Association Between High Serum Anion Gap and All-Cause Mortality in Non-Traumatic Subarachnoid Hemorrhage: A Retrospective Analysis of the MIMIC-IV Database.

Background: High serum anion gap (AG) on admission is often correlated with poor outcomes in critically ill patients; however, data in patients with non-traumatic subarachnoid hemorrhage (SAH) are lacking. Herein, we aimed to identify the association between serum AG and all-cause mortality in patients with non-traumatic SAH. Methods: A retrospective analysis of data from the Medical Information Mart for Intensive Care (MIMIC-IV) database was performed on critically ill patients with non-traumatic SAH. Serum AG was collected on Intensive Care Unit (ICU) admission, and ICU and hospital all-cause mortality were analyzed. The multivariate Cox proportional hazard regression model and Kaplan-Meier survival curve analysis were used to analyze the correlation of serum AG with ICU and hospital all-cause mortality. Furthermore, interaction and subgroup analyses were evaluated for the consistency of these correlations. Results: A total of 893 patients with non-traumatic SAH were included in this study. The all-cause mortality in ICU and hospital were 14.8% (132/893), and 18.9% (169/893), respectively. Multivariate analysis after adjusting for potential confounders indicated that high serum AG levels (≥16 mmol/L) were associated with increased risk of ICU and hospital all-cause mortality as compared to that with low serum AG levels (<16mmol/L), (hazards ratio (HR): 2.31 [95% CI: 1.58-3.38]) and HR: 1.91 [95% CI: 1.36-2.67)], respectively). Similarly, the Kaplan-Meier (K-M) survival curve also showed that patients with high serum AG levels presented with a lower survival rate. Stratified analyses further showed that depending on the variable testes, an association between higher serum AG levels and hospital all-cause mortality in different subgroups was observed. Conclusion: Among patients with non-traumatic SAH, high serum AG level at ICU admission was associated with increased ICU and hospital all-cause mortality.

A hybridization chain reaction amplification strategy for fluorescence imaging of human telomerase activity in living cells.

A hybridized chain reaction (HCR)-based biosensing method has been developed for the imaging detection of intracellular telomerase activity. The telomerase-targeting responder-transmitter DNA complex (HPT) consisting of telomerase primer sequence (HP) and a HCR initiator (trigger) is transfected into cell plasma. In the presence of telomerase, HPT can be recognized and extended, producing plenty of triggers which initiate HCR amplification reaction. Finally, a long nicked dsDNA with a lot of outstretched single chains was formed by hybridizing with Q of the reporter complex, generating an enhanced fluorescence signal. The developed biosensing approach can be used for the detection of telomerase activity in cell lysate with the detection limit of 578 cells/100 µl. In addition, this strategy has been successfully applied not only for the sensitive and specific imaging of telomerase activity in living cells but also for comparing of telomerase activity among different cell lines. Therefore, the method might become a potential alternative tool for telomerase-related cancer diagnosis and therapy in medical research and early clinical diagnosis.

[Metabolic Characteristics of Leucocyte-deplated and Suspended RBC Produced by Using Lipid Whole Blood During Routine Storage].

OBJECTIVE: To investigate the differences of metabolic pathways of leucocyte-deplated RBCs prepared by using lipid whole blood and nomal blood during routine storage so as to provide some reference for clinical blood use. METHODS: Twenty U whole blood from 20 donors, including 10 U lipid blood and 10 U normal whole blood, were selected for preparing leukodepleted red blood cells, red blood cells were taken from storage bags on day 0, 14 and 35, respectively. Metabolites in the red blood cells were analyzed, red blood cell metabolic extracts were detected by UPLC-MS/MS. The metabolite data of RBC from 2 groups were analyzed by SIMCA-P 13.0 software using OPLS-DA and by SPSS 19.0 using Mann-Whitney U test. Difference of metabolic pathways was described according to different metabolites. RESULTS: The glucose, adenine, pyruvic acid, GSH, GSSG and niacinamide levels on day 0 in lipid RBCs were higher than those in the control group(P<0.05). The glucose, pyruvic acid and GSH levels on day 14 in lipid RBCs were lower than those in the control group (P<0.05), and the levels of adenine, GSSG and niacinamide were higher than that in the control group (P<0.05). The glucose level on day 0 was lower than that in the control group (P<0.05), and the levels of adenine and niacinamide were higher than those in the control group (P<0.05). but the pyruvic acid, GSH and GSSG levels were not significantly different between 2 groups (P>0.05). CONCLUSION: Compared with the normal red blood cells, the energy metabolism pathway decreases in lipid red blood cells within the storage period and pentose phosphate pathway increases.

Cisplatin-induced autophagy protects breast cancer cells from apoptosis by regulating yes-associated protein.

