Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration.

PURPOSE: GPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown. METHODS: Immunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry. RESULTS: The GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8. CONCLUSION: The findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Development of a deep pathomics score for predicting hepatocellular carcinoma recurrence after liver transplantation.

BACKGROUND AND PURPOSE: Tumor recurrence after liver transplantation (LT) impedes the curative chance for hepatocellular carcinoma (HCC) patients. This study aimed to develop a deep pathomics score (DPS) for predicting tumor recurrence after liver transplantation using deep learning. PATIENTS AND METHODS: Two datasets of 380 HCC patients who underwent LT were enrolled. Residual convolutional neural networks were used to identify six histological structures of HCC. The individual risk score of each structure and DPS were derived by a modified DeepSurv network. Cox regression analysis and Concordance index were used to evaluate the prognostic significance. The cellular exploration of prognostic immune biomarkers was performed by quantitative and spatial proximity analysis according to three panels of 7-color immunofluorescence. RESULTS: The overall classification accuracy of HCC tissue was 97%. At the structural level, immune cells were the most significant tissue category for predicting post-LT recurrence (HR 1.907, 95% CI 1.490-2.440). The C-indices of DPS achieved 0.827 and 0.794 in the training and validation cohorts, respectively. Multivariate analysis for recurrence-free survival (RFS) showed that DPS (HR 4.795, 95% CI 3.017-7.619) was an independent risk factor. Patients in the high-risk subgroup had a shorter RFS, larger tumor diameter and a lower proportion of clear tumor borders. At the cellular level, a higher infiltration of intratumoral NK cells was negatively correlated with recurrence risk. CONCLUSIONS: This study established an effective DPS. Immune cells were the most significant histological structure related to HCC recurrence. DPS performed well in post-LT recurrence prediction and the identification of clinicopathological features.

CD36+ cancer-associated fibroblasts provide immunosuppressive microenvironment for hepatocellular carcinoma via secretion of macrophage migration inhibitory factor.

Hepatocellular carcinoma (HCC) is an immunotherapy-resistant malignancy characterized by high cellular heterogeneity. The diversity of cell types and the interplay between tumor and non-tumor cells remain to be clarified. Single cell RNA sequencing of human and mouse HCC tumors revealed heterogeneity of cancer-associated fibroblast (CAF). Cross-species analysis determined the prominent CD36+ CAFs exhibited high-level lipid metabolism and expression of macrophage migration inhibitory factor (MIF). Lineage-tracing assays showed CD36+CAFs were derived from hepatic stellate cells. Furthermore, CD36 mediated oxidized LDL uptake-dependent MIF expression via lipid peroxidation/p38/CEBPs axis in CD36+ CAFs, which recruited CD33+myeloid-derived suppressor cells (MDSCs) in MIF- and CD74-dependent manner. Co-implantation of CD36+ CAFs with HCC cells promotes HCC progression in vivo. Finally, CD36 inhibitor synergizes with anti-PD-1 immunotherapy by restoring antitumor T-cell responses in HCC. Our work underscores the importance of elucidating the function of specific CAF subset in understanding the interplay between the tumor microenvironment and immune system.

Cystathionine ß-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine ß-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of CBS in HCC development and immune evasion have not been systematically investigated, and their underlying mechanisms remain elusive. Here, we investigated the roles of CBS in tumor cells and tumor microenvironment of HCC. METHODS: 236 HCC samples were collected to detect the expression of CBS, cleaved Caspase-3 and paired related homeobox 2 (PRRX2) and the number of immune cells. HCC cell lines were employed to examine the effects of CBS on cellular viability, apoptosis and signaling in vitro. Cbs heterozygous knockout mice, C57BL/6 mice, nude mice and non-obese diabetic severe combined immunodeficiency mice were used to investigate the in vivo functions of CBS. RESULTS: Downregulation of CBS was observed in HCC, and low expression of CBS predicted poor prognosis in HCC patients. CBS overexpression dramatically promoted cellular apoptosis in vitro and inhibited tumor growth in vivo. Activation of the Cbs/H2S axis also reduced the abundance of tumor-infiltrating Tregs, while Cbs deficiency promoted Tregs-mediated immune evasion and boosted tumor growth in Cbs heterozygous knockout mice. Mechanistically, CBS facilitated the expression cleaved Caspase-3 in tumor cells, and on the other hand, suppressed Foxp3 expression in Tregs via inactivating IL-6/STAT3 pathway. As a transcription factor of IL-6, PRRX2 was reduced by CBS. Additionally, miR-24-3p was proven to be an upstream suppressor of CBS in HCC. CONCLUSIONS: Our results indicate the antitumor function of CBS in HCC by inactivation of the PRRX2/IL-6/STAT3 pathway, which may serve as a potential target for HCC clinical immunotherapy.

