Downregulation of Tumor Promotor Genes in Oryza Sativa Linn.-Induced Antiproliferative Activity of Human Squamous Carcinoma Cells.

OBJECTIVES: Oral cancer represents the third leading cause of death in Southeast Asia and targeted therapy could prevent or delay disease etymology. Oryza sativa Linn. (OS) extract has been implicated as an antitumor agent in many cancer types, however none has been investigated in human squamous carcinoma-2 (HSC-2) cells, thus we aim to investigate the effects of OS on HSC-2 cells. METHODS: Our study investigated the growth inhibitory effects of an ethanolic extract of OS on HSC-2 cells by BrdU ELISA and MTT assays, as well as changes in tumor promoter genes using RT-qPCR and western blotting. RESULTS: We found that OS was able to induce cell cytotoxicity and inhibit HSC-2 proliferation. OS also decreased the expression of genes involved in the TGF-ß/Smads signaling pathway and genes involved in cell motility such as GPNMB, ITGB6, and E2F1 by RT-qPCR. Western blotting confirmed the downregulation of TGF-ß1 by OS. Co-treatment of OS and 5-Flurouracil also reversed Snail and Slug overexpression caused by HSC-2 exposure to 5-Flurouracil. CONCLUSION: Together, these results indicate that OS can inhibit HSC-2 cell proliferation and this may involve TGF-ß1 downregulation. Thus, this study shows OS could be useful for the treatment of patients with squamous carcinoma.

Gene Profiling of Cannabis-sativa-mediated Apoptosis in Human Melanoma Cells.

BACKGROUND/AIM: Malignant melanoma is an aggressive skin cancer, accounting for the majority of skin cancer deaths. Prognosis is often poor and finding effective treatment remains a challenge. Tetrahydrocannabinol (THC) and cannabidiol (CBD) are main bioactive components of Cannabis sativa plant extracts that have been shown to exert anti-tumor effects. In this study, we aimed to perform gene expression analysis of human melanoma A375 cells following stimulation with C. sativa extracts. MATERIALS AND METHODS: Gene expression profiles of A375 human melanoma and Vero (control) cell lines were evaluated by RNA sequencing and quantitative real-time PCR. RESULTS: Flow cytometry showed that the THC+CBD cannabis fractions induced apoptosis on A375 cells. Induction of apoptosis was accompanied by a notable up-regulation of DNA damage inducible transcript 3 (DDIT), nerve growth factor receptor (NGFR), colony-stimulating factor 2 (CSF2), growth arrest and DNA damage inducible beta (GADD45B), and thymic stromal lymphopoietin (TSLP) genes and down-regulation of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), cyclin E2 (CCNE2), integrin subunit alpha 9 (ITGA9), proliferating cell nuclear antigen (PCNA) and E2F transcription factor 1 (E2F1) genes. Treatment of A375 cells with the THC+CBD fraction inhibited the phosphorylation of ERK1/2 signaling pathway, which regulates melanoma cell proliferation. We showed that the THC+CBD combination disrupted melanoma cell migration. CONCLUSION: Use of C. sativa-derived extracts containing equal amounts of THC and CBD is proposed as a potential treatment of melanoma.

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE-/-) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.