NRF2/HO-1 axis, BIRC5, and TP53 expression in ESCC and its correlation with clinical pathological characteristics and prognosis.

BACKGROUND: Many types of cancer exhibit high nuclear factor erythroid 2-related factor 2 (NRF2), which is effective in resisting drugs and radiation. However, the role of NRF2 gene expression in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: The association between NRF2, heme oxygenase-1 (HO-1), baculovirus IAP repeat 5 (BIRC5), P53 gene expression levels and their relationship to immune-infiltrating cells were assessed using the Cancer Genome Atlas dataset, the Human Protein Atlas and the TISDB database. The expression of NRF2, HO-1, BIRC5, and TP53 in 118 ESCC patients was detected by immunohistochemistry, and the relationship between their expression level and clinicopathological parameters and prognosis was analyzed. RESULTS: In ESCC, NRF2 overexpression was significantly associated with Han ethnicity, lymph node metastasis, and distant metastasis. HO-1 overexpression was significantly associated with differentiation, advanced clinical staging, lymph node metastasis, nerve invasion, and distant metastasis. BIRC5 overexpression was significantly associated with Han ethnicity and lymph node metastasis. TP53 overexpression was significantly associated with Han ethnicity and T staging. The NRF2/HO-1 axis expression was positively correlated with BIRC5 and TP53. Kaplan-Meier and multivariate Cox regression analysis showed that NRF2, BIRC5, and TP53 genes co-expression was an independent prognostic risk factor. TISIDB dataset analysis showed that immune-infiltrating cells were significantly negatively correlated with NRF2 and BIRC5. CONCLUSION: NRF2, BIRC5, and TP53 axis gene expressions are predictors of poor prognosis for ESCC. The overexpression of the NRF2/HO-1/BIRC5 axis may not be related to immune-infiltrating cells.

Prepackaged formula low-residue diet vs. self-prepared low-residue diet before colonoscopy: A multicenter randomized controlled trial.

Background and aims: Compared with self-prepared LRD, a prepackaged low-residue diet (LRD) can improve patient compliance, but whether it can further improve the quality of bowel preparation is uncertain. The study aimed to compare the application of the prepackaged formula LRD with self-prepared LRD in bowel preparation for colonoscopy. Methods: A multicenter randomized controlled trial was conducted in 15 centers. The eligible subjects were randomly assigned to one of two groups: the formula LRD group and the self-prepared LRD group. On the day before the colonoscopy, subjects in the self-prepared LRD group were instructed to consume a restricted LRD prepared by themselves, while subjects in the formula LRD group were given six bags of prepackaged formula LRD and instructed to consume them according to their individual need. The primary outcome was an adequate bowel preparation rate. Secondary outcomes mainly included Boston Bowel Preparation Scale (BBPS) scores, dietary restriction compliance rate, tolerance, satisfaction, adenoma detection rate (ADR), and adverse reactions. The trial was registered at ClinicalTrials.gov under the identifier NCT03943758. Results: A total of 550 subjects were recruited. Compared with the self-prepared LRD group, the formula LRD group showed a higher adequate bowel preparation rate (94.5 vs. 80.4%; P < 0.01), BBPS scores (7.87 ± 1.13 vs. 6.75 ± 1.47; P < 0.01), dietary compliance rate (92.4 vs. 78.9%; P < 0.01), tolerance (P < 0.01 in degree of hunger, intensity of physical strength, and negative influence on daily activities), satisfaction (8.56 ± 1.61 vs. 7.20 ± 2.02; P < 0.01), and ADR (25.6 vs. 16.0%; P < 0.01). There was no significant difference in adverse reactions. Conclusion: Compared with self-prepared LRD, the formula LRD showed similar safety and higher bowel preparation quality, compliance, and tolerance in bowel preparation. More formula LRDs could be designed according to different dietary habits and ethnic populations, and further researches are warranted to confirm their effect. Clinical trial registration: https://register.clinicaltrials.gov, identifier: NCT03943758.