Adjuvant Transarterial chemoembolization does not influence recurrence-free or overall survival in patients with combined hepatocellular carcinoma and Cholangiocarcinoma after curative resection: a propensity score matching analysis.

BACKGROUND: The prognosis of patients with combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma (CHC) is usually poor, and effective adjuvant therapy is missing making it important to investigate whether these patients may benefit from adjuvant transarterial chemoembolization (TACE). We aimed to evaluate the efficiency of adjuvant TACE for long-term recurrence and survival after curative resection before and after propensity score matching (PSM) analysis. METHODS: In this retrospective study, of 230 patients who underwent resection for CHC between January 1994 and December 2014, 46 (18.0%) patients received adjuvant TACE. Univariate and multivariate regression analyses were used to identify the independent predictive factors of survival. Cox regression analyses and log-rank tests were used to compare overall survival (OS) and disease-free survival (DFS) between patients who did or did not receive adjuvant TACE. RESULTS: A total of 230 patients (mean age 52.2 ± 11.9 years; 172 men) were enrolled, and 46 (mean age 52.7 ± 11.1 years; 38 men) patients received TACE. Before PSM, in multivariate regression analysis, γ-glutamyl transpeptidase (γ-GT), tumour nodularity, macrovascular invasion (MVI), lymphoid metastasis, and extrahepatic metastasis were associated with OS. Alanine aminotransferase (ALT), MVI, lymphoid metastasis, and preventive TACE (HR: 2.763, 95% CI: 1.769-4.314, p < 0.001) were independent prognostic factors for DFS. PSM created 46 pairs of patients. Before PSM, adjuvant preventive TACE was not associated with an increased risk of OS (HR: 0.911, 95% CI: 0.545-1.520, p = 0.720) or DFS (HR: 3.345, 95% CI: 1.686-6.638, p = 0.001). After PSM, the 5-year OS and DFS rates were comparable in the TACE group and the non-TACE group (OS: 22.7% vs 14.9%, respectively, p = 0.75; DFS: 11.2% vs 14.4%, respectively, p = 0.06). CONCLUSIONS: The present study identified that adjuvant preventive TACE did not influence DFS or OS after curative resection of CHC.

Age-adjusted Charlson Comorbidity Index predicts survival in intrahepatic cholangiocarcinoma patients after curative resection.

BACKGROUND: Comorbidity among cancer patients is prevalent and influential to prognosis after operation. Limited data are available on comorbidity evaluations in patients with intrahepatic cholangiocarcinoma (ICC). This study aimed to assess the comorbidity distribution in ICC patients and to adapt the Charlson Comorbidity Index (CCI) or the age-adjusted CCI (ACCI) for survival prediction. METHODS: The study cohort included 268 ICC patients treated with curative surgery from January 2000 to December 2007 at the Department of Liver Surgery, Zhongshan Hospital. The association between the comorbidity index and overall survival (OS) or disease-free survival (DFS). was analyzed by the Kaplan-Meier method. Multivariable analysis was established to select the determinant parameters. RESULTS: Major comorbid conditions of ICC patients included liver disease, hypertension, diabetes and ulcer. The median follow-up time was 25.5 months in the whole data set. Among the entire cohort, the 1-, 3- and 5-year OS rates were 55.3%, 26.0% and 15.6%, respectively. In multivariate analysis, the ACCI correlated with OS, and higher scores were associated with poorer prognosis (hazard ratio =1.134, 95% confidence interval: 1.015-1.267 and P value =0.026). CCI was not an independent predictive factor for OS or DFS. CONCLUSIONS: In contrast to CCI, ACCI was a more promising model to accurately predict OS in ICC patients who underwent liver resection. Further research should be focused on the impact of comorbidity therapies.

