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Shikonin is the main component of the traditional Chinese medicine comfrey, which can inhibit the activity of PKM2 by regulating glycolysis and ATP production. Rheumatoid arthritis synovial cells (RA-FLSs) have been reported to increase glycolytic activity and have other similar hallmarks of metabolic activity. In this study, we investigated the effects of shikonin on glycolysis, mitochondrial function, and cell death in RA-FLSs. The results showed that shikonin induced apoptosis and autophagy in RA-FLSs by activating the production of reactive oxygen species (ROS) and inhibiting intracellular ATP levels, glycolysis-related proteins, and the PI3K-AKT-mTOR signaling pathway. Shikonin can significantly reduce the expression of apoptosis-related proteins, paw swelling in rat arthritic tissues, and the levels of inflammatory factors in peripheral blood, such as TNF-α, IL-6, IL-8, IL-10, IL-17A, and IL-1β while showing less toxicity to the liver and kidney., (© 2021. The Author(s).)

Objective: To investigate the effect of chloroquine in inducing apoptosis of human hepatocellular carcinoma cells and explore the possible mechanism., Methods: MTT assay and flow cytometry were used to evaluate chloroquine-induced growth inhibition and apoptosis in human hepatocellular carcinoma HepG2 cells, respectively. The ATP levels in chloroquine-treated cells were detected using an ATP assay kit. PCR and Western blotting were used to detect the expression levels of miR-26b and Mcl-1 in the cells, respectively., Results: Chloroquine inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner. Treatments with 80 µmol/L chloroquine for 24, 48, and 72 h induced survival rates of (71.59∓0.2)%, (45.40∓0.5)%, and (26.34∓1.4)% in the cells. Treatments with chloroquine at 40, 80, and 160 µmol/L for 5 h resulted in obviously lowered intracellular ATP levels in the cells to 87.80%, 71.29%, and 38.02% of the control level, respectively. At 80 µmol/L, chloroquine significantly increased the expression of miR-26b and down-regulated the expression of Mcl-1 in HepG2 cells, and the application of the miR-26b inhibitor increased the cellular expression of Mcl-1., Conclusion: s Chloroquine can inhibit the cell proliferation, reduce ATP level and induce apoptosis in HepG2 cells possibly through miR-26b-mediated regulation of Mcl-1.

Granulosa cells (GCs) are essential somatic cells in the ovary and play an important role in folliculogenesis. Brain-derived neurotropic factor (BDNF) and the TGF-β pathway have been identified as a critical hormone and signalling pathway, respectively, in GCs. In this study, we found that a conserved microRNA family that includes miR-10a and miR-10b repressed proliferation and induced apoptosis in human, mouse, and rat GCs (hGCs, mGCs and rGCs, respectively). Moreover, essential hormones and growth factors in the follicle, such as FSH, FGF9 and some ligands in the TGF-β pathway (TGFβ1, Activin A, BMP4 and BMP15), inhibited miR-10a and miR-10b expression in GCs. In contrast, the miR-10 family suppressed many key genes in the TGF-β pathway, suggesting a negative feedback loop between the miR-10 family and the TGF-β pathway in GCs. By using bioinformatics approaches, RNA-seq, qPCR, FISH, immunofluorescence, Western blot and luciferase reporter assays, BDNF was identified as a direct target of the miR-10 family in GCs. Additionally, reintroduction of BDNF rescued the effects of miR-10a and miR-10b in GCs. Collectively, miR-10a and miR-10b repressed GC development during folliculogenesis by repressing BDNF and the TGF-β pathway. These effects by the miR-10 family on GCs are conserved among different species.

