Bao-Song Zhu

Blocking NF-κB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells. METHODS MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment. Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α. RESULTS The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α. CONCLUSION The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.

LY294002 induces p53-dependent apoptosis of SGC7901 gastric cancer cells.

AbstractAim:To study the effects of LY294002, an inhibitor of class I phosphatidylinositol 3-kinase (PI3K), on proliferation and apoptosis of SGC7901 gastric cancer cells.Methods:The MTT assay was used to determine the cytotoxic effects of LY294002. Cell cycle distribution was analyzed using flow cytometry and apoptosis was assessed using flow cytometry analysis after staining DNA with propidium iodide. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Expression of p53 and PUMA was determined using real-time RT-PCR and Western blotting analysis.Results:The viability of SGC7901 cells was significantly reduced by LY294002 treatment. Expression of p53 and PUMA was induced, and mitochondrial membrane potential collapsed after treatment with LY294002. LY294002 induced apoptotic cell death.Conclusion:Activation of the p53 pathway is involved in LY294002-induced SGC7901 cell death.

SN50 enhances the effects of LY294002 on cell death induction in gastric cancer cell line SGC7901.

Introduction In the previous study, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 induced SGC7901 cell death in vitro. We did not know whether SN50, which is a specific inhibitor of nuclear factor κB (NF-κB), could increase the cell death induction of gastric cancer of LY294002 in vitro, and we also wanted to know the mechanism of it, which might be applied to clinical tumor therapy. Material and methods The 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxic effects of the drugs. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Hoechst 33258 staining was used to detect apoptosis and necrosis morphological changes after LY294002 and/or SN50 treatment. Expression of p53, PUMA and Beclin1 were determined with real-time polymerase chain reaction (RT-PCR) analysis. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after LY294002 and/or SN50 treatment. Results In this study, we found that treating the human gastric cancer cells SGC7901 with SN50 could significantly enhance the effects of LY294002 on inducing cell death after 24 h, compared to the control group (p < 0.05). Detection of mitochondrial potential and transmission electron microscopic examination indicated that the rate of cell death increased progressively. The expression of p53, PUMA and Beclin1 was up-regulated. Conclusions The NF-κB inhibitor SN50 could enhance the role of LY294002 on inducing cell death of human gastric cancer cells SGC7901, which might be a promising new approach to gastric cancer therapy.

Effects of small interfering RNA inhibit Class I phosphoinositide 3-kinase on human gastric cancer cells

AIM To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class I phosphoinositide 3-kinase (Class I PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells. METHODS We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant. RESULTS The viability of gastric cancer cells was inhibited after siRNA targeting to the Class I PI3K blocked Class I PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class I PI3K induced apoptosis in SGC7901 cells. The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity, indicating increased formation of autophagosomes. The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low. After incubating with adenovirus PI3K(I)-RNAi-GFP (50 MOI), Beclin-1, LC3, and p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)-RNAi-GFP (50 MOI) treatment. Control cells showed a round shape and contained normal-looking organelles, nucleus, and chromatin, while adenovirus PI3K(I)-RNAi-GFP (50 MOI)-treated cells exhibited the typical signs of autophagy. CONCLUSION After the Class I PI3K signaling pathway has been blocked by siRNA, the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

A comparative study of small intestinal perforation secondary to foreign body and other non-traumatic causes

BACKGROUND Little was known about gastrointestinal perforation secondary to foreign body in adults, which was only documented by several case series reports. The aim of this study was to characterize it with comparative methods. METHODS A retrospective study was conducted on twenty patients with the diagnosis of gastrointestinal perforation secondary to foreign body between January 2003 and October 2013. The perforations were all located in the small intestine and compared to eighty-seven patients with non-traumatic small intestinal perforation. RESULTS 35% of the patients in the foreign body group were over 65 years of age, which is much higher than the local elderly population ratio (p=0.002). In the foreign body group, more patients presented without diffuse abdominal physical signs (p=0.008) and preoperational CT scans had higher accuracy (p=0.027). Perforation repair was performed more often (p=0.024). Mean MPI was 19.9 and the morbidity rate was 35%, significantly lower than in the cases of other causes (p=0.001, 0.041). Mean duration of hospitalization was 11.5 days and was shorter compared to other causes (p=0.038). CONCLUSION Clinical performance of small intestinal perforation secondary to foreign body is atypical, and preoperative diagnosis relies on CT scans. Primary perforation closure is safe and effective, and relatively better outcomes can be achieved.