Elimination of cells with abnormal nuclei in human epidermoid carcinoma cell line A431 by α-lipoic acid

Skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and malignant transformation. Antioxidants are able to affect these processes and eliminate transformed cells. The purpose of this work was to investigate the effect of α-lipoic acid (ALA) on human epidermoid carcinoma cell line A431. It was found that 100, 200, 300, 500 μM ALA added for 24, 48, 72 h inhibited cell proliferation and stimulated apoptosis. Most dying cells have abnormal nuclei (micronuclei, giant nuclei, nuclei with buds). Electron microscopy showed that cells with normal nuclear phenotypes after treatment with 200 μM ALA for 48 h had ultrastructural organizations typical for control cells. Thus, α-ALA not only triggers the apoptosis of carcinoma cells, but it may also activate the mechanism for eliminating cells with abnormal numbers of chromosomes.     

Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR–NF-κB signalling pathway in human epidermoid carcinoma A431 cells

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR–NF-κB signaling pathways.     

Nuclear factor-κB signaling pathway is involved in phospholipase Cε-regulated proliferation in human renal cell carcinoma cells

Glioblastoma is the most aggressive cerebral gliomas. Despite advances in therapies, the prognosis is still very poor. Therefore, novel therapeutic strategies are required. As a proteasome inhibitor, bortezomib has shown its efficacy as an active antitumor agent against a variety of tumors. However, inhibition of proteasome activity leads to cell death and also induces cell autophagy, and due to the dual roles of autophagy in the survival and death of tumor cells, the effect of inhibition of autophagy on glioblastoma cells remains to be explored. We therefore assessed whether bortezomib is capable of inducing autophagy, and investigated the antitumor effect of bortezomib combined with autophagy inhibitors on human glioblastoma U251 and U87 cells. Cell viability was measured by MTT assay. The expressions of autophagy and apoptosis-related proteins were determined by Western blot analysis. U251 and U87 cells proliferation was inhibited in a dose-dependent manner. Both apoptosis and autophagy induced by bortezomib were observed in human glioblastoma U87 and U251 cells. However, when U251 and U87 cells were co-treated with bortezomib and autophagy inhibitors 3-MA or Atg7 siRNA, the autophagy inhibitors blocked the autophagy in the cells and resulted in a further inhibition of cell proliferation and a further increase in cell apoptosis as compared with that treated with bortezomib alone. These findings indicated that combination of bortezomib and autophagy inhibitors may shed new light on glioblastoma treatment.     

Mechanism of programmed cell death factor 4/nuclear factor-κB signaling pathway in porcine coronary micro-embolization-induced cardiac dysfunction

The aim of this study was to investigate the role of the programmed cell death factor 4 (PDCD4)/nuclear factor-κB (NF-κB) signaling pathway in coronary micro-embolism (CME)-induced inflammatory responses and cardiac dysfunction in a porcine model. Bama miniature pigs were randomly divided into four groups (n = 5 per group). Micro-embolization balls or saline were infused through a microcatheter in the left anterior descending (LAD) artery in the CME and Sham groups, respectively. PDCD4 siRNA or control siRNA mixed with transfection reagent was infused via the LAD artery 72 h before CME induction in the CME + siRNA-PDCD4 and siRNA-control groups, respectively. Cardiac function was evaluated with ultrasound. Tissue biopsy was stained with hematoxylin–eosin (HE) and hematoxylin basic fuchsin picric acid (HBFP) to measure infarction area. Myocardial PDCD4 and tumor necrosis factor-α (TNF-α) mRNA and protein expression were analyzed by quantitative PCR and Western blotting. NF-κB activity was evaluated in gel electrophoretic mobility shift assay. Echocardiographic parameters showed that compared with the sham group, the CME group had impaired heart function, manifested as systolic dysfunction and left ventricular dilatation (reduced left ventricular ejection fraction [LVEF], left ventricular fractional shortening [FS], and cardiac output [CO] [P < 0.05] and increased left ventricular end-diastolic diameter [LVEDd] [P < 0.05]). Compared with the CME group, the CME + siRNA-PDCD4 group had attenuated CME-induced cardiac function damage (increased LVEF, FS and CO [P < 0.05] and reduced LVEDd [P < 0.05]). Compared with the sham group, the CME group had significantly increased PDCD4 and TNF-α mRNA and protein expression and increased NF-κB activity (P < 0.05). These effects were significantly inhibited in the CME + siRNA-PDCD4 group (P < 0.05). In conclusion, PDCD4/NF-κB signaling pathway activation is an important mechanism for CME-induced cardiac dysfunction, suggesting that inhibition of PDCD4/NF-κB signaling pathway may be a potential target for the prevention and treatment of CME.     

