Bcl‐2 hijacks the arsenic trioxide resistance in SH‐SY5Y cells

Aresenic trioxide (ATO) is proven to be active against leukaemia cells by inducing apoptosis and differentiation. Even though ATO could effectively induce remissions of leukaemia cells, the drug resistance was observed occasionally. To further dissect the mechanism of ATO resistance, we selected the ATO‐resistant SH‐SY5Y cells and found that Bcl‐2 controlled the sensitivity of ATO in SH‐SY5Y cells. We report that necroptosis, autophagy, NF‐ƘB and MAPK signalling pathway are not involved in ATO‐induced apoptosis. Moreover, the ATO‐resistant cells showed distinct mitochondrial morphology compared with that of ATO‐sensitive cells. Intriguingly, nude mice‐bearing ATO‐sensitive cells derived xenograft tumours are more sensitive to ATO treatment compared with that of ATO‐resistant cells. These data demonstrate that cancer cells can acquire the ATO‐resistance ability by increasing the Bcl‐2 expression.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

Bcl‐xL expression following articular cartilage injury and its effects on the biological function of chondrocytes

This study aimed to investigate the expression of B‐cell lymphoma‐extra large (Bcl‐xL) in cartilage tissues following articular cartilage injury and to determine its effects on the biological function of chondrocytes. A total of 25 necrotic cartilage tissue samples and 25 normal tissue samples were collected from patients diagnosed with osteoarthritis at our hospital from December 2015 to December 2018. The mRNA expression levels of Bcl‐xL, caspase‐3, and matrix metalloproteinase‐3 (MMP‐3) in the normal and necrotic tissues were examined via quantitative polymerase chain reaction, and their protein expression levels were detected via western blotting. The expression levels of Bcl‐xL, insulin‐like growth factor‐1 (IGF‐1), and bone morphogenetic protein (BMP) were significantly lower but those of caspase‐3, MMP‐3, interleukin‐1β (IL‐1β), and chemokine‐like factor 1 (CKLF1) levels were markedly higher in necrotic cartilage tissues than in normal tissues. Following cell transfection, the expression levels of Bcl‐xL, IGF‐1, and BMP were remarkably higher but those of caspase‐3, MMP‐3, IL‐1β, and CKLF1 were notably lower in the Si‐Bcl‐xL group than in the NC group. The Si‐Bcl‐xL group showed significantly lower cell growth and noticeably higher apoptosis rate than the NC group (normal control group). The expression of Bcl‐xL is reduced following articular cartilage injury, and this reduction promotes the proliferation and inhibits the apoptosis of chondrocytes. Therefore, Bcl‐xL could serve as a relevant molecular target in the clinical practice of osteoarthritis and other diseases causing cartilage damage.     

Alpha‐fetoprotein accelerates the progression of hepatocellular carcinoma by promoting Bcl‐2 gene expression through an RA‐RAR signalling pathway

Previous studies have found that alpha‐fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA‐RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA‐induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl‐2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down‐regulation of Bcl‐2; the opposite effect was observed in AFP gene‐transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non‐cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl‐2. Our data reveal a novel mechanism through which AFP regulates Bcl‐2 expression and further suggest that AFP may be used as a novel target for treating HCC.     

circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

Renal ischaemia‐reperfusion (RI/R) injury is one major pathological state of acute kidney injury (AKI) with a mortality rate ranking 50% to 80%. MiR‐144‐5p acts as a molecular trigger in various diseases. We presumed that miR‐144‐5p might be involved RI/R injury progression. We found that RI/R injury decreased miR‐144‐5p expression in rat models. MiR‐144‐5p downregulation promoted cell apoptosis rate and activated Wnt/β‐catenin signal in RI/R injury rats. By performing bioinformatic analysis, RIP, RNA pull‐down, luciferase reporter experiments, we found that circ‐AKT3 sponged to miR‐144‐5p and decreased its expression in RI/R injury rats. Moreover, we found that circ‐AKT3 promoted cell apoptosis rate and activated Wnt/β‐catenin signal, and miR‐144‐5p mimic reversed the promotive effect of circ‐AKT3 in rat models. We also found that circ‐AKT3 increased the oxidative stress level in rat models. In conclusion, our study suggests that the circAKT3 is involved RI/R injury progression through regulating miR‐144‐5p/Wnt/β‐catenin pathway and oxidative stress.     

BCL‐XL exerts a protective role against anemia caused by radiation‐induced kidney damage

Studies of gene‐targeted mice identified the roles of the different pro‐survival BCL‐2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti‐cancer agents. We investigated the role of BCL‐XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL‐XL exclusively in non‐hematopoietic tissues to prevent anemia caused by BCL‐XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ‐irradiation (TBI) and genetic loss of Bcl‐x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL‐XL in the adult kidney and inform on the use of BCL‐XL inhibitors in combination with DNA damage‐inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage‐inducing anti‐cancer therapy plus a BCL‐XL inhibitor could be tolerated in mice, at least when applied sequentially.     

