Suberoylanilide hydroxamic acid upregulates reticulophagy receptor expression and promotes cell death in hepatocellular carcinoma cells

BACKGROUND Hepatocellular carcinoma(HCC)is a common clinical condition with a poor prognosis and few effective treatment options.Potent anticancer agents for treating HCC must be identified.Epigenetics plays an essential role in HCC tumorigenesis.Suberoylanilide hydroxamic acid(SAHA),the most common histone deacetylase inhibitor agent,triggers many forms of cell death in HCC.However,the underlying mechanism of action remains unclear.Family with sequence similarity 134 member B(FAM134B)-induced reticulophagy,a selective autophagic pathway,participates in the decision of cell fate and exhibits anticancer activity.This study focused on the relationship between FAM134B-induced reticulophagy and SAHA-mediated cell death.AIM To elucidate potential roles and underlying molecular mechanisms of reticulophagy in SAHA-induced HCC cell death.METHODS The viability,apoptosis,cell cycle,migration,and invasion of SAHA-treated Huh7 and MHCC97L cells were measured.Proteins related to the reticulophagy pathway,mitochondria-endoplasmic reticulum(ER)contact sites,intrinsic mitochondrial apoptosis,and histone acetylation were quantified using western blotting.ER and lysosome colocalization,and mitochondrial Ca^(2+)levels were characterized via confocal microscopy.The level of cell death was evaluated through Hoechst 33342 staining and propidium iodide colocalization.Chromatin immunoprecipitation was used to verify histone H4 lysine-16 acetylation in the FAM134B promoter region.RESULTS After SAHA treatment,the proliferation of Huh7 and MHCC97L cells was significantly inhibited,and the migration and invasion abilities were greatly blocked in vitro.This promoted apoptosis and caused G1 phase cells to increase in a concentration-dependent manner.Following treatment with SAHA,ER-phagy was activated,thereby triggering autophagy-mediated cell death of HCC cells in vitro.Western blotting and chromatin immunoprecipitation assays confirmed that SAHA regulated FAM134B expression by enhancing the histone H4 lysine-16 acetylation in the FAM134B promoter region.Further,SAHA disturbed the Ca^(2+)homeostasis and upregulated the level of autocrine motility factor receptor and proteins related to mitochondria-endoplasmic reticulum contact sites in HCC cells.Additionally,SAHA decreased the mitochondrial membrane potential levels,thereby accelerating the activation of the reticulophagy-mediated mitochondrial apoptosis pathway and promoting HCC cell death in vitro.CONCLUSION SAHA stimulates FAM134B-mediated ER-phagy to synergistically enhance the mitochondrial apoptotic pathway,thereby enhancing HCC cell death.

Effect of Danshao Huaxian capsule on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibrotic liver of rats

AIM： To investigate the effects of Danshao Huaxian （DSHX） capsules, a preparation of traditional Chinese medicine, on the expression of matrix metalloproteinase-1 （MMP-1）, and tissue inhibitor of metalloproteinase-1 （TIMP-1） in the fibrous livers of rats. METHODS： Eighty male Wistar rats were randomly divided into normal control group （group A）, CCl4-induced hepatic fibrosis group （group B）, non-DSHX-treated group （group C）, low dose-treated group （group D）, and high dose-treated group （group E）. Fibrous liver models in rats were induced by subcutaneous injection of CCI4, oral administration of alcohol and high-lipid/low-protein diet for 8 wk. After the models were established, the rats in groups D and E were orally given a low dose （0.5 g/kg） and a high dose （1.0 g/kg） of DSHX daily for 8 wk, respectively. Then, the liver indexes, serum hyaluronic acid （HA） and alanine aminotransferase （ALT） were examined. The degree of hepatic fibrosis was evaluated by optical microscopy. Hydroxyproline （Hyp） in the urine was determined, and the expression of MMP-1 and TIMP-1 was detected by immunohistochemical techniques. RESULTS： In groups D and E, the liver indexes, levels of serum HA and ALT reduced and development of hepatic fibrosis weakened significantly. The urinary Hyp and expression of MMP-1 in the liver tissues elevated, but the expression of TIMP-1 decreased obviously, as compared to groups B and C.CONCLUSION： DSHX enhances the expression of MMP-1 but decreases that of TIMP-1 in liver tissues of CCI4-induced hepatic fibrotic rats, which may result in its elevated activity that contributes to fighting against hepatic fibrosis.

