A Novel Single Nucleotide T980C Polymorphism in the Human Carboxypeptidase E Gene Results in Loss of Neuroprotective Function.

Report of a human with a homozygous truncating null mutation of the Carboxypeptidase E (CPE) gene with endocrinological and neurological deficits prompted us to search for other mutations in the human CPE gene that might be linked to disease. We searched an EST database and identified from a small population of patients, a novel T to C single nucleotide polymorphism (SNP) in the CPE gene at bp980 of exon 4, herein called TC-CPE. This introduces a tryptophan to arginine (W235R) mutation in the catalytic domain of human CPE protein. Over-expression of TC-CPE in N2A cells, a neuroendocrine cell line, showed that it was synthesized, but was found in lesser amounts compared to over-expressed WT-CPE in these cells. Furthermore, TC-CPE was secreted poorly from these N2A cells. The levels of TC-CPE were significantly increased after the N2A cells were treated with MG132 (a proteasome inhibitor), suggesting that TC-CPE was targeted to proteasomes for degradation in N2A cells. In addition, TC-CPE induced ER stress as demonstrated by the increased expression of CHOP in N2A cells. Double labeling of CPE and calnexin (and ER marker) suggested the accumulation of TC-CPE in the ER, and the accumulation appears to be enhanced by the treatment of MG132 in the cells. Moreover, the secreted levels of TC-CPE were not affected by the treatment of MG132 in the cells. Over-expression studies revealed that while N2A cells transfected with WT-CPE showed reduced cytotoxicity when challenged with H2O2 compared to cells expressing an empty vector, cells transfected with TC-CPE had no effect. Furthermore, WT-CPE condition medium showed protective effect against oxidative stress, but not TC-CPE condition medium. Although co-expression of WT-CPE and TC-CPE in N2A cells resulted in the reduction in secretion of WT-CPE, co-expression of WT-CPE and TC-CPE did not significantly affect the protective effect of WT-CPE. Taken together, we have identified a novel SNP in the CPE gene which results in the loss of its neuroprotective function in cells and may confer neurological disorders in humans

The synergistic effect of Canady Helios cold atmospheric plasma and a FOLFIRINOX regimen for the treatment of cholangiocarcinoma in vitro

Cholangiocarcinoma (CCA) is a rare biliary tract cancer with a low five-year survival rate and high recurrence rate after surgical resection. Currently treatment approaches include systemic chemotherapeutics such as FOLFIRINOX, a chemotherapy regimen is a possible treatment for severe CCA cases. A limitation of this chemotherapy regimen is its toxicity to patients and adverse events. There exists a need for therapies to alleviate the toxicity of a FOLFIRINOX regimen while enhancing or not altering its anticancer properties. Cold atmospheric plasma (CAP) is a technology with a promising future as a selective cancer treatment. It is critical to know the potential interactions between CAP and adjuvant chemotherapeutics. In this study the aim is to characterize the efficacy of FOLFIRINOX and CAP in combination to understand potential synergetic effect on CCA cells. FOLFIRINOX treatment alone at the highest dose tested (53.8 microM fluorouracil, 13.7 microM Leucovorin, 5.1 microM Irinotecan, and 3.7 microM Oxaliplatin) reduced CCA cell viability to below 20% while CAP treatment alone for 7 min reduced viability to 3% (p \u3c 0.05). An analysis of cell viability, proliferation, and cell cycle demonstrated that CAP in combination with FOLFIRINOX is more effective than either treatment alone at a lower FOLFIRINOX dose of 6.7 microM fluorouracil, 1.7 microM leucovorin, 0.6 microM irinotecan, and 0.5 microM oxaliplatin and a shorter CAP treatment of 1, 3, or 5 min. In conclusion, CAP has the potential to reduce the toxicity burden of FOLFIRINOX and warrants further investigation as an adjuvant therapy

Carboxypeptidase E-ΔN, a Neuroprotein Transiently Expressed during Development Protects Embryonic Neurons against Glutamate Neurotoxicity

