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Objective To evaluate the chemopreventive effects of 8-allyl garcinol on oral squamous cell carcinoma(OSCC).Methods OSCC cell line CAL27 were cultured and treated with different concentrations of garcinol or 8-allyl garcinol. Their effects on the biological behaviors of OSCC cell line CAL27 were measured by MTT assay,clony formation assay,scratch migration assay,and flow cytometry with Annexin V-FITC/PI staining assay. We established DMBA-induced hamster cheek pouch models of dysplasia. While the negative control group was not treated,the positive group was treated with 0.5% DMBA solution tropically to the left cheek pouch three times per week for three consecutive weeks. The other four groups received 0.5 mmol/L or 1.0 mmol/L garcinol or 8-allyl garcinol respectively three times within the following two weeks after DMBA treatment. Hamsters were sacrificed at the fifth week to obtain tissue samples of the left cheek pouch. The samples were examined by histopathology and BrdU immunohistochemisty.Results MTT assay showed that both garcinol and 8-allyl garcinol inhibited the proliferation of CAL27 cells in a concentration-and time-dependent manner. The half maximal inhibitory concentration(IC 50 )of 8-allyl garcinol[(13.13±2.55)μmol/L] was significantly lower than garcinol[(32.20±3.24)μmol/L;t=8.008,P=0.001]. Comparing the two grougs of medicine in the same concentration,the inhibiting proliferation effects 8-allyl garcinol had significantly stronger effect in inhibiting proliferation than garcinol when the same dose was applied,and the difference was largest at the concentrations of 10(24 h:t=8.012,P=0.001;48 h:t=5.939,P=0.001;72 h:t=12.551,P=0.001)and 20 μmol/L(24 h:t=8.887,P=0.001;48 h:t=9.324,P=0.002;72 h:t=5.361,P=0.002). The clone formation assay showed the clone formation rates after the treatment with 20 μmol/L garcinol and 20 μmol/L 8-allyl garcinol were(44.1±0.4)% and(23.6±0.6)%,respectively,which were significantly lower than those after treatment with 10 μmol/L garcinol[(55.6±2.8)%;t=6.894,P=0.019] and 10 μmol/L 8-allyl garcinol[(31.0±0.6)%;t=15.556,P=0.001]. The inhibiting effects of 8-allyl garcinol at the concentrations of 10 μmol/L(t=14.682,P=0.003)and 20 μmol/L(t=51.514,P=0.001)were significantly stronger than garcinol.Scratch test showed the relative cell migration rates after treatment with 10 and 20 μmol/L garcinol for 12 hours were(16.00±4.55)%(t=3.139,P=0.026)and(3.00±3.16)%(t=6.608,P=0.001),respectively,which were lower than negative control [(30.33±7.64)%]. The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 12 hours were(16.25±3.86)%(t=3.245,P=0.023)and(6.00±2.65)%(t=5.214,P=0.006),respectively,which were also lower than negative control[(30.33±7.64)%]. In addition,the relative cell migration rates after treatment with 10 and 20 μmol/L garcinol for 24 hours were(23.75±4.57)%(t=4.718,P=0.005)and(5.75±1.50)%(t=10.432,P=0.001),respectively,which were lower than negative control[(45.33±7.64)%]. The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 24 hours were(23.50±2.38)%(t=5.529,P=0.003)and(11.67±2.31)%(t=7.308,P=0.002),respectively,which were also lower than negative control[(45.33±7.64)%]. Furthermore,the relative cell migration rate after treatment with 20 μmol/L garcinol for 24 hours was significantly lower than after treatment with 8-allyl garcinol(t=4.151,P=0.009). The apoptosis experiments showed that the early apoptosis rate of CAL27 cells was(5.00±0.10)% after treatment with 10 μmol/L garcinol,which was significantly higher than negative control[(1.57±0.21)%;F=70.950,P=0.001]. The early and late apoptosis rates of CAL27 cells were(5.90±0.78)%(t=39.384,P=0.001)and(9.73±1.67)%(t=10.101,P=0.001),respectively,after treatment with 20 μmol/L garcinol,which were also significantly higher than negative control. The early apoptosis rate of CAL27 cells was(4.63±1.16)% after treatment with 8-allyl garcinol,which was significantly higher than negative control(t=4.511,P=0.041). The effects of 8-allyl garcinol in promoting cell apoptosis were weaker than garcinol(10 μmol/L:t=5.982,P=0.004;20 μmol/L:t=8.578,P=0.001). The histopathological test also showed that the hyperplastic areas of oral mucosal epithelium in hamsters after treatment with 