Emmanuel Ansong

Molecular cross‐talk between members of distinct families of selenium containing proteins

Dietary intake of selenium has been associated with reduced risk of several cancer types, and this is likely due to its role as a specific constituent of selenium containing proteins. One of these, selenium-binding protein 1 (SBP1), is a protein of unknown function that has been shown to be reduced in tumors of diverse tissue types as compared to the corresponding normal tissue. More importantly, SBP1 has also been reported to be a predictor of clinical outcome. Levels of SBP1 are inversely associated with the levels of another protein representative of a different class of selenoproteins, glutathione peroxidase1 (GPx-1). GPx-1 is an anti-oxidant, selenocysteine containing enzyme implicated in several diseases, including cancer, due to the association of specific alleles with disease risk. The relationship between SBP1 and GPx-1 represents a unique example of a molecular interaction between selenium containing proteins with a likely significant impact on human health and disease.

A sequence variant in the phospholipase C epsilon C2 domain is associated with esophageal carcinoma and esophagitis.

A single‐nucleotide polymorphism (rs2274223: A5780G:His1927Arg) in the phospholipase C epsilon gene (PLCϵ) was recently identified as a susceptibility locus for esophageal cancer in Chinese subjects. To determine the underlying mechanisms of PLCϵ and this SNP in esophageal carcinogenesis, we analyzed PLCϵ genotypes, expression, and their correlation in esophageal cancer cell lines, non‐transformed esophageal cells, 58 esophageal squamous cell carcinomas and 10,614 non‐cancer subjects from China. We found that the G allele (AG or GG) was associated with increased PLCϵ mRNA and protein expression in esophageal cancer tissues and in esophageal cancer cell lines. G allele was also associated with higher enzyme activity, which might be associated with increased protein expression. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance was strongly linked to esophageal malignancy. Moreover, the analysis of 10,614 non‐cancer subjects demonstrated that the G allele was strongly associated with moderate to severe esophagitis in the subjects from the high‐incidence areas of China (OR 6.03, 95% CI 1.59–22.9 in high‐incidence area vs. OR 0.74, 95% CI 0.33–1.64 in low‐incidence area; P = 0.008). In conclusion, the PLCϵ gene, particularly the 5780G allele, might play a pivotal role in esophageal carcinogenesis via upregulating PLCϵ mRNA, protein, and enzyme activity, and augmenting inflammatory process in esophageal epithelium. Thus, 5780G allele may constitute a promising biomarker for esophageal squamous cell carcinoma risk stratification, early detection, and progression prediction.

Evidence that selenium binding protein 1 is a tumor suppressor in prostate cancer.

Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.

Quantitative Proteomic Analysis Reveals That Anti-Cancer Effects of Selenium-Binding Protein 1 In Vivo Are Associated with Metabolic Pathways

Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

Abstract Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H2O2), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H2O2 determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

Analgesic effect of perineural magnesium sulphate for sciatic nerve block for diabetic toe amputation: A randomized trial

Background and objectives High concentrations of local anesthetics may be neurotoxic for diabetic patients. Additive perineural administration of magnesium was reported to decrease the consumption of local anesthetics for nerve block. It was hypothesized that MgSO4 added to dilute ropivacaine was equianalgesic to more concentrated ropivacaine for toe amputations in diabetic patients. Methods Seventy diabetic patients were allocated into 3 groups: 1) perineural 200 mg MgSO4 added to 0.25% ropivacaine, 2) 0.25% ropivacaine alone, and 3) 0.375% ropivacaine alone. All patients underwent popliteal sciatic nerve block that was guided by ultrasonography using the respective regimens. Time of onset, duration of motor and sensory block were recorded. Spontaneous and evoked pain score, worst pain score, additional analgesic consumption, satisfaction score and initial time of analgesic requirement of each patient were documented up to 48 hours postoperatively. Results In comparison with 0.25% ropivacaine alone, magnesium supplement prolonged the duration of sensory block (p = 0.001), as well as better evoked pain score at 6 hour postoperatively (p = 0.001). In comparison with evoked pain score (1.6/10) in group of 0.375% ropivacaine, magnesium plus 0.25% ropivacaine presented a little higher score (2.5/10) at 6 hour postoperatively (p = 0.001), while lower worst pain score (p = 0.001) and less postoperative total analgesic consumption (p = 0.002). Conclusions The regimen of adding 200mg MgSO4 to 0.25% ropivacaine for sciatic nerve block yields equal analgesic effect in comparison with 0.375% ropivacaine. These findings have suggested that supplemental MgSO4 could not improve analgesic quality except reducing the total amount of local anesthetics requirement in diabetic toe amputations with sciatic nerve blocks.

