Whole serum as cell protection reagent in cell shipment at low or room temperature.

To maintain cell viability and characteristics physiological over long time or long-distance cell shipment is critical to promote the collaborative efforts in the research field of cell biology. Herein, we investigated the possibility of different concentration of fetal bovine serum as transport cell reagent. Cell suspension of three different mammalian cell lines were transported in 1.5 mL tube under different temperature conditions. Cell viability was closely related to environmental temperature and shipment time. No significant difference of cell survival rate was observed between 2-8 â„ƒ and 8-16 â„ƒ groups, under these two temperature conditions, reagent containing above 50 % FBS showed the best protection effect to maintain over 80 % cell survival rate. After 10 days of cell shipment under 2-16 â„ƒ environmental temperature, C3H10 cells exhibited the same multiple differentiation ability, 143B cells had the same capability of proliferation, migration and invasion, LX-2 cells showed the same activation state with TGF-ß stimulation. Three cell lines maintained their primary characteristics after long time cell shipment. This entire shipment process does not require the maintenance of specific temperatures, humidity and container, providing a low-costing and convenient way for cell shipment between long distance laboratories.

A case report of a giant ileocecal cystic prolapse through the anus and literature review.

Intussusception refers to the invagination of a proximal loop of the bowel into an adjacent distal segment. This condition is rare in adults, especially when it involves a complete folding of the ileocecal area out of the body cavity. Meanwhile, enterogenous cysts are congenital malformations that are largely identified in childhood following symptoms of bowel obstruction. While surgical treatment is ultimately required for both diseases, deciding on the type of surgery and the right time to operate can be a challenge for clinicians. It is especially difficult to decide on treatment for an adult with the coincidental occurrence of both conditions and no definitive pathologic diagnosis prior to surgery. Here, we present the case study of a 19-year-old female patient who presented with a prolapsed anus due to intussusception caused by a large ileocecal mass. The patient was admitted to the emergency department with a "massive anal mass." She remained symptomatic after receiving conventional conservative treatment and had to undergo emergency surgery after developing an intestinal obstruction. While the patient's intraoperative condition also confirmed the preoperative CT findings, the situation became more complicated during surgery. The postoperative pathological report indicated the presence of an enterogenous cyst. After recovery from surgery, the patient was successfully discharged. Intussusception or intestinal obstruction caused by an intestinal mass is a surgical indication, and removal is the only way to cure the condition. This case study provides a helpful reference for general surgeons, especially anorectal surgeons, imaging physicians, and pathologists, and informs the diagnosis and treatment of this patient population.

[Retracted] Hypoxia­induced mitochondrial translocation of DNM1L increases mitochondrial fission and triggers mPTP opening in HCC cells via activation of HK2.

Subsequently to the publication of the above paper, the authors have retrospectively realized that they used the human normal liver cell, line L­02, for the experiments reported in this study instead of the intended hepatocellular cell line, Huh­7. Consequently, the results and conclusions reported in this article must be considered to lack reliability. Therefore, the authors have requested that the article be retracted from the publication. The authors apologize to the Editor and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 42: 1125­1132, 2019; DOI: 10.3892/or.2019.7213].

Hypoxia­induced mitochondrial translocation of DNM1L increases mitochondrial fission and triggers mPTP opening in HCC cells via activation of HK2.

Disturbed mitochondrial dynamics are closely associated with the progression of different types of cancer including hepatocellular carcinoma (HCC). However, the manner in which mitochondrial dynamics are regulated in HCC remains largely unclear. In the present study, via immunofluorescence, real­time PCR and western blot analysis, the effects of dynamin­1­like (DNM1L) on mitochondrial translocation and the mitochondrial permeability transition pore (mPTP) were investigated in HCC cells under hypoxic conditions, and the underlying molecular mechanisms were explored. Our data revealed that hypoxic treatment decreased the mitochondrial membrane potential, elevated generation of reactive oxygen species, and promoted mitochondrial fission in a DNM1L­dependent manner. Instead of changing the levels of DNMlL, hypoxia increased the translocation of DNM1L to mitochondria, leading to excessive mitochondrial fission and decreased the viability of HCC cells. In addition to the effects on mitochondrial dynamics, DNM1L also regulated mPTP opening in HCC. IP analysis revealed that DNM1L interacted with the enzyme hexokinase 2 (HK2), and was involved in its phosphorylation, resulting in HK2 detachment from the mitochondria and consequently mPTP opening.