0.5 mmol/L garcinol(t=2.546,P=0.031),0.5 mmol/L 8-allyl garcinol(t=3.485,P=0.008),1.0 mmol/L garcinol(t=4.556,P=0.001),and 1.0 mmol/L 8-allyl garcinol(t=5.393,P=0.001)were significantly smaller than positive control. The dysplasia areas of oral mucosal epithelium in hamsters after treatment with 0.5 mmol/L 8-allyl garcinol(t=2.130,P=0.046),1.0 mmol/L garcinol(t=3.434,P=0.010),and 1.0 mmol/L 8-allyl garcinol(t=4.518,P=0.004)were also smaller than positive control;1.0 mmol/L garcinol group(t=2.793,P=0.023)and 1.0 mmol/L 8-allyl garcinol group(t=4.997,P=0.001)were smaller than 0.5 mmol/L garcinol treatment group. Immunohistochemical staining of BrdU showed that the BrdU-labeled indicators were significantly lower in negative control group(t=7.563,P=0.001),0.5 mmol/L garcinol(t=2.862,P=0.029),0.5 mmol/L 8-allyl garcinol(t=4.693,P=0.002),1.0 mmol/L garcinol(t=5.071,P=0.002),and 1.0 mmol/L 8-allyl garcinol(t=5.133,P=0.001)when compared with the positive control. The BrdU-labeled indicators in 0.5 mmol/L 8-allyl garcinol(t=3.724,P=0.007),1.0 mmol/L garcinol(t=7.000,P=0.001),and 1.0 mmol/L 8-allyl garcinol(t=4.413,P=0.003)were also significantly lower than in 0.5 mmol/L garcinol group.Conclusions 8-allyl garcinol could inhibit the proliferation and migration of OSCC cell line CAL27 and promotes apoptosis. It also has prominent inhibitory effects on DMBA-induced hamster cheek pouch dysplasia. However,the specific effects are slightly different from garcinol.

Garcinol from the fruit rind of Garcinia indica shows anti-carcinogenic and anti-inflammatory properties, but its mechanism and key functional groups were still need to be identified. Our previous computer modeling suggested that the C8 side chain of Garcinol is so large that it may influence the bioactivity of the compound. 8-Me Garcinol, a derivative of Garcinol in which the bulky side chain at the C8 position of Garcinol is replaced with a much smaller methyl group, was synthesized through a 12-step procedure starting from 1,3-cyclohexanedione. The antitumor activity of Garcinol and 8-Me Garcinol was evaluated in vitro by MTT, cell cycle and cell apoptosis assays. The results showed that 8-Me Garcinol had weaker inhibitory activity on cells proliferation, and little effects on cell cycle and apoptosis in oral cancer cell line SCC15 cells when compared with Garcinol. All of the results indicated 8-Me Garcinol exerts weaker antitumor activity than Garcinol, and the C8 side chain might be an important active site in Garcinol. Changing the C8 side chain will affect the inhibitory effect of Garcinol., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the presence of K2CO3/Cs2CO3 (molar ratio 10:1), garcinol was subjected to methylation by reaction with iodomethane at room temperature to afford 13,14-dimethoxy garcinol. The methylated garcinol derivative was screened against oral cancer cell line SCC15 for cell proliferation and apoptosis. 13,14-Dimethoxy garcinol showed weaker inhibitory activity on SCC15 cell growth than garcinol, and had little effect on cell cycle and apoptosis of SCC15, whereas garcinol effectively induced cell cycle arrest and cell apoptosis. Meanwhile, the ELISA data showed that the inhibitory effect of garcinol on 5-Lox pathway was more potent than 13,14-dimethoxy garcinol (P<0.05). All these results have confirmed the important role of 13,14-dihydroxy groups for anti-cancer effects of garcinol., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Zengshengping (ZSP) tablets had inhibitory effects on oral precancerous lesions by reducing the incidence of oral cancer. However, the severe liver toxicity caused by systemic administration of ZSP limits the long-term use of this anti-cancer drug. The purpose of this study was to evaluate the tumor inhibitory effects due to the topical application of extracts from ZSP, a Chinese herbal drug, on 7, 12-dimethlbenz(a)anthracene (DMBA) induced oral tumors in hamsters. The study also investigated the anti-cancer mechanisms of the ZSP extracts on oral carcinogenesis., Methods: DMBA (0.5%) was applied topically to the buccal pouches of Syrian golden hamsters (6 - 8 weeks old) three times per week for six weeks in order to induce the development of oral tumors. Different fractions of ZSP were either applied