Honokiol improved chondrogenesis and suppressed inflammation in human umbilical cord derived mesenchymal stem cells via blocking nuclear factor-κB pathway

Background

Cartilage degradation is the significant pathological process in osteoarthritis (OA). Inflammatory cytokines, such as interleukin-1β (IL-1β), activate various downstream mediators contributing to OA pathology. Recently, stem cell-based cartilage repair emerges as a potential therapeutic strategy that being widely studied, whereas, the outcome is still far from clinical application. In this study, we focused on an anti-inflammatory agent, honokiol, which is isolated from an herb, investigated the potential effects on human umbilical cord derived mesenchymal stem cells (hUC-MSCs) in IL-1β stimulation.

Methods

Second passage hUC-MSCs were cultured for multi-differentiation. Flow cytometry, qRT-PCR, von Kossa stain, alcian blue stain and oil red O stain were used for characterization and multi-differentiation determination. Honokiol (5, 10, 25, 50 μM) and IL-1β (10 ng/ml) were applied in hUC-MSCs during chondrogenesis. Analysis was performed by MTT, cell apoptosis evaluation, ELISA assay, qRT-PCR and western blot.

Results

hUC-MSC was positive for CD73, CD90 and CD105, but lack of CD34 and CD45. Remarkable osteogenesis, chondrogenesis and adipogenesis were detected in hUC-MSCs. IL-1β enhanced cell apoptosis and necrosis and activated the expression of caspase-3, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-1, ?9, 13 in hUC-MSCs. Moreover, the expression of SRY-related high-mobility group box 9 (SOX-9), aggrecan and col2α1 was suppressed. Honokiol relieved these negative impacts induced by IL-1β and suppressed Nuclear factor-κB (NF-κB) pathway by downregulating expression of p-IKKα/β, p-IκBα and p-p65 in dose-dependent and time-dependent manner.

Conclusions

Honokiol improved cell survival and chondrogenesis of hUC-MSCs and inhibited IL-1β-induced inflammatory response, which suggested that combination of anti-inflammation and stem cell can be a novel strategy for better cartilage repair.

     

Nitric oxide (•NO) generation but not ROS plays a major role in silibinin-induced autophagic and apoptotic death in human epidermoid carcinoma A431 cells

Abstract

Silibinin, a major active constituent of silymarin, is clinically used as a hepatoprotectant, and in recent years, it has been used for the treatment of cancer in China. Because the mechanism of silibinin action on cancer cells was still unclear, we investigated the contribution of silibinin to the induction of apoptosis and autophagy via generation of reactive oxygen species (ROS) and nitric oxide (?NO) in human epidermoid carcinoma A431 cells. Silibinin inhibited the cell growth in a dose‐and time-dependent manner. Obvious autophagy was observed after treatment with different doses of silibinin. At a high dose (400 μM), silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. Loss of mitochondrial membrane potential by silibinin led to mitochondrial dysfunction and decreased ROS levels, suggesting that silibinin might act as an antioxidant in this process. Furthermore, silibinin induced ?NO generation in a time‐and dose-dependent manner. The ?NO scavenger PTIO could effectively clear ?NO and exerted a minor cell protection effect through partial inhibition of silibinin-induced apoptosis and autophagy.     