Protective effect of apoptosis signal-regulating kinase 1 inhibitor against mice liver injury.

OBJECTIVE: To explore the protective effect and its molecular mechanism of apoptosis signal-regulating kinase 1 (ASK1) inhibitor (GS-459679) on acetaminophen-induced liver injury in mice. METHODS: The model of liver injury was established by administration of acetaminophen (APAP) (300 mg/kg, i.p.) on C57BL/6 mice. Forty-eight male C57BL/6 mice were randomly divided into four groups, consisting of control group, GS group (GS-459679, 30 mg/kg, i.p.), APAP-induced group, and GS combined with APAP-induced group. For GS combined with APAP-induced group, mice were treated with GS 30 min prior to administration of APAP. After mice were euthanized at 6 h or 12 h, respectively, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed, and mRNA levels of TNF-α, IL-6 and IL-1ß were tested. The activity of glutathione (GSH), oxidized GSH (GSSG) and malondialdehyde were quantified. In addition, ASK1, P-ASK1, JNK and P-JNK protein levels were tested in all groups. RESULTS: The ASK1 and P-ASK1 levels were up-regulated in APAP-induced group. Compared to the control group, serum levels of ALT and AST, and mRNA levels of TNF-α, IL-6 and IL-1ß were increased in APAP-induced group. Meanwhile, the levels of MAD and GSSG, and the ratio of GSSG/GSH were higher and the JNK was activatedin APAP-induced group compared with that in control group. However, compared to APAP-induced group, GS combined with APAP-induced group displayed a decrease of protein expression levels of ASK1, P-ASK1 and P-JNK, a reduction of serum levels of ALT and AST, a decrease in TNF-α, IL-6 and IL-1ß mRNA levels, and a low ration of GSSG/GSH. CONCLUSIONS: GS-459679 treatment effectively down-regulates ASK1 and P-ASK1 expression. Addition of GS-459679 decreases the generation of liver metabolites and inflammatory factors, reduces oxidative stress reaction, inhibits JNK activation, and then protects the responsiveness to APAP-induced liver injury.

Saturated free fatty acid sodium palmitate-induced lipoapoptosis by targeting glycogen synthase kinase-3ß activation in human liver cells.

BACKGROUND: Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD). AIM: The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms. METHODS: Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48 h with or without lithium chloride, a glycogen synthase kinase-3ß (GSK-3ß) inhibitor, or GSK-3ß shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression. RESULTS: The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3ß protein, which was indicated by dephosphorylation of GSK-3ß at Ser-9. However, inhibition of GSK-3ß activity with lithium chloride treatment or knockdown of GSK-3ß expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3ß expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3ß inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response. CONCLUSIONS: The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3ß activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3ß inhibition may be a potential therapeutic target to control NAFLD.

Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells.

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.

Silence of p15 expression by RNAi enhances cisplatin resistance in hepatocellular carcinoma cells.

The insensitivity of hepatocellular carcinoma to chemotherapy is associated with alternation in tumor cell cycling. This current study was designed to investigate the impact of p15 silencing on the sensitivity of Human hepatocellular carcinoma HepG2 cells to cisplatin. HepG2/CDDP/1.6 and HepG2/CDDP/2.0 cells were induced by culture with increased doses of cisplatin and their sensitivities to cis-Diamine dichloroplatinum (CDDP) were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The impacts of p15 silencing on the cell cycling and P-gp expression were characterized by flow cytometry, RT-PCR and Western blot assays, respectively. Knockdown of p15 expression dramatically reduced the relative levels of p15 expression and the frequency of phase G1, promoting cell cycling. On the other hand, knockdown of p15 expression significantly up-regulated the expression of P-glycoprotein (P-gp) in HepG2/CDDP/2.0 cells, associated with the increased resistance of HepG2 cells to CDDP in vitro. In conclusion, the p15 may be a critical regulator of the development of CDDP resistance in HepG2 cells.