The effect of MTH1 inhibition on cancer cell survival has been elusive. Here we report that although silencing of MTH1 does not affect survival of melanoma cells, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homolog of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a reactive oxygen species scavenger or an antioxidant attenuated the apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of reactive oxygen species. Collectively, these results indicate that the cytotoxicity of TH588 toward melanoma cells is not associated with its inhibitory effect on MTH1, although it is mediated by cellular production of ROS., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells., Methods: Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays., Results: Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G)., Conclusion: Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+)]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca(2+)]i and inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities. This Cd-induced elevation of [Ca(2+)]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+) is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+) chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+) homeostasis, by compromising ATPases activities and ER-regulated Ca(2+), and this elevation in Ca(2+) triggers the activation of the Ca(2+)-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca(2+)]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Dysregulation of microRNA (miRNA) biogenesis is implicated in cancer development and progression. Dicer and Drosha are established regulators of miRNA biogenesis. In this study, we used a miRNA array to evaluate the miRNA expression profiles in nasopharyngeal carcinoma (NPC) samples. The significance analysis of microarrays showed a global downregulation of miRNA expression in NPC samples compared with normal nasopharyngeal epithelial tissues. Notably, miR-18a, a member of the oncogenic miR-17-92 cluster, was upregulated in the NPC samples and cell lines. Clinical parameter studies showed that higher levels of miR-18a correlated with NPC advanced stage, lymph node metastasis, Epstein-Barr virus infection and a higher death rate from NPC, indicating oncogenic roles in NPC development. The expression levels of miR-18a and Dicer1 were inversely related in NPC tissues. Further studies demonstrated that miR-18a negatively regulated Dicer1 by binding to the 3' untranslated regions of Dicer1. In vitro and in vivo biological function assays showed that miR-18a promoted the growth, migration and invasion of NPC cells by regulating Dicer1 expression, which caused the global downregulation of miRNA expression levels including miR-200 family and miR-143. Furthermore, we found that the epithelial mesenchymal transition marker E-cadherin and the oncogene K-Ras were aberrantly expressed after miR-18a transduction, and these alterations were directly induced by downregulation of the miR-200 family and miR-143. Collectively, our findings indicate that miR-18a plays an oncogenic role in the development of NPC by widespread downregulation of the miRNome and could be a potential therapeutic target for NPC.

Purpose: To examine mechanisms that determine long-term responses of B-RAF(V600E) melanoma cells to B-RAF inhibitors., Experimental Design: B-RAF(V600E) melanoma cells were exposed to the B-RAF inhibitor PLX4720 for prolonged periods to select for cells resistant to apoptosis induced by the inhibitor. The resultant cells were analyzed for activation of extracellular signal regulated kinase (ERK), MAP/ERK kinase (MEK), and Akt, and related signals. Their roles in survival of the cells were also examined., Results: B-RAF(V600E) melanoma cells selected for resistant to PLX4720-induced apoptosis retained the V600E mutation in B-RAF, and proliferated steadily in the presence of the inhibitor, albeit with slow growth rate. These cells displayed high levels of ERK activation, that is, at least in part, independent of the conventional RAF/MEK/ERK pathway, as MEK activation was low and inhibition of MEK did not significantly block activation of ERK. In contrast, extracellular signals appeared involved. This was associated with elevated activation of the phosphoinositide 3-kinase (PI3k)/Akt pathway and could be inhibited by serum starvation and inhibition of PI3k/Akt. Inhibition of MEK did not impact on survival of these cells, whereas serum starvation, inhibition of PI3K/Akt, and inhibition of ERK1/2 reduced their viability., Conclusions: These results indicate that sensitivity to induction of apoptosis may be a major determinant of long-term responses of B-RAF(V600E) melanomas to specific inhibitors and suggest that rebound melanoma growth after initial treatment with the inhibitors may not be responsive to MEK inhibitors, but may be susceptible to inhibition of the PI3k/Akt pathway., (©2010 AACR.)

Wild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligase MDM2. However, we found in this study that blockade of the interaction of p53 and MDM2 by the MDM2 antagonist nutlin-3 in melanoma cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless, nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, nutlin-3 upregulated Mcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as nutlin-3 upregulated the Mcl-1 protein to a much greater extent than the Mcl-1 mRNA, and this was associated with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in fresh melanoma isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and, importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in addition to docetaxel in melanoma., (©2010 AACR.)

Background: Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells., Results: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1 alpha, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells., Conclusion: These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1 alpha/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1-deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Up-regulation of Mcl-1 was also due to increased transcription that involved the IRE1alpha and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress.

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8-mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress-induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress-induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress-induced caspase-4 activation, ARC seems to be critical in blocking activation of caspase-8 in melanoma cells subjected to ER stress.