Nine-factor-based immunohistochemistry classifier predicts recurrence for early-stage hepatocellular carcinoma after curative resection.

BACKGROUND: Immunoscore have shown a promising prognostic value in many cancers. We aimed to establish and validate an immune classifier to predict survival after curative resection of hepatocellular carcinoma (HCC) patients who have undergone curative resection. METHODS: The immunohistochemistry (IHC) classifier assay was performed on 664 patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A HCC. A nine-feature-based HCC-IHC classifier was then constructed by the least absolute shrinkage and selection operator method. The associations between the HCC-IHC classifier and patient outcomes were assessed. Herein, a nomogram was generated from the Cox regression coefficients and evaluated by decision curve analysis. RESULTS: We constructed an HCC-IHC classifier based on nine features; significant differences were found between the low-HCC-IHC classifier patients and high-HCC-IHC classifier patients in the training cohort in the 5-year relapse-free survival rates (46.7% vs. 26.7%, respectively; P < 0.001). The HCC-IHC classifier-based nomogram presented better accuracy than traditional staging systems. CONCLUSIONS: In conclusion, the HCC-IHC classifier could effectively predict recurrence in early-stage HCC patients and supplemented the prognostic value of the BCLC staging system. The HCC-IHC classifier may facilitate patient decision-making and individualise the management of postoperative patients with early-stage HCC.

Daily decrease of post-operative alpha-fetoprotein by 9% discriminates prognosis of HCC: A multicenter retrospective study.

BACKGROUND: Mixed evidence challenges preoperative alpha-fetoprotein (AFP) as an independent prognostic factor for patients with hepatocellular carcinoma (HCC) after hepatectomy. RESULTS: Daily post-operative decrease of AFP by 9% as compared to the preoperative level (A09) were selected as the Cut-off. The Kaplan-Meier curve showed that A09 was significantly different for OS (P=0.043) and RFS (P=0.03). A decrease in risk by 54% was observed for OS and 32% for RFS in the at-risk population (A09>9%). A better concordance was observed after adding A09 into TNM and BCLC staging systems. Moreover, a consistent concordance was observed in the internal (FDZS5:0.63; FDZS3:0.608) and external (FDZS5:0.85; FDZS3:0.762) validation cohorts, suggesting its prognostic value in HCC population with elevated AFP. CONCLUSIONS: Decrease in perioperative serum AFP rather than preoperative AFP is an independent prognostic factor for HCC patients after hepatectomy. Cut-off A09 significantly discriminates overall and recurrence-free survival and could be interpret into TNM and BCLC staging systems to improve the stratification power for HCC patients with elevated AFP. METHODS: Kaplan-Meier curve depicted the differences of overall survival (OS) and recurrence-free survival (RFS). Nomogram and concordance were employed to evaluate the superiority of the current staging system.

Histopathology-based immunoscore predicts recurrence for intrahepatic cholangiocarcinoma after hepatectomy.

Intrahepatic cholangiocarcinoma (ICC) is a rare malignancy with poor prognosis. The evaluation of recurrence risk after liver resection is of great importance for ICCs. We aimed to assess the prognostic value of intra- and peritumoral immune infiltrations and to establish a novel histopathology-related immunoscore (HRI) associated with ICC recurrence. A total of 280 ICC patients who received curative resection between February 2005 and July 2011 were enrolled in our study. Patients were randomly assigned to the derivation cohort (n = 176) or the validation cohort (n = 104). Sixteen immune biomarkers in both intra- and peritumoral tissues were examined by immunohistochemistry. The least absolute shrinkage and selection operator (LASSO) Cox model was used to establish the HRI score. Cox regression analysis was used for multivariate analysis. Nine recurrence-related immune features were identified and integrated into the HRI score. The HRI score was used to categorize patients into low-risk and high-risk groups using the X-tile software. Kaplan-Meier analysis presented that the HRI score showed good stratification between low-risk and high-risk groups in both the derivation cohort (P < 0.001) and the validation cohort (P = 0.014), respectively. Multivariate analysis demonstrated that serum γ-glutamyl transpeptidase, carbohydrate antigen 19-9, lymphoid metastasis, tumor numbers, and the HRI score were independent risk factors associated with recurrence-free survival (RFS). The combination of Shen's model and HRI score provided better performance in recurrence prediction compared with traditional staging systems. The HRI score might serve as a promising RFS predictor for ICC with prognostic values.

