lactoferrin and Mouth-Neoplasms

The oral cavity harbors different taxonomic groups, the evolutionary coexistence of which develops the oral ecosystem. These resident microorganisms can alter the balance between the physiologic and pathologic conditions that affect the host, both locally and systemically. This highly sophisticated nature of the oral cavity poses a significant therapeutic challenge. Numerous human and animal studies have been conducted to potentiate the efficacy and competence of current treatments of pathologic conditions as well as to develop novel therapeutic modalities. One of these studies is the use of the potent antimicrobial agent lactoferrin (LF), which was originally derived from the host immune system. LF is an 80-kDa glycoprotein that has a free iron sequestration mechanism with evident antimicrobial, anti-tumor, and immunomodulatory properties. A wide range of active peptides have been isolated from the N-terminal region of LF, which possess antimicrobial activities. In this review, we discuss the role of LF and LF-derived peptides under a heterogeneous group of oral and maxillofacial conditions, including bacterial, fungal, viral infections; head and neck cancers; xerostomia; and implantology-bone-related manifestations.

Topics: Animals; Bacteria; Candida albicans; Carcinogenesis; Dental Caries; Humans; Lactoferrin; Mouth Neoplasms; Peptides; Periodontal Diseases; Polymorphism, Single Nucleotide; Streptococcus mutans; Virus Physiological Phenomena

Lactoferrin (LF), a member of the transferrin family, recently has been demonstrated to have anticancer effects on various cancers including oral squamous cell carcinoma (OSCC). However, little is known about the underlying mechanisms of its effects on OSCC. Therefore, we aimed to investigate the mechanism of the suppressive effects of bovine LF (bLF) on the growth of OSCC cells.. In the current study, HSC2, HSC3, HSC4 and normal human oral keratinocytes (RT7) cell lines were tested with bLF 1, 10, and 100 μg/ml. The effects and detail mechanisms of bLF on proliferation and apoptosis of cells were investigated using flow cytometry and western blotting.. We found that bLF (1, 10, and 100 μg/ml) induced activation of p53, a tumor suppressor gene, is associated with the induction of cell cycle arrest in G1/S phase and apoptosis in OSCC. Moreover, bLF downregulated the phosphorylation of Akt and activated suppressor of cytokine signaling 3 (SOCS3), thereby attenuating multiple signaling pathways including mTOR/S6K and JAK/STAT3. Interestingly, we revealed that bLF exerted its effect selectively against HSC3 but not on RT7 via different effects on the phosphorylation status of NF-κB and Akt.. This is the first report showing that bLF selectively suppresses proliferation through mTOR/S6K and JAK/STAT3 pathways and induction of apoptosis in OSCC. This study provides important new findings, which might be useful in the prevention and treatment of OSCC.

Topics: Animals; Carcinoma, Squamous Cell; Cattle; Cell Cycle; Cell Line, Transformed; Cell Proliferation; Humans; Lactoferrin; Mouth Neoplasms

Epithelial-to-mesenchymal transition (EMT) is a biological process of invasion and metastasis in cancers, including in oral squamous cell carcinoma (OSCC). However, an effective anticancer drug that directly targets EMT has not yet been discovered. Therefore, we aimed to investigate the repressive effects of bovine lactoferrin (bLF) on EMT to achieve mesenchymal-to-epithelial transition (MET) in OSCC. OSCC cell lines, HOC313 (EMT-induced) and SCCVII (without EMT induction), were treated with bLF. The effects of bLF on EMT in OSCC were identified histologically by haematoxylin and eosin staining and observed morphologically and immunohistochemically using an anti-E-cadherin antibody. Expression levels of E-cadherin and vimentin were investigated using RT-PCR and western blotting. Immuno-expression of E-cadherin was examined in vivo in tumour tissues of C3H/HeN mice, transplanted with SCCVII cells, with or without bLF administration. We found that bLF changed the spindle-like mesenchymal cells to cuboidal-like epithelial cells and enhanced the affinity of membrane-bound E-cadherin in HOC313 cells. The transformation of EMT-MET in HOC313 cells was confirmed by the upregulation of E-cadherin and suppression of vimentin. Moreover, bLF suppressed TWIST expression through downregulation of ERK1/2 phosphorylation. Additionally, the inhibition tumour cell infiltration and increase in E-cadherin expression were observed in xenografts of the mice orally administered with bLF. Thus, based on the results from in vitro and in vivo studies, we concluded that bLF caused the restoration of epithelial properties through MET. Importantly, this finding is novel and is the first report indicating that bLF inhibited EMT and induced MET in OSCC, suggesting that bLF may provide a novel therapeutic strategy in OSCC.