UBE2C triggers HIF‐1α‐glycolytic flux in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy in Taiwan. Therefore, refining the diagnostic sensitivity of biomarkers for early‐stage tumours and identifying therapeutic targets are critical for improving the survival rate of HNSCC patients. Metabolic reprogramming contributes to cancer development and progression. Metabolic pathways, specifically, play a crucial role in these diverse biological and pathological processes, which include cell proliferation, differentiation, apoptosis and carcinogenesis. Here, we investigated the role and potential prognostic value of the ubiquitin‐conjugating enzyme E2 (UBE2) family in HNSCC. Gene expression database analysis followed by tumour comparison with non‐tumour tissue showed that UBE2C was upregulated in tumours and was associated with lymph node metastasis in HNSCC patients. Knockdown of UBE2C significantly reduced the invasion/migration abilities of SAS and CAL27 cells. UBE2C modulates glycolysis pathway activation and HIF‐1α expression in SAS and CAL27 cells. CoCl₂ (HIF‐1α inducer) treatment restored the expression of glycolytic enzymes and the migration/invasion abilities of UBE2C knockdown cells. Based on our findings, UBE2C expression mediates HIF‐1α activation, increasing glycolysis pathway activation and the invasion/migration abilities of cancer cells. UBE2C may be an independent prognostic factor and a therapeutic target in HNSCC.     

Inhibition of PKC‐δ reduce rhabdomyolysis‐induced acute kidney injury

Despite extensive research, the mechanisms underlying rhabdomyolysis‐induced acute kidney injury (AKI) remain largely elusive. In this study, we established both cell and murine models of rhabdomyolysis‐induced AKI by using myoglobin and glycerin, respectively, and provided evidence that protein kinase Cδ (PKC‐δ) was activated in both models and subsequently promoted cell apoptosis. Moreover, we found that this detrimental effect of PKC‐δ activation can be reversed by its pharmaceutical inhibitor rottlerin. Furthermore, we detected and confirmed the existence of PKC‐δ‐mediated myoglobin‐induced cell apoptosis and the expression of TNF‐α and IL1‐β via regulation of the p38MAPK and ERK1/2 signalling pathways. In summary, our research revealed the role of PKC‐δ in renal cell apoptosis and suggests that PKC‐δ is a viable therapeutic target for rhabdomyolysis‐induced AKI.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Sildenafil improves radiation‐induced oral mucositis by attenuating oxidative stress,NF‐κB,ERK and JNK signalling pathways

Radiation‐induced oral mucositis is a common and dose‐limiting complication of head and neck radiotherapy with no effective treatment. Previous studies revealed that sildenafil, a phosphodiesterase 5 inhibitor, has anti‐inflammatory and anti‐cancer effects. In this study, we investigated the effect of sildenafil on radiation‐induced mucositis in rats. Two doses of radiation (8 and 26 Gy X‐ray) were used to induce low‐grade and high‐grade oral mucositis, separately. A control group and three groups of sildenafil citrate‐treated rats (5, 10, and 40 mg/kg/day) were used for each dose of radiation. Radiation increased MDA and activated NF‐κB, ERK and JNK signalling pathways. Sildenafil significantly decreased MDA level, nitric oxide (NO) level, IL1β, IL6 and TNF‐α. The most effective dose of sildenafil was 40 mg/kg/day in this study. Sildenafil also significantly inhibited NF‐κB, ERK and JNK signalling pathways and increased bcl2/bax ratio. In addition, high‐dose radiation severely destructed the mucosal layer in histopathology and led to mucosal cell apoptosis in the TUNEL assay. Sildenafil significantly improved mucosal structure and decreased inflammatory cell infiltration after exposure to high‐dose radiation and reduced apoptosis in the TUNEL assay. These findings show that sildenafil can improve radiation‐induced oral mucositis and decrease the apoptosis of mucosal cells via attenuation of inflammation and oxidative stress.     