Dynamics of differentiation in human epidermoid squamous carcinoma cells (A431) with continuous,long-term γ-IFN treatment

Summary We investigated the long-term effects of continuous gamma interferon (γ-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses (“tumoroids”) exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin “pearls,” intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of γ-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by γ-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types.     

Inhibition of ROS-NF-κB-dependent autophagy enhances Hypocrellin A united LED red light-induced apoptosis in squamous carcinoma A431 cells

Cutaneous squamous cell carcinoma (cSCC) is a type of malignant skin tumor derived from epidermal Malpighian cells. Photodynamic therapy is regarded as a crucial method in oncology. Hypocrellin A (HA), an efficient natural photosensitizer, has been reported to exert excellent light induced antiviral, antimicrobial and anticancer activity through mediating multiple signaling pathways. The purpose of the present study is to examine the effects of HA united red light irradiation on human squamous carcinoma A431 cells and further reveal the underlying regulatory mechanisms. The results showed that synergistic treatment of HA and red light irradiation inhibited cell proliferation and induced cell apoptosis and autophagy. Moreover, HA united red light irradiation caused a significant accumulation of reactive oxygen species (ROS), and induced the activation of c-Jun NH 2 terminal kinases (JNKs) which was inhibited by the antioxidant N-Acetyl-cysteine (NAC). Furthermore, HA united red light irradiation activated the nuclear factor-kappa B (NF-κB) pathway, and inhibition of NF-κB activity exacerbated HA united red light irradiation-induced apoptosis but suppressed cell autophagy. In addition, the inhibition of autophagy promoted HA united red light irradiation-induced apoptosis and facilitated the NF-κB activity. Over all, our results revealed that HA united red light irradiation could inhibit A431 cell proliferation by inducing apoptosis and autophagy via the activation of the ROS mediated JNK and NF-κB pathways, providing prospective for HA as a potential therapeutic for the treatment of cSCC.     

Sources of cell-to-cell variability in canonical nuclear factor-κB (NF-κB) signaling pathway inferred from single cell dynamic images

The canonical nuclear factor-κB (NF-κB) signaling pathway controls a gene network important in the cellular inflammatory response. Upon activation, NF-κB/RelA is released from cytoplasmic inhibitors, from where it translocates into the nucleus, subsequently activating negative feedback loops producing either monophasic or damped oscillatory nucleo-cytoplasmic dynamics. Although the population behavior of the NF-κB pathway has been extensively modeled, the sources of cell-to-cell variability are not well understood. We describe an integrated experimental-computational analysis of NF-κB/RelA translocation in a validated cell model exhibiting monophasic dynamics. Quantitative measures of cellular geometry and total cytoplasmic concentration and translocated RelA amounts were used as priors in Bayesian inference to estimate biophysically realistic parameter values based on dynamic live cell imaging studies of enhanced GFP-tagged RelA in stable transfectants. Bayesian inference was performed on multiple cells simultaneously, assuming identical reaction rate parameters, whereas cellular geometry and initial and total NF-κB concentration-related parameters were cell-specific. A subpopulation of cells exhibiting distinct kinetic profiles was identified that corresponded to differences in the IκBα translation rate. We conclude that cellular geometry, initial and total NF-κB concentration, IκBα translation, and IκBα degradation rates account for distinct cell-to-cell differences in canonical NF-κB translocation dynamics.     

Inhibition of the nuclear factor-κB pathway prevents beta cell failure and diet induced diabetes in Psammomys obesus

Background

High doses of anti-inflammatory drugs, such as aspirin and salicylates, improve glucose metabolism in insulin resistant and type 2 diabetic patients. It has also been shown that the glucose lowering effect is related to the unspecific ability of these drugs to inhibit inhibitor kinaseβ (IKKβ). In this study we have investigated the effect of a selective IKKβ-inhibitor on beta cell survival and the prevention of diet induced type 2 diabetes in the gerbil Psammomys obesus (P. obesus).