<div><p>Neuroprotective proteins expressed in the fetus play a critical role during early embryonic neurodevelopment, especially during maternal exposure to alcohol and drugs that cause stress, glutamate neuroexcitotoxicity, and damage to the fetal brain, if prolonged. We have identified a novel protein, carboxypeptidase E-ΔN (CPE-ΔN), which is a splice variant of CPE that has neuroprotective effects on embryonic neurons. CPE-ΔN is transiently expressed in mouse embryos from embryonic day 5.5 to postnatal day 1. It is expressed in embryonic neurons, but not in 3 week or older mouse brains, suggesting a function primarily <i>in utero</i>. CPE-ΔN expression was up-regulated in embryonic hippocampal neurons in response to dexamethasone treatment. CPE-ΔN transduced into rat embryonic cortical and hippocampal neurons protected them from glutamate- and H₂O₂-induced cell death. When transduced into embryonic cortical neurons, CPE-ΔN was found in the nucleus and enhanced the transcription of FGF2 mRNA. Embryonic cortical neurons challenged with glutamate resulted in attenuated FGF2 levels and cell death, but CPE-ΔN transduced neurons treated in the same manner showed increased FGF2 expression and normal viability. This neuroprotective effect of CPE-ΔN was mediated by secreted FGF2. Through receptor signaling, FGF2 activated the AKT and ERK signaling pathways, which in turn increased BCL-2 expression. This led to inhibition of caspase-3 activity and cell survival.</p></div

The Granger Causal Effects of Canady Helios Cold Plasma on the Inhibition of Breast Cancer Cell Proliferation

Cold atmospheric plasma (CAP) has become a promising tool for modern medicine. With its recent applications in oncology, regenerative medicine, and immunotherapy, CAP can be used for a myriad of different clinical treatments. When using CAP specifically for the treatment of tumors, it is known to elicit an oxidative response within malignant cancer cells, inducing cell cycle arrest and apoptosis. In this study, data of intracellular reactive oxygen species (ROS), caspase activity, Ki-67 expression, and cell cycle activity in the G1 phase were acquired to determine the causal relationships these intermediates have with cell proliferation and death after Canady Helios Cold Plasma (CHCP) treatment. The data were derived from four different subtypes of breast cancer cell lines: BT-474, MCF-7, MDA-MB-231, and SK-BR-3. Data transformation techniques were conducted on the time-series data for the input into the causal model code. The models were created on the basis of Granger causality principles. Our results demonstrated that there was a Granger causal relationship among all potentially causal variables (ROS, caspase, Ki-67, and G1 activity) and cell proliferation after 5 min CHCP treatment; however, not all variables were causal for the 3 min models. This same pattern did not exist for cell death models, which tested all potentially causal variables (ROS, Ki-67, and G1 activity) vs. caspase activity. All models were validated through a variety of statistical tests and forecasting accuracy metrics. A pseudo data set with defined causal links was also created to test R&rsquo;s ability in picking up known causal relationships. These models, while nonexhaustive, elucidated the effects cold plasma has on cell activity regulators. Research in causal modeling is needed to help verify the exact mechanism of cold plasma for the ultimate optimization of its application in the treatment of cancers

The Granger Causal Effects of Canady Helios Cold Plasma on the Inhibition of Breast Cancer Cell Proliferation

Cold atmospheric plasma (CAP) has become a promising tool for modern medicine. With its recent applications in oncology, regenerative medicine, and immunotherapy, CAP can be used for a myriad of different clinical treatments. When using CAP specifically for the treatment of tumors, it is known to elicit an oxidative response within malignant cancer cells, inducing cell cycle arrest and apoptosis. In this study, data of intracellular reactive oxygen species (ROS), caspase activity, Ki-67 expression, and cell cycle activity in the G1 phase were acquired to determine the causal relationships these intermediates have with cell proliferation and death after Canady Helios Cold Plasma (CHCP) treatment. The data were derived from four different subtypes of breast cancer cell lines: BT-474, MCF-7, MDA-MB-231, and SK-BR-3. Data transformation techniques were conducted on the time-series data for the input into the causal model code. The models were created on the basis of Granger causality principles. Our results demonstrated that there was a Granger causal relationship among all potentially causal variables (ROS, caspase, Ki-67, and G1 activity) and cell proliferation after 5 min CHCP treatment; however, not all variables were causal for the 3 min models. This same pattern did not exist for cell death models, which tested all potentially causal variables (ROS, Ki-67, and G1 activity) vs. caspase activity. All models were validated through a variety of statistical tests and forecasting accuracy metrics. A pseudo data set with defined causal links was also created to test R’s ability in picking up known causal relationships. These models, while nonexhaustive, elucidated the effects cold plasma has on cell activity regulators. Research in causal modeling is needed to help verify the exact mechanism of cold plasma for the ultimate optimization of its application in the treatment of cancers

Expression and secretion of TC-CPE in N2A cells.