Prognostic value of Nrf2/HO-1 expression and its correlation with occurrence in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is thought to be started and developed by genes associated with inflammation. A cancer's ability to spread and grow can be aided by nuclear factor erythroid-2 related factor 2 (Nrf2) hyperactivation, which can also make a tumor more resistant to chemotherapy and radiation treatment. However, it is still unknown how Nrf2 gene expression affects ESCC prognosis and controls function throughout ESCC advancement. OBJECTIVE: The expression of Nrf2 and HO-1 in ESCC and precancerous esophageal precancerous lesions was analyzed, and their relationship with esophageal squamous cell carcinoma was analyzed. METHODS: Immunohistochemistry (IHC) was used to confirm the expression of Nrf2 and heme oxygenase-1 (HO-1) proteins in tissue microarrays from Chinese populations with ESCC. We looked at the connections between Nrf2/HO-1 expression and invading immune cells using the TIMER database. RESULTS: Ethnicity and N stage are associated with Nrf2 overexpression. Differentiation, N stage, vascular invasion, distant metastasis, and American Joint Committee on Cancer (AJCC) staging are all associated with HO-1 overexpression. The expression of Nrf2 and HO-1 had a favorable correlation. Patients with elevated Nrf2 and HO-1 expression had lower progression-free survival (PFS) and overall survival (OS). In high-grade intraepithelial neoplasia, Nrf2 and HO-1 expression generally occurred, partially in low-grade intraepithelial neoplasia specimens, and rarely in normal mucosa. We further show that Nrf2 suppression is linked to higher immunological marker expression and lower immune cell infiltration. CONCLUSION: The prognosis of ESCC may be improved by inhibiting the expression of Nrf2 and HO-1. A lack of immune cells was seen in ESCC with Nrf2 impairment.

Construction of a prognostic model for radical esophagectomy based on immunohistochemical prognostic markers combined with clinicopathological factors.

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis and lacks effective biomarkers to evaluate prognosis and treatment. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a protein highly expressed in ESCC tissues screened by isobaric tags for relative and absolute quantitation proteomics, which has significant prognostic value in a variety of malignant tumors, but its relationship with ESCC remains unclear. By immunohistochemical staining of 266 ESCC samples, we analyzed the relationship between GPNMB and ESCC. To explore how to improve the ability of ESCC prognostic assessment, we established a prognostic model of GPNMB and clinicopathological features. The results suggest that GPNMB expression is generally positive in ESCC tissues and is significantly associated with poorer differentiation, more advanced American Joint Council on Cancer (AJCC) stage, and higher tumor aggressiveness (P < .05). Multivariate Cox analysis indicated that GPNMB expression was an independent risk factor for ESCC patients. A total of 188 (70%) patients were randomly selected from the training cohort and the four variables were automatically screened by stepwise regression based on the AIC principle: GPNMB expression, nation, AJCC stage and nerve invasion. Through the weighted term, we calculate the risk score of each patient, and by drawing the receiver operating characteristic curve, we show that the model has good prognostic evaluation performance. The stability of the model was verified by test cohort. Conclusion: GPNMB is a prognostic marker consistent with the characteristics of tumor therapeutic targets. For the first time, we constructed a prognostic model combining immunohistochemical prognostic markers and clinicopathological features in ESCC, which showed higher prognostic efficacy than AJCC staging system in predicting the prognosis of ESCC patients in this region.

Decomposition of Clinical Significance of FSH, LH, E2, AMH, and AFC Standards in Females at Lofty Elevation Based on HIF1α.

Based on hypoxia-inducible factor-1α (HIF-1α), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Mullerian hormone (AMH), the antral follicle is explored. The expression of count (AFC) in females at lofty elevation and its clinical significance are analyzed. A total of 82 females in lofty elevation areas and in low-elevation areas who received health checks from April 2020 to May 2021 are selected as the lofty elevation set and the low-elevation set, respectively. In addition, 76 females are served as the routine set. By comparing the serum HIF-1α standards, the standards of sex hormone indexes FSH, LH, and E2, and the expressions of AMH and AFC between the two sets of females, the correlation between HIF-1α and sex hormone indexes and ovarian reserve function is analyzed. The experimental results show that the lofty standard of HIF-1α in females at lofty elevation may lead to abnormal standards of sex hormones and weakened ovarian reserve. The detection of HIF-1α in females in lofty elevation areas is of great significance for evaluating their sex hormone standards, ovarian function, and preventing the occurrence of female gynecological diseases.

Prognostic Value of GPNMB, EGFR, p-PI3K, and Ki-67 in Patients with Esophageal Squamous Cell Carcinoma.

