JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein σ1 and membrane penetration protein μ1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L × T3D reassortant viruses revealed that the μ1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.     

LncRNA HOXB-AS1 promotes cell growth in multiple myeloma via FUT4 mRNA stability by ELAVL1

Multiple myeloma (MM) is defined as the second most common hematological tumor in the globe. Long noncoding RNAs (lncRNAs) have been reported to play stimulative or suppressive role in the progression of different carcinomas. The investigation of lncRNAs in MM is still inadequate. LncRNA HOXB cluster antisense RNA 1 (HOXB-AS1) was once revealed to facilitate glioma progression by affecting cellular activities of glioma cells. However, whether HOXB-AS1 participates in the development of MM still remains an enigma. In this study, we unveiled that HOXB-AS1 was highly expressed in MM and loss-of-function assays certified that HOXB-AS1 obstruction suppressed MM cell proliferation, and stimulated cell apoptosis. In addition, HOXB-AS1 could modulate fucosyltransferase 4 (FUT4) and FUT4-mediated Wnt/β-catenin pathway. In subsequence, it was observed from mechanism assays that HOXB-AS1 enhanced the interaction between ELAVL1 and FUT4 so as to stabilize FUT4 messenger RNA. In the end, rescue experiments affirmed that HOXB-AS1 affected the cell growth through FUT4 in MM. In conclusion, the whole modulation mechanism of HOXB-AS1/ELAVL1/FUT4 axis in MM was validated in this study, which suggested that HOXB-AS1 might function as a powerful and promising therapeutic biomarker for the clinical treatment of patients with MM.     

Reovirus infection and tissue injury in the mouse central nervous system are associated with apoptosis.

Reovirus serotype 3 strains infect neurons within specific regions of the neonatal mouse brain and produce a lethal meningoencephalitis. Viral replication and pathology colocalize and have a predilection for the cortex, hippocampus, and thalamus. We have shown previously that infection of cultured fibroblasts and epithelial cells with reovirus type 3 Dearing (T3D) and other type 3 reovirus strains results in apoptotic cell death, suggesting that apoptosis is a mechanism of cell death in vivo. We now report that T3D induces apoptosis in infected mouse brain tissue. To determine whether reovirus induces apoptosis in neural tissues, newborn mice were inoculated intracerebrally with T3D, and at various times after inoculation, brain tissue was assayed for viral antigen by immunostaining and apoptosis was identified by DNA oligonucleosomal laddering and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cells were also stained with cresyl violet to detect morphological changes characteristic of apoptosis, including chromatin condensation and cell shrinkage. DNA laddering was detected in T3D- but not in mock-infected brain tissue. Apoptotic cells were restricted to the same regions of the brain in which infected cells and tissue damage were observed. These findings suggest that virus-induced apoptosis is a mechanism of cell death, tissue injury, and mortality in reovirus-infected mice. The correlation between apoptosis and pathogenesis in vivo identifies apoptosis as a potential target for molecular and pharmacological strategies designed to curtail or prevent diseases resulting from induction of this cell death pathway.     

Apoptosis-induced by TRAIL AND TNF-alpha in human multiple myeloma cells is not blocked by BCL-2

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor is a member of the tumour necrosis factor (TNF) family of death signal tranduction proteins with a mechanism of cell death, similar to the Fas and Fas ligand (Fas-L) system. Here, we provide first time evidence that TRAIL and TNF-alpha are potent inducers of apoptosis in multiple myeloma (MM) cell lines and freshly isolated myeloma cells. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner reaching 80% within 48 h of treatment with a dose of 160 ng/ml. Bcl-2 transfected 8226 and ARP-1 cells were equally sensitive to apoptosis by TRAIL. Apoptosis with TNFalpha, reached >60% within 48 h of treatment with a dose of 160 ng/ml. In addition to MM cell lines, freshly isolated, flow-sorted myeloma cells from 8 different MM patients expressing variable levels of bcl-2 were equally sensitive to both TRAIL and TNF-alpha. We have previously shown that anti-Fas-induced apoptosis is not blocked by endogenous or ectopic bcl-2 in MM cell lines. Here we extend our observation with Fas to include TNF-alpha and TRAIL to the apoptotic signals that are not be blocked by bcl-2, in MM cells.     

Targeting Poly (ADP-Ribose) Polymerase Partially Contributes to Bufalin-Induced Cell Death in Multiple Myeloma Cells

Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM) remains an incurable disease. Recently, ploy(ADP-ribose) polymerase 1 (PARP1) has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine (“Chan Su”), might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA–damage-induced poly(ADP-ribosyl)ation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S) and primary CD138⁺ MM cells could be inhibited by bufalin, mainly via apoptosis and G₂-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.     

Bendamustine overcomes resistance to melphalan in myeloma cell lines by inducing cell death through mitotic catastrophe

Melphalan has been a mainstay of multiple myeloma (MM) therapy for many years. However, following treatment with this alkylator, malignant plasma cells usually escape both apoptosis and cell cycle control, and acquire drug-resistance resulting in tumor progression. Bendamustine is being used in MM patients refractory to conventional DNA-damaging agents, although the mechanisms driving this lack of cross-resistance are still undefined. Here, we investigated the molecular pathway of bendamustine-induced cell death in melphalan-sensitive and melphalan-resistant MM cell lines. Bendamustine affected cell survival resulting in secondary necrosis, and prompted cell death primarily through caspase-2 activation. Also, bendamustine blocked the cell cycle in the G2/M phase and induced micronucleation, erratic chromosome spreading and mitotic spindle perturbations in melphalan-resistant MM cells. In these cells, both Aurora kinase A (AURKA) and Polo-like kinase-1 (PLK-1), key components of the spindle-assembly checkpoint, were down-regulated following incubation with bendamustine, whereas levels of Cyclin B1 increased as a consequence of the prolonged mitotic arrest induced by the drug. These findings indicate that, at least in vitro, bendamustine drives cell death by promoting mitotic catastrophe in melphalan-resistant MM cells. Hence, activation of this alternative pathway of cell death may be a novel approach to the treatment of apoptosis-resistant myelomas.     

