Luteolin inhibits the Nrf2 signaling pathway and tumor growth in vivo

Nuclear factor erythroid 2-related factor 2 (Nrf2) is over-expressed in many types of tumor, promotes tumor growth, and confers resistance to anticancer therapy. Hence, Nrf2 is regarded as a novel therapeutic target in cancer. Previously, we reported that luteolin is a strong inhibitor of Nrf2 in vitro. Here, we showed that luteolin reduced the constitutive expression of NAD(P)H quinone oxidoreductase 1 in mouse liver in a time- and dose-dependent manner. Further, luteolin inhibited the expression of antioxidant enzymes and glutathione transferases, decreasing the reduced glutathione in the liver of wild-type mice under both constitutive and butylated hydroxyanisole-induced conditions. In contrast, such distinct responses were not detected in Nrf2^−/− mice. In addition, oral administration of luteolin, either alone or combined with intraperitoneal injection of the cytotoxic drug cisplatin, greatly inhibited the growth of xenograft tumors from non-small-cell lung cancer (NSCLC) cell line A549 cells grown subcutaneously in athymic nude mice. Cell proliferation, the expression of Nrf2, and antioxidant enzymes were all reduced in tumor xenograft tissues. Furthermore, luteolin enhanced the anti-cancer effect of cisplatin. Together, our findings demonstrated that luteolin inhibits the Nrf2 pathway in vivo and can serve as an adjuvant in the chemotherapy of NSCLC.     

Depletion of HDAC6 Enhances Cisplatin-Induced DNA Damage and Apoptosis in Non-Small Cell Lung Cancer Cells

Histone deacetylase inhibitors (HDACi) are promising therapeutic agents which are currently used in combination with chemotherapeutic agents in clinical trials for cancer treatment including non-small cell lung cancer (NSCLC). However, the mechanisms underlying their anti-tumor activities remain elusive. Previous studies showed that inhibition of HDAC6 induces DNA damage and sensitizes transformed cells to anti-tumor agents such as etoposide and doxorubicin. Here, we showed that depletion of HDAC6 in two NSCLC cell lines, H292 and A549, sensitized cells to cisplatin, one of the first-line chemotherapeutic agents used to treat NSCLC. We suggested that depletion of HDAC6 increased cisplatin-induced cytotoxicity was due to the enhancement of apoptosis via activating ATR/Chk1 pathway. Furthermore, we showed that HDAC6 protein levels were positively correlated with cisplatin IC(50) in 15 NSCLC cell lines. Lastly, depletion of HDAC6 in H292 xenografts rendered decreased tumor weight and volume and exhibited increased basal apoptosis compared with the controls in a xenograft mouse model. In summary, our findings suggest that HDAC6 is positively associated with cisplatin resistance in NSCLC and reveal HDAC6 as a potential novel therapeutic target for platinum refractory NSCLC.     

Crude alkaloid extract of Rhazya stricta inhibits cell growth and sensitizes human lung cancer cells to cisplatin through induction of apoptosis

There is an urgent need to improve the clinical management of non-small cell lung cancer (NSCLC), one of the most frequent causes of cancer-related deaths in men and women worldwide. Rhazya stricta, an important medicinal plant used in traditional Oriental medicine, possesses anti-oxidant, anti-carcinogenic and free radical scavenging properties. This study was done to explore the potential anticancer activity of a crude alkaloid extract of R. stricta (CAERS) against the NSCLC line A549. CAERS markedly suppressed the growth of A549 cells and considerably enhanced the anti-proliferative potential of cisplatin. CAERS-mediated inhibition of A549 cell growth correlated with the induction of apoptosis that was accompanied by numerous morphological changes, DNA fragmentation, an increase in the Bax/Bcl-2 ratio, the release of mitochondrial cytochrome c, activation of caspases 3 and 9 and cleavage of poly(ADP-ribose)-polymerase. CAERS reduced the constitutive expression of anti-apoptotic proteins (Bcl-2, Bcl-X_L, Mcl-1 and Survivin) and cell cycle regulating proteins (cyclin D1 and c-Myc), but enhanced expression of the proapoptotic proteins Noxa and BAD. These observations indicate that CAERS induced apoptosis and sensitized NSCLC to cisplatin via a mitochondria-mediated apoptotic pathway. These data provide a rationale for using a combination of CAERS and CDDP to treat NSCLC and other CDDP-resistant tumors.     

RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage

The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis and necroptosis in the presence of caspase inhibitors. Transfection of RIP3 in the WEHI-3b leukemia cell line or in the mouse embryonic fibroblasts also resulted in increased cell death. Surprisingly, re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-kinase dead (RIP3-KD)) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3-WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD but not RIP3-WT prolonged the survival of mice injected with leukemia cells. The expression of RIP3-KD induced p65/RelA nuclear factor-κB (NF-κB) subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued these cells from apoptosis. p65/RelA cleavage appears to be at least partially mediated by caspase-6. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and NF-κB activity.Impairment in cell death pathways represents a general characteristic of most cancer cells. Cells can die through several mechanisms; two such cell death pathways include apoptosis and necrosis, which display distinct characteristics.¹ Necrosis can occur in either an incidental or intentional manner as a result of defined signals, and the term necroptosis has been proposed to describe this programmed necrosis.² Activation of the receptor-interacting protein kinase 1 (RIP1) and 3 (RIP3) proteins in the necrosome complex can induce apoptosis, necroptosis, or cell proliferation after the activation of death receptors, including TNFR1, TRAIL, and FAS.^{3, 4} RIP1 and RIP3 are serine threonine kinases with strong homology.⁵ Both proteins are composed of a kinase domain at the N-terminus and a RIP homotypic interaction motif (RHIM) at the C-terminus of RIP3. The RIP1/RIP3 complex can induce necroptosis initiated by cell death receptors of the tumor necrosis factor family. RIP3 binds to RIP1 via their respective RHIM domains, and these proteins form a filamentous structure with characteristics similar to β-amyloids and can cross phosphorylate each other and several downstream targets involved in necroptosis, apoptosis, or nuclear factor-κB (NF-κB) activation.⁶The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood. A previous study in chronic lymphocytic leukemia (CLL) showed that malignant lymphoid cells were resistant to tumor necrosis factor-α (TNFα+Z-VAD-induced (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) necroptosis and expressed reduced levels of RIP3 and cylindromatosis (CYLD), which regulates RIP1.⁷ Another study on childhood acute lymphoblastic leukemia reported that RIP1 was necessary to mediate the inhibitor of apoptosis protein-mediated sensitization of blast cells to chemotherapy.⁸ Autocrine TNFα loops that activate NF-κB through RIP1 have also been described in various cancer cell lines.^{9, 10}Here we report that the expression of RIP3 was decreased in the majority of acute myeloid leukemia (AML) patients examined, whereas the expression of RIP1 remained unaffected. The expression of a RIP3 mutant with an inactivated kinase domain (RIP3-kinase dead (RIP3-KD)) in myeloid cell lines resulted in massive and early apoptosis and the caspase-mediated cleavage of p65/RkelA at a caspase-6 putative consensus site. Moreover, only RIP3-KD prolonged the survival of leukemic mice. Our results show that RIP3 activity regulates the apoptosis/necroptosis switch via its kinase activity in leukemia cells, and that other functions of RIP3 that are independent of its kinase domain modulate apoptosis and NF-κB activity.     

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.     

Centipeda minima extract sensitizes lung cancer cells to DNA-crosslinking agents via targeting Fanconi anemia pathway

BackgroundIntrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear.PurposeWe aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms.MethodsCell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP.ResultsCombined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMC-induced DNA damage and apoptosis, as evidenced by increased expression of γ-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDP-mediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues.ConclusionOur results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.     

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.     

Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.     

Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

Twist1 is highly expressed in primary and metastatic non-small cell lung cancer (NSCLC), and thus acts as a critical target for lung cancer chemotherapy. In the current study, we investigated the underlying mechanism initiated by silencing of Twist1 that sensitizes NSCLC cells to cisplatin. Silencing of Twist1 triggered ATP depletion, leading to AMP-activated protein kinase (AMPK)-activated mammalian target of rapamycin (mTOR) inhibition in NSCLC cells. AMPK-induced mTOR inhibition, in turn, resulted in downregulation of ribosome protein S6 kinase 1 (S6K1) activity. Downregulation of mTOR/S6K1 reduced Mcl-1 protein expression, consequently promoting sensitization to cisplatin. Overexpression of Mcl-1 reduced PARP cleavage induced by cisplatin and Twist1 siRNA, suggesting that this sensitization is controlled through Mcl-1 expression. Interestingly, cells treated with Twist1 siRNA displayed upregulation of p21^Waf1/CIP1, and suppression of p21^Waf1/CIP1 with specific siRNA further enhanced the cell death response to cisplatin/Twist1 siRNA. In conclusion, silencing of Twist1 sensitizes lung cancer cells to cisplatin via stimulating AMPK-induced mTOR inhibition, leading to a reduction in Mcl-1 protein. To our knowledge, this is the first report to provide a rationale for the implication of cross-linking between Twist1 and mTOR signaling in resistance of NSCLC to anticancer drugs.     

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.     

Dexamethasone Reduces Sensitivity to Cisplatin by Blunting p53-Dependent Cellular Senescence in Non-Small Cell Lung Cancer

Introduction

Dexamethasone (DEX) co-treatment has proved beneficial in NSCLC patients, improving clinical symptoms by the reduction of side effects after chemotherapy. However, recent studies have shown that DEX could render cancer cells more insensitive to cytotoxic drug therapy, but it is not known whether DEX co-treatment could influence therapy-induced senescence (TIS), and unknown whether it is in a p53-dependent or p53-independent manner.

Methods

We examined in different human NSCLC cell lines and detected cellular senescence after cisplatin (DDP) treatment in the presence or absence of DEX. The in vivo effect of the combination of DEX and DDP was assessed by tumor growth experiments using human lung cancer cell lines growing as xenograft tumors in nude mice.

Results

Co-treatment with DEX during chemotherapy in NSCLC resulted in increased tumor cell viability and inhibition of TIS compared with DDP treated group. DEX co-treatment cells exhibited the decrease of DNA damage signaling pathway proteins, the lower expression of p53 and p21^CIP1, the lower cellular secretory program and down-regulation of NF-κB and its signaling cascade. DEX also significantly reduced DDP sensitivity in vivo.

Conclusions

Our results underscore that DEX reduces chemotherapy sensitivity by blunting therapy induced cellular senescence after chemotherapy in NSCLC, which may, at least in part, in a p53-dependent manner. These data therefore raise concerns about the widespread combined use of gluocorticoids (GCs) with antineoplastic drugs in the clinical management of cancer patients.     

Neoalbaconol induces energy depletion and multiple cell death in cancer cells by targeting PDK1-PI3-K/Akt signaling pathway

Many natural compounds derived from plants or microbes show promising potential for anticancer treatment, but few have been found to target energy-relevant regulators. In this study, we report that neoalbaconol (NA), a novel small-molecular compound isolated from the fungus, Albatrellus confluens, could target 3-phosphoinositide-dependent protein kinase 1 (PDK1) and inhibit its downstream phosphoinositide-3 kinase (PI3-K)/Akt-hexokinase 2 (HK2) pathway, which eventually resulted in energy depletion. By targeting PDK1, NA reduced the consumption of glucose and ATP generation, activated autophagy and caused apoptotic and necroptotic death of cancer cells through independent pathway. Necroptosis was remarkably induced, which was confirmed by several necroptosis-specific markers: the activation of autophagy, presence of necrotic morphology, increase of receptor-interacting protein 1 (RIP1)/RIP3 colocalization and interaction and rescued by necroptosis inhibitor necrostatin-1. The possibility that Akt overexpression reversed the NA-induced energy crisis confirmed the importance of the PDK1-Akt-energy pathway in NA-mediated cell death. Moreover, NA shows the capability to inhibit PI3-K/Akt signaling and suppress tumor growth in the nasopharyngeal carcinoma (NPC) nude mouse model. These results supported the feasibility of NA in anticancer treatments.     

