The potential preventive effect of dietary phytochemicals In Vivo

Cancer remains a leading cause of mortality, characterized by dysregulated proliferation and impaired programmed cell death. Despite therapeutic advances, incidence and mortality remain high, highlighting the need for effective, less toxic approaches such as chemoprevention. Phytochemicals—bioactive plant compounds abundant in fruits, vegetables, teas, and spices—are associated with reduced cancer risk and can modulate key carcinogenic pathways. Green tea catechins (notably EGCG) and curcumin (from Curcuma longa) are well-studied phenolic phytochemicals with anti-proliferative, pro-apoptotic, anti-angiogenic, and anti-invasive actions. Given evidence for complementary mechanisms and pathway crosstalk, the study hypothesized that combining green tea and curcumin could provide synergistic chemoprevention by enhancing apoptosis and suppressing proliferation and angiogenesis in oral squamous cell carcinoma (OSCC). The hamster buccal pouch carcinoma (HBPC) model induced by DMBA serves as a preclinical proxy for tobacco-related oral carcinogenesis and enables evaluation of clinical and molecular responses to chemopreventive agents.

Prior studies demonstrate that plant-rich diets correlate with lower cancer risk. Green tea polyphenols (EGCG, EGC, ECG, EC) inhibit pathways linked to cancer growth, survival, and metastasis; EGCG’s cancer-preventive activity has been recognized since 1987. Curcumin exhibits anti-inflammatory, antioxidant, chemopreventive, and anti-tumorigenic properties, reduces DNA adducts, repairs DNA, and deactivates carcinogens. Both agents modulate proliferation, apoptosis, and angiogenesis via multiple signaling pathways and can protect normal cells during therapy. Combination chemoprevention may enhance efficacy and reduce toxicity by targeting multiple pathways simultaneously. In HBPC and other models, green tea and curcumin have shown efficacy individually and suggested synergistic interactions, including cell cycle arrest at different phases, downregulation of cyclins, and modulation of Bcl-2 family proteins and VEGF. However, curcumin’s solubility and bioavailability pose challenges for clinical translation.

Design and ethics: Controlled comparative in vivo experimental study approved by Alexandria University IRB (IRB#00010556-IORG0008839). Animals: 50 healthy male Syrian golden hamsters (Mesocricetus auratus), 5 weeks old, 80–129 g, housed individually under standardized conditions (23 ± 1°C, 50 ± 5% RH, 12:12 light/dark), acclimatized 7 days, with ad libitum conventional feed and water. Grouping: Randomized into 5 groups (n=10 each): A) normal control; B) DMBA only (positive control); C) DMBA + green tea; D) DMBA + curcumin; E) DMBA + green tea + curcumin. Carcinogenesis induction: Left buccal pouch painted with 0.5% 7,12-dimethylbenz[a]anthracene (DMBA; 0.5 g in 100 ml liquid paraffin) using brush, three times per week for 18 weeks. Treatments: - Green tea (Group C): classic Darjeeling tea solids prepared fresh (6 mg tea solids/ml) provided as sole drinking source via gastric lavage throughout 18 weeks. - Curcumin (Group D): curcumin powder 10 mmol (in 100 ml paraffin oil) applied topically to left buccal pouch three times/week on alternate days with DMBA for 18 weeks. - Combination (Group E): same green tea and curcumin regimens together alongside DMBA. Clinical assessment: After 18 weeks, left buccal pouch examined for lesions (e.g., leukoplakia, erythroplakia, exophytic masses). Animals sacrificed under anesthesia (ketamine 30 mg/kg, i.p.) by cervical decapitation. Tissue processing and histology: Lesion biopsies fixed in 10% buffered formalin, paraffin-embedded; 4 µm sections stained with H&E. Blinded evaluation by two independent pathologists to confirm dysplasia/carcinoma. Immunohistochemistry (angiogenesis): CD34 staining to assess microvessel density (MVD). Image analysis via Leica Qwin 500; microvessels counted in 10 hot-spot fields at 400x, mean per sample recorded. Immunofluorescence (proliferation): PCNA immunofluorescence on deparaffinized sections using standard protocol: permeabilization (PBS + 0.1% Triton X), blocking (1% BSA), primary anti-PCNA (10 µg/mL) overnight at 4°C, Alexa Fluor 555-conjugated secondary (1:400), Hoechst 33342 nuclear counterstain. Imaging by confocal laser scanning microscopy (Leica TCS SPE II/DMi8) in DIC and fluorescence modes. Flow cytometry (apoptosis): Fresh tumor tissue minced in RPMI-1640, enzymatically dissociated (trypsin/EDTA), filtered (100 µm), ethanol-fixed (70% ice-cold), rehydrated, and stained with Annexin V-FITC and propidium iodide (PI). Acquisition on FACSCalibur (Becton Dickinson) with CellQuest software; 10,000 events per sample; duplicate runs; analysis within 4 h. Data analysis: SPSS; one-way ANOVA for group comparisons; significance at p < 0.05; data reported as mean ± SD.

