The Histone Deacetylase (HDAC) Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1562-1562 ◽  
Author(s):  
Noor M Khaskhely ◽  
Daniela Buglio ◽  
Jessica Shafer ◽  
Catherine M. Bollard ◽  
Anas Younes
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Protein Expression ◽  
Cell Lines ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Apoptosis Pathway ◽  
Gene And Protein Expression ◽  
Combination Studies

Abstract Abstract 1562 Poster Board I-585 Purpose SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay. Results SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced. Conclusions Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing. Disclosures Younes: MethylGene: Honoraria, Research Funding.

The Histone Deacetylase Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2850-2850 ◽  
Author(s):  
Adam Jona ◽  
Noor Khaskhely ◽  
Daniela Buglio ◽  
Jessica A. Shafer ◽  
Enrico Derenzini ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Hodgkin Lymphoma ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Dependent Manner ◽  
Gene And Protein Expression ◽  
Combination Studies ◽  
20 Nm

Abstract Abstract 2850 Introduction: Based on recent favorable in vitro and in vivo activity of several HDACi (histone deacetylase inhibitors) in HL (Hodgkin lymphoma), we investigated the in vitro activity of SNDX-275, an oral, class 1 isoform of selective HDACi in HL-derived cell lines. Materials and methods: Proliferation and cell death were examined by MTS assay, Annexin-V/PI and FACS analysis. For combination studies, cells were incubated with SNDX-275 (0.1-2 μM) and either ABT-737 (0.01-0.2 μM), Obatoclax (0.1-2 μM), Gemcitabine (1-20 nM) or Bortezomib (1-20 nM) for 72 hours. Gene and protein expression were measured by RT-PCR, Western blot, and immunohistochemistry. A multiplex assay was used to determine 30 cytokines and chemokines. Results: SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased histone-3 acetylation, up-regulated p21 expression, and activated the intrinsic apoptosis pathway by down-regulating the XIAP (X-linked inhibitor of apoptosis protein), which was associated with activation of Caspase 9 and 3. Similarly to other HDACis, SNDX-275 decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, while level of Mcl-1 and pro-apoptotic Bax remained the same level. Combination studies demonstrated that SNDX-275 had more synergistic effect when combined with Bcl-2 inhibitors ABT-737 or Obatoclax and less when combined with Gemcitabine or Bortezomib. Dysregulated cytokine/chemokine production has been shown previously to contribute to HL pathology, including immune tolerance of the cancer cells. Hence, we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Increased IL12 p40-70, IP10, and RANTES, and decreased IL13, IL4 and TARC levels were found, thus favoring Th1-type cytokines/chemokines. Recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of CTAs (cancer testis antigens), leading to a favorable immune response. SNDX-275 was able to induce CTA expression of SSX2 and NY-ESO only in one cell line whereas MAGE-A4 was induced in both HL cell lines. Conclusion: Our studies demonstrate that SNDX-275 has a dual effect on apoptotic and immunomodulatory pathways in HL, which can be enhanced by the addition of agents targeting cell survival pathways. Phase II studies with SNDX-275 in HL are ongoing, future clinical studies should investigate combinations with SNDX-275. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TGF-β Promotes the Proliferation of Microglia In Vitro

2019 ◽  
Vol 10 (1) ◽  
pp. 20 ◽  
Author(s):  
Costansia Bureta ◽  
Takao Setoguchi ◽  
Yoshinobu Saitoh ◽  
Hiroyuki Tominaga ◽  
Shingo Maeda ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Microglial Cell ◽  
Transforming Growth Factor ◽  
Dependent Manner ◽  
In Vitro Proliferation ◽  
Inhibition Of Proliferation ◽  
Significant Difference

