Murine Fibrosarcoma L929 Cells Research Articles

Anti-proliferation and anti-inflammation effects of corilagin in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways

Ethnopharmacological relevanceThe dried aboveground part of Geranium Wilfordii Maxim. (G. Wilfordii) is a traditional Chinese herbal medicine named lao-guan-cao. It has long been used for dispelling wind-dampness, unblocking meridians, and stopping diarrhea and dysentery. Previous investigations have revealed that 50% ethanolic extract of G. Wilfordii has anti-inflammatory and anti-proliferation activities on TNF-α induced murine fibrosarcoma L929 cells. Corilagin (COR) is a main compound in G. Wilfordii with the content up to 1.69 mg/g. Pharmacology study showed that COR has anti-inflammatory, anti-tumor, anti-microorganism, anti-oxidant, and hepatoprotective effects. However, there is no any investigation on its anti-proliferation and anti-inflammation effects in rheumatoid arthritis (RA). Aim of the studyThe present study aimed to evaluate the potential pharmacological mechanisms of anti-proliferation and anti-inflammation effects of COR in RA. Materials and methodsIn vitro, MH7A cells model induced by IL-1β was used. The anti-proliferation activity of COR was assessed by Cell Counting Kit-8 (CCK-8) assay, and the anti-migration and anti-invasion activity of COR was determined by wound healing assay and transwell assay, respectively. Furthermore, apoptosis assay by flow cytometer was used to measure the pro-apoptotic effect of COR. The mRNA expressions of Bax, Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS were measured by qRT-PCR, and related protein were further verified by ELISA kits or Western blot. Moreover, protein levels associated with NF-κB and MAPK signaling pathways of p65, P-p65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK1/2/3, p38, and P-p38 were determined by Western blot. The nuclear translocation of NF-κB-p65 was detected by immunofluorescent staining. In vivo, adjuvant-induced arthritis (AIA) rat model was used, and the body weight, paw swelling, and arthritis score during the entire period were measured. Histopathological analysis of joints of synovial tissues was also determined. The expression of pro-inflammatory cytokines in serum including IL-6, TNF-α, IL-1β, and IL-17 were measured. ResultsThe in vitro results showed that COR could dose-dependently inhibit the proliferation, migration, and invasion of IL-1β-induced MH7A cells, as well as promote its apoptosis. Moreover, it also suppressed the over-expression of Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS while up-regulated the level of Bax. Besides, the ratios of P-p65/p65, P-IκBα/IκBα, P-ERK/ERK, P-JNK/JNK, and P-p38/p38 were decreased, and the nuclear translocation of p65 induced by IL-1β was blocked by COR. In vivo results indicated that COR significantly reduced the paw swelling and arthritis score in AIA rats, and inhibited synovial tissue hyperplasia and erosion, as well as inflammatory cells infiltration. It also decreased the serum pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, and IL-17) production. ConclusionThese results revealed that COR exerted anti-rheumatoid arthritis effect, and its underlying mechanisms may be related to inhibiting the proliferation, migration, and invasion of synovial fibroblasts, enhancing cell apoptosis, and suppressing inflammatory responses via downregulating NF-κB and MAPK signaling pathways.

Layered double hydroxide-galactose as an excellent nanocarrier for targeted delivery of curcumin to hepatocellular carcinoma cells

