Drug-Drug Interaction between Metformin and Sorafenib Alters Antitumor Effect in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is one of the leading causes of cancer-related deaths worldwide. The multitarget inhibitor sorafenib is a first-line treatment of patients with advanced unresectable HCC. Recent clinical studies have evidenced that patients treated with sorafenib together with the antidiabetic drug metformin have a survival disadvantage compared with patients receiving sorafenib only. Here, we examined whether a clinically relevant dose of metformin (50 mg/kg per day) could influence the antitumoral effects of sorafenib (15 mg/kg per day) in a subcutaneous xenograft model of human HCC growth using two different sequences of administration, i.e., concomitant versus sequential dosing regimens. We observed that the administration of metformin 6 hours prior to sorafenib was significantly less effective in inhibiting tumor growth (15.4% tumor growth inhibition) than concomitant administration of the two drugs (59.5% tumor growth inhibition). In vitro experiments confirmed that pretreatment of different human HCC cell lines with metformin reduced the effects of sorafenib on cell viability, proliferation, and signaling. Transcriptomic analysis confirmed significant differences between xenografted tumors obtained under the concomitant and the sequential dosing regimens. Taken together, these observations call into question the benefit of parallel use of metformin and sorafenib in patients with advanced HCC and diabetes, as the interaction between the two drugs could ultimately compromise patient survival. SIGNIFICANCE STATEMENT: When drugs are administered sequentially, metformin alters the antitumor effect of sorafenib, the reference treatment for advanced hepatocellular carcinoma, in a preclinical murine xenograft model of liver cancer progression as well as in hepatic cancer cell lines. Defective activation of the AMP-activated protein kinase pathway as well as major transcriptomic changes are associated with the loss of the antitumor effect. These results echo recent clinical work reporting a poorer prognosis for patients with liver cancer who were cotreated with metformin and sorafenib.

Molecular analyses indicate profuse bacterial diversity in primary and post- treatment endodontic infections within a cohort from the United Arab Emirates-A preliminary study.

OBJECTIVE: Endodontic microbiota appears to undergo evolutionary changes during disease progression from inflammation to necrosis and post-treatment. The aim of this study was to compare microbiome composition and diversity in primary and post-treatment endodontic infections from a cohort of patients from the UAE. DESIGN: Intracanal samples were collected from primarily infected (n = 10) and post-treatment infected (n = 10) root canals of human teeth using sterile paper points. Bacterial DNA was amplified from seven hypervariable regions (V2-V4 and V6-V9) of the 16S rRNA gene, then sequenced using next-generation sequencing technology. The data was analyzed using appropriate bioinformatic tools. RESULTS: Analyses of all the samples revealed eight major bacterial phyla, 112 genera and 260 species. Firmicutes was the most representative phylum in both groups and was significantly more abundant in the post-treatment (54.4%) than in primary (32.2%) infections (p>0.05). A total of 260 operational taxonomic units (OTUs) were identified, of which 126 (48.5%) were shared between the groups, while 83 (31.9%) and 51 (19.6%) disparate species were isolated from primary and post-treatment infections, respectively. A significant difference in beta, but not alpha diversity was noted using several different indices (p< 0.05). Differential abundance analysis indicated that, Prevotella maculosa, Streptococcus constellatus, Novosphigobium sediminicola and Anaerococcus octavius were more abundant in primary infections while Enterrococcus faecalis, Bifidobacterium dentium, Olsenella profusa and Actinomyces dentalis were more abundant in post-treatment infections (p <0.05). CONCLUSION: Significant differences in the microbiome composition and diversity in primary and post-treatment endodontic infections were noted in our UAE cohort. Such compositional differences of microbiota at various stages of infection could be due to both intrinsic and extrinsic factors impacting the root canal ecosystem during disease progression, as well as during their therapeutic management. Identification of the key microbiota in primarily and secondarily infected root canals can guide in the management of these infections.