CircSAMD4A aggravates H/R‐induced cardiomyocyte apoptosis and inflammatory response by sponging miR‐138‐5p

Hypoxia/reoxygenation (H/R)‐induced myocardial cell injury is the main cause of acute myocardial infarction (AMI). Many proofs show that circular RNA plays an important role in the development of AMI. The purpose of this study was to investigate the role of circSAMD4A in H/R‐induced myocardial injury. The levels of circular SAMD4A (circSAMD4A) were detected in the heart tissues of AMI mice and H/R‐induced H9C2 cells, and the circSAMD4A was suppressed in AMI mice and H/R‐induced H9C2 cells to investigate its’ function in AMI. The levels of circSAMD4A and miR‐138‐5p were detected by real‐time quantitative PCR, and MTT assay was used to detect cell viability. TUNEL analysis and Annexin V‐FITC were used to determine apoptosis. The expression of Bcl‐2 and Bax proteins was detected by Western blot. IL‐1β, TNF‐α and IL‐6 were detected by ELISA kits. The study found that the levels of circSAMD4A were up‐regulated after H/R induction and inhibition of circSAMD4A expression would reduce the H/R‐induced apoptosis and inflammation. MiR‐138‐5p was down‐regulated in H/R‐induced H9C2 cells. circSAMD4A was a targeted regulator of miR‐138‐5p. CircSAMD4A inhibited the expression of miR‐138‐5p to promote H/R‐induced myocardial cell injury in vitro and vivo. In conclusion, CircSAMD4A can sponge miR‐138‐5p to promote H/R‐induced apoptosis and inflammatory response.     

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad,MAPK and PI3K signalling pathway in asthma

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.     

Telmisartan anti‐cancer activities mechanism through targeting N‐cadherin by mimicking ADH‐1 function

This study aimed to investigate if Telmisartan as a novel N‐cadherin antagonist, can overcome cell migration of cancer cells. We investigated the mechanism and influence of Docetaxel and Telmisartan (as an analogous to ADH‐1, which is a well‐known N‐cadherin antagonist) on cancer cells. The effect of ADH‐1 and Telmisartan on cell attachment in PC3, DU145, MDA‐MB‐468 cell lines using recombinant human N‐cadherin was studied. Cell viability assay was performed to examine the anti‐proliferative effects of Telmisartan, ADH‐1 and Docetaxel. Migration was examined via wound healing assay, and apoptosis was determined by flow cytometry. The expression of AKT‐1 as a downstream gene of N‐cadherin signalling pathway was assayed by real‐time PCR. Treatment of PC3, MDA‐MB‐468 and DU145 cells with Telmisartan (0.1 µM) and ADH‐1 (40 µM) resulted in 50%, 58% and approximately 20% reduction in cell attachment to N‐cadherin coated plate respectively. It shows reduction of cell attachment in PC3 and MDA‐MB‐468 cell lines appeared to be more sensitive than that of DU145 cells to the Telmisartan and ADH‐1 treatments. Telmisartan (0.1 µM) and Docetaxel (0.01 nM) significantly reduced cell migration in PC3 and MDA‐MB‐468 cell lines compared with the control group. Using Real‐time PCR, we found that Telmisartan, Docetaxel and ADH‐1 had significant influence on the AKT‐1 mRNA level. The results of the current study for the first time suggest that, Telmisartan, exerts anti‐proliferation and anti‐migration effects by targeting antagonistically N‐cadherin. Also, these data suggest that Telmisartan as a less expensive alternative to ADH‐1 could potentiate Docetaxel anticancer effects.     

PAK4 suppresses motor neuron degeneration in hSOD1G93A‐linked amyotrophic lateral sclerosis cell and rat models

ObjectivesAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MN). CREB pathway‐mediated inhibition of apoptosis contributes to neuron protection, and PAK4 activates CREB signalling in diverse cell types. This study aimed to investigate PAK4’s effect and mechanism of action in ALS.MethodsWe analysed RNA levels by qRT‐PCR, protein levels by immunofluorescence and Western blotting, and apoptosis by flow cytometry and TUNEL staining. Cell transfection was performed for in vitro experiment. Mice were injected intraspinally to evaluate PAK4 function in vivo experiment. Rotarod test was performed to measure motor function.ResultsThe expression and activation of PAK4 significantly decreased in the cell and mouse models of ALS as the disease progressed, which was caused by the negative regulation of miR‐9‐5p. Silencing of PAK4 increased the apoptosis of MN by inhibiting CREB‐mediated neuroprotection, whereas overexpression of PAK4 protected MN from hSOD1^G93A‐induced degeneration by activating CREB signalling. The neuroprotective effect of PAK4 was markedly inhibited by CREB inhibitor. In ALS models, the PAK4/CREB pathway was inhibited, and cell apoptosis increased. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1^G93A mice suppressed MN degeneration, prolonged survival and promoted the CREB pathway.ConclusionsPAK4 protects MN from degeneration by activating the anti‐apoptotic effects of CREB signalling, suggesting it may be a therapeutic target in ALS.

Schematic representation of the mechanism of PAK4 protecting MN from apoptosis in ALS. PAK4 increases CREB levels and activation, leading to the upregulation of PGC‐1a and Bcl‐2, thereby decreasing cleaved‐caspase3 levels, and inhibiting MN degeneration. miR‐9‐5p is responsible for the decreased expression of PAK4 in ALS.     

Effect of MiR‐100‐5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.