Targeted drug delivery systems have been developed as a promising strategy for targeted delivery of the drugs, genes, and biomolecules to the target tissue. In this study, a drug delivery system based on layered double hydroxide (LDH), as a carrier, and galactose (Gal), as a ligand, was designed for targeted delivery of curcumin (Cur) to hepatocellular carcinoma cells. Gal was conjugated onto Cur/LDH nanohybrid using SiO2 nanodot-coating and amine-functionalization strategy to fabricate a Gal-Cur/LDH delivery system. The physicochemical properties were determined using field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM), zeta potential, Fourier transform infrared spectrometry (FTIR) and X-ray powder diffraction (XRD). The cell viability, cellular uptake, and apoptotic effects of the Gal-Cur/LDH nanoparticles for human hepatocellular carcinoma cell line (HepG2 cells) were evaluated. Additionally, in the cell viability test, to investigate the specific targeting efficiency of Gal-Cur/LDH nanoparticles into hepatoma cells, Murine fibrosarcoma L929 cells were utilized as control cells for studying the specific targeting efficiency of Gal-Cur/LDH nanoparticles for HepG2 cells. Results demonstrated the successful synthesis of LDH and the intercalation of Cur in LDH with 31.0 ± 0.0% loading efficiency. According to the cyto assay results, LDH did not significantly induce cytotoxicity in both L929 and HepG2 cells. Gal-Cur/LDH nanoparticles showed higher cytotoxicity effects against HepG2 cells than L929 cells, compared with free Cur and Cur/LDH nanohybrid. Additionally, Gal-Cur/LDH nanoparticles significantly exhibited a higher rate of apoptosis than the other groups, which might be attributed to higher cellular uptake efficiency via ASGP-receptors in HepG2 cells surface. Our findings suggest the Gal-Cur/LDH nanoparticles as an excellent carrier system for targeted delivery of Cur to the hepatocellular carcinoma cells.

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB

ABSTRACTPurpose: Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. Material and methods: We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. Results: L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. Conclusion: NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Dynamic quantitative proteomics characterization of TNF-α-induced necroptosis

Emerging evidence suggested that necroptosis has essential functions in many human inflammatory diseases, but the molecular mechanisms of necroptosis remain unclear. Here, we employed SILAC quantitatively dynamic proteomics to compare the protein changes during TNF-α-induced necroptosis at different time points in murine fibrosarcoma L929 cells with caspase-8 deficiency, and then performed the systematical analysis on the signaling networks involved in the progress using bioinformatics methods. Our results showed that a total of 329, 421 and 378 differentially expressed proteins were detected at three stages of necroptosis, respectively. Gene ontology and ingenuity pathway analysis (IPA) revealed that the proteins regulated at early stages of necroptosis (2, 6h) were mainly involved in mitochondria dysfunction, oxidative phosphorylation and Nrf-2 signaling, while the expression levels of the proteins related to ubiquitin, Nrf-2, and NF-κB pathways were found to have changes at last stages of necroptosis (6, 18h). Taken together, we demonstrated for the first time that dysfunction of mitochondria and ubiquitin-proteasome signaling contributed to the initiation and execution of necroptosis. These findings may provide clues for the identification of important regulators in necroptosis and the development of novel therapeutic strategies for the related diseases.

Identification of anti-inflammatory fractions of Geranium wilfordii using tumor necrosis factor-alpha as a drug target on Herbochip® – an array-based high throughput screening platform

BackgroundGeranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays.MethodsA microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vitro model.ResultsThe in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity.ConclusionWe have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.

Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death.

Quantitative Phosphoproteomic Analysis of RIP3‐dependent Protein Phosphorylation in the Course of TNF‐induced Necroptosis (LB175)

Tumor necrosis factor (TNF)‐induced cell death in murine fibrosarcoma L929 cells is a model system in studying programmed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine‐threonine kinase, is known to play an essential role in TNF‐induced necroptosis ; however, the phosphorylation events initiated by RIP3 activation in the necroptotic process is still largely unknown. Here, we performed a quantitative mass spectrometry‐based analysis to compare TNF‐induced changes in the global phosphoproteome of wildtype (RIP3+/+) and RIP3‐knockdown L929 cells at different time points after TNF treatment. A total of 8,058 phosphopeptides spanning 6,892 phosphorylation sites in 2,762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 hours, respectively. By comparing the phosphorylation sites in wildtype and RIP3‐knockdown L929 cells, 174, 167 and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2 and 4 hour time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3 dependent phosphorylation in LPS‐stimulated peritoneal macrophages and TNF‐treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report is highly relevant to the study of TNF‐induced necroptosis of L929 cells.

Quantitative phosphoproteomic analysis of RIP3‐dependent protein phosphorylation in the course of TNF‐induced necroptosis

Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells.