<p><b>(A)</b> Representative Western blot analysis of CPE expression after EV, WT-CPE or TC-CPE transfection for 24 h in N2A cells. <b>(B)</b> Bar graphs show the quantification of CPE expression normalized to actin. One-way ANOVA (F_(2,6) = 85.04, p<0.001, n = 3) followed by Tukey’s multiple comparison test. <b>(C)</b> Representative Western blot analysis of secreted CPE after transfection of EV, WT-CPE or TC-CPE for 24 h in N2A cells. (D) N2A cells were treated with EV, WT-CPE or TC-CPE condition medium for 48 h in the presence of 100 μM H₂O₂. LDH release assay demonstrated that WT-CPE condition medium protected against oxidative stress, but not TC-CPE condition medium.One-way ANOVA analysis followed by Tukey’s multiple comparison test: F_(2,12) = 5.842, p <0.05, n = 5.</p

Neuroprotection by CPE-ΔN is mediated by FGF2.

<p>A. Bar graph shows the quantification by qRT-PCR of <i>FGF2</i> mRNA in primary cultured rat cortical neurons after transduction with CPE-ΔN or LacZ (control) viral vectors. Data are normalized against 18S RNA and presented as a % compared to control (LacZ) cells. Values are the mean ± SEM (n = 5), <i>t</i> test, ** p<0.01. At least three independent experiments were done. Data shown represent all the experiments combined. B. Bar graph shows the levels of secreted FGF2 by ELISA in primary cultured rat cortical neurons after transduction with CPE-ΔN or LacZ (control) viral vectors. Values are the mean ± SEM (n = 4), <i>t</i> test, * p<0.05. Two independent experiments were done. Data shown represent one experiment. C. Top panel: Western blot analysis of FGF2 protein in primary cortical neurons, transduced with CPE-ΔN or LacZ viral vectors and subsequently challenged with or without glutamate for 24 h. Actin was also analyzed and served as an internal control for protein load; Bottom panel: Bar graphs show the quantification of FGF2 protein normalized to actin and expressed as a % compared to vehicle treated control cells. Note that CPE-ΔN significantly inhibited the glutamate-induced decrease in FGF2 protein in primary cultured cortical neurons. At least three independent experiments were done. Data shown represent all the experiments combined. D. Bar graphs show WST activity, indicative of cell viability of rat cortical neurons with and without transduction of CPE-ΔN construct and treated with and without glutamate in the continued presence or absence of FGF receptor inhibitor, PD166285. Note the neuroprotective effect of CPE-ΔN was blocked by PD166285 in primary cultured cortical neurons, indicating that FGF2 mediates the neuroprotective effect of CPE-ΔN. Two independent experiments were done. Data shown represent one experiment. n = 6/group (C); 5/group (D). Values are mean ± SEM, one-way ANOVA followed by Tukey test, *p<0.05.</p

CPE-ΔN protects primary cortical neurons against glutamate-induced neurotoxicity.

<p>In A–D, rat primary cortical neurons were transduced with CPE-ΔN or LacZ viral vectors and subsequently treated with or without with glutamate for 24 h. A. Bar graphs show WST activity, indicative of cell viability. Note that the reduced cell viability after glutamate treatment was significantly increased in neurons transduced with the CPE-ΔN construct. At least three independent experiments were done. Data shown represent one experiment. B. Bar graphs show LDH release, indicative of cytotoxicity of cortical neurons. Note that the glutamate-induced cytotoxicity was significantly attenuated by the transduction of CPE-ΔN construct. At least three independent experiments were done. Data shown represent one experiment. C, D. Photomicrographs and bar graphs showing cortical neurons stained with TUNEL (green) and DAPI (blue). Note that the number of dead cells (green) increased significantly after glutamate treatment and that transduction of CPE-ΔN construct protected the neurons. The bar graphs represent the quantification of dead cells as a % of the total number of cells determined by the DAPI staining. At least 500 cells were counted in each of 3 different dishes generated from embryos from two independent experiments. Data shown represent one experiment. Bar = 100 microns, n = 6/group (A); 5/group (B) and 3/group (C). Values are mean ± SEM, one-way ANOVA followed by Tukey test, *p<0.05.</p

CPE mutation and the effect on enzymatic activity.

<p><b>(A)</b>: Nucleotide and (<b>B)</b>: amino acid alignment of <i>WT-hCPE</i> and a human <i>CPE</i> mutant identified from an EST database. The mutant was named as <i>TC-CPE</i> due to the T to C mutation of <i>hCPE</i> gene at nucleotide 980 of exon 4. <b>C:</b> X-ray crystal structure of the catalytic domain of duck carboxypeptidase D (CPD, Protein Data Bank ID #1qmu), a homologous protein to CPE. The TC mutation occurs in the a-helix 4of CPD (see arrow). Condition medium from <b>(D)</b>WT-CPE and <b>(E)</b> TC-CPE transfected COS-7 cells were collected. The enzymatic activity of CPE from the conditioned medium was analyzed by HPLC. Condition medium from untransfected COS-7 cells were also analyzed and found to have no CPE activity (data not shown).</p