Topics: Animals; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cattle; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lactoferrin; Male; MAP Kinase Signaling System; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Invasiveness; Nuclear Proteins; Twist-Related Protein 1; Vimentin

Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-25)4, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25), for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(a)anthracene, and tumors were treated with one of the following peptides: LfcinB(20-25)4, LfcinB(20-25), or vehicle (control). Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-25)4 and LfcinB(20-25) was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively). Also, LfcinB(20-25)4 caused acellularity in the parenchymal tumor compared with LfcinB(20-25) and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-25)4 and LfcinB(20-25) induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively). Moreover, although the lowest inflammatory response was achieved when LfcinB(20-25)4 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25). In addition the cellular damage and selectivity of the LfcinB(20-25)4 peptide was evaluated in vitro. These assays showed that LfcinB(20-25)4 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-25)4 could be considered as a new therapeutic agent for the treatment of OSCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cattle; Cell Proliferation; Humans; Jurkat Cells; Lactoferrin; Mouth Neoplasms; Peptides

Lactotransferrin (LTF) has been confirmed to act as a tumor suppressor in multiple cancers; however, its roles in oral squamous cell carcinoma (OSCC), one of malignant head and neck carcinomas, has not been explored.. Here, the expression of LTF in OSCC tissues and TCA8113 cells was detected with RT-PCR, qPCR, and IHC. And the correlation between LTF expression and OSCC metastasis was assessed. MS-PCR was performed to reveal the methylation status in promoter regions of LTF both in OSCC tissue samples and cells. The influences of 5-Aza-Cdc treatment to the methylation status and expression levels of LTF were also analyzed. At last, the functions of LTF in OSCC progression were demonstrated by MTT analysis, clone formation assay, and cell cycle analysis in TCA8113 cells with forced ectopic expression of LTF.. LTF showed a low or null expression pattern in OSCC tissues and cells, at least partially, due to the hypermethylated status in promoter regions for 5-Aza-Cdc, a methyltransferase inhibitor, could restore the expression of LTF in TCA8113 cells. And the expression level of LTF exhibited a negative correlation with OSCC metastasis.. Re-expression of LTF inhibited the growth, proliferation, as well as cell cycle progression of TCA8113 cells. In conclusion, hypermethylation contributes much to LTF inactivation in OSCC. And LTF can partially reverse the malignant phenotypes of OSCC cells and may be served as a potential target for diagnosis and therapy of OSCC in future.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Gene Silencing; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lactoferrin; Molecular Sequence Data; Mouth Neoplasms; Neoplasm Metastasis; Polymerase Chain Reaction; Promoter Regions, Genetic; Squamous Cell Carcinoma of Head and Neck

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

Topics: Animals; Carcinoma, Squamous Cell; Cattle; Cell Line, Tumor; Cell Membrane Permeability; Cytotoxins; Humans; Lactoferrin; Mouth Neoplasms; Necrosis; Peptides

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antioxidants; Aryl Hydrocarbon Hydroxylases; Carcinogens; Cell Proliferation; Cricetinae; Cyclin D1; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; DNA Damage; Free Radical Scavengers; Lactoferrin; Male; Matrix Metalloproteinase 9; Mesocricetus; Mouth Neoplasms; Neovascularization, Pathologic; Oxidation-Reduction; Phenols; Proliferating Cell Nuclear Antigen; Random Allocation; Tissue Inhibitor of Metalloproteinase-2; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Combination chemoprevention is a promising approach for oral cancer prevention. The authors evaluated the combined chemopreventive effects of bovine milk lactoferrin (bLF) and black tea polyphenols (Polyphenon-B) in a clinically relevant in vivo model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Although dietary administration of bLF and Polyphenon-B alone significantly reduced the tumor incidence, combined administration of bLF and polyphenon-B was more effective in inhibiting DMBA-induced genotoxicity and development of HBP carcinomas by modulation of carcinogen-metabolizing enzymes and cellular redox status. These results suggest that a "designer item" approach will be useful for human oral cancer prevention strategies.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cattle; Cheek; Chemoprevention; Cricetinae; Drug Therapy, Combination; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Lactoferrin; Lipid Peroxidation; Male; Mesocricetus; Micronucleus Tests; Milk; Mouth Neoplasms; NAD(P)H Dehydrogenase (Quinone); Phenols; Tea; Thiobarbituric Acid Reactive Substances

