Glaucocalyxin A impairs tumor growth via amplification of the ATF4/CHOP/CHAC1 cascade in human oral squamous cell carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural extract glaucocalyxin A (GLA), purified from the aboveground sections of the Chinese traditional medicinal herb Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, has various pharmacological benefits, such as anti-bacterial, anti-coagulative, anti-neoplastic, and anti-inflammatory activities. Although GLA has shown anti-tumor activity against various cancers, the therapeutic potential and biological mechanisms of GLA remain to be further explored in oral squamous cell carcinoma (OSCC). AIM OF THE STUDY: This study aimed to elucidate the therapeutic potential and regulatory mechanisms of GLA in OSCC. MATERIALS AND METHODS: The cell proliferation and apoptosis effects of GLA were analyzed by CCK-8, clone formation, Annexin V/PI staining, and apoptotic protein expression in vitro. An OSCC xenograft model was applied to confirm the anti-neoplastic effect in vivo. Furthermore, the changes of reactive oxygen species (ROS) were determined by DCFH-DA probe and GSH/GSSG assay, and inhibited by the pan-caspase inhibitor Z-VAD(OMe)-FMK and the ROS scavenger N-acetylcysteine (NAC). The modulation of GLA on mitochondria and ER-dependent apoptosis pathways was analyzed by JC-1 probe, quantitative real-time PCR, and Western blot. Finally, public databases, clinical samples, and transfection cells were analyzed to explore the importance of GLA's indirect targeting molecule CHAC1 in OSCC. RESULTS: GLA significantly inhibited cell proliferation and induced apoptosis in vitro and in vivo. GLA perturbed the redox homeostasis, and cell apoptosis was totally rescued by Z-VAD(OMe)-FMK and NAC. Furthermore, GLA activated the mitochondrial apoptosis pathway. Simultaneously, the overexpression and knockdown of CHAC1 dramatically affected GLA-mediated apoptosis. The endoplasmic reticulum stress-associated ATF4/CHOP signal was identified to participate in GLA-upregulated CHAC1 expression. Finally, we found that CHAC1 expression was lower in OSCC compared with normal tissues and positively correlated with 4-Hydroxynonenal (4-HNE) level. High CHAC1 expression also indicated better overall survival. Moreover, CHAC1 selectively regulated the viability of oral cancer cells. CONCLUSION: GLA is a promising therapeutic agent that activates the ROS-mediated ATF4/CHOP/CHAC1 axis in OSCC patients.

Common genetic variants of microsomal epoxide hydrolase affect warfarin dose requirements beyond the effect of cytochrome P450 2C9.

BACKGROUND: Warfarin dose response is partially explained by the polymorphisms in the cytochrome P450 (CYP) 2C9 gene, affecting S -warfarin clearance, as well as by age and body weight. We examined the influence on warfarin dose requirements of candidate genes encoding microsomal epoxide hydrolase (mEH), as well as glutathione S -transferase A1 (GSTA1) components of vitamin K epoxide reductase and the gamma-glutamylcarboxylase (GGCX) gene. METHODS: We studied the effects of CYP2C9, mEH, GSTA1, and GGCX genotypes on warfarin maintenance doses, accounting for age, weight, vitamin K plasma concentrations and concurrent medications, in 100 patients undergoing therapeutic anticoagulation. RESULTS: Allele frequencies were 76.5%, 12.5%, and 11% for CYP2C9*1 , *2 , and *3 , respectively; 75% and 25% for mEH T 612 C; 75.8% and 24.2% for mEH A 691 G; 73.5% and 26.5% for GSTA1 T 631 G; and 70.5% and 29.5% for GGCX G 8762 A. Warfarin doses differed among the CYP2C9 ( 2C9*1 , 2C9*2 , and 2C9*3 ) genotype groups: 6.3 +/- 1.9 mg/d, 5.3 +/- 1.8 mg/d, and 3.8 +/- 1.7 mg/d, respectively (F = 4.83, P < .01). There were no differences in any of the other genotype groups. Among the 62 wild-type CYP2C9 patients, variant mEH T 612 C homozygotes required higher doses than heterozygotes and wild-type patients (7.5 +/- 2.9 mg/d, 6.5 +/- 4.2 mg/d, and 6.0 +/- 2.6 mg/d, respectively [F = 3.57, P = .03]). The odds ratio for requiring greater than 7 mg/d in variant mEH T 612 C patients versus wild-type patients was 3.14 (95% confidence interval, 1.47-6.67), accounting for CYP2C9. CONCLUSIONS: Variant mEH T 612 C genotypes are associated with warfarin doses of greater than 50 mg/wk beyond the effect of CYP2C9.