Azithromycin downregulates ICOS (CD278) and OX40 (CD134) expression and mTOR activity of TCR-activated T cells to inhibit proliferation.

The precise mechanism of macrolide antibiotic azithromycin (AZM) mediated CD4+ T cell suppression is not fully understood. Given the crucial role of co-stimulatory signaling in T-lymphocyte function, we tested in vitro effects of AZM on two of the most extensively investigated costimulatory molecules, ICOS and OX40 in context to CD4+ T cell proliferation. Using multi-color flow cytometry approach on TCR-activated healthy donor peripheral blood mononuclear cells, we observed a marked reduction in the frequencies and surface expression of ICOS and OX40 receptors following AZM treatment. Functionally, in contrast to ICOS- and OX40- CD3+ CD4+ T cells, AZM treated ICOS+ and OX40+ displayed profound reduction in cell proliferation. Furthermore, AZM treated T cells displaying reduced levels of ICOS and OX40 found to be associated with suppressed mTOR activity as detected by phosphorylation levels of S6 ribosomal protein. This study provides new insights on potential mechanism of AZM mediated inhibition of T cell proliferation by targeting costimulatory pathways.

Diversity of site-specific microbes of occlusal and proximal lesions in severe- early childhood caries (S-ECC).

BACKGROUND: Severe-early childhood caries (S-ECC) a global problem of significant concern, commonly manifest on the occlusal, and proximal surfaces of affected teeth. Despite the major ecological differences between these two niches the compositional differences, if any, in the microbiota of such lesions is unknown. METHODS: Deep-dentine caries samples from asymptomatic primary molars of children with S-ECC (n 19) belonging to caries-code 5/6, (ICDAS classification) were evaluated. Employing two primer pools, we amplified and compared the bacterial 16S rRNA gene sequences of the seven hypervariable regions (V2-V4 and V6-V9) using NGS-based assay. RESULTS: Bray-Curtisevaluation indicated that occlusal lesions (OL) had a more homogeneous community than the proximal lesions (PL) with significant compositional differences at the species level (p = 0.01; R- 0.513). Together, the occlusal and proximal niches harbored 263 species, of which 202 (76.8%) species were common to both , while 49 (18.6%) and 12 (4.6%) disparate species were exclusively isolated from the proximal and occlusal niches, respectively. The most commonl genera at both niches included Streptococcus, Prevotella, and Lactobacillus. S. mutans was predominant in PL (p ≤ 0.05), and Atopobium parvulum (p = 0.01) was predominant in OL. CONCLUSIONS: Distinct differences exist between the caries microbiota of occlusal and proximal caries in S-ECC.

HER2 overexpression is a putative diagnostic and prognostic biomarker for late-stage colorectal cancer in North African patients.

Aim: Colorectal cancer (CRC) is one of the leading cancers in the world. Even though its mortality and pathophysiology are well documented in the US and the European countries, it is seldom studied in North African population. Recent studies have shown link of HER2 overexpression in oesophageal and gastric cancers. The aim of this study is to assess the HER2 protein and mRNA expression and its correlation with tumor pathogenesis in Libyan CRC patients.Methodology: A total of 17 FFPE tissue blocks were collected from patients with primary CRC. The HER2 protein expression was assessed by immunohistochemistry and the mRNA expression was assessed using qRT-PCR. Survival analysis of the role of HER2 overexpression on rectal adenocarcinoma was carried out on additional 165 patients.Results: From the CRC cohort, adenocarcinoma was found to be more frequent accounting for 88.2%, and 11.8% for mucinous adenocarcinomas. Almost 47% of the cases were positive for HER2 (score ≥ 2+) and about 50% adenocarcinoma cases with tumor grade II were positive for HER2. Moreover, 57.4% adenocarcinoma patients with grade-II tumor had undergone right hemicolectomy. Furthermore, significant correlation (p = 0.03) between the HER2 mRNA expression with the tumor grade was observed. In addition, poor overall all survival was observed with high HER2 expression in rectum adenocarcinoma.Conclusion: To our knowledge, this is the first study that HER2 overexpression correlates with more aggressive colorectal cancer in North African population. Our study shows that HER2 overexpression associates with right colon surgeries. Also, the correlation of mRNA and protein expression could warrant the implementation of a nationwide screening program for HER2 positivity in CRC patients. Taken together, stratifying patients according to HER2 expression can help in the diagnosis and prognosis of CRC patients from North African origin.