We have previously reported that sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is largely correlated with the levels of expression of TRAIL death receptors, in particular, TRAIL-R2 on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express, in general, low levels of death receptors for TRAIL. We show in this study that the endoplasmic reticulum stress inducer, thapsigargin (TG), selectively up-regulated TRAIL-R2 and enhanced TRAIL-induced apoptosis in melanoma cells. However, the TRAIL-R2 pathway did not appear to be involved in induction of apoptosis by TG alone. Up-regulation of TRAIL-R2 appeared to be cooperatively mediated by the inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha)- and activation of transcription factor (ATF)-6-signaling pathways of the unfolded protein response (UPR) and the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). The latter played a critical role in the initial phase of the increase in TRAIL-R2 as small interfering RNA (siRNA) knockdown of CHOP blocked up-regulation of TRAIL-R2 only at a relatively early stage (16 h) after exposure to TG. In contrast, IRE1alpha and ATF6 appeared to be crucial in maintaining the increased levels of TRAIL-R2 in that siRNA knockdown of IRE1alpha or ATF6 had no effect on the increase in TRAIL-R2 at the initial phase, but blocked TRAIL-R2 up-regulation at a relatively late stage (36 h). Our results indicate that modulation of the UPR may be useful in sensitizing melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-R2.

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Purpose: Given that inhibitors of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) are being introduced into treatment for melanoma, the present study was carried out to better understand the mechanism by which they may induce apoptosis of melanoma cells., Experimental Design: A panel of human melanoma cell lines and fresh melanoma isolates was assessed for their sensitivity to apoptosis induced by the MEK inhibitor U0126. The apoptotic pathways and regulatory mechanisms involved were examined by use of the inhibitor and small interfering RNA (siRNA) techniques., Results: Inhibition of MEK induced apoptosis in the majority of melanoma cell lines through a mitochondrial pathway that was associated with the activation of Bax and Bak, release of mitochondrial apoptogenic proteins, and activation of caspase-3. However, apoptosis was independent of caspases and instead was associated with mitochondrial release of AIF as shown by the inhibition of apoptosis when AIF was knocked down by siRNA. Inhibition of MEK resulted in the up-regulation of the BH3-only proteins PUMA and Bim and down-regulation of the antiapoptotic protein Mcl-1. These changes were critical for the induction of apoptosis by U0126 as siRNA knockdown of PUMA or Bim inhibited apoptosis, whereas siRNA knockdown of Mcl-1 increased apoptosis particularly in the apoptosis-resistant cell lines., Conclusions: Apoptosis of melanoma cells induced by the inhibition of the MEK/ERK pathway is mediated by the up-regulation/activation of PUMA and Bim and down-regulation of Mcl-1. Release of AIF rather than the activation of caspases seems to be the mediator of apoptosis. Our results suggest that cotargeting Mcl-1 and the MEK/ERK pathway may further improve treatment results in melanoma.

We have reported previously low expression of death receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in fresh isolates and tissue sections of melanoma. This seemed to correlate with relative resistance of freshly isolated melanoma cells to TRAIL-induced apoptosis. We show in this study that the endoplasmic reticulum (ER) stress inducer, tunicamycin, selectively up-regulated the cell surface expression of TRAIL-R2, but not other members of the TNF receptor family, and enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. Tunicamycin-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was associated with increased activation of the caspase cascade and reduction in mitochondrial membrane potential and was inhibited by a recombinant TRAIL-R2/Fc chimeric protein. Up-regulation of TRAIL-R2 on the melanoma cell surface was associated with increased transcription of TRAIL-R2 and its total protein levels. Two signaling pathways of the ER stress-induced unfolded protein response mediated by inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1alpha pathway, and small interfering RNA (siRNA) knockdown of IRE1alpha substantially reduced the up-regulation of TRAIL-R2. Similarly, there was evidence for the activation of the ATF6 pathway, and siRNA knockdown of ATF6 had a delayed effect on TRAIL-R2 expression in one but not another melanoma cell line. Moreover, the transcription factor CCAAT/enhancer-binding protein homologous protein seemed to be involved in the up-regulation of TRAIL-R2 by tunicamycin, but its role varied between different melanoma lines. Taken together, our results suggest that agents that induce ER stress may enhance TRAIL-R2 expression and increase the therapeutic response to TRAIL in melanoma.