Negatively-regulated necroptosis by autophagy required caspase-6 activation in TNFα-treated murine fibrosarcoma L929 cells

Autophagy and necroptosis have been known to be interconnected, while the relationship between autophagy and necroptosis remains unclear. Here, we demonstrated that pan-caspase inhibitor z-VAD-fmk (zVAD) exacerbated TNFα-induced necroptosis and autophagy in murine fibrosarcoma L929 cells. And the RIP-1 inhibitor necrostatin-1 inhibited TNFα+zVAD-induced necroptosis and autophagy. Inhibition of autophagy by 3-methyladenine (3MA) or small interfering RNA (siRNA) against Beclin 1 augmented TNFα-induced necroptosis, while, autophagy inhibition did not influence TNFα+zVAD-induced necroptosis. These results suggested that autophagy was a downstream consequence of necroptosis, and had a negative-feedback function to necroptosis in TNFα-treated L929 cells, but not in the presence of zVAD. Subsequently, TNFα administration was accompanied with caspase-6 activation. Inhibition of caspase-6 activity by z-V-E(OMe)-I-D(OMe)-fmk (zVEID) or caspase-6 (p20) siRNA had no effect on necroptosis but promoted TNFα-induced autophagy. Meanwhile, autophagy inhibition further increased caspase-6 activation. Caspase-6 (p20) siRNA sequestered the increased necroptotic ratio by 3MA pretreatment in TNFα-treated L929 cells. In addition, caspase-6 activation induced by TNFα administration was inhibited by zVAD. Further, autophagy induced by higher concentration of zVAD did not negatively regulate necroptosis because caspase-6 was not activated. Collectively, our data indicated that autophagy was a downstream consequence of necroptosis, and negatively regulated necroptosis when caspase-6 was activated in TNFα-treated L929 cells.

MTOR inactivation by ROS-JNK-p53 pathway plays an essential role in Psedolaric acid B induced autophagy-dependent senescence in murine fibrosarcoma L929 cells

Pseudolaric acid B (PAB), the primary biologically active compound isolated from the root bark of P. kaempferi Gordon, has been reported to exhibit anti-tumor effect primarily via cell cycle arrest and apoptosis. Our previous study demonstrated that PAB triggered mitotic catastrophe in L929 cells. In addition, a small percentage of the cells undergoing mitotic catastrophe displayed an apoptotic phenotype. Therefore, we continued to investigate the fate of the other cells. The results indicated that PAB induced senescence through p19–p53–p21 and p16–Rb pathways in L929 cells. PAB also triggered autophagy via inhibiting Akt-mammalian target of rapamycin (mTOR) activity in L929 cells. In addition, autophagy was demonstrated to reinforce senescence through regulating the senescence pathways. Thus, we focused on the detailed molecular mechanisms whereby autophagy promoted senescence. Reactive oxygen species (ROS) plays an important in autophagy and senescence. We found that PAB triggered a ROS-JNK-p53 positive feedback loop and this feedback loop played a crucial role in autophagy via repressing the activation of mTOR. Furthermore, ROS-JNK-p53 positive feedback loop was demonstrated to regulate senescence. Tuberous sclerosis proteins1 and 2, also known as TSC1 and TSC2, form a protein-complex. TSC1/TSC2 heterodimer is a downstream target of growth factor-phosphoinositide 3-kinase-Akt signaling which negatively regulates mTOR activity. Activation of mTOR by insulin or inhibition of endogenous TSC2 levels by siRNA obviously delayed PAB-induced senescence. In conclusion, mTOR inactivation by ROS-JNK-p53 pathway played an important role in autophagy-dependent senescence in PAB-treated L929 cells.

Microbial synthesis of poly-3-hydroxybutyrate and its application as targeted drug delivery vehicle

Arsenic trioxide loaded biocompatible PHB-PVA(1) nanoparticles (<100 nm in size) with folate functionalized surface were synthesized using poly-[(R)-3-hydroxybutyric acid] (PHB) produced by Bacillus firmus NII 0830. Folate functionalization was carried using dicyclohexyl carbodiimide (DCC) as a catalyst and 10-bromodecanol as a linker to conjugate glutamic acid terminal of folate with the hydroxylate groups present on the surface of PHBA-PVA(2) nanotrojans. The effect of fabrication parameters on shape, size distribution and PDI of the PHB nanoparticles were also investigated. It was observed that increase in sonication time and polyvinyl alcohol (PVA) concentration greatly reduced the size of nanoparticles. The drug release studies on arsenic trioxide incorporated PHB-PVA nanoparticles were conducted at physiological pH and temperature. FOL-PHBA-PVA(3) nanoparticles showed greater extent of cytotoxicity towards murine fibrosarcoma L929 cells than PHBA-PVA nanoparticles alone without conjugated folate, indicating the significance of folate as ligand for specific targeting of FR+ cancer cells.

RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy

Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine–threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death.

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H2O2 in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.

Function without form: an ongoing search for maxi-anion channel proteins. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

<i>Function without form: an ongoing search for maxi-anion channel proteins</i>. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

Oridonin induces apoptosis and autophagy in murine fibrosarcoma L929 cells partly via NO-ERK-p53 positive-feedback loop signaling pathway

To investigate the role of nitric oxide (NO) in oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells and the underlying molecular mechanisms. Cell viability was measured using MTT assay. Intracellular NO level, SubG(1) cell ratio and autophagy cell ratios were analyzed with flow cytometry after diaminofluorescein-2 diacetate (DAF-2DA), propidium iodide (PI) and monodansylcadaverine (MDC) staining, respectively. Protein expression was examined using Western blot analysis. Exposure of L929 cells to oridonin (50 μmol/L) for 24 h led to intracellular NO production. Pretreatment with NOS inhibitor 1400w or L-NAME inhibited oridonin-induced apoptosis and autophagy in L929 cells. The pretreatment decreased the apoptosis-related protein Bax translocation and cytochrome c release, increased Bcl-2 level, reversed the autophagy-associated protein Beclin 1 increase and conversion of LC3 I to LC3 II. Furthermore, pretreatment with NO scavenger DTT completely inhibited oridonin-induced apoptosis and autophagy in L929 cells. In addition, oridonin (50 μmol/L) activated ERK and p53 in L929 cells, and the interruption of ERK and p53 activation by PD 98059, pifithrin-α, or ERK siRNA decreased oridonin-induced apoptosis and autophagy. The inhibition of NO production reduced oridonin-induced ERK and p53 activation, and NO production was down-regulated by blocking ERK and p53 activation. NO played a pivotal role in oridonin-induced apoptosis and autophagy in L929 cells. Taken together with our previous finding that ERK contributes to p53 activation, it appears that NO, ERK, and p53 form a positive feedback loop. Consequently, we suggest that oridonin-induced apoptosis and autophagy are modulated by the NO-ERK-p53 molecular signaling mechanism in L929 cells.

Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells

The maxi-anion channel plays a classically recognized role in controlling the membrane potential through the chloride conductance. It also has novel functions as a regulated pathway for the release of the anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Because hemichannels formed by pannexins and connexins have been reported to mediate ATP release from a number of cell types, these hemichannels may represent the molecular correlate of the maxi-anion channel. Here, we found that L929 fibrosarcoma cells express functional maxi-anion channels which mediate a major portion of swelling-induced ATP release, and that ATP released via maxi-anion channels facilitates the regulatory volume decrease after osmotic swelling. Also, it was found that the cells express the mRNA for pannexin 1, pannexin 2, and connexin 43. Hypotonicity-induced ATP release was partially suppressed not only by known blockers of the maxi-anion channel but also by several blockers of pannexins including the pannexin 1-specific blocking peptide (10)Panx1 and small interfering (si)RNA against pannexin 1 but not pannexin 2. The inhibitory effects of maxi-anion channel blockers and pannexin 1 antagonists were additive. In contrast, maxi-anion channel activity was not affected by pannexin 1 antagonists and siRNAs against pannexins 1 and 2. Although a connexin 43-specific blocking peptide, Gap27, slightly suppressed hypotonicity-induced ATP release, maxi-anion channel activity was not affected by Gap27 or connexin 43-specific siRNA. Thus, it is concluded that the maxi-anion channel is a molecular entity distinct from pannexin 1, pannexin 2, and connexin 43, and that the maxi-anion channel and the hemichannels constitute separate pathways for swelling-induced ATP release in L929 cells.