Breast cancer is a common cause of cancer­related deaths in women. Treatment with cisplatin exhibits some therapeutic efficacy. However, treatment optimization is required, and the mechanisms underlying the cisplatin's proapoptotic effects remain unclear. In the present study, we demonstrated that cisplatin induced apoptosis and autophagy in breast cancer cells. Autophagy induced by cisplatin played a protective role in breast cancer cells, which impaired its proapoptotic effect. Mechanistically, for the first time, we found that cisplatin treatment activated the MAPK signaling pathway and promoted autophagy via the ERK signaling pathway. Notably, we found that nuclear translocation of yes-associated protein (YAP) was regulated by cisplatin-induced autophagy, and we identified YAP as a survival input that promoted survival in cisplatin-treated breast cancer cells. These findings revealed that administration of cisplatin along with an autophagy inhibitor is a promising therapeutic strategy for treating breast cancer.

Leptin promotes the migration and invasion of breast cancer cells by upregulating ACAT2.

BACKGROUND: Previously, it has been shown that obesity may be considered as a risk factor for breast cancer in postmenopausal women. Leptin, a hormone whose level is elevated in obesity, has been suggested to be involved in the development of breast cancer, and univariate survival analyses have shown that over-expression of ACAT2, an enzyme that is involved in the production of cholesteryl esters, may be associated with a poor prognosis. Here, we aimed to investigate the effect of leptin on the proliferation, migration and invasion of breast cancer cells, as well as to elucidate its underlying mode of action. METHODS: Gene expression changes in leptin treated breast cancer-derived MCF-7, T47D and BT474 cells were assessed using PCR array, qRT-PCR and Western blot analyses. The expression patterns of Ob-R (leptin receptor) and ACAT2 in breast cancer cells and primary breast cancer tissue samples were analyzed using immunofluorescence and immunohistochemistry, respectively. Leptin-induced proliferation of breast cancer cells was assessed using a CCK8 assay, and scratch wound and Transwell assays were used to assess breast cancer cell invasion and migration. RESULTS: We found that, among the genes tested, ACAT2 expression exhibited the most significant changes in the leptin treated cells. In addition, we found that inhibition of ACAT2 expression using pyripyropene A (PPPA) or siRNA-mediated gene silencing significantly decreased leptin-induced proliferation, migration and invasion of MCF-7 and T47D cells. Subsequent Western blot analyses strongly indicated that the PI3K/AKT/SREBP2 signaling pathway was involved in leptin-induced ACAT2 upregulation in both MCF-7 and T47D cells. Finally, through the analysis of primary breast cancer tissue samples we found that ACAT2 may affect cancer progression through activation of the Ob-R. CONCLUSIONS: Our data indicate that leptin may enhance the proliferation, migration and invasion of breast cancer cells via ACAT2 up-regulation through the PI3K/AKT/SREBP2 signaling pathway. Therefore, the leptin/ACAT2 axis may represent an attractive therapeutic target for breast cancer, particularly in postmenopausal and/or obese women.

Pyruvate kinase M2 interacts with mammalian sterile 20-like kinase 1 and inhibits tamoxifen-induced apoptosis in human breast cancer cells.

Tamoxifen has been reported to be associated with antagonism of estrogen-mediated cell growth signaling and activation of estrogen receptor-independent apoptosis events. It has been demonstrated that mammalian sterile 20-like kinase 1 is a direct target of Caspases to amplify the apoptotic signaling pathway. Here, we presented that breast cancer MCF-7 and SKBR3 cells under treatment with 4-hydroxytamoxifen displayed decreased level of pyruvate kinase M2. Western blot results also showed that 4-hydroxytamoxifen induced the activity of pro-apoptotic protein Caspase-3 in MCF-7 and SKBR3 cells, as evidenced by the cleavage of mammalian sterile 20-like kinase 1 substrate in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence experiments were performed to clarify the relationship between pyruvate kinase M2 and mammalian sterile 20-like kinase 1. The results indicated that mammalian sterile 20-like kinase 1 was associated with pyruvate kinase M2 in cultured mammalian cells, and the interaction between mammalian sterile 20-like kinase 1 and pyruvate kinase M2 was decreased in response to 4-hydroxytamoxifen treatment. In addition, knockdown of pyruvate kinase M2 upregulated the level of cleaved Caspase-3 and subsequently facilitated the nuclear translocation of mammalian sterile 20-like kinase 1. Our data further supplemented the extensive functions of pyruvate kinase M2 in mediating breast cancer cell viability by substantially abating the mammalian sterile 20-like kinase 1-mediated apoptosis. In summary, our results identified that mammalian sterile 20-like kinase 1 is a novel downstream target of pyruvate kinase M2, and knockdown of pyruvate kinase M2 contributes apoptosis via promoting nuclear translocation of mammalian sterile 20-like kinase 1 by enhancing Caspase-3-dependent cleavage.