Background: GPNMB is a newly discovered tumour-promoting factor that may promote tumour cell progression by activating the PI3K/AKT pathway by EGFR. However, there are insufficient studies about GPNMB in ESCC. This study investigated the relationship between GPNMB and EGFR/PI3K pathway genes in ESCC. Methods: The expression levels of GPNMB, EGFR, p-PI3K, and Ki-67 were examined using immunohistochemistry. Statistical analysis was done by SPSS 22.0 and R. Results: GPNMB mRNA expression is higher in ESCC compared with paracancerous tissues. The expression of EGFR, PIK3CA, PIK3CB, and AKT1 was increased in GPNMB upregulated samples. GPNMB expression was positively correlated with EGFR, p-PI3K, and Ki-67 expression. GPNMB was expressed higher in the AJCC III stage, lymph node metastasis, and moderately poorly differentiated patients. EGFR was higher expressed in patients with vascular invasion; p-PI3K expression in Kazak was higher than that in Han; Ki-67 expression was higher in tumour size ≥ 3 cm. Patients with high expression of GPNMB, p-PI3K, and Ki-67 had worse OS. p-PI3K, Ki-67, nerve invasion, and lymphatic metastasis were independent risk factors, and postoperative adjuvant therapy was a protective factor in ESCC. Conclusion: As a tumour-promoting factor, GPNMB is expected to be a potential target for ESCC.

Machine Learning-Based Prediction Model of Preterm Birth Using Electronic Health Record.

Objective: Preterm birth (PTB) was one of the leading causes of neonatal death. Predicting PTB in the first trimester and second trimester will help improve pregnancy outcomes. The aim of this study is to propose a prediction model based on machine learning algorithms for PTB. Method: Data for this study were reviewed from 2008 to 2018, and all the participants included were selected from a hospital in China. Six algorisms, including Naive Bayesian (NBM), support vector machine (SVM), random forest tree (RF), artificial neural networks (ANN), K-means, and logistic regression, were used to predict PTB. The receiver operating characteristic curve (ROC), accuracy, sensitivity, and specificity were used to assess the performance of the model. Results: A total of 9550 pregnant women were included in the study, of which 4775 women had PTB. A total of 4775 people were randomly selected as controls. Based on 27 weeks of gestation, the area under the curve (AUC) and the accuracy of the RF model were the highest compared with other algorithms (accuracy: 0.816; AUC = 0.885, 95% confidence interval (CI): 0.873-0.897). Meanwhile, there was positive association between the accuracy and AUC of the RF model and gestational age. Age, magnesium, fundal height, serum inorganic phosphorus, mean platelet volume, waist size, total cholesterol, triglycerides, globulins, and total bilirubin were the main influence factors of PTB. Conclusion: The results indicated that the prediction model based on the RF algorithm had a potential value to predict preterm birth in the early stage of pregnancy. The important analysis of the RF model suggested that intervention for main factors of PTB in the early stages of pregnancy would reduce the risk of PTB.

The Relationship between MACC1/c-Met/Cyclin D1 Axis Expression and Prognosis in ESCC.

BACKGROUND: Esophageal cancer is one of the most common malignant tumors of the digestive system, with high incidence and mortality. METHODS: Immunohistochemical method was used to detect the expression of MACC1, c-Met, and cyclin D1 in ESCC and its adjacent tissues. Statistical analysis was done by SPSS 23.0. RESULTS: The high expression of MACC1 and cyclin D1 was significantly correlated with tumor size. High c-Met expression was associated with patient ethnicity. MACC1 expression was positively correlated with both c-Met and cyclin D1. c-Met expression was also positively correlated with cyclin D1. Patients with high expression of MACC1 and c-Met had worse OS; patients with high c-Met expression also had worse PFS. CONCLUSION: MACC1, c-Met, and cyclin D1 proteins are closely related to the occurrence and development of esophageal squamous cell carcinoma. MACC1 may affect the prognosis of ESCC by regulating the expression of the c-Met/cyclin D1 axis.

Expression and function analysis of CRABP2 and FABP5, and their ratio in esophageal squamous cell carcinoma.