Methodology/Principal Findings

P. obesus were fed a diabetes inducing high energy diet for one month in the absence or presence of the IKKβ-inhibitor. Body mass, blood glucose, HbA_1C, insulin production and pancreatic insulin stores were measured. The effects on beta cell survival were also studied in INS-1 cells and primary islets. The cells were exposed to IL-1β and subsequently reactive oxygen species, insulin release and cell death were measured in the absence or presence of the IKKβ-inhibitor. In primary islets and beta cells, IL-1β induced the production of reactive oxygen species, reduced insulin production and increased beta cell death, which were all reversed by pre-treatment with the IKKβ-inhibitor. In P. obesus the IKKβ-inhibitor prevented the development of hyperglycaemia and hyperinsulinaemia, and maintained pancreatic insulin stores with no effect on body weight.

Conclusions/Significance

Inhibition of IKKβ activity prevents diet-induced diabetes in P. obesus and inhibits IL-1β induced reactive oxygen species, loss of insulin production and beta cell death in vitro.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Alcohol enhances Aβ42-induced neuronal cell death through mitochondrial dysfunction

Mitochondrial dysfunction is a hallmark of beta-amyloid (Aβ)-induced neuronal toxicity in Alzheimer’s disease (AD). Epidemiological studies have indicated that alcohol consumption plays a role in the development of AD. Here we show that alcohol exposure has a synergistic effect on Aβ-induced neuronal cell death. Aβ-treated cultured neurons displayed spontaneous generation of reactive oxygen species (ROS), disruption of their mitochondrial membrane potential, induction of caspase-3 and p53 activities, and loss of cell viability. Alcohol exposure facilitated Aβ-induced neuronal cell death. Our study shows that alcohol consumption enhances Aβ-induced neuronal cell death by increasing ROS and mitochondrial dysfunction.     

High cholesterol inhibits tendon-related gene expressions in tendon-derived stem cells through reactive oxygen species-activated nuclear factor-κB signaling

Clinical studies have indicated that increased serum cholesterol levels raised the risk of tendinopathy in hypercholesterolemia, but the effect of cholesterol on tendon-derived stem cells (TDSCs) and its underlying mechanism have not been studied. The purpose of this study is to investigate the association between cholesterol and tendinopathy in vitro and in vivo, and its underlying molecular mechanism as well. In TDSCs, the effect of cholesterol was assessed by quantitative polymerase chain reaction, western blot analysis, and immunofluorescence staining. Intracellular levels of reactive oxygen species (ROS) was detected, using flow cytometry. The link between nuclear factor (NF)-κB signaling and the effect of cholesterol was evaluated using a representative IκB kinase (IKK) inhibitor, BAY 11-7082. In addition, Achilles tendons from apolipoprotein E mice fed with a high-fat diet were histologically assessed using hematoxylin and eosin staining and immunohistochemistry. We found that high cholesterol apparently lowered the expression of tendon cell markers (collagen 1, scleraxis, tenomodulin), and elevated ROS levels via the NF-κB pathway both in vitro and in vivo. The ROS scavenger N-acetylcysteine (NAC) and BAY 11-7082 reversed the inhibiting effect of cholesterol on the tendon-related gene expressions of TDSCs. Moreover, NAC blocked cholesterol-induced phosphorylation of IκBα and p65. Significant histological alternation in vivo was shown in Achilles tendon in the hypercholesterolemic group. These results indicated that high cholesterol may inhibit the tendon-related gene expressions in TDSCs via ROS-activated NF-кB signaling, implying pathogenesis of tendinopathy in hypercholesterolemia and suggesting a new mechanism underlying hypercholesterolemia-induced tendinopathy.