Development and validation of a prognostic score predicting recurrence in resected combined hepatocellular cholangiocarcinoma.

Purpose: To develop and validate a decision aid to help make individualized estimates of tumor recurrence for patients with resected combined hepatocellular cholangiocarcinoma (CHC). Patients and methods: Risk factors of recurrence were identified in the derivation cohort of 208 patients who underwent liver resection between 1995 and 2014 at Zhongshan Hospital to develop a prediction score. The model was subsequently validated in an external cohort of 101 CHC patients using the C concordance statistic and net reclassification index (NRI). Results: On multivariate analysis, five independent predictors associated with tumor recurrence were identified, including sex, γ-glutamyl transferase, macrovascular invasion, hilar lymphoid metastasis and adjuvant transcatheter arterial chemoembolization. The prediction score was constructed using these 5 variables, with scores ranging from 0 to 5. A patient with a score of 0 had a predicted 1- and 5-year recurrence risk of 11.1% and 22.2%, respectively. In the validation cohort, the NRIs of prediction score vs American Joint Committee on Cancer 7th TNM staging system at 1-year and 5-year were 0.185 (95% CI, 0.090-0.279, P<0.001) and 0.425 (95% CI, 0.044-0.806, P=0.03), respectively. Conclusion: Our developed and validated prediction score might be a simple and reliable method in postoperative CHC patients and help clinicians identify candidates who may benefit from future adjuvant therapies.

Tissue-infiltrating lymphocytes signature predicts survival in patients with early/intermediate stage hepatocellular carcinoma.

BACKGROUND: Intratumoral immune infiltrates have manifested a robust prognostic signature in patients with hepatocellular carcinoma (HCC). We hypothesized that a novel tissue-related immune signature (TRIS) could improve the prediction of postoperative survival for patients diagnosed with early/intermediate HCC. METHODS: Twenty-eight immune features were immunohistochemically examined on 352 HCC specimens. The LASSO Cox regression model was used to construct a five-feature-based TRIS. The univariate and multivariate Cox analyses were performed. Based on independent predictors, the immune-clinical prognostic index (ICPI) was established. Performance assessment was measured with C-index and compared with seven traditional staging systems. The independent validation cohort (n = 393) was included to validate the model. RESULTS: By using the LASSO method, the TRIS were constructed on the basis of five immune features, CD3intratumoral (T), CD27T, CD68peritumoral (P), CD103T, and PD1T. Multivariate Cox analysis showed that the TRIS was an independent prognostic predictor. In the training cohort, γ-glutamyl transferase, tumor diameter, tumor differentiation, and TRIS were incorporated into the ICPI. The ICPI presented satisfactory discrimination ability, with C-index values of 0.691 and 0.686 in the training and validation cohorts, respectively. Compared with seven conventional staging systems (C-index, training cohort, 0.548-0.597; validation cohort, 0.519-0.610), the ICPI exhibited better performance for early/intermediate-stage HCCs. Further, the patients were categorized into three subgroups with X-tile software, and the stratified ICPI presented a superior corrected Akaike information criterion and homogeneity in both cohorts. CONCLUSIONS: Our ICPI was a useful and reliable prognostic tool which may offer good individualized prediction capability for HCC patients with early/intermediate stage.

Regulating ferroportin-1 and transferrin receptor-1 expression: A novel function of hydrogen sulfide.