Methylation status of IGFBP-3 as a useful clinical tool for deciding on a concomitant radiotherapy

The methylation status of the IGFBP-3 gene is strongly associated with cisplatin sensitivity in patients with non-small cell lung cancer (NSCLC). In this study, we found in vitro evidence that linked the presence of an unmethylated promoter with poor response to radiation. Our data also indicate that radiation might sensitize chemotherapy-resistant cells by reactivating IGFBP-3-expression through promoter demethylation, inactivating the PI3K/AKT pathway. We also explored the IGFBP-3 methylation effect on overall survival (OS) in a population of 40 NSCLC patients who received adjuvant therapy after R0 surgery. Our results indicate that patients harboring an unmethylated promoter could benefit more from a chemotherapy schedule alone than from a multimodality therapy involving radiotherapy and platinum-based treatments, increasing their OS by 2.5 y (p = .03). Our findings discard this epi-marker as a prognostic factor in a patient population without adjuvant therapy, indicating that radiotherapy does not improve survival for patients harboring an unmethylated IGFBP-3 promoter.     

Toxin-Induced Necroptosis Is a Major Mechanism of Staphylococcus aureus Lung Damage

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. Rip3 ^-/- mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.     

Icariside II enhances cisplatin-induced apoptosis by promoting endoplasmic reticulum stress signalling in non-small cell lung cancer cells

Although cisplatin is the most effective first-line drug in the management of advanced non-small cell lung cancer (NSCLC), drug resistance remains a major clinical challenge. There is increasing evidence that icariside II (IS) exhibits antitumour activity in a variety of cancers. In the current study, we investigated the anticancer effects of icariside II combined with cisplatin and elucidated the underlying mechanism in NSCLC. Here, we showed that cotreatment with IS and cisplatin inhibited cell proliferation and induced cellular apoptosis. Using mRNA sequencing (mRNA-seq), we identified differentially expressed genes (DEGs) in which there was an enrichment in PERK-mediated unfolded protein response (UPR) signalling. The western blot results revealed that IS activated endoplasmic reticulum (ER) stress, including three branches of UPR signalling, PERK, IRE1 and ATF6, and the downstream PERK-eIF2α-ATF4-CHOP pathway, thus potentiating the apoptosis induced by cisplatin. In addition, the combination of IS with cisplatin significantly reduced xenograft tumour growth in C57BL/6 and BALB/c nude mice in vivo. Notably, the combination therapy displayed no evident toxicity. Taken together, IS enhances cisplatin-induced apoptosis partially by promoting ER stress signalling in NSCLC, suggesting that combination treatment with IS and cisplatin is a novel potential therapeutic strategy for NSCLC.     

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q₁₀ (N5/C₁₀)_, which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C₁₀ protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.     

Knockdown of LncRNA PVT1 inhibits tumorigenesis in non-small-cell lung cancer by regulating miR-497 expression

Plasmacytoma variant translocation1 (PVT1) was reported to be upregulated in non-small-cell lung cancer (NSCLC) tissues, serve as a promising biomarker for diagnosis and prognosis of NSCLC, and promoted NSCLC cell proliferation. However, the detailed molecular mechanism of PVT1 involved in the pathogenesis and development of NSCLC remains largely unknown. In this study, the expression levels of PVT1 and miR-497 in NSCLC cells were determined by qRT-PCR. Cell viability, invasion and apoptosis were detected by MTT assay, cell invasion assay and flow cytometry analysis, respectively. RNA immunoprecipitation (RIP) and luciferase reporter assay were performed to confirm whether PVT1 directly interacts with miR-497. A xenograft mouse model was established to confirm the effect of PVT1 on tumor growth in vivo and the underlying molecular mechanism. Our findings indicated that PVT1 was significantly upregulated and miR-497 was markedly downregulated in NSCLC cell lines. si-PVT1 effectively decreased the expression of PVT1 and increased the expression of miR-497. PVT1 knockdown remarkably inhibited cell viability, invasion and promoted apoptosis in NSCLC cells. RIP and luciferase reporter assay demonstrated that PVT1 could directly interact with miR-497. Moreover, PVT1 overexpression reversed the inhibitory effect of miR-497 on cell viability, invasion and promotion effect on apoptosis of NSCLC cells. Furthermore, in vivo experiment showed that knockdown of PVT1 inhibited tumor growth in vivo and promoted miR-497 expression. In conclusion, knockdown of PVT1 inhibited cell viability, invasion and induced apoptosis in NSCLC by regulating miR-497 expression, elucidating the molecular mechanism of the oncogenic role of PVT1 in NSCLC and providing an lncRNA-directed target for NSCLC.