Invalidation of mitophagy by FBP1-mediated repression promotes apoptosis in breast cancer.

Fructose-1,6-bisphosphatase 1, a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor. However, the functions of fructose-1,6-bisphosphatase 1 in the regulation of mitophagy and apoptosis remain unknown. Here, we investigated the effects of fructose-1,6-bisphosphatase 1 on mitophagy and apoptosis as well as their underlying mechanisms in breast cancer cells. In this work, the messenger RNA and protein expression of various molecules were determined by quantitative realtime polymerase chain reaction and western blot, respectively. Gene-expression correlations were obtained from The Cancer Genome Atlas Breast Cancer database and analyzed using cBioPortal. The levels of cellular reactive oxygen species and apoptotic index were detected by flow cytometry. The mitochondrial membrane potentials were assessed with a JC-1 fluorescent sensor. Subcellular structures were observed under a transmission electron microscope. The intracellular distribution of translocase of outer membrane 20 was detected by immunofluorescence staining. Protein-protein interactions were analyzed by immunoprecipitation. Our results indicated that fructose-1,6-bisphosphatase 1 expression was negatively correlated with autophagy level in breast cancer. Fructose-1,6-bisphosphatase 1 restrained autophagy activity by increasing the level of p62 and decreasing the levels of LC3 and Beclin 1. Additionally, fructose-1,6-bisphosphatase 1 promoted cell apoptosis by upregulating the levels of intracellular ROS and expression of pro-apoptotic proteins such as cleaved PARP, cleaved Caspase 3, and Bax and downregulating the levels of anti-apoptotic proteins such as PARP, Caspase 3, and Bcl-2. Finally, fructose-1,6-bisphosphatase 1 limited the efficient removal of diseased mitochondria and reduced the messenger RNA and protein expressions of HIF-1α, BNIP3L/NIX, and BNIP3. More importantly, fructose-1,6-bisphosphatase 1 facilitated co-action between Bcl-2 and Beclin 1, which may be important in the mechanism of fructose-1,6-bisphosphatase 1-mediated mitophagy inhibition. In summary, loss of mitophagy by fructose-1,6-bisphosphatase 1-mediated repression promotes apoptosis in breast cancer.

miR520c blocks EMT progression of human breast cancer cells by repressing STAT3.

Breast cancer is one of the most malignant diseases world-wide and it ranks the first among female cancers. Masses of intrinsic and extrinsic factors, especially the inflammatory factors can lead to breast cancer. Aberrant activation and accumulation of key molecules can lead to inflammation associated carcinogenesis. The signal transducers and activators of transcription 3 (STAT3) is one of them. Therefore, to evaluate the novel molecular mechanisms, STAT3 has become our focus for breast cancer targeted therapy. At present, many tumor suppressing microRNAs have been validated, and are the highlights in research on microRNAs. Thus, we predicted microRNAs which could putatively regulate STAT3 through databases and selected six to screen with Dual-luciferase assay. The result hinted that miR520c could bind with STAT3 3'UTR. We mutated the seed sequence of miR520c on STAT3 3'UTR, which illustrated a reverse effect compared with wild-type of STAT3 3'UTR. Subsequently, STAT3, p-STAT3 and miR520c were assessed in three different grades of breast cancer cells, with the degree of malignancy, we found an escalating trend of STAT3 and p-STAT3, on the contrary, a downward trend of miR520c. We observed STAT3 was deactivated by miR520c. Epithelial to mesenchymal transition (EMT) is a fatal transfer of cancer progression. To find out whether the downregulation of STAT3 can repress breast cancer motility and invasion ability, we detected EMT markers. The result implied a suppression effect on EMT. We overexpressed STAT3 to conduct rescue experiments, the result showed a recovery of STAT3 and EMT characteristics. Cell motility and invasion property were regained as well. In the study, we elucidated miR520c could inhibit breast cancer EMT by targeting STAT3. It can enrich the mechanism of breast cancer and may lay the foundation for breast cancer targeted treatment.

Leptin promotes epithelial-mesenchymal transition of breast cancer via the upregulation of pyruvate kinase M2.