Hydrogen sulfide (H2 S) has a significant effect on the regulation of interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) activities, while IL-6 directly regulates hepcidin expression via STAT3. We therefore hypothesized that H 2 S has a role in body iron homeostasis by regulating the expression of iron transport proteins via the IL-6/STAT3/Hepcidin pathway. Here, we investigated the effects of two H 2 S donors sodium hydrosulfide and GYY4137 on the expression of ferroportin-1 (Fpn1), transferrin receptor-1 (TfR1), hepcidin, IL-6 and pSTAT3 in the spleen of mice in vivo and peritoneal macrophage in vitro. We also examined the effects of H 2 S on serum iron, transferrin saturation, and ferritin light chain contents in the spleen, and on nitrite content, nuclear factor erythroid 2-related factor-2 (Nrf2) and iron regulatory protein 1 (IRP1) in the macrophages. We demonstrated that H 2 S regulates the expression of TfR1 and Fpn1 in the spleen in vivo and in peritoneal macrophages in vitro predominantly via the IL-6/pSTAT3/hepcidin pathway, under the conditions of inflammation induced by lipopolysaccharides. We also provide evidence that under uninflamed conditions, the regulation of Fpn1 and TfR1 expression by H 2 S, both in vivo and in vitro, are mediated by the nitric oxide (NO)/Nrf2 and iron regulatory protein/iron responsive element pathways, respectively, which are independent of IL-6/pSTAT3/hepcidin signals. These findings show that H 2 S is a key player in iron homeostasis under not only the inflamed conditions but also uninflamed conditions.

[Effects of Targeting lncRNA HOTAIR on the Invasion and Nude Mouse Tumorigenicity of Epithelial Ovarian Cancer Cells].

OBJECTIVE: To explore the effects of shRNA-mediated downregulating lncRNA HOTAIR on the invasion,nude mouse tumorigenicity and snail expression of epithelial ovarian cancer SKOV3 cells. METHODS: The expression of lncRNA HOTAIR was detected by RT-PCR in SKOV3 cells. The shRNA targeting the lncRNA HOTAIR gene was cloned into RNA interference plasmid. The construction shHOTAIR vector was transfected into ovarian cancer SKOV3 cells by lipofectamine 2000,and the stably transfected cells were isolated by G418 and single clone selection. The downregulating expression of lncRNA HOTAIR was detected by quantitative real time PCR(qRT-PCR). The characteristics of shHOTAIR transfected SKOV3 cells were analyzed from the assays of invasion and nude mouse tumorigenicity,as well as the expression of snail and E-cadherin mRNA detected by qRT-PCR,and snail detected by immunohistochemistry and Western blot methods in xenograft tumor,respectively. RESULTS: The lncRNA HOTAIR expression was proved by RT-PCR in SKOV3 cells. The lncRNA HOTAIR expression in shHOTAIR transfected SKOV3 cells was significantly lower than the scramble control (P<0.01). The shHOTAIR transfected SKOV3 cells show that the invasion ability was significantly decreased compared with the scramble control (P<0.01). The nude mouse tumorigenicity,including tumorigenicity mouse number and tumor volume,was significantly decreased compared with the control group (P<0.01). The snail protein expression detected by IHC and Western blot in shHOTAIR-SKOV3 xenograft tumor was significantly decreased compared with the control scramble- SKOV3 group (P<0.05). The lncRNA HOTAIR low expression resulted in increasing E-cadherin and decreasing snail expression detected by qRT-PCR in the shHOTAIR transfected SKOV3 cells of xenograft tumor,compared with the scramble control (P<0.05). CONCLUSION: Targeting lncRNA HOTAIR expression in SKOV3 cells with RNA interference can decrease snail,increase E-cadherin and significantly reduce the invasion and tumorigenicity of epithelial ovarian cancer SKOV3 cells. These results suggest that the lncRNA HOTAIR could be an effective therapeutic target for human epithelial ovarian caner treatment.

Fasting up-regulates ferroportin 1 expression via a Ghrelin/GHSR/MAPK signaling pathway.

The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft-L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O-acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft-L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft-L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre-treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway.

Cystathionine ß-synthase is required for body iron homeostasis.