OBJECTIVE: The purpose of this study was to explore the effect of CRABP2 and FABP5, and their ratio on prognosis in esophageal squamous cell carcinoma. METHODS: The expression data of CRABP2 in esophageal cancer in TCGA and GEO were collected by the public database GEPIA. The expression levels of CRABP2 and FABP5 were examined using immunohistochemistry. The relationship between the two proteins and related clinicopathological parameters were analyzed by χ 2 test. Survival analysis was used to investigate the effect of CRABP2 and FABP5, and their ratio on prognosis. RESULTS: Compared with normal esophageal mucosal epithelium, there was lower CRABP2 gene mRNA in the esophageal cancer tissue, and the difference was statistically significant (p < 0.01). For the expression level, no significant difference was observed in patients with stages I-IV in esophageal cancer. Immunohistochemistry showed that CRABP2 and FABP5 were both highly expressed in normal esophageal squamous epithelial cells at 100 and 94.1%, while lower in ESCC (75.6 and 58.7%). There was a significant difference in the expression between cancer and adjacent tissues (p < 0.001). No inherent relationship was manifested between the CRABP2 expression and the clinical parameters of the ESCC. The expression of FABP5 was related to lymph node metastasis (p = 0.032), the depth of invasion (p = 0.041), and the AJCC stage (p = 0.013). The ratio of CRABP2 and FABP5 was related to ethnicity (p = 0.001), nerve invasion (p = 0.031), and postoperative treatment (p = 0.038). CRABP2 is positively associated with FABP5 (r = 0.156, p = 0.041) and the ratio (r = 0.334, p = 0.000), while there was a negative correlation between FABP5 and the ratio (r = -0.269, p = 0.000). Patients with CRABP2-positive expression had a significantly longer overall survival than patients with CRABP2-negative expression (p = 0.025). CONCLUSION: CRABP2 as a suppressor factor is expected to be a potential prognosis marker for esophageal squamous cell carcinoma.

Clinical efficacy analysis of different therapeutic methods in patients with cesarean scar pregnancy.

OBJECTIVE: The purpose of this study was to analyze the clinical efficacy of five therapeutic strategies in patients with CSP. MATERIALS AND METHODS: A total of 135 CSP patients were included and divided into five groups based on the treatment they received, including transvaginal resection (Group A), laparoscopic resection (Group B), uterine arterial embolization (UAE) combined with hysteroscopic curettage (Group C), UAE combined with uterine curettage (Group D), and hysteroscopic curettage (Group E). To investigate the clinical efficacy of these strategies, intraoperative bleeding, serum ß-hCG levels and recovery time, menstruation recovery time, hormone levels at 1 month after treatment. RESULTS: Patients in group A had the lowest postoperative serum ß-hCG levels, and the shortest recovery times of both serum ß-hCG and menstruation, followed by patients in group B. Group C and D had small amount of blood loss. The hospital stays and costs were low in group E. In addition, the sex hormone levels showed no significant difference among the five groups. CONCLUSION: Our results indicated that resection surgery and UAE have good curative effects, but high hospital costs in CSP treatment. The selection of an optimal treatment regimen for CSP should be carried out based on specific conditions of the patients.

Expression and prognosis of CyclinA and CDK2 in patients with advanced cervical cancer after chemotherapy.

This study aimed to investigate the expression and prognosis of CyclinA and CDK2 in patients with advanced cervical cancer after chemotherapy. The patient history of 108 patients with advanced cervical cancer admitted to our hospital from December 2013 to January 2016 was selected as a cervical cancer group. 54 normal healthy people admitted to our hospital for physical examination in the same period were selected as the control group. Western blotting and RT-PCR were used to detect the difference between CyclinA and CDK2 proteins and mRNA expression between the two groups and the correlation between them was analyzed. The expressions of CyclinA and CDK2 in serum and the changes in detection index level of squamous cell carcinoma antigen (SCCA), carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) were observed in cervical cancer group at different stages of treatment. The correlation between the two indexes and SCCA, CEA, VEGF and the 3-year survival and prognostic significance of cervical cancer patients with different CyclinA and CDK2 expressions were analyzed. The relative expressions of CyclinA and CDK2 proteins and mRNA in the cervical cancer group were significantly higher than those in the control group (P< 0.05). Pearson correlation analysis showed a positive correlation between CyclinA and CDK2 proteins and mRNA expressions. After treatment, the expressions of CyclinA, CDK2 mRNA and SCCA, CEA and VEGF were significantly lower than those before treatment (P< 0.05). The 3-year survival rate of CyclinA and CDK2 in the high expression group was significantly lower than that of the low expression group. CyclinA and CDK2 are highly expressed in advanced cervical cancer. The expression is decreased after chemotherapy. The prognosis of both low expressions is higher and the expression is good. It can be used to predict the efficacy and prognosis of cervical cancer in the clinic.