topically to the oral tumor lesions or fed orally at varying dosages to animals with oral tumors for 18 weeks. Tumor volume was measured by histopathological examination. Tumor cell proliferation was evaluated by counting BrdU labeled cells and by Western blotting for mitogen-activated protein kinase (MAPK) protein levels. The protein levels of apoptosis marker Caspase-3 and regulator Bcl-2 protein were also measured by Western blotting., Results: Topical application of DMBA to the left pouch of hamsters induced oral tumor formation. Animals treated with DMBA showed a loss in body weight while animals treated with ZSP maintained normal body weights. Both the ZSP n-butanol fraction and water fraction significantly reduced tumor volume by 32.6% (P < 0.01) and 22.9% (P < 0.01) respectively. Topical application of ZSP also markedly decreased the BrdU-positive cell numbers in oral tumor lesions and reduced the expression level of MAPK. In addition, ZSP promoted tumor cell apoptosis by increasing Caspase-3 expression but decreasing Bcl-2 protein production., Conclusion: The n-butanol and water fractions of ZSP are effective at inhibiting tumor cell proliferation and stimulating apoptosis in oral cancer suggesting that these fractions have chemopreventive effects on DMBA induced oral carcinogenesis.

Objective: To evaluate the chemopreventive effects of boswellic acid and curcumin on 7,12-dimethyl benzanthracene(DMBA)-induced oral carcinogenesis in the hamster cheek pouch model., Methods: Male Syrian golden hamsters (6 - 8 weeks old, 80 - 130 g in weight) were randomly divided into seven groups, with group A serving as the untreated negative control. The left cheek pouch of the remaining hamsters was topically treated with 0.5% DMBA in mineral oil three times a week for 6 weeks. They were then randomized to six groups with group B serving as a positive control and receiving no further treatment. Groups C-G were treated topically with 5, 10 mg/L boswellic acid, 5, 10 µmol/L curcumin, or the combination of 5 mg/L boswellic acid and 5 µmol/L curcumin three times per week for 18 weeks. The animals were injected with bromodeoxyuridine intraperitoneally at 50 mg/kg 2 h prior to killing. At the 25 th week all the hamsters were sacrificed and cheek pouch tissue was harvested. One half of the tissue was snap frozen in liquid nitrogen for analysis of arachidonic acid metabolites, and the other half was fixed in 10% phosphate-buffered saline(PBS)-buffered formalin for histopathological examination., Results: Six-weeks of DMBA followed by 18-weeks of topical application of boswellic acid and curcumin, both boswellic acid (5, 10 mg/L) and curcumin (5, 10 µmol/L) significantly inhibited the incidence from 93.8% to 73.9% (P > 0.05), numbers from 2.19 ± 0.98 to 1.13 ± 0.81 (P < 0.01) and size of visible tumors. Microscopically the incidence of squamous cell carcinoma and BrdU index were also significantly suppressed by boswellic acid and curcumin., Conclusions: Both boswellic acid and curcumin were effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model.

Objective: To investigate the roles of surviving and caspase-3 in the development of oral cancer., Methods: Archival tissue sections of 17 oral squamous cell carcinoma (OSCC), 28 oral leukoplakia with dysplasia, 10 normal oral mucosa were obtained from Capital Medical University School of Stomatology for immunohistochemical staining of markers of survivin and caspase-3. The cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated nucleotide shift enzyme (TdT) mediated d-UTP end labeling (TUNEL). Positively stained cells were counted and analyzed statistically to determine potential relationship between survivin, caspase-3 and cell apoptosis., Results: The expression of survivin was faint or negative in normal epithelial cells. The average positive rate of survivin was (1.05 ± 1.21)% in control group and (21.89 ± 10.45)% in OSCC. Caspase-3 was expressed in all the normal mucosa,but it obviously down-regulated in dysplasia and OSCC. The apoptosis index (AI) decreased from (0.89 ± 0.46)% in normal mucosa to (0.21 ± 0.12)% in OSCC., Conclusions: Both survivin and caspase-3 are associated with carcinogenesis of the oral mucosa. Survivin may restrain cell apoptosis by inhibiting caspase-3.