administration & dosage , Neovascularization, Pathologic/drug therapy , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Antimetabolites, Antineoplastic/chemistry , Antimetabolites, Antineoplastic/therapeutic use , Bone Marrow/drug effects , Cell Line , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/therapeutic use , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Fluorouracil/chemistry , Fluorouracil/therapeutic use , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/chemistry , Humans , Male , Mice, Inbred BALB C , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/pathology , Spleen/drug effects , Tumor Burden/drug effects , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/therapeutic useABSTRACT
There is no clear evidence to show the direct causal relationship between passive cigarette smoking and osteoporosis. Furthermore, the underlying mechanism is unknown. The objective of this study is to demonstrate the effects of long-term passive cigarette smoking on bone metabolism and microstructure by a mouse model and cell culture systems. BALB/c mice were exposed to 2 or 4 % cigarette smoke for 14 weeks. The bone turnover biochemical markers in urine and serum and also the bone micro-architecture by micro-CT were compared with the control group exposed to normal ambient air. In the cell culture experiments, mouse MC3T3-E1 and RAW264.7 cell lines to be employed as osteoblast and osteoclast, respectively, were treated with the sera obtained from 4 % smoking or control mice. Their actions on cell viability, differentiation, and function on these bone cells were assessed. The urinary mineral and deoxypyridinoline (DPD) levels, and also the serum alkaline phosphatase activity, were significantly higher in the 4 % smoking group when compared with the control group, indicating an elevated bone metabolism after cigarette smoking. In addition, femoral osteopenic condition was observed in the 4 % smoking group, as shown by the decrease of relative bone volume and trabecular thickness. In isolated cell studies, osteoblast differentiation and bone formation were inhibited while osteoclast differentiation was increased. The current mouse smoking model and the isolated cell studies demonstrate that passive cigarette smoke could induce osteopenia by exerting a direct detrimental effect on bone cells differentiation and further on bone remodeling process.
Subject(s)
Bone and Bones/drug effects , Cell Differentiation/drug effects , Osteoblasts/drug effects , Osteoclasts/drug effects , Tobacco Smoke Pollution/adverse effects , Animals , Bone Diseases, Metabolic/etiology , Bone Remodeling/drug effects , Female , Mice , Mice, Inbred BALB CABSTRACT
Chromatin remodeling has emerged as a hallmark of gastric cancer, but the regulation of chromatin regulators other than genetic change is unknown. Helicobacter pylori causes epigenetic dysregulation to promote gastric carcinogenesis, but the roles and functions of microRNAs (miRNA) in this multistage cascade are not fully explored. In this study, miRNA expression in preneoplastic and neoplastic lesions in murine stomachs induced by H. pylori and N-methyl-N-nitrosourea (MNU) was profiled by miRNA expression array. miR-490-3p exhibited progressive downregulation in gastritis, intestinal metaplasia, and adenocarcinoma during H. pylori and MNU-induced gastric carcinogenesis. Significant downregulation of miR-490-3p was confirmed in human gastric cancer tissues in which its regulatory region was found to be hypermethylated. miR-490-3p exerted growth- and metastasis-suppressive effects on gastric cancer cells through directly targeting SMARCD1, a SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex subunit. Knockdown of SMARCD1 significantly attenuated the protumorigenic effects of miR-490-3p inhibitor, whereas enforced expression of SMARCD1 promoted in vitro and in vivo oncogenic phenotypes of gastric cancer cells. SMARCD1 was markedly upregulated in gastric cancer in which its high expression was associated with shortened patients' survival independent of TNM staging. In conclusion, hypermethylation-mediated silencing of miR-490-3p reactivates SMARCD1 to confer malignant phenotypes, mechanistically linking H. pylori, chromatin remodeling, and gastric carcinogenesis.
Subject(s)
Adenocarcinoma/genetics , Carcinogenesis , MicroRNAs/genetics , Stomach Neoplasms/genetics , Adenocarcinoma/chemically induced , Adenocarcinoma/pathology , Animals , Cell Line, Tumor , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Gene Silencing , Helicobacter pylori/pathogenicity , Humans , Metaplasia/chemically induced , Methylnitrosourea/toxicity , Mice , Stomach Neoplasms/chemically induced , Stomach Neoplasms/pathologyABSTRACT
OBJECTIVE: Multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine. METHODS: The inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones. RESULTS: Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity. CONCLUSIONS: Taken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.