Cystathionine ß-synthase (CBS) catalyzes the transsulfuration pathway and contributes, among other functions, to the generation of hydrogen sulfide. In view of the exceptionally high expression of CBS in the liver and the common interleukin-6 pathway used in the regulatory systems of hydrogen sulfide and hepcidin, we speculate that CBS is involved in body iron homeostasis. We found that CBS knockout (CBS-/- ) mice exhibited anemia and a significant increase in iron content in the serum, liver, spleen, and heart, along with severe damage to the liver, displaying a hemochromatosis-like phenotype. A high level of hepatic and serum hepcidin was also found. A major cause of the systemic iron overload is the reduced iron usage due to suppressed erythropoiesis, which is consistent with an increase in interleukin-6 and reduced expression of erythropoietin. Importantly, in the liver, absence of CBS caused both a reduction in the transcriptional factor nuclear factor erythroid 2-related factor-2 and an up-regulation of hepcidin that led to a decrease in the iron export protein ferroportin 1. The resulting suppression of iron export exacerbates iron retention, causing damage to hepatocytes. Finally, administration of CBS-overexpressing adenovirus into CBS mutant mice could partially reverse the iron-related phenotype. CONCLUSION: Our findings point to a critical role of CBS in iron homeostasis of the body, and the liver in particular; it is likely that a hemochromatosis-like phenotype in patients can be induced by aberration not only in the expression of key molecules in the hepcidin pathway but also of those related to CBS. (Hepatology 2018;67:21-35).

Hepcidin Protects Neuron from Hemin-Mediated Injury by Reducing Iron.

Hemin plays a key role in mediating secondary neuronal injury after intracerebral hemorrhage (ICH) and the cell toxicity of hemin is thought to be due to iron that is liberated when hemin is degraded. In a recent study, we demonstrated the iron regulatory hormone hepcidin reduces brain iron in iron-overloaded rats. Therefore, we hypothesized that hepcidin might be able to reduce iron and then protect neurons from hemin or iron-mediated neurotoxicity in hemin-treated neuronal cells. Here, we tested the hypothesis and demonstrated that ad-hepcidin and hepcidin peptide both have the ability to suppress the hemin-induced increase in LDH release and apoptotic cell numbers, to reduce cell iron and ferritin contents, and to inhibit expression of transferrin receptor 1, divalent metal transporter 1, and ferroportin 1 in hemin-treated neurons. We conclude that hepcidin protects neuron from hemin-mediated injury by reducing iron via inhibition of expression of iron transport proteins.

Bi-directionally protective communication between neurons and astrocytes under ischemia.

The extensive existing knowledge on bi-directional communication between astrocytes and neurons led us to hypothesize that not only ischemia-preconditioned (IP) astrocytes can protect neurons but also IP neurons protect astrocytes from lethal ischemic injury. Here, we demonstrated for the first time that neurons have a significant role in protecting astrocytes from ischemic injury. The cultured medium from IP neurons (IPcNCM) induced a remarkable reduction in LDH and an increase in cell viability in ischemic astrocytes in vitro. Selective neuronal loss by kainic acid injection induced a significant increase in apoptotic astrocyte numbers in the brain of ischemic rats in vivo. Furthermore, TUNEL analysis, DNA ladder assay, and the measurements of ROS, GSH, pro- and anti-apoptotic factors, anti-oxidant enzymes and signal molecules in vitro and/or in vivo demonstrated that IP neurons protect astrocytes by an EPO-mediated inhibition of pro-apoptotic signals, activation of anti-apoptotic proteins via the P13K/ERK/STAT5 pathways and activation of anti-oxidant proteins via up-regulation of anti-oxidant enzymes. We demonstrated the existence of astro-protection by IP neurons under ischemia and proposed that the bi-directionally protective communications between cells might be a common activity in the brain or peripheral organs under most if not all pathological conditions.

Nifedipine Increases Iron Content in WKPT-0293 Cl.2 Cells via Up-Regulating Iron Influx Proteins.