APOPTOSIS-INDUCED BY TRAIL AND TNF-α IN HUMAN MULTIPLE MYELOMA CELLS IS NOT BLOCKED BY BCL-2

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor is a member of the tumour necrosis factor (TNF) family of death signal tranduction proteins with a mechanism of cell death, similar to the Fas and Fas ligand (Fas-L) system. Here, we provide first time evidence that TRAIL and TNF-α are potent inducers of apoptosis in multiple myeloma (MM) cell lines and freshly isolated myeloma cells. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner reaching 80% within 48 h of treatment with a dose of 160 ng/ml. Bcl-2 transfected 8226 and ARP-1 cells were equally sensitive to apoptosis by TRAIL. Apoptosis with TNFα, reached >60% within 48 h of treatment with a dose of 160 ng/ml. In addition to MM cell lines, freshly isolated, flow-sorted myeloma cells from 8 different MM patients expressing variable levels of bcl-2 were equally sensitive to both TRAIL and TNF-α. We have previously shown that anti-Fas-induced apoptosis is not blocked by endogenous or ectopic bcl-2 in MM cell lines. Here we extend our observation with Fas to include TNF-α and TRAIL to the apoptotic signals that are not be blocked by bcl-2, in MM cells.     

Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors

The cellular pathways of apoptosis have not been fully characterized; however, calpain, a cytosolic calcium-activated cysteine protease, has been implicated in several forms of programmed cell death. Reoviruses induce apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of calpain in reovirus-induced apoptosis in vitro by measuring calpain activity as well as evaluating the effects of calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and calpain activity, measured as cleavage of the fluorogenic calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of apoptosis by calpain inhibitors is independent of effects on viral replication. We conclude that calpain activation is a component of the regulatory cascade in reovirus-induced apoptosis.     

Activation of p53 by Chemotherapeutic Agents Enhances Reovirus Oncolysis

Mammalian reovirus is a benign virus that possesses the natural ability to preferentially infect and kill cancer cells (reovirus oncolysis). Reovirus exploits aberrant Ras signalling in many human cancers to promote its own replication and spread. In vitro and in vivo studies using reovirus either singly or in combination with anti-cancer drugs have shown very encouraging results. Presently, a number of reovirus combination therapies are undergoing clinical trials for a variety of cancers. Previously we showed that accumulation of the tumor suppressor protein p53 by Nutlin-3a (a specific p53 stabilizer) enhanced reovirus-induced apoptosis, and resulted in significantly higher levels of reovirus dissemination. In this study, we examined the role of p53 in combination therapies involving reovirus and chemotherapeutic drugs. We showed that sub-lethal concentrations of traditional chemotherapy drugs actinomycin D or etoposide, but not doxorubicin, enhanced reovirus-induced apoptosis in a p53-dependent manner. Furthermore, NF-κB activation and expression of p53-target genes (p21 and bax) were important for the p53-dependent enhancement of cell death. Our results show that p53 status affects the efficacy of combination therapy involving reovirus. Choosing the right combination partner for reovirus and a low dosage of the drug may help to both enhance reovirus-induced cancer elimination and reduce drug toxicity.     

Reovirus-induced apoptosis is mediated by TRAIL

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.     

Serotype-dependent induction of pulmonary neutrophilia and inflammatory cytokine gene expression by reovirus.

Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3D-infected compared with those of T1IL-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model.     

Potent induction of apoptosis by beta-lapachone in human multiple myeloma cell lines and patient cells

BACKGROUND: Human multiple myeloma (MM) remains an incurable hematological malignancy. We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity. MATERIALS AND METHODS: We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay. To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays. The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis. To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay. RESULTS: We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5). Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors. In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis. Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM. CONCLUSIONS: Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.     

Bortezomib induces autophagic death in proliferating human endothelial cells

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.     

EV71 infection induces cell apoptosis through ROS generation and SIRT1 activation

Several studies have substantiated the correlation between reactive oxygen species (ROS) and Sirtuin 1 (SIRT1). Normally, enterovirus 71 (EV71) is associated with severe clinical manifestations and death. However, the effect of EV71 on the induction of cellular death and the interplay between ROS/SIRT1 in cell death has not been confirmed yet. In the current study, an increase in the number of apoptotic cells was observed as soon as the EV71 infection was initiated in cells and mice. Furthermore, EV71 infection also promoted a rise in the levels of three commonly known proinflammatory cytokines, interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-α. During EV71-induced apoptosis in the different cell lines, ROS generation and SIRT1 downregulation were observed. Further investigations showed that the administration of ROS inhibitor, N-acetyl- l -cysteine (NAC), reduced the level of apoptosis and inflammation, reduced EV71 propagation, and increased SIRT1 expression in EV71-infected cells. In addition, combined administration of NAC and EX527 (SIRT1 inhibitor) restored apoptosis in the EV71-infected cells, which was reduced due to NAC. This data demonstrated that ROS generation is positively associated with EV71-induced apoptosis and inflammation, while this effect could be reversed by SIRT1 inhibition. Collectively, we have shown that EV71 induces apoptosis and inflammation by promoting ROS generation and reducing SIRT1 expression.