Subject(s)
Abietanes/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Phenanthrenes/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , Caco-2 Cells , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Digoxin/pharmacology , Doxorubicin/pharmacology , Drugs, Chinese Herbal/pharmacology , Furans , Gene Expression Regulation, Neoplastic , Humans , Irinotecan , Quinones , Salvia miltiorrhiza/chemistryABSTRACT
Inflammation and cancer are the two major disorders in the gastrointestinal tract. They are causally related in their pathogenesis. It is important to study animal models' causal relationship and, in particular, to discover new therapeutic agents for such diseases. There are several criteria for these models in order to make them useful in better understanding the etiology and treatment of the said diseases in humans. In this regard, animal models should be similar as possible to human diseases and also be easy to produce and reproducible and also economic to allow a continuous replication in different laboratories. In this review, we summarize the various animal models for inflammatory and cancerous disorders in the upper and lower gastrointestinal tract. Experimental approaches are as simple as by giving a single oral dose of alcohol or other noxious agents or by injections of multiple dosages of ulcer inducing agents or by parenteral administration or in drinking water of carcinogens or by modifying the genetic makeups of animals to produce relatively long-term pathological changes in particular organs. With these methods they could induce consistent inflammatory responses or tumorigenesis in the gastrointestinal mucosa. These animal models are widely used in laboratories in understanding the pathogenesis as well as the mechanisms of action for therapeutic agents in the treatment of gastrointestinal inflammation and cancer.
Subject(s)
Gastrointestinal Diseases/physiopathology , Inflammation/physiopathology , Neoplasms/pathology , Animals , Disease Models, Animal , Gastric Mucosa/pathology , Gastrointestinal Diseases/etiology , Humans , Inflammation/etiology , Intestinal Mucosa/pathology , Neoplasms/etiology , Reproducibility of Results , Species SpecificityABSTRACT
Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF-/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Caspases/metabolism , Cathelicidins/pharmacology , Peptide Fragments/pharmacology , Apoptosis Inducing Factor/metabolism , Colonic Neoplasms , Drug Screening Assays, Antitumor , Endodeoxyribonucleases/metabolism , Enzyme Activation , HCT116 Cells , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Listeria monocytogenes, a food-borne bacterial pathogen causing significant human mortality, propagates by expressing genes in response to environmental signals, such as temperature and pH. Listeria gene (lmo0799) encodes a protein homologous to the Bacillus subtilis YtvA, which has a flavin-light, oxygen or voltage (LOV) domain and a Sulfate Transporters Anti-Sigma factor antagonist (STAS) output domain that regulates transcription-initiation factor Sigma B in the bacterial stress response upon exposure to light. This could be significant for the pathogenesis of listeriosis because Sigma B has been linked to virulence of Listeria, and the Listeria Lmo0799 protein has recently been identified as a virulence factor activated by blue light. We have cloned, expressed heterologously in Escherichia coli and purified the full-length LM-LOV-STAS protein. Although it exhibits photochemical activity similar to that of YtvA, LM-LOV-STAS lacks an almost universally conserved arginine in the flavin-binding site, as well as another positively charged residue, a lysine in YtvA. The absence of these positive charges was found to destabilize retention of the flavin mononucleotide (FMN) chromophore in the LM-LOV-STAS protein, particularly at higher temperatures. The unusual sequence of the LM-LOV-STAS protein alters both spectral features and activation/deactivation kinetics, potentially expanding the sensory capacity of this LOV domain, e.g. to detect light plus cold.
Subject(s)
Bacterial Proteins/chemistry , Gene Expression Regulation, Bacterial/radiation effects , Listeria monocytogenes/radiation effects , Virulence Factors/chemistry , Amino Acid Sequence , Arginine/chemistry , Arginine/genetics , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Flavin Mononucleotide/chemistry , Kinetics , Light , Listeria monocytogenes/chemistry , Listeria monocytogenes/genetics , Lysine/chemistry , Lysine/genetics , Molecular Sequence Data , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Homology, Amino Acid , Virulence Factors/geneticsABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. They have been found to be dysregulated in many pathological conditions including cancer and play an important role during the progression of such disease. Recent efforts have been directed in translating the primary findings of miRNAs into clinical uses. This article gives a general overview on the potential of miRNAs as diagnostic and prognostic markers and also as therapeutic targets for gastrointestinal and liver cancers in animals and humans. Other contributors in this special series would focus in discussing the roles of specific miRNAs and their pathogenic mechanisms and therapeutic applications in different types of cancer in the gastrointestinal tract and liver.
Subject(s)
Gastrointestinal Neoplasms/genetics , Liver Neoplasms/genetics , MicroRNAs/physiology , Biomarkers, Tumor/genetics , Humans , MicroRNAs/genetics , Oncogenes , PrognosisABSTRACT
Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin-sensitive G-protein-coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG-mediated apoptosis in colon cancer cells.