Nifedipine was reported to enhance urinary iron excretion in iron overloaded mice. However, it remains unknown how nifedipine stimulates urinary iron excretion in the kidney. We speculated that nifedipine might inhibit the TfR1/ DMT1 (transferrin receptor 1/divalent metal transporter1)-mediated iron uptake by proximal tubule cells in addition to blocking L-type Ca2+ channels, leading to an increase in iron in lumen-fluid and then urinary iron excretion. To test this hypothesis, we investigated the effects of nifedipine on iron content and expression of TfR1, DMT1 and ferroportin1 (Fpn1) in WKPT-0293 Cl.2 cells of the S1 segment of the proximal tubule in rats, using a graphite furnace atomic absorption spectrophotometer and Western blot analysis, respectively. We demonstrated for the first time that nifedipine significantly enhanced iron content as well as TfR1 and DMT1 expression and had no effect on Fpn1 levels in the cells. We also found that ferric ammonium citrate decreased TfR1 levels, increased Fpn1 expression and had no effect on DMT1 content, while co-treatment with nifedipine and FAC increase TfR1 and DMT1 expression and also had no effect on Fpn1 levels. These findings suggest that the nifedipine-induced increase in cell iron may mainly be due to the corresponding increase in TfR1 and DMT1 expression and also imply that the effects of nifedipine on iron transport in proximal tubule cells can not explain the increase in urinary iron excretion.

Targeted inhibition of Notch1 gene enhances the killing effects of paclitaxel on triple negative breast cancer cells.

OBJECTIVE: To study the influence of targeted inhibition of Notch1 gene on the killing effects of paclitaxel on triple negative breast cancer cells. METHODS: The triple negative [estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (Her2)] breast cancer cell line MDA-MB-231 and ER/PR/HER-2-positive breast cancer cell line MCF-7 were cultured, transfected with Notch1-siRNA-overexpression plasmid and blank plasmid, and treated with different concentrations of paclitaxel, and then the cell proliferation activity and apoptosis rate as well as the mRNA expression of Caspase-3, Caspase-9 and Bcl-2 were determined. RESULTS: Paclitaxel could decrease the MDA-MB-231 and MCF-7 cell proliferation activity as well as Bcl-2 mRNA expression, and increase MDA-MB-231 and MCF-7 cell apoptosis rate as well as Caspase-3 and Caspase-9 mRNA expression in dose-dependent manners; with the same dose of paclitaxel treatment, the inhibitory effects on MDA-MB-231 cell proliferation activity and Bcl-2 mRNA expression as well as the promoting effects on MDA-MB-231 cell apoptosis and mRNA expression of Caspase-3 and Caspase-9 were weaker than those on MCF-7 cell; after 0.5 µM paclitaxel combined with Notch1-siRNA treatment, MDA-MB-231 cell proliferation activity and Bcl-2 mRNA expression were significantly lower than those after 0.5 µM paclitaxel combined with control plasmid treatment while cell apoptosis rate and mRNA expression of Caspase-3 and Caspase-9 were higher than those after 0.5 µM paclitaxel combined with control plasmid treatment. CONCLUSIONS: Targeted inhibition of Notch1 gene may enhance the killing effects of paclitaxel on triple negative breast cancer cells by up-regulating the expression of Caspase-3 and Caspase-9 and inhibiting the expression of Bcl-2.

Effects of alpha-lipoic acid on expression of iron transport and storage proteins in BV-2 microglia cells.

BACKGROUND: The antioxidant properties of alpha-lipoic acid (ALA) are associated with its ability to reduce iron in cells and tissues, which is partly due to its inhibiting effect on iron uptake from transferrin and its promoting effect on iron deposition into ferritin. However, the relevant mechanisms are unknown. METHODS: We therefore investigated the effects of ALA on the expression of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and ferritin in BV-2 microglia cells. RESULTS: We demonstrated that ALA significantly inhibited DMT1 expression, lowered ferritin-light-chain (Ft-L) and ferritin-heavy-chain (Ft-H) content, and had no effect on TfR1 and Fpn1 in BV-2 microglia cells. This indicated that the inhibiting effect of ALA on DMT1 might be one of the causes of the ALA-induced reduction in cellular transferrin-bound-iron uptake. We also demonstrated that ALA enhanced DMT1 and TfR1 expression in ferric ammonium citrate (FAC)-treated cells. FAC treatment led to a significant increase in Ft-L, Ft-H and Fpn1, and pre-treatment with ALA resulted in a further increase in the contents of Ft-L and Ft-H but not Fpn1 in cells. CONCLUSIONS: ALA could up-regulate TfR1, DMT1 and ferritin expression when iron is increased outside of the cell, promoting iron deposition into ferritin by increasing cell iron uptake, and then reducing free iron both inside and outside of the cell.