Clinical and histological outcomes after 18 weeks: - Positive control (DMBA only): 100% developed exophytic oral lesions; histology showed 4/10 well-differentiated SCC and 6/10 moderately differentiated SCC with pleomorphism, hyperchromatism, abnormal mitoses. - Green tea (DMBA + green tea): Clinically, 7/10 white patches (70%), 2/10 small exophytic lesions (20%), 1/10 no pathology (10%). Histology: 7/10 dysplasia; 2/10 well-differentiated SCC; 1/10 normal epithelium. - Curcumin (DMBA + curcumin): Clinically, 5/10 red patches (50%), 3/10 small exophytic lesions (30%), 2/10 no pathology (20%). Histology: 5/10 moderate–severe dysplasia; 3/10 invasive carcinoma; 2/10 normal epithelium. - Combination (DMBA + green tea + curcumin): Clinically, 7/10 white patches (70%), 3/10 tiny red exophytic lesions (30%). Histology: 7/10 moderate–severe dysplasia/carcinoma in situ; 3/10 well-differentiated SCC. Proliferation (PCNA immunofluorescence): Positive control showed strong red nuclear/cytoplasmic fluorescence in SCC cells indicating high proliferation; treated groups (green tea, curcumin, combination) showed decreased red nuclear fluorescence consistent with reduced proliferation; normal controls lacked red fluorescence. Angiogenesis (CD34 IHC, MVD mean ± SD): Positive control 28.20 ± 6.41; green tea 7.20 ± 0.92; curcumin 7.80 ± 2.90; combination 5.0 ± 0.94. Treated groups exhibited lower MVD than positive control; differences among treated groups were not statistically significant. Apoptosis (Annexin V-FITC/PI flow cytometry; total apoptosis = early + late): - Normal control: 1.72% (0.81% early, 0.91% late); necrosis 0% reported. - Positive control: 11.57% (3.59% early, 7.98% late); necrosis 8.16%. - Green tea: 82.22% (48.86% early, 33.36% late); necrosis 0.55%. - Curcumin: 78.91% (4.25% early, 74.66% late); necrosis 5.87%. - Combination: 96.63% (14.70% early, 81.93% late); necrosis 0.20%. Overall, green tea and curcumin each enhanced apoptosis and reduced proliferation and angiogenesis, with the combination showing the highest apoptotic effect and lowest MVD.

Findings support the hypothesis that green tea and curcumin exert chemopreventive effects in DMBA-induced HBPC, with enhanced efficacy when combined. Across modalities, treated groups demonstrated reduced proliferation (lower PCNA fluorescence), decreased angiogenesis (lower CD34-assessed MVD), and markedly increased apoptosis compared with positive controls, consistent with complementary mechanisms. The combination yielded the highest apoptosis (96.63%) and lowest MVD (mean 5.0), suggesting synergistic or at least additive effects on apoptotic and anti-angiogenic pathways. Clinically and histologically, combination treatment improved outcomes relative to DMBA alone, although histologic conversion to non-malignant states was only moderate, indicating room for optimization. These results align with literature showing that EGCG and curcumin target multiple cell cycle and survival pathways (e.g., S and G2/M phase arrest, cyclin D1/B1 suppression; modulation of Bcl-2 family proteins, VEGF, MMPs, JAK2/STAT3), potentially explaining synergy through multi-target effects. The HBPC model, which mirrors aspects of tobacco-related oral carcinogenesis, proved useful for evaluating chemopreventive efficacy at clinical, histologic, and molecular levels. Future work should refine dosing, timing, and delivery to further enhance histologic regression and address pharmacokinetic limitations.

Green tea combined with curcumin exhibits synergistic growth-inhibitory, pro-apoptotic, anti-proliferative, and anti-angiogenic effects in vivo in a DMBA-induced hamster buccal pouch carcinoma model, outperforming either agent alone. The study demonstrates significant increases in apoptosis, decreases in proliferation and microvessel density, and improved clinical/histologic profiles with combination therapy. Future research should optimize dosing, duration, and delivery (e.g., formulations to improve curcumin bioavailability) and further investigate molecular mechanisms to translate these findings into preventive strategies for OSCC.

• Angiogenesis differences among treated groups were not statistically significant despite lower mean MVDs versus positive control, possibly reflecting variability and/or sample size. - Combination therapy produced only moderate histological improvement (dysplasia/carcinoma in situ predominated), suggesting that dosing, duration, or administration routes may need optimization. - Curcumin’s poor solubility and bioavailability may limit therapeutic impact and warrant improved formulations. - The study is preclinical in a single animal model; generalizability to humans requires further investigation.