Pseudolaric acid B induces mitotic catastrophe followed by apoptotic cell death in murine fibrosarcoma L929 cells

Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Previous studies have demonstrated that PAB arrests cells in G2/M phase in several cancer cell lines without significantly perturbing the G2/M transition-associated proteins. CylinB1, a marker for mitotic phase arrest, was up-regulated in cells treated with PAB. Therefore, we investigated whether PAB affects cell cycle progression at the mitotic phase. The mitotic index increased during a 24h treatment with PAB, suggesting that PAB arrested cell cycle progression at mitosis. In addition, after a prolonged mitotic arrest, the cells underwent mitotic catastrophe. After an extended treatment with PAB (longer than 24h), the protein levels of cylinB1 and cdc2 significantly decreased in both nuclear and cytosolic extracts. According to these results, we concluded that mitotic slippage could be due to the inactivation of the cylinB1–cdc2 complex resulting from prolonged treatment with PAB. The cells undergoing mitotic catastrophe died via apoptosis.

TNFα-Induced Necroptosis and Autophagy via Supression of the p38–NF-κB Survival Pathway in L929 Cells

Tumor necrosis factor alpha (TNFα) has been reported to induce necroptosis and autophagy, but its mechanisms remain unclear. In this study, we found that TNFα significantly induced necroptosis and autophagy in murine fibrosarcoma L929 cells. The necroptosis inhibitor necrostatin-1 (Nec-1) completely blocked TNFα-induced necroptosis and autophagy, but inhibition of autophagy with 3-methyladenine (3MA) or Beclin 1 small interfering RNA (siRNA) promoted necroptosis, indicating that autophagy acted as a negative regulator of TNFα-induced necroptosis. The cytotoxicity of TNFα was accompanied by decreased expressions of phosphorylated p38 mitogen-activated protein kinase (p-p38) and nuclear factor-kappa B (NF-κB), and inhibition of p38 and NF-κB activation by chemical inhibitors or siRNA augmented these necroptotic and autophagic responses to TNFα in the cells. The pan-caspase inhibitor z-VAD-fmk (zVAD) exacerbated TNFα-induced necroptosis and autophagy. Combined treatment with TNFα and zVAD further decreased the expressions of p-p38 and NF-κB compared with TNFα alone treatment. Consequently, these results indicated that suppression of the p38-NF-κB survivial signaling pathway promoted necroptotic and autophagic cell death in TNFα-treated L929 cells.

Survivin S81A Enhanced TRAIL's Activity in Inducing Apoptosis

BACKGROUND: Survivin is rarely expressed in normal healthy adult tissues, however, it is upregulated in the majority of cancers. Survivin, which belongs to IAPs family, has been widely reported to protect cells from apoptosis by inhibiting caspases pathway. Survivin’s mitotic activity is modulated by many kinases, and its phosphor status can also influence its ability to inhibit apoptosis. There are several important survivin’s phosphorylation sites, such as S20 and T34. We have continued our investigation on other potential survivin’s phosphorylation sites that could be important site for regulating survivin’s cyto-protection.METHODS: By assuming that S81 could be a potential target to modify activity of survivin, wild-type survivin (Survivin), antisense survivin (Survivin-AS), mutated-survivin Thr34Ala (Survivin-T34A) and mutated-survivin Ser81Ala (Survivin-S81A) were constructed and inserted into pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter. Each retroviral product was produced in BOSC23 cells. LY294002 pretreatment and TRAIL treatment along with infection of retroviral products were performed in murine fibrosarcoma L929 cells. For analysis, flow cytometric apoptosis assay and western blot were performed.RESULTS: In our present study, survivin for providing cytoprotection was regulated by PI3K. The results showed that LY294002, an inhibitor of PI3K, effectively suppressed survivin-modulated cytoprotection in a TRAIL-induced apoptotic model. In addition, mutated survivin S81A showed marked suppression on survivin’s cytoprotection. Along with that, TRAIL’s apoptotic activity was enhanced for inducing apoptosis.CONCLUSION: We suggested that survivin could inhibit apoptosis through PI3K and S81A could be another potential target in order to inhibit Survivin-modulated cytoprotection as well as to sensitize efficacy of TRAIL or other related apoptotic inducers.KEYWORDS: apoptosis, survivin, TRAIL, S81A, L929, LY294002

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras–Raf and PI3K–Akt signaling pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras–Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K–Akt signaling pathways.