TRANSFORMING GROWTH FACTOR-β1 AND SMAD4 SIGNALING PATHWAY DOWN-REGULATES RENAL EXTRACELLULAR MATRIX DEGRADATION IN DIABETIC RATS

Objective To investigate the role of transforming growth factor-131 （TGF-β1）/Smad4 pathway in development of renal fibrosis in streptozotocin （STZ）-induced diabetic nephropathy （DN） rats and explore its possible mechanism. Methods Male Wistar rats weighing 180-220 g were divided into 5 groups： group A （ normal control）, group B [ diabetes mellitus （DM） 2 weeks ], group C （ DM 4 weeks）, group D （ DM 8 weeks）, and group E （ DM 16 weeks）. Except for the normal control group, other groups were induced DM by single injection of STZ （55 mg/kg） respectively. Blood glucose level, serum creatinine, and 24-hour urine protein were examined. Expressions of TGF-β1 and Smad4 protein and mRNA in kidney were detected using immunohistochemical technique, Western blot, and real-time PCR. mRNA expressions of stromelysin-1 （ MMP-3 ）, tissue inhibitor of metalloproteinase-1 （ TIMP-1 ）, and collagen Ⅲ in kidney were also detected by real-time PCR. Results The levels of blood glucose, serum creatinine, and 24-hour urine protein in rats of group B, C, D, and E were higher than those of the control group. With the progression of renal fibrosis, the expressions of TGF-β1 and Smad4 protein and mRNA in kidney of diabetic rats elevated. In addition, the renal MMP-3 mRNA expression diminished in diabetic rats, while TIMP-1 and collagen Ⅲ mRNA increased. Conclusions In STZ-induced diabetic rats, the TGF-β1/Smad4 appears to play an important role in renal fibrosis of DN. The increased expression of TGF-β1 and Smad4 might result in the transcriptional regulation of downstream target genes of TGF-β1/Smad4 pathway, which contributes to the progression of renal fibrosis in diabetic rats.

Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes

BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum(ER)stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODS BRL-3A cells were treated with varying doses of dithiothreitol(DTT),and their viability and apoptosis were quantified by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry.The expression of ER stress-and apoptosis-related proteins was detected by Western blot analysis.The protease activity of calpain-2 was determined using a fluorescent substrate,Nsuccinyl-Leu-Leu-Val-Tyr-AMC.Intracellular Ca^2+content,and ER and calpain-2 co-localization were characterized by fluorescent microscopy.The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTS DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis.DTT treatment significantly upregulated 78 kDa glucose-regulated protein,activating transcription factor 4,C/EBP-homologous protein expression by>2-fold,and enhanced PRKR-like ER kinase phosphorylation,caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence.DTT treatment also significantly increased intracellular Ca^2+content,calpain-2 expression,and activity by>2-fold in BRL-3A cells.Furthermore,immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2.Moreover,calpain-2 silencing to decrease calpain-2 expression by 85%significantly mitigated DTT-enhanced calpain-2 expression,caspase-12 cleavage,and apoptosis in BRL-3A cells.CONCLUSION The data indicated that Ca^2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol

BACKGROUND Endoplasmic reticulum(ER)stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases.Previous studies have revealed that endoplasmic reticulophagy(ER-phagy)promotes the selective clearance of damaged ER fragments during ER stress,playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis.Family with sequence similarity 134 member B(FAM134B)is a receptor involved in ER-phagy that can form a complex with calnexin(CNX)and microtubule-associated protein 1 light chain 3(LC3).The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis.However,the precise regulatory mechanisms remain unclear.AIM To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A(BRL-3A)rat hepatocytes and the potential regulatory mechanisms.METHODS ER stress-related hepatocyte apoptosis was induced using dithiothreitol(DTT).Proteins related to ER stress and autophagy were measured with western blotting.Protein complex interactions with FAM134B were isolated by co-immunoprecipitation.ER-phagy was evaluated in immunofluorescence experiments.Cell cycle distribution and apoptosis were measured by flow cytometry.Mitochondrial Ca^(2+) levels were evaluated by the co-localization of intracellular Ca^(2+)-tracker and Mitotracker.The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells.RESULTS ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment.Furthermore,co-immunoprecipitation confirmed an interaction between FAM134B,CNX,FAM134B,and LC3 in BRL-3A cells.Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment,but increased after long-term treatment.Mitochondrial Ca2+levels and apoptotic rates were dramatically elevated,and more cells were arrested in the G1 stage after short-term DTT treatment;however,these decreased 48 h later.Moreover,FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress.CONCLUSION FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway.Our findings provide new evidence highlighting the importance of FAM134Bmediated ER-phagy in attenuating hepatocyte apoptosis.