Correction to: Angiotensin II receptor blocker LCZ696 attenuates cardiac remodeling through the inhibition of the ERK signaling pathway in mice with pregnancy­associated cardiomyopathy.

[This corrects the article DOI: 10.1186/s13578-019-0348-1.].

Angiotensin II receptor blocker LCZ696 attenuates cardiac remodeling through the inhibition of the ERK signaling pathway in mice with pregnancy-associated cardiomyopathy.

Pregnancy-associated cardiomyopathy (PAH) represents a pregnancy-associated myocardial disease that is characterized by the progression of heart failure due to marked left ventricular systolic dysfunction. Compelling evidence has highlighted the potential of angiotensin (Ang) receptor inhibitors as therapeutic targets in PAH treatment. The present study aims to elucidate the molecular mechanisms underlying Ang II receptor inhibitor LCZ696 treatment in PAH. Initially, a PAH mouse model was induced, followed by intraperitoneal injection of LCZ696. Subsequently, cardiomyocytes and fibroblasts were isolated, cultured, and treated with Ang II and LCZ696, followed by detection of the total survival rate, cardiac injury, cardiac fibrosis and apoptosis. Moreover, in order to quantify the cardiac hypertrophy and fibrosis degree of cardiac fibroblasts, the expression levels of markers of cardiac hypertrophy (ANP, ßMHC and TIMP2) and markers of fibrosis (collagen I, collagen III and TGF-ß) were evaluated. Furthermore, the potential effect of LCZ696 on the extracellular signal-regulated kinase (ERK) signaling pathway was examined. The acquired findings revealed that LCZ696 increased the total survival rate of PAH mice, but decreased cardiac injury, cardiac fibrosis, and apoptosis in vitro. LCZ696 attenuated cardiac injury induced by Ang II through the inhibition the expression of markers of cardiac hypertrophy, fibrosis and apoptosis by inhibiting ERK phosphorylation in vivo and in vitro. Altogether, LCZ676 could potentially alleviate cardiac remodeling in mice with PAH via blockade of the ERK signaling pathway activation. Our findings suggest that LCZ696 could be a potential target for PAH therapy.

EPHA3 enhances macrophage autophagy and apoptosis by disrupting the mTOR signaling pathway in mice with endometriosis.

Background: Endometriosis is a chronic fibrotic disease characterized by agonizing pelvic pain and low fertility, mainly affecting middle-aged women. The aim of the present study is to assess the potential effects of erythropoietin-producing hepatocellular carcinoma A3 (EPHA3) on endometriosis, with emphasis on the autophagy and apoptosis of macrophages via inhibition of the mammalian target of rapamycin (mTOR) signaling pathway.Methods: The mouse models of endometriosis were established followed by culturing the macrophages and macrophage transfection via the EPHA3 vector, siRNA EPHA3, and RAPA (an inhibitor of the mTOR signaling pathway). The expression of EPHA3, related factors in the mTOR signaling pathway, macrophage autophagy (autophagy-related gene 3 (Atg3), light chain 3-I (LC3-I), light chain 3-II (LC3-II) and Beclin1) and apoptosis (B-cell lymphoma-2 (bcl-2), bax and fas) were all detected and documented, respectively. The changes of autophagic lysosomes and the apoptosis of macrophages in each group following transfection were also inspected and detected.Results: The results of the in silico analysis ascertained EPHA3 to be a candidate gene of endometriosis. After successful modeling, the uterine tissues of endometriosis mice presented with a low expression of EPHA3 and activated mTOR signaling pathway. Overexpression of EPHA3 inhibited the activation of the mTOR signaling pathway, down-regulated bcl-2 expression, up-regulated the expression of Atg3, LC3-II/LC3-I, Beclin1, bax and fas, and also promoted the autophagy and apoptosis of macrophages in endometriosis mice.Conclusion: Altogether, EPHA3 could potentially promote the autophagy and apoptosis of macrophages in endometriosis via inhibition of the mTOR signaling pathway, highlighting the potential of EPHA3 as the target to treat endometriosis.

Inhibitory effect of AQP1 silencing on adhesion and angiogenesis in ectopic endometrial cells of mice with endometriosis through activating the Wnt signaling pathway.