Clinical significance of the correlation between PLCE 1 and PRKCA in esophageal inflammation and esophageal carcinoma

Esophagitis and Barretts esophagus are linked to esophageal squamous cell carcinoma and adenocarcinoma, respectively. However, the underlying mechanisms are still unclear. This study analyzed the expression levels of and correlation between PLCE1 and PRKCA in human esophagitis, carcinogen NMBA-induced rat esophagus, PLCE1 genetic deficient mouse esophageal epithelial tissues and human esophageal cancer cell line, integrated with Online oncology data sets. We found that the expression levels of both PLCE1 and PRKCA were significantly elevated in human esophagitis, esophageal squamous cell carcinoma, Barretts esophagus, esophageal adenocarcinoma and in NMBA-treated rat esophageal epithelia. However, PRKCA and cytokines were significantly downregulated in PLCE1-deficient mouse esophageal epithelia, and knockdown of PLCE1 in human esophageal cancer cells led to reduction of PRKCA and cytokines. Finally, high expression of both PLCE1 and PRKCA is significantly associated with poor outcomes of the patients with esophageal cancers. In conclusion, this study defined the initiation and progression of esophageal inflammation and malignant transformation, in which the positive correlation of PLCE1 and PRKCA exhibits critical clinical significance.

A Critical Role for Cysteine 57 in the Biological Functions of Selenium Binding Protein-1.

The concentration of selenium-binding protein1 (SBP1) is often lower in tumors than in the corresponding tissue and lower levels have been associated with poor clinical outcomes. SBP1 binds tightly selenium although what role selenium plays in its biological functions remains unknown. Previous studies indicated that cysteine 57 is the most likely candidate amino acid for selenium binding. In order to investigate the role of cysteine 57 in SBP1, this amino acid was altered to a glycine and the mutated protein was expressed in human cancer cells. The SBP1 half-life, as well as the cellular response to selenite cytotoxicity, was altered by this change. The ectopic expression of SBP1GLY also caused mitochondrial damage in HCT116 cells. Taken together, these results indicated that cysteine 57 is a critical determinant of SBP1 function and may play a significant role in mitochondrial function.

Correlations of SELENOF and SELENOP genotypes with serum selenium levels and prostate cancer.

Selenium status is inversely associated with the incidence of prostate cancer. However, supplementation trials have not indicated a benefit of selenium supplementation in reducing cancer risk. Polymorphisms in the gene encoding selenoprotein 15 (SELENOF) are associated with cancer incidence/mortality and present disproportionately in African Americans. Relationships among the genotype of selenoproteins implicated in increased cancer risk, selenium status, and race with prostate cancer were investigated.

Abstract 225: Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins

MnSOD detoxifies superoxide and impacts tumor biology by generating H 2 O 2 which can diffuse through the mitochondrial membrane and affect metabolism and apoptosis. The selenium-dependent enzyme GPx-1 localizes to both the cytoplasm and the mitochondria and reduces H 2 O 2 to water and may therefore modulate the impact of MnSOD on carcinogenesis. A val to ala polymorphism in codon 16 of the MnSOD gene has been shown to be associated with increased prostate cancer risk in men with the lowest level of dietary antioxidant intake and individuals who consumed less dietary antioxidants, including selenium, had the greatest risk of prostate cancer (Li et al, 2005). A polymorphism in the GPx-1 gene resulting in a leucine (L) instead of a proline (P) at position 198 has frequently been reported to be associated with elevated cancer risk. To examine the molecular mechanism behind the epidemiological observation that genotypes in GPx-1 gene modify elevated risk of cancer associated with MnSOD genotypes, MCF-7 human breast cancer cells null for GPx-1 and having negligible levels of endogenous MnSOD were transfected with GPx-1 and MnSOD allele-specific expression constructs to determine outcomes related to cellular signaling and metabolism. MnSOD and GPx-1 alleles differentially interacted to modulate the expression of the anti-oxidant stress response regulator Nrf2, the cell adhesion protein E-Cadherin, the cell signaling protein pAkt, the anti-apoptotic protein Bcl-2 and the mitochondria located deacetylase Sirt3. Also, co-expression of the GPx-1 A7L and either MnSOD val or MnSOD ala increased oxidative phosphorylation as measured by O 2 uptake using the Seahorse XF24 XF analyzer, which simultaneously determines relative mitochondrial respiration and glycolysis. In order to assess whether GPx-1 and MnSOD allelic variants modulate mitochondrial membrane potential, CMX-ROS fluorescence was measured. Independent and irrespective of which allele was evaluated, MnSOD and GPx-1 individually decreased mitochondrial potential as compared to that seen using MCF-7 control cells. In addition, co-expression of GPx-1 A7L with either MnSOD val or MnSOD ala further decreased membrane potential above that observed for either MnSOD and GPx-1 alone. In order to determine if MnSOD and GPx-1 levels are associated with a higher risk for biochemical recurrence of prostate cancer after radical prostatectomy, immunohistochemistry of human prostate tissue cores will be performed. Preliminary results indicate that a high MnSOD/ GPx-1 ratio was observed in prostate cancer tissue compared to adjacent normal tissue. Citation Format: Dede N. Ekoue, Soumen Bera, Emmanuel Ansong, Peter Hart, Virgilia Macias, Andre Kajdacsy-Balla, Marcelo Bonini, Alan M. Diamond. Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 225.