We investigated the anticancer effects of green and black tea polyphenols alone and in combination with bovine milk lactoferrin (bLF) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells. Both green (Polyphenon-E;P-E) and black tea polyphenols (Polyphenon-B;P-B) preferentially inhibit the growth of CAL-27 cells in a dose-dependent manner. Based on the IC(50) values, P-E was found to be more effective than P-B and the combination of P-E and bLF (1:2 ratio) exhibited synergistic inhibition of CAL-27 cells. Analysis of the mechanism revealed nuclear fragmentation and condensation with appearance of the A(o) peak indicative of apoptosis. Furthermore, tea polyphenols transduced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3. Overall, the potency of cytotoxic and apoptosis inducing effects of dietary agents on CAL-27 cells was in the order P-E and bLF combination (1:2 ratio)>P-E>P-B. These results suggest that a "designer" approach may be useful for oral cancer prevention strategies.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Catechin; Cattle; Cell Cycle; Cell Death; Cell Line, Tumor; Drug Synergism; Fibroblasts; Flavonoids; Humans; Lactoferrin; Membrane Potential, Mitochondrial; Mouth Neoplasms; Phenols; Polyphenols; Reactive Oxygen Species; Tea

Alpha-defensin or human neutrophil peptide-1 (HNP1) is a neutrophil-derived antimicrobial peptide with cytotoxic effects towards cancer cells. Lactoferrin is also stored in human neutrophils and is a glycoprotein involved in mediating cytotoxicity towards tumour cells. This study investigated the sensitivity of normal oral keratinocyte and oral squamous cell carcinoma (OSCC) cells to HNP1 and lactoferrin in various combinations. A concentration of 100 microg/ml HNP1 induced the most significant cytotoxic effect on both normal and OSCC cells. Lactoferrin (12.5, 25 and 250 microg/ml) also significantly induced cell death in OSCC cells after 72 h. Of note, a combination of 10 microg/ml HNP1 and 50 microg/ml lactoferrin induced a differential effect, not observed with either concentration alone, which stimulated proliferation in normal cells, but induced cell death in OSCC cells throughout the study. These results indicate a potentially important co-operative role for HNP1 and lactoferrin in facilitating a selective cytotoxic effect on tumour cells.

Topics: alpha-Defensins; Carcinoma, Squamous Cell; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Drug Screening Assays, Antitumor; Electrophoresis, Polyacrylamide Gel; Humans; Keratinocytes; Lactoferrin; Mouth Mucosa; Mouth Neoplasms; Tumor Cells, Cultured

Combination chemoprevention using tea polyphenols as one of the components has received growing consideration in recent years. The present study was designed to evaluate the antiproliferative and apoptosis inducing effects of bovine lactoferrin (bLF) and black tea polyphenol (Polyphenon-B: P-B) combination on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Topical application of DMBA for 14 weeks induced buccal pouch tumours that showed aberrant expression of cytokeratins, a marker for epithelial carcinomas. This was associated with increased cell proliferation and evasion of apoptosis as revealed by upregulation of proliferating cell nuclear antigen, NF-kappaB, mutant p53, Bcl-2 and downregulation of Bax, Fas and caspase 3 protein expression. Although dietary administration of bLF and Polyphenon-B alone significantly reduced tumour incidence, combined administration of bLF and Polyphenon-B was more effective in inhibiting HBP carcinogenesis by restoring normal cytokeratin expression, inhibiting cell proliferation and inducing apoptosis. These findings suggest that a "designer item" approach will be useful for human oral cancer prevention strategies.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Proliferation; Cheek; Chemoprevention; Cricetinae; Lactoferrin; Male; Mesocricetus; Mouth Neoplasms; Phenols; Tea

Chemoprevention by dietary constituents has emerged as a novel approach to control oral cancer incidence. We therefore evaluated the chemopreventive efficacy of bovine milk lactoferrin (bLF) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis.. Hamsters were divided into four groups. The right buccal pouches of animals in groups 1 and 2 were painted with 0.5% DMBA three times a week for 14 wk. Animals in group 2, received in addition, basal diet containing 0.2% bLF. Group 3 animals were given 0.2% bLF alone. Group 4 animals served as control. The status of carcinogen-metabolizing enzymes, the extent of lipid peroxidation and glutathione-dependent antioxidants in the buccal pouch and liver as well as bone marrow micronuclei incidence were used as biomarkers.. All the hamsters painted with DMBA alone for 14 wk, developed HBP carcinomas that showed diminished lipid peroxidation and increased activities of carcinogen-metabolizing enzymes and antioxidants with enhanced bone marrow micronuclei. In the liver of tumor bearing animals, the increase in phase I enzymes and lipid peroxidation was accompanied by reduced activities of antioxidant and phase II detoxification enzymes. Administration of bLF decreased the incidence of DMBA-induced micronuclei and HBP carcinomas by decreasing phase I enzymes, modulating lipid peroxidation and enhancing antioxidant and phase II enzyme activities.. The chemopreventive effects of bLF is mediated by reducing DMBA-induced genotoxicity and modulating carcinogen-metabolizing enzymes and oxidant-antioxidant profile in the target organ as well as in the liver.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cattle; Chemoprevention; Cricetinae; Cytochrome P-450 Enzyme System; Disease Models, Animal; Glutathione Reductase; Lactoferrin; Lipid Peroxidation; Liver; Male; Mesocricetus; Mouth Neoplasms; Oxidation-Reduction; Random Allocation; Thiobarbituric Acid Reactive Substances; Xenobiotics