The development mechanism of endometriosis remains unknown. Water channel aquaporin-1 (AQP1) enhances water flux across cell membranes, which is highly expressed and associated with cell migration, metastasis and angiogenesis in some human cancers. In this study, the role of the Wnt signaling pathway mediated by AQP1 in endometriosis was investigated, in a bid to provide new therapeutic targets for endometriosis. Microarray expression profiles were screened to acquired differentially expressed genes related to endometriosis. Mouse models with endometriosis were established and grouped. The level of endometriosis was evaluated by measurement of the volume of ectopic region. The expression of AQP1, pathway-related factors (Wnt1 and Wnt4), adhesion molecules (VCAM-1 and ICAM-1), invasive factors (MMP-2, MMP-9, TIMP-1 and TIMP-2), angiogenic factors (VEGF-A, VEGFR1 and VEGFR2) and apoptotic factors (Caspase-3, Caspase-9, Bax and BcL-2) was measured by RT-qPCR and western blot analysis. Furthermore, the role of AQP1 in adhesion, invasion, angiogenesis, and apoptosis of ectopic endometrial cells was determined by transfection of si-AQP1 plasmid. AQP1 was robustly expressed in endometriosis. AQP1 gene silencing alleviated the progression of endometriosis by activating the Wnt signaling pathway in mice with endometriosis. Specifically, silencing of AQP1 gene inhibited ectopic endometrial cell adhesion and invasion abilities, suppressed angiogenesis while promoted apoptosis. Collectively, the present study highlights the role of AQP1 in the regulation of the Wnt signaling pathway in endometriosis mouse models, suggesting that AQP1 could represent a new target aimed at improving the survival of patients with endometriosis.

[Assessment of two standards for diagnosis of dental caries in epidemiological survey].

OBJECTIVE: To investigate the difference between the results of using two standards(CPI and No.5 sharp probes) in the diagnosis of clinical dental caries during epidemiological survey. METHODS: The CPI and No. 5 sharp probes were used respectively for the diagnosis of clinical dental caries in the same group of children aged 12 by the two examiners. This group of children were reexamined after 14 days to compare the consistency and the rate of coincidence of the location of teeth and caries average and the prevalence rate of dental caries diagnosed by the two kinds of probes. RESULTS: The difference between the location of teeth with caries examined by the two different standards respectively by the same examiner was not statistically significant. The difference between caries average and the prevalence rate of dental caries examined with the two different standards respectively by the same examiner was also not statistically significant. The difference between the outcomes of the crossover comparison by the two examiners showed no statistical significance. CONCLUSIONS: There was no significant difference between the two diagnostic standards for caries. The outcomes of the diagnosis based on both standards shows a similarity. CPI could replace No. 5 sharp probes due to its convenience in large scale of oral health survey.

Regulation of caspase-3 activity by insulin in skeletal muscle cells involves both PI3-kinase and MEK-1/2.

A hallmark of skeletal muscle atrophy is increased activities of several proteolytic systems, including caspase-3. We have previously shown that conditions involving insulin deficiency or insulin resistance increase both overall protein degradation and caspase-3-mediated actin cleavage. In the present experiments, we examined how insulin regulates caspase-3 activity in L6 myotubes. Reducing the serum concentration in the culture media from 2 to 0.5% overnight increased caspase-3 activity and actin cleavage. Addition of insulin to proteolytically active cells attenuated both responses within 4 h. Individually, inhibitors of either phosphatidylinositide 3-kinase (PI3K) or MEK1/2 partially blocked the insulin-induced reduction in caspase-3 activity; in combination, the inhibitors completely prevented insulin from attenuating caspase-3 activity. Insulin suppressed caspase-3 activity by a complex mechanism that included direct inhibition due to an increased interaction between caspase-3 and cellular inhibitor of apoptosis-1 and indirect inhibition via phosphorylation (i.e., inactivation) of the proapoptotic protein Bad, which participates in the intrinsic (i.e., mitochondrial) apoptosis activation cascade. Unlike other cell types, the phosphorylation of Bad Ser112 was mediated by the PI3K/Akt pathway rather than the MEK/ERK/ribosomal S6 protein kinase pathway. In summary, our findings indicate that insulin regulates caspase-3 activity by a multistep process that is unique to skeletal muscle, thus providing insights about the muscle-specific nature of the atrophy process.