Zataria multiflora Boiss. Essential Oil Induce Apoptosis in Two Human Colon Cancer Cell Lines (HCT116 & SW48).

BACKGROUND: Zataria multiflora Boiss. is known by the common Persian name "Avishan-e-Shirazi", is one of the best-known medicinal herbs belonging to the Labiatae (Lamiaceae) family. The aim of this study was to evaluate the anticancer effects and the underlying mechanisms of how Z. multiflora Boiss., essential oil induced apoptosis in the human colorectal tumor cell lines (HCT116 & SW48). METHODS: This study was conducted in National Institute of Genetic Engineering and Biotechnology (NIGEB) (Tehran, Iran) from 2017 to 2019. The cytotoxicity of this essential oil was assessed by 3- (4,5-di-methylthiazol-2-yl)- 2,5-diphenyltetra-zolium bromide (MTT) assay, trypan blue exclusion, and colony formation assays. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Then gene expression was analyzed by Quantitative Real-Time RT-PCR. RESULTS: Z. multiflora Boiss., essential oil time- and dose-dependently inhibits cell proliferation and also induced apoptosis in both cell lines via UCP2-related mitochondrial pathway by the induction of intracellular ROS. CONCLUSION: Z. multiflora Boiss., essential oil could be a good candidate for use as an inhibitor of the growth of colorectal tumor cells.

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells.

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL 2 ) genes are downregulated, and BCL 2 associated X protein, BCL 2 antagonist/killer, BCL 2 interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.

Overexpression of enhancer of zeste human homolog 2 (EZH2) gene in human cytomegalovirus positive glioblastoma multiforme tissues.

Glioblastoma multiforme (GBM) is considered to be one of the most invasive human cancers, characterized by a high mortality rate and an average survival is <1 year. These tumors are highly aggressive and insensitive to conventional radio and chemotherapy. An interesting aspect of glioblastoma is the association of active human cytomegalovirus (HCMV) infection, which is evident by the presence of viral DNA, mRNA and protein level in most glioblastoma tissues. Although the presence of the HCMV infection in glioblastoma is well established, but the oncomodulatory role of HCMV is not defined yet. Enhancer of zeste human homolog 2 (EZH2) is a key protein of the polycomb repressive complex 2, epigenetic gene silencers. There have been several reports that EZH2 activity is essential in GBM pathogenesis. In our previous research, we have found a high rate of HCMV infection in a cohort of Iranian glioblastoma patients. In this study, we investigated the expression of EZH2 in HCMV-negative versus HCMV-positive GBM tissues in comparison to non-tumor tissues. The level of expression was determined by real time PCR and the differences were calculated using the Livac or 2(-ΔΔCt) and analysis of variance (ANOVA). Relative expression of EZH2 in HCMV-negative glioblastoma tissues were increased 6.053-fold compared to non-neoplastic brain tissues, while EZH2 gene expression was increased 41.098-fold in HCMV-positive glioblastoma tissues. ANOVA test showed that there is a significant difference in EZH2 expression between normal brain tissue, HCMV-negative and HCMV-positive glioblastoma tumors (p value = 0.0001). Our data indicate that EZH2 expression can be considered a risk factor in glioblastoma and EZH2 inhibitors may serve as potential new treatment in glioblastoma. This would be an interesting new field to investigate in more detail.

Expression of insulin-like growth factor binding protein-2 (IGFBP-2) gene in negative and positive human cytomegalovirus glioblastoma multiforme tissues.

Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma, is the most common and lethal primary brain tumor in adults and has but few treatments. The median survival of glioblastoma patients is 12 months. The (possible) relationship between human cytomegalovirus (HCMV) infection and cancer has been investigated for decades. Detection of viral DNA, mRNA and/or antigens in tumor tissues suggests that HCMV infection has a role to play in the etiology of several human malignancies. HCMV gene products can promote the various signaling pathways critical to tumor growth, including platelet derived growth factor receptor, phosphatidyl inositol 3-kinases (PI3K/AKT), signal transducer and activator of transcription 3 and glycogen synthase kinase 3 beta that are involved in apoptosis, angiogenesis, invasion and immune evasion. Insulin-like growth factor binding protein 2 (IGFBP2) is a biomarker of the PI3K/AKT pathway so we decided to evaluate the expression of this gene in 3 groups: HCMV-negative GBM tissues, HCMV-positive GBM tissues and non-tumor tissues. The presence of HCMV was assessed according to our previous article. HCMV was present in %75 of glioblastoma tissues. Then RNA was extracted, cDNA was synthesized, and real-time PCR was performed. Then, the rate of increased expression was calculated using the Livac or 2(-ΔΔCt). ΔCt of samples in the three groups were compared using analysis of variance (ANOVA). The expression of IGFBP2 gene relative to GAPDH gene in HCMV-negative glioblastoma tissues and HCMV-positive glioblastoma tissues, respectively, was increased 5.486 and 15.032 times compared to non-neoplastic brain tissues. ANOVA tests showed that the difference of mean ΔCt for IGFBP2 gene between healthy subjects and patients with HCMV-positive and HCMV-negative glioblastoma tumors statistically significant.