Purpose: Our studies have shown variable sensitivity of cultured melanoma cells to docetaxel. To better understand this response, we studied the role of signal transduction pathways in modulating docetaxel-induced melanoma killing., Experimental Design: Involvement of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and Akt signaling was studied by evaluating their extent of activation in melanoma cells after treatment with docetaxel. The effect of their activation on docetaxel-induced apoptosis was assessed using biochemical inhibitors of the pathways and Western blot analysis of proteins involved., Results: Docetaxel induced activation of both JNK and ERK1/2 but not p38 mitogen-activated protein kinase or Akt kinases. Apoptosis was dependent on activation of JNK and mediated through activation of caspase-2 and caspase-dependent changes in Bax and Bak. The levels of activated JNK in individual lines showed a close correlation with the levels of apoptosis. In contrast, activation of ERK1/2 by docetaxel inhibited apoptosis and the levels of activation in individual lines were inversely correlated to the degree of apoptosis. Studies on the Bcl-2 family proteins seemed to reflect changes induced by activation of JNK and ERK1/2 pathways. Docetaxel-induced JNK activation was required for Bcl-2 phosphorylation as well as caspase-2-dependent activation of Bax and Bak and subsequent mitochondrial release of apoptosis-inducing factor and cytochrome c. In contrast, activation of ERK1/2 resulted in degradation of BH3-only protein Bim and phosphorylation of Bad., Conclusions: These studies provide further insights into sensitivity of melanoma cells to taxanes and provide a basis for the current rationale of combining taxanes with inhibitors of the Raf-ERK1/2 pathway.

Micro(nano)plastic, as a new type of environmental pollutant, have become a potential threat to the life and health of various stages of biology. However, it is not yet clear whether they will affect brain development in the fetal stage. Therefore, this study aims to explore the potential effects of nanoplastics on the development of fetal rat brains. To assess the allocation of NPs (25 nm and 50 nm) in various regions of the fetal brain, pregnant rats were exposed to concentrations (50, 10, 2.5, and 0.5 mg/kg) of PS-NPs. Our results provided evidence of the transplacental transfer of PS-NPs to the fetal brain, with a prominent presence observed in several cerebral regions, notably the cerebellum, hippocampus, striatum, and prefrontal cortex. This distribution bias might be linked to the developmental sequence of each brain region. Additionally, we explored the influence of prenatal exposure on the myelin development of the cerebellum, given its the highest PS-NP accumulation in offspring. Compared with control rats, PS-NPs exposure caused a significant reduction in myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) expression, a decrease in myelin thickness, an increase in cell apoptosis, and a decline in the oligodendrocyte population. These effects gave rise to motor deficits. In conclusion, our results identified the specific distribution of NPs in the fetal brain following prenatal exposure and revealed that prenatal exposure to PS-NPs can suppress myelin formation in the cerebellum of the fetus.

Background: EBV-associated lymphoma is a neoplasm with a poor prognosis, highly aggressive, and progressive rapidly. There is no standard clinical treatment protocol. Decitabine and gemcitabine are known to have anticancer properties against cells of various cancer, respectively. However, the effect of the combination medication on NK/T cell lymphoma cells and potential mechanisms have not been thoroughly investigated.Methods: Human NK/T cell lymphoma cells NK92MI were treated with decitabine and gemcitabine alone or in combination. Experiments, including the Cell Counting Kit-8 and flow cytometry, were performed to investigate how the combination of decitabine and gemcitabine affects the biological behavior of NK92MI cells in vitro. mRNA sequencing, RT-PCR, and western blotting were used to detect changes in the related signal pathway, mRNA, and protein expressions.Results: Decitabine and gemcitabine significantly inhibited the viability and proliferation of NK92MI cells in a dose-dependent manner. The combination index was less than 1 after treating with two drugs, which was a significant synergistic effect. The decitabine concentration with the best synergistic effect was 4.046 µM, and the gemcitabine concentration was 0.005 µM. Flow cytometry showed that combining two drugs could significantly promote apoptosis and arrest the cell cycle at the S phase. In the combined DAC and GEM group, caspase3 protein levels were higher than in either group alone or the control group. The transcriptome sequence, KEGG, and PPI analysis showed that the differential genes after combined treatment were mainly enriched in signal pathways related to cell proliferation, adhesion, and migration compared with using alone and control groups. Based on the sequencing results, we further investigated the role of DAC and GEM in ferroptosis-related signaling molecules using RT-PCR and Western blot techniques. RT-PCR and western blotting showed that the expression levels of HMOX1 and EBV cleavage gene BRLF1 were higher in the group with combined DAC and GEM than in the group alone and the control group, while the protein and mRNA expression levels of SLC7A11 were lower than the others. In addition, the GPX4 protein expression level in the combination group was lower than in the drug-alone and control groups. In addition, the combination treatment increased the ROS level of NK92MI cells.Conclusion: Our current findings suggested that decitabine had an inhibitory effect on the proliferation of NK92MI cells when co-treated with gemcitabine. This combination may increase the expression of ferroptosis-related signaling molecules, thus inhibiting the proliferation of NK92MI cells. It also promoted apoptosis in NK/T cell lymphoma. For patients with NK/T cell lymphoma, this novel combination may provide clinical benefits.