Azithromycin Differentially Alters TCR-Activated Helper T Cell Subset Phenotype and Effector Function.

In addition to their antibiotic activities, azithromycin (AZM) exhibits anti-inflammatory effects in various respiratory diseases. One of the potent anti-inflammatory mechanisms is through inhibition of CD4+ helper T (Th) cell effector function. However, their impact on specific Th subset is obscure. Herein, we demonstrate the cellular basis of phenotypic and functional alterations associated with Th subsets following AZM treatment in vitro. Using well-characterized Th subset specific chemokine receptors, we report significant suppression of T cell receptor (TCR)-stimulated hyperactivated CCR4+CXCR3+ (Th0) expansion compared to CCR4-CXCR3+ (Th1-like) and CCR4+CXCR3- (Th2-like) cells. Interestingly, this effect was associated with diminished cell proliferation. Furthermore, AZM significantly inhibited the inflammatory cytokines IFN-γ and IL-4 production, CCR4 and CXCR3 receptor expression, and viability of Th0, Th1-like, and Th2-like subsets. Our findings suggest that AZM differentially affects TCR-activated Th subsets phenotype and function, and CCR4 and CXCR3 downregulation and suppressed Th0 subset expansion could potentially influence their trafficking and differentiation into cytokine-producing effector cells.

Correlation of securin and Ki67 in invasive breast carcinoma.

AIMS: To identify the role of securin (PTTG) as a prognostic marker in invasive breast carcinoma and its possible relation to ki67 and to evaluate the use of ImmunoRatio® as a tool for calculating ki67 and securin labelling indices. METHODS: Securin and ki67 immunohistochemical staining were performed on tissue microarray sections representative of 118 patients diagnosed with invasive breast carcinoma from 2005 to 2011. Assessment of immunohistochemical staining was carried out using both visual counting and ImmunoRatio®. The 118 cases were categorized into 2 groups according to their clinical outcome; the first group (G1) (n=77) comprised patients who were disease-free while the second group (G2) (n=41) included patients who developed either recurrence and/or metastasis at the end of 24 months follow-up duration. RESULTS: Both securin and ki67 labelling indices (LIs) obtained by visual counting were significantly higher in G2, while only securin LIs acquired by ImmunoRatio® were significantly higher in G2. Securin assessment by visual counting was the most accurate (AUC=0.775) in identifying patients who will likely suffer from recurrence and/or distant metastasis. Pearson correlation showed r=0.638, p<0.001 for Ki67 and r=0.671, p<0.001 for securin. Linear regression analysis showed a significant correlation between ki67 and securin, B=1.75, p<0.001. CONCLUSION: The present results suggest that securin may add to the prognostic value of ki67 in highlighting intra-tumoural heterogeneity in invasive breast carcinoma patients with poor clinical outcome. In addition, the study showed that since securin has a visual counting cutoff with more than 1%, making it easier to use as a breast cancer biomarker in conjunction with ki67 to predict the outcome of the cases more accurately than using only ki67. However, a multivariate analysis on a larger cohort of patients is mandatory to test its potential prognostic value.

Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1ß Release From NK92 Cells.

INTRODUCTION: Although natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1ß (IL-1ß), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity. METHODS: NK92 cells were examined for the synthesis and release of IL-1ß utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)1, HCA2 and HCA3 was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses. RESULTS: LPS-induced the release of IL-1ß from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis. DISCUSSION: Our results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1ß. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients. .