Apoptosis-related Molecules Caspase-3 Research Articles

Modulation of proliferation, apoptosis and inflammation of Caco-2 epithelial cells and THP-1 macrophage-like monocytes in LPS stimulated co-culture model.

Epithelial cells and macrophages play major roles in modulating the state of inlammatory response regulated by intracellular signaling pathways. The aim of this study was to characterize changes in cell proliferation, apoptosis and inflammation related intracellular signalling pathways MAPKs and NF-κB in Caco-2 epithelial cells and THP-1 macrophage-like monocytes contacted and filter-seperated co-cultures in the presence of LPS stimulation. We assessed the apoptosis and inflammation by measuring caspase-3 activity, TNF-α and IL-10 cytokines, total and phosphorylated forms of intracellular signalling pathway molecules p53, JNK, Jun, ERK, p38, NF-κB p65 and IkB. The contacted co-culture of Caco-2 and THP-1 cells represented higher levels of JNK, jun and p38 MAPK pathway proteins associated with cell proliferation, whereas apoptosis related molecule caspase-3 and p53 increased in the filter-separated co-culture condition. Also, the contacted co-culture condition led to proinflammatory changes in NF-κB signalling pathways and cytokine responses with high phosphorylated NF-κB p65 and TNF-α, and low I-κB and IL-10 levels. Epithelia - monocytes co-culture stimulated with LPS regulated cell proliferation and inflammation in a contact dependent manner, whereas apoptosis was associated with non-contacted cell culture condition. Thus, co-culture models are important models for explaining the immunopathologies in the mucosal areas (Fig. 5, Ref. 30).

Infusion of Mesenchymal Stem Cells Combined with Epithelial Progenitor Cells Promotes Injured Intestinal Repairing after Hematopoietic Cell Transplantation

Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis

A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

Maspin enhances cisplatin chemosensitivity in bladder cancer T24 and 5637 cells and correlates with prognosis of muscle-invasive bladder cancer patients receiving cisplatin based neoadjuvant chemotherapy.

BackgroundMaspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Chemotherapeutic insensitivity is one of major obstacles to effectively treating muscle invasive bladder cancer (MIBC). This study was conducted to investigate the role and probable mechanism of Maspin enhancing cisplatin chemosensitivity of bladder cancer in vitro and MIBC patients.MethodsMaspin expression was quantified by qRT-PCR in two MIBC cell lines (T24 and 5637). After successful established Maspin overexpression model by lipidosome transfection, MTT and cell apoptosis assay were used to assess the MIBC’s cisplatin sensitivity. Western blot method was used to test PI3K/ AKT/mTOR signal passway and apoptosis related molecules Caspase3 and Bcl-2. Additionally, we evaluated Maspin expression and prognosis in 62 MIBC cases who underwent cisplatin based neoadjuvant chemotherapy (NACT) using immunohistochemistry.ResultUpregulate Maspin expression could enhance the chemosensitivity induced by cisplatin in T24 and 5637 cell lines. The cell viability, cloning ability and IC50 were reduced while apoptosis rate was upregulated when cells were transfected Maspin. Phospho(p)-AKT, PI3K, mTOR, and Bcl-2 expression were significantly decreased, whereas Caspase3 was greatly increased in the Maspin group. In the clinic study, there was significant correlation between Maspin expression and overall survival (OS) and progression-free survival (PFS) rate in MIBC patients who received cisplatin based NACT.ConclusionMaspin could enhance cisplatin chemosensitivity in T24 and 5637 cell lines. Its expression correlated with prognosis of MIBC patients who received cisplatin based neoadjuvant chemotherapy.

Abstract 138: Novel PAK4 allosteric modulators (PAMs) provide potential therapeutic options in human gastric cancer

Abstract Expression of PAK4 (p21-activated serine/threonine kinase 4) is associated with gastric tumorigenesis. Overexpression of PAK4 is correlated with metastasis of gastric cancer and in part confers cisplatin resistance in gastric cancer patients. We transduced the human gastric cancer cell line AGS with CRISPR/cas9 targeting PAK4, and observed reduced cell proliferation and motility. Novel PAK4 allosteric modulators (PAMs) were evaluated for their potential therapeutic use for gastric cancer. A panel of human gastric cancer cell lines was screened with four PAMs (KPT-7189, -7349, -7657, -8752). MTS assay showed inhibition of cell viability by PAMs in 6 of 10 gastric cancer cell lines (IC50 ranged from 50 to 5000 nM). Cell cycle analysis and annexin V assay indicated a combination of cell growth arrest at G1 phase and cell apoptosis after treatment with PAMs. Furthermore, PAMs diminished expression levels of PAK4, induced apoptosis-related molecule caspase-3, and suppressed PI3K/Akt and MEK/Erk signaling. Wound assay showed inhibition of cell migration and soft agar assay demonstrated suppression of colony formation for gastric cancer cells treated with PAMs. Mouse xenograft models are currently under investigation. Remarkably, PAMs in combination with cisplatin induced cell death in cisplatin-resistant gastric cancer cells in a synergistic manner. This study demonstrates PAMs have anti-tumor activity against gastric cancer cells and suggests their potential use for the treatment of gastric cancer. Citation Format: Wenwen Chien, Jinfen Xiao, Ling-Wen Ding, Muhammad Ikhsan B. Muzakar, Qiao-Yang Sun, Sigal Gery, William Senapedis, Sharon Shacham, Erkan Baloglu, H. Phillip Koeffler. Novel PAK4 allosteric modulators (PAMs) provide potential therapeutic options in human gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 138. doi:10.1158/1538-7445.AM2015-138

TIPE2 Inhibits Lung Cancer Growth Attributing to Promotion of Apoptosis by Regulating Some Apoptotic Molecules Expression.

Recent studies found that TIPE2 was involved in cancer development. However, little is known about TIPE2 in lung cancer. Our study aims to clarify the role of TIPE2 in lung carcinogenesis. We examined the expression of TIPE2 in lung squamous cancer (LSC), small cell lung cancer and lung adenocarcinoma (AdC) tissues and found that TIPE2 expression was lost in small cell lung cancer, compared with adjacent non-tumor tissues. Overexpression of TIPE2 significantly inhibited the growth of lung cancer cell H446 in vitro and even suppressed tumor formation in vivo. Flow cytometry analysis found TIPE2 overexpression promoted apoptosis of H446. In TIPE2 over-expression cells, caspase-3, caspase-9, and Bax were significantly up-regulated while Bcl-2 was down-regulated. Moreover, coincident results were shown by immunohistochemistry in tumors from nude mice. TIPE2 inhibited the phosphorylation of Akt, while promoting the phosphorylation of P38, but had no effect on IκBα and ERK pathway. Taken together, TIPE2 promoted lung cancer cell apoptosis through affecting apoptosis-related molecules caspase-3, caspase-9, Bcl-2 and Bax, possibly via regulating P38 and Akt pathways, indicating that TIPE2 might be a novel marker for lung cancer diagnosis and therapy.