Abstract Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH1) responses and regulatory T (Treg) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) ‐γ and tumor necrosis factor (TNF) ‐α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency‐mediated TH1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI.

Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata, hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata. It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

Background Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown. Methods In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells. Results Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells. Conclusions Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future. [ABSTRACT FROM AUTHOR]

The activation of caspase-3 is an important hallmark in Parkinson’s disease. It could induce neuron death by apoptosis and microglia activation by inflammation. As a result, inhibition the activation of caspase-3 would exert synergistic dual effect in brain in order to prevent the progress of Parkinson’s disease. Silencing caspase-3 genes by RNA interference could inhibit the activation of caspase-3. We developed a brain-targeted gene delivery system based on non-viral gene vector, dendrigraft poly-L-lysines. A rabies virus glycoprotein peptide with 29 amino-acid linked to dendrigraft poly-L-lysines could render gene vectors the ability to get across the blood brain barrier by specific receptor mediated transcytosis. The resultant brain-targeted vector was complexed with caspase-3 short hairpin RNA coding plasmid DNA, yielding nanoparticles. In vivo imaging analysis indicated the targeted nanoparticles could accumulate in brain more efficiently than non-targeted ones. A multiple dosing regimen by weekly intravenous administration of the nanoparticles could reduce activated casapse-3 levels, significantly improve locomotor activity and rescue dopaminergic neuronal loss and in Parkinson’s disease rats’ brain. These results indicated the rabies virus glycoprotein peptide modified brain-targeted nanoparticles were promising gene delivery system for RNA interference to achieve anti-apoptotic and anti-inflammation synergistic therapeutic effects by down-regulation the expression and activation of caspase-3. [ABSTRACT FROM AUTHOR]

Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma. [ABSTRACT FROM PUBLISHER]

Skin cancers are by far the most common human malignancies. Retinoids have shown promising preventive and therapeutic effects against a variety of human malignancies. The aim of this study was to investigate the apoptosis-inducing effect of acitretin on human skin squamous cell carcinoma (SCC) SCL-1 cells. We found that acitretin preferentially inhibited the growth of SCL-1 cells in a dose- and time-dependent manner, but not of non-malignant keratinocyte HaCaT cells. This inhibition appeared to be due to induction of apoptosis as revealed by enzyme-linked immunosorbent assay. AnnexinV/propidium iodide assay and morphological observation confirmed the pro-apoptotic effect of acitretin on SCL-1 cells. We further demonstrated that apoptosis was induced within 1–2 days and involved activation of caspases-8, -9, -3 and poly (ADP-ribose) polymerase (PARP). Caspase-8 inhibitor effectively suppressed acitretin-induced apoptosis whereas caspase-9 inhibitor did not. Acitretin increased the levels of CD95 (Fas), CD95-ligand and Fas-associated death domain. Neutralizing ZB4 anti-Fas antibody significantly inhibited the apoptosis in SCL-1 cells induced by acitretin. These results suggest that acitretin is able to induce apoptosis in skin cancer cells possibly via death receptor CD95 apoptosis pathway without affecting the viability of normal keratinocyte. [ABSTRACT FROM AUTHOR]

We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention. Oncogene (2000) 19, 2324–2330 [ABSTRACT FROM AUTHOR]