Lactoferrin, a member of the transferrin family, is iron-binding and a strongly cationic 76 kDa glycoprotein. In breast milk it is secreted in high concentrations from glandular epithelia and is also present in other exocrine fluids including saliva. In the present study, we examined the biological mechanisms of apoptosis induced by pepsin-digested-lactoferrin peptide (Lfn-p) in the human oral squamous cell carcinoma cell line SAS. We found that treatment with Lfn-p induced cell death with apoptotic nuclear changes, preceded by the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in the apoptotic cells. Treatment with Lfn-p induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), a member of the MAP kinase family, at early stages of apoptosis. Another MAP kinase, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), was also phosphorylated by treatment with Lfn-p. Pretreatment of SAS cells with SP600125, a JNK/SAPK inhibitor, diminished Lfn-induced apoptosis, as assessed by determining released lactate dehydrogenase activity. On the other hand, the MEK1 inhibitors PD98059 or U0126 showed no effect on repression of cell death, but rather an increase. These results suggest that JNK/SAPK activation may play an important role in Lfn-p-induced apoptotic cell death of human oral squamous cell carcinoma cells.

Topics: Animals; Anthracenes; Apoptosis; Cattle; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Humans; JNK Mitogen-Activated Protein Kinases; Lactoferrin; Mouth Neoplasms; Pepsin A

We analyzed the radiation-induced changes in the flow rate and protein composition of stimulated whole saliva in eleven patients treated for malignant conditions of the head and neck. In all patients the radiation field covered all major salivary glands and a large area of the oral mucosa. Paraffin-stimulated whole saliva samples were collected once 2 to 21 days before therapy and then after 20, 40, and 60 gray (Gy) cumulative dose of irradiation. Five patients also provided samples 6 months after the therapy. Hyposalivation or xerostomia occurred in all patients, although the pretreatment secretion rates were already relatively low. Salivary amylase activities decreased with increasing dose of radiation, especially when expressed as the amount of enzyme secreted per minute. Unusually high salivary concentrations of albumin, lactoferrin, lysozyme, salivary peroxidase, myeloperoxidase, and total protein were observed during the therapy, but most values slowly returned to pretreatment levels after cessation of radiation. It is concluded that the observed qualitative changes in whole saliva components are net effects caused by the cancer itself, radiation therapy given, systemic diseases, or medications, as well as mucosal inflammations.

Topics: Adult; Aged; Amylases; Female; Humans; Isoenzymes; Lactoferrin; Male; Middle Aged; Mouth Neoplasms; Muramidase; Peroxidase; Peroxidases; Pharyngeal Neoplasms; Saliva; Salivary Proteins and Peptides; Secretory Rate

In oral dysplasias and squamous cell carcinomas, relationships exist between the presence of keratin filaments and cell differentiation. The keratinized areas of high differentiated carcinomas are carcinoembryonic antigen (CEA) positive. The labeling of the higher molecular keratins is similar to the distribution of lectin receptors so that lectins represent membrane-oriented markers of differentiation. In dysplasias a gradual loss of blood group substances A and B can be observed. Squamous cell carcinomas possess no substances A or B. H antigen as precursor of A and B is increased in preneoplasias and absent in carcinomas. In oral papillomas, leukoplakias, and carcinomas virogen koilocytotic cell changes, papilloma viruses and viral antibodies can be demonstrated. In salivary gland tumors a distinct pattern of distribution for keratin, vimentin, CEA, tissue polypeptide antigen (TPA), metalloproteins, and enzymes can be observed. The cellular stromal reaction (lymphocytes, Langerhans cells, and so forth) can be defined more exactly by monoclonal antibodies.

Topics: Antigens, Viral; Blood Group Antigens; Carcinoembryonic Antigen; Cell Differentiation; Histocytochemistry; Humans; Intermediate Filament Proteins; Keratins; Lactoferrin; Mouth Neoplasms; Peptides; Receptors, Mitogen; Salivary Gland Neoplasms; Tissue Polypeptide Antigen