BACKGROUND: Accumulating researches have shown that epithelial-mesenchymal transition (EMT) contributes to tumor metastasis. Leptin, a key adipokine secreted from adipocytes, shapes the tumor microenvironment, potentiates the migration of breast cancer cells and angiogenesis, and is also involved in EMT. However, the potential mechanism remains unknown. This study aims to explore the effect of leptin on EMT in breast cancer cells and the underlying mechanism. METHODS: With the assessment of EMT-associated marker expression in MCF-7, SK-BR-3, and MDA-MB-468 cells, the effect of leptin on breast cancer cells was analyzed. Besides, an array of pathway inhibitors as well as RNA interference targeting pyruvate kinase M2 (PKM2) were used to clarify the underlying mechanism of leptin-mediated EMT in vitro and in vivo. RESULTS: The results demonstrated that leptin promoted breast cancer cells EMT, visibly activated the PI3K/AKT signaling pathway, and upregulated PKM2 expression. An antibody against the leptin receptor (anti-ObR) and the PI3K/AKT signaling pathway inhibitor LY294002 significantly abolished leptin-induced PKM2 expression and EMT-associated marker expression. SiRNA targeting PKM2 partially abolished leptin-induced migration, invasion, and EMT-associated marker expression. In vivo xenograft experiments indicated that RNA interference against PKM2 suppressed breast cancer growth and metastasis. CONCLUSIONS: Our data suggest that leptin promotes EMT in breast cancer cells via the upregulation of PKM2 expression as well as activation of PI3K/AKT signaling pathway, and PKM2 might be one of the key points and potential targets for breast cancer therapy.

Leptin promotes breast cancer cell migration and invasion via IL-18 expression and secretion.

In recent years, crosstalk between tumor microenvironment and cancer cells have received increasing attention. Accumulating research data suggests that leptin, a key adipokine secreted from adipocytes, plays important roles in breast cancer development. In our study, the effects of leptin on polarization of tumor-associated macrophages (TAMs) and promotion of the invasiveness of tumor cells were investigated. THP1 cells were used to differentiate M2 polarization macrophages. After stimulated by leptin, we established a co-culture system of tumor cells and macrophages to evaluate the function of leptin-induced macrophages in the migration and invasion of breast cancer cells. The gene and protein expressions were analyzed and the underlying mechanisms were evaluated. Moreover, pathological human specimens, and xenografts in nude mice, were detected to strengthen the in vitro results. Leptin elevated the expression of an array of cytokines in TAMs, IL-18 was the most increased, with an activation of the NF-κB/NF-κB1 signalling pathway. Additionally, after treated with leptin, TAMs significantly promoted the migration and invasion of breast cancer cells. However, these effects of leptin were abolished by the co-incubation of Bay11­7082, a pharmacological NF-κB inhibitor. Leptin also directly stimulated IL-18 expression in breast cancer cells, which, differently, was via the PI3K/AKT-ATF-2 signaling pathway. In vivo studies showed that malignant breast carcinoma exhibited strong higher expression of Leptin, IL-8, and TAMs markers. Xenograft tumor-bearing mouse models showed that leptin significantly increased tumor volume, enhanced lung metastases, and increased expression of IL-8 and TAM markers, which were abolished by depletion of macrophages by clophosome-clodronate liposomes (CCL). Leptin could induce IL-18 expression both in TAMs and breast cancer cells. Leptin-induced IL-18 expression was regulated via NF-κB/NF-κB1 signaling in TAMs, while via PI3K-AKT/ATF-2 signaling in breast cancer cells, which, eventually, lead to invasion and metastasis of breast cancer cells.

[BAY11-7082 inhibits proliferation and promotes apoptosis in breast carcinoma MCF-7 cells by inhibiting phosphorylation of ATP citrate lyase].

OBJECTIVE: To investigate the effect of NF-κB inhibitor BAY11-7082 on proliferation and apoptosis of breast carcinoma MCF-7 cells and the underlying mechanism. METHODS: MCF-7 cells in the logarithmic growth phase were divided into control group, 5 µmol/L BAY11-7082 group and 10 µmol/L LY294002 group. After the treatment of BAY11-7082 and LY294002, the protein levels of ATP citrate lyase (ACL), phosphated-ACL (p-ACL), phosphated-Akt (p-Akt) and phosphated nuclear factor κB (p-NF-κB) were determined by Western blotting. The proliferation of MCF-7 cells treated with BAY11-7082 or siACL were detected by CCK-8 assay. The apoptosis of MCF-7 cells treated with BAY11-7082 or siACL were observed by flow cytometry combined with annexin V-FITC/PI staining. RESULTS: The proliferation of MCF-7 cells was inhibited by BAY11-7082 in a dose-dependent manner. Compared with the control group, the expressions of p-ACL and p-NF-κB protein in MCF-7 cells treated with BAY11-7082 were lowered. The expressions of p-ACL and p-NF-κB protein in MCF-7 cells treated with 10 µmol/L LY294002 were also reduced significantly. The proliferation of MCF-7 cells treated with BAY11-7082 or siACL for 48 hours was inhibited and the apoptosis was promoted significantly, as shown by CCK-8 assay and flow cytometry. CONCLUSION: BAY11-7082 could inhibit the proliferation of MCF-7 breast carcinoma cells and promote the apoptosis by inhibiting the phosphorylation of ACL.