Chemoresistance remains a formidable hurdle against cancer therapy. Seeking for novel therapy strategies is an urgent need for those who no longer benefit from chemotherapy. Chemoresistance is usually associated with the dysfunction of intrinsic apoptosis. Targeting extrinsic apoptosis via TRAIL signaling and the death receptors could be a potential solution to treat chemoresistant cancer. A highly biocompatible nano system for codelivery of the TRAIL DNA and the death receptor sensitizer monensin was developed, in which low-molecular-weight PEI (LMW-PEI) was crosslinked by the sulfhydryl cyclodextrin via disulfide bonds, and then bound with DNA, thus forming the bioreducible polyplex cores. In addition, the cyclodextrin also functioned as a carrier for the hydrophobic monensin via host-guest inclusion. Poly-γ-glutamic acid (γ-PGA) was used to modify the polyplex core via charge interaction. The γ-PGA corona can specifically bind with the tumor-associated gamma-glutamyl transpeptidase (GGT) overexpressed on the tumor cells, and achieve tumor-targeting delivery. Moreover, the tumor-homing peptide RGD-modified γ-PGA was also prepared as the surface coating materials for further improving gene delivery efficiency. This gene delivery system was characterized by the dual ligand-targeting, dual stimuli-responsive features. The ligands of RGD and γ-PGA can target the tumor-associated receptors (i.e., integrin and GGT). The conformation of γ-PGA is pH-sensitive, and the tumor acidic micro environments could trigger the detachment of surface-coating γ-PGA. The disulfide crosslinking LMW-PEI is redox-sensitive, and its fast disassembling in the tumor cells could favor the efficient gene delivery. The anti-tumor efficacy was demonstrated both in vitro and in vivo. Moreover, MYC-mediated synthetic lethality could be an important mechanism for overcoming the drug resistance. An important finding of our studies is the demonstration of the in vivo treatment efficacy of TRAIL/monensin, thus providing a potential novel therapeutic strategy for overcoming drug-resistant cancer. [ABSTRACT FROM AUTHOR]

Abstract: Gene therapy offers a promising cure of brain glioma and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce cell apoptosis of glioma selectively without affecting the normal cells. In this study, the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), was selected as the principal vector. Angiopep-2, which can target to the low-density lipoprotein receptor-related protein-1 (LRP1) expressed on BCECs and glial cells, was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethyleneglycol (PEG) and then complexed with the DNA, designated as PAMAM-PEG-Angiopep/DNA nanoparticles (NPs). The cellular uptake mechanism explored in glial cells showed that the DNA of PAMAM-PEG-Angiopep/DNA NPs entered into the nuclei through the endosome/lysosome pathway. The in vivo biodistribution of PAMAM-PEG-Angiopep/DNA NPs in the brain especially the tumor site was higher than that of PAMAM-PEG/DNA NPs and PAMAM/DNA NPs. Furthermore, the TUNEL analysis showed a more wide-extended apoptosis in the PAMAM-PEG-Angiopep/pORF-TRAIL NPs treated group, compared to other groups including commercial Temozolomide-treated one. The median survival time of PAMAM-PEG-Angiopep/pORF-TRAIL NPs and Temozolomide treated on brain tumor-bearing mice was 61 and 49 days respectively, significantly longer than that of other groups. Besides, the NPs suggested low cytotoxicity after in vitro transfection. Thus, the results showed that Angiopep-2 could be exploited as a specific ligand to cross the BBB and targeted to glial cells, and PAMAM-PEG-Angiopep/DNA NPs can be a potential non-viral delivery system for gene therapy of glial tumor. [Copyright &y& Elsevier]

Abstract: A combination cancer therapy was investigated via co-delivery of therapeutic gene encoding human tumor necrosis factor-related apoptosis-inducing ligand (pORF-hTRAIL) and doxorubicin (DOX) using a tumor-targeting carrier, peptide HAIYPRH (T7)-conjugated polyethylene glycol-modified polyamidoamine dendrimer (PAMAM-PEG-T7). T7, a transferrin receptor-specific peptide, was chosen as the ligand to target the co-delivery system to the tumor cells expressing transferrin receptors. The result of fluorescence scanning showed that about 375 DOX molecules were bound to one pORF-hTRAIL molecule. The co-delivery system was constructed based on the electrostatic interactions between pORF-hTRAIL–DOX complex and cationic PAMAM-PEG-T7. T7-modified co-delivery system showed higher efficiency in cellular uptake and gene expression than unmodified co-delivery system in human liver cancer Bel-7402 cells, and accumulated in tumor more efficiently in vivo. In comparison with single DOX or pORF-hTRAIL delivery system, co-delivery system induced apoptosis of tumor cells in vitro and inhibited tumor growth in vivo more efficiently. In mice bearing Bel-7402 xenografts, lower doses of co-delivery system (4 μg DOX/mouse, about 0.16 mg/kg) effectively inhibited tumor growth comparable to high doses (5 mg/kg) of free doxorubicin (77% versus 69%). These results suggested that T7-mediated co-delivery system of DOX and pORF-hTRAIL was a simply prepared, combined delivery platform which can significantly improve the anti-tumor effect. This co-delivery system might widen the therapeutic window and allow for the selective destruction of cancer cells. [ABSTRACT FROM AUTHOR]