Anti-Cancer Effect of Quercetin in Xenograft Models with EBV-Associated Human Gastric Carcinoma

Abstract

Licorice extracts have been widely used in herbal and folk medications. Glycyrrhiza contains diverse range of biological compounds including triterpenes (glycyrrhizin, glycyrrhizic acid) and flavonoids (quercetin, liquiritin, liquiritigenin, glabridin, licoricidin, isoliquiritigenin). The flavonoids in licorice are known to have strong anti-cancer activities. Quercetin, the most abundant flavonoid, has been shown to have anti-ulcer, anti-cancer, antioxidant, and anti-inflammatory properties. Latent Epstein-Barr virus (EBV) infection can lead to serious malignancies, such as, Burkitt's lymphoma, Hodgkin's disease and gastric carcinoma(GC), and (Epstein-Barr virus associated gastric carcinoma) EBVaGC is one of the most common EBV-associated cancers. In this study, the authors first examined the anti-cancer effects of quercetin and isoliquiritigenin in vivo xenograft animal models implanted with EBV(+) human gastric carcinoma (SNU719) or EBV(-) human gastric carcinoma (MKN74), and then explored the molecular mechanisms responsible for their anti-cancer activities. The results obtained showed that anti-cancer effect of quercetin was greater than isoliquiritigenin in mice injected with EBV(+) human gastric carcinoma (SNU719) cells. On the other hand, quercetin and isoliquiritigenin had similar anti-cancer effects in mice injected with EBV(-) human gastric carcinoma (MKN74) cells. Interestingly, quercetin inhibited EBV viral protein expressions, including EBNA-1 and LMP-2 proteins in tumor tissues from mice injected with EBV(+) human gastric carcinoma. Quercetin more effectively induced p53-dependent apoptosis than isoliquiritigenin in EBV(+) human gastric carcinoma, and this induction was correlated with increased expressions of the cleaved forms of caspase-3, -9, and Parp. In EBV(-)human gastric carcinoma (MKN74), both quercetin and isoliquiritigenin induced the expressions of p53, Bax, and Puma and the cleaved forms of caspase-3 and -9 and Parp at similar levels.

Keywords: EBV; SNU719; human gastric carcinoma; p53; quercetin.

Figure 1

Anti-tumor effects of quercetin and isoliquiritigenin in xenograft NOD/SCID mice bearing EBV(+) or EBV(−) human gastric carcinoma (SNU719 or MKN74) NOD/SCID mice were randomly divided into two groups. One group was implanted with EBV(+) human gastric carcinoma, SNU719, and the other group was implanted with EBV(−) human gastric carcinoma, MKN74. These two groups were then subdivided into three subgroups (n = 5/subgroup). Two weeks after implantation, mice were orally administrated (in drinking water) quercetin (QC, 30 mg/kg/day) or isoliquiritigenin (ISL, 30 mg/kg/day). (A) The structures of quercetin and isoliquiritigenin; (B) The study design of the animal experiment; (C) Tumor sizes in animals injected with EBV(+) positive carcinoma (SNU719) or (D) EBV(−) carcinoma (MKN74).

Figure 2

Expressions of EBNA1, LMP-2, and BZLF-1 proteins in tumor tissues from mice implanted with EBV(+) human gastric carcinoma (SNU719) EBV(+) human gastric carcinoma tumor tissue was excised from each animal fed quercetin (QC) or isoliquiritigenin (ISL) and prepared for western blot analysis. (A) The protein expression of EBNA1, LMP-2, and BZLF-1 were identified and (B) relative intensities were measured. GAPDH was used as the loading control.

Figure 3

Expression of p53, p21, PUMA, and Bax in tumor tissues from mice implanted with EBV(+) human gastric carcinoma (SNU719) EBV(+) human gastric carcinoma tumor tissue was excised from each animal fed quercetin (QC) or isoliquiritigenin (ISL) and prepared for western blot analysis. The protein expressions of (A) p53, p21; (B) PUMA and Bax were identified and the relative intensities were measured. β-Actin was used as the loading control.

Figure 4

Expressions of (cleaved) caspase-3, -9, and (cleaved) Parp proteins in tumor tissues from mice implanted with EBV(+) human gastric carcinoma (SNU719) EBV(+) human gastric carcinoma tumor tissue was excised from each animal fed quercetin (QC) or isoliquiritigenin (ISL) and prepared for western blot analysis. (A) The expressions (cleaved) caspase-3, -9, and (cleaved) Parp were identified and (B) relative intensities were measured. β-Actin was used as the loading control.

Figure 5

Expressions of p53, p21, Bax, PUMA, (cleaved) caspase-3, -9 and (cleaved) Parp proteins in tumor tissues from mice implanted with EBV(−) human gastric carcinoma (MKN74) EBV(−) tumor tissue was excised from each animal and prepared for western blot analysis. The protein expressions of (A) p53, p21; (B) Bax, PUMA; (C,D) (Cleaved) caspase-3, -9, and Parp were identified and relative intensities were measured. β-Actin was used as the loading control.

Figure 5

Expressions of p53, p21, Bax, PUMA, (cleaved) caspase-3, -9 and (cleaved) Parp proteins in tumor tissues from mice implanted with EBV(−) human gastric carcinoma (MKN74) EBV(−) tumor tissue was excised from each animal and prepared for western blot analysis. The protein expressions of (A) p53, p21; (B) Bax, PUMA; (C,D) (Cleaved) caspase-3, -9, and Parp were identified and relative intensities were measured. β-Actin was used as the loading control.