Y. Hara

In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells

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 IC50 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 Ao 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.

Intrinsic apoptosis and NF-κB signaling are potential molecular targets for chemoprevention by black tea polyphenols in HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo

Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO-EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague-Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKβ and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death.

Comparative evaluation of antiproliferative, antiangiogenic and apoptosis inducing potential of black tea polyphenols in the hamster buccal pouch carcinogenesis model.

Background To evaluate the relative chemopreventive efficacy of two black tea polyphenols, Polyphenon-B [P-B] and BTF-35 on 7,12-dimethylbenz [a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Methods Hamsters were divided into 6 groups. The right buccal pouches of animals in groups 1–3 were painted with 0.5% of DMBA three times a week for 14 weeks. While hamsters in group 1 received no further treatment, animals in groups 2 and 3 received diet containing 0.05% P-B and BTF-35 respectively, four weeks before DMBA painting that was continued until the end of the experiments. Animals in groups 4 and 5 were given P-B and BTF-35 alone respectively as in groups 2 and 3. Group 6 animals served as the untreated control. All the animals were sacrificed after 18 weeks. The expression of p21, cyclin D1, glutathione S-transferase pi (GST-P), nuclear factor kappa B (NF-κB), Bcl-2, Bax, cytochrome C, caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP), cytokeratins and vascular endothelial growth factor (VEGF) was analysed by RT-PCR, immunohistochemical and Western blot analyses. Results DMBA treated animals developed buccal pouch carcinomas that displayed increased expression of p21, cyclin D1, GST-P, NF-κB, cytokeratins, VEGF and Bcl-2 with decreased expression of Bax, cytochrome C, caspase-3, caspase-9, and PARP. Dietary administration of both P-B and BTF-35 reduced the incidence of DMBA-induced HBP carcinomas by modulating markers of cell proliferation, cell survival, tumour infiltration, angiogenesis, and apoptosis. Conclusion The results of the present study provide a mechanistic basis for the chemopreventive potential of black tea polyphenols. The greater efficacy of BTF-35 in inhibiting HBP carcinogenesis and modulating multiple molecular targets may have a potential role in the prevention of oral cancer.

Black tea polyphenols protect against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis.

Dietary chemoprevention has emerged as a cost-effective approach for cancer control. We evaluated the chemopreventive effects of black tea polyphenols (Polyphenon-B) administration during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. The expression of proliferating cell nuclear antigen (PCNA) in the buccal pouch and the concentration of lipid peroxides, protein carbonyl, and the antioxidant status in the buccal pouch, liver and erythrocytes were used as biomarkers of chemoprevention. All the hamsters painted with DMBA alone for 14 weeks developed buccal pouch carcinomas associated with increased expression of PCNA, diminished lipid and protein oxidation, and enhanced antioxidant status. In the liver and erythrocytes of tumor-bearing animals, enhanced oxidation of lipids and proteins was accompanied by compromised antioxidant defenses. Dietary administration of Polyphenon-B effectively suppressed DMBA-induced HBP carcinogenesis as revealed by decreased incidence of tumours and PCNA expression. In addition, Polyphenon-B modulated lipid and protein oxidation and enhanced the antioxidant status in the pouch, liver, and erythrocytes. We suggest that Polyphenon-B exerts its chemopreventive effects by inhibiting cell proliferation in the target tissue and modulating the oxidant-antioxidant status in the target as well as in host tissues.

Modulatory effects of black tea polyphenols on oxidant–antioxidant profile and expression of proliferation, apoptosis, and angiogenesis-associated proteins in the rat forestomach carcinogenesis model

BackgroundChemoprevention by dietary constituents has emerged as a novel approach to control stomach cancer incidence. We therefore evaluated the chemopreventive effects of black tea polyphenols (Polyphenon-B) on oxidant–antioxidant status, cell proliferation, apoptosis, and angiogenesis during N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis.MethodsMale Wistar rats were divided into four groups. Rats in group 1 and 2 were given MNNG (150 mg/kg body weight) by intragastric intubation three times at 2 week intervals and followed for 26 weeks. Rats in group 2 received in addition a basal diet containing 0.05% Polyphenon-B. Group 3 animals were given 0.05% Polyphenon-B alone. Group 4 animals served as controls. The status of lipid peroxidation and antioxidants and the expression of the lipid peroxidation marker 4-hydroxy nonenal (4-HNE), proliferating cell nuclear antigen (PCNA), glutathiones-transferase (GST)-π, Bcl-2, Bax, cytochrome C, caspase 3, cytokeratins, and vascular endothelial growth factor (VEGF) were used as biomarkers.ResultsIntragastric administration of MNNG induced well-differentiated squamous cell carcinomas that showed diminished lipid and protein oxidation and an increase in antioxidant status. This was associated with increased cell proliferation, angiogenesis, and invasive potential coupled with apoptosis evasion as revealed by upregulation of PCNA, GST-π, Bcl-2, cytokeratins, and VEGF and downregulation of Bax, cytochrome C, and caspase 3 protein expression. Dietary administration of Polyphenon-B effectively suppressed MNNG-induced gastric carcinogenesis, as evidenced by modulation of oxidant–antioxidant status, inhibition of cell proliferation and infiltration, and angiogenesis associated with apoptosis induction.ConclusionsThe present study provides evidence that Polyphenon-B exerts multifunctional inhibitory effects on MNNG-induced gastric carcinogenesis and suggests that it can be developed as a potential chemopreventive agent.

Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model

The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of ρ-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b5, cytochrome b5 reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis.

Pretreatment with black tea polyphenols modulates xenobiotic-metabolizing enzymes in an experimental oral carcinogenesis model.

The objective of this study was to evaluate the chemopreventive potential of the black tea polyphenols Polyphenon-B and BTF-35 during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Hamsters were divided into six groups. Animals in groups 2 and 3 received diet containing Polyphenon-B and BTF-35, respectively, 4 weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, animals in groups 1, 2, and 3 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 4 and 5 were given Polyphenon-B and BTF-35 alone, respectively, as in groups 2 and 3. Animals in group 6 served as control. All the animals were sacrificed after an experimental period of 18 weeks. Phase I and phase II xenobiotic-metabolizing enzymes and 8-hydroxy-deoxyguanosine (8-OH-dG) in the buccal pouch and liver were used as biomarkers of chemoprevention. Hamsters painted with DMBA showed increased expression of 8-OH-dG and enhanced activities of phase I (CYP450; total as well as CYP1A1, 1A2, and 2B isoforms and cytochrome b5) and phase II (GST and quinone reductase) xenobiotic-metabolizing enzymes with increased immunohistochemical expression of CYP1A1, and CYP1B1 isoforms in the buccal pouch. This was accompanied by increased phase I and decreased phase II enzyme activities in the liver. Administration of Polyphenon-B and BTF-35 significantly decreased tumor incidence, oxidative DNA damage, phase I enzyme activities as well as expression of CYP1A1 and CYP1B1 isoforms, while enhancing phase II enzyme activities in the buccal pouch and liver. Our results provide a mechanistic basis for the chemopreventive potential of black tea polyphenols. Furthermore, the greater efficacy of BTF-35 in chemoprevention of HBP carcinomas via inhibition of oxidative DNA damage and modulation of xenobiotic-metabolizing enzymes may have a major impact in human oral cancer prevention.

Combination Chemoprevention of Hamster Buccal Pouch Carcinogenesis by Bovine Milk Lactoferrin and Black Tea Polyphenols

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.