The activation and proliferation of microglia is characteristic of the early stages of brain pathologies. In this study, we aimed to identify a factor that promotes microglial activation and proliferation and examined the in vitro effects on these processes. We cultured microglial cell lines, EOC 2 and SIM-A9, with various growth factors and evaluated cell proliferation, death, and viability. The results showed that only transforming growth factor beta (TGF-β) caused an increase in the in vitro proliferation of both microglial cell lines. It has been reported that colony-stimulating factor 1 promotes the proliferation of microglia, while TGF-β promotes both proliferation and inhibition of cell death of microglia. However, upon comparing the most effective doses of both (assessed from the proliferation assay), we identified no statistically significant difference between the two factors in terms of cell death; thus, both have a proliferative effect on microglial cells. In addition, a TGF-β receptor 1 inhibitor, galunisertib, caused marked inhibition of proliferation in a dose-dependent manner, indicating that inhibition of TGF-β signalling reduces the proliferation of microglia. Therefore, galunisertib may represent a promising therapeutic agent for the treatment of neurodegenerative diseases via inhibition of nerve injury-induced microglial proliferation, which may result in reduced inflammatory and neuropathic and cancer pain.

scholarly journals IMMU-14. ONCOLYTIC VIRUS EXPRESSING A POSITIVE IMMUNE CHECKPOINT MODULATOR AS A THERAPEUTIC APPROACH FOR DIPG

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi122-vi122
Author(s):  
Virginia Laspidea ◽  
Montse Puigdelloses ◽  
Ignacio Iñigo-Marco ◽  
Marc Garcia-Moure ◽  
Iker Ausejo ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Oncolytic Virus ◽  
Murine Models ◽  
Antitumoral Effect ◽  
Infected Cells

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

scholarly journals Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation

2021 ◽  
Author(s):  
Tayla M. Olsen ◽  
Wei Hong Tan ◽  
Arne C. Knudsen ◽  
Anthony Rongvaux
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Type I ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
A Cell

AbstractRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

Immunomodulatory Drugs Inhibit H2O2 Decomposition in Multiple Myeloma Cells and Its Mediated Cytotoxicity Is Determined By Cellular Antioxidative Capacity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2475-2475
Author(s):  
Sinto Sebastian Chirackal ◽  
Yuan Xiao Zhu ◽  
Esteban Braggio ◽  
Chang-Xin Shi ◽  
Sonali Panchabhai ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Oxidative Capacity ◽  
Antioxidative Capacity ◽  
H2o2 Decomposition ◽  
Gene And Protein Expression ◽  
Induced Apoptosis

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

MMP7 Induces T-DM1 Resistance and Leads to the Poor Prognosis of Gastric Adenocarcinoma via a DKK1-Dependent Manner

2019 ◽  
Vol 18 (14) ◽  
pp. 2010-2016 ◽  
Author(s):  
Hua Li ◽  
Xin Xu ◽  
Yang Liu ◽  
Shunle Li ◽  
Di Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Gastric Adenocarcinoma ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Statistical Significance ◽  
Dependent Manner ◽  
Who Grade ◽  
Knock Down ◽  
The Poor

Background: Gastric adenocarcinoma is one of the most common and lethal cancer types and is known as the second leading cause of cancer-related death of Asian adults, early diagnosis based on either pathology or molecular biology could be one of the most efficient ways to improve the outcomes of gastric adenocarcinoma patients. Methods: Quantitative Real-Time PCR and Western-blot were used in detection of mRNA and protein expression. Lentivirus infection was used to overexpression or knock down target gene. Alarma blue assay was used to monitor cells proliferation. Flow cytometry analysis was performed to test protein expression and apoptosis level. Immunohistochemistry was used to identify protein expression in tissue. Statistical differences between two groups are evaluated by two-tailed t-tests. The comparison among multiple groups is performed by one-way Analysis of Variance (ANOVA) followed by Dunnett’s posttest. The statistical significance of the Kaplan-Meier survival plot is determined by log-rank analysis. Results: MMP7 as one of the most up-regulated genes in T-DM1 resistant NCI-N87 gastric adenocarcinoma cells compared to matched naïve cell lines. T-DM1 resistant NCI-N87 cell lines by exposed to T-DM1 in vitro. Exogenous overexpression of MMP7 promotes T-DM1 resistance and tumor growth in NCI-N87 cell lines while MMP7 knockdown enhanced sensitivity to T-DM1 in T-DM1 resistant NCI-N87 cell lines established previously. MMP7 was enriched in high WHO grade GC samples and implies poor outcomes for these patients. DKK1 as one of the most correlated genes to MMP7 in gastric adenocarcinoma and knock-down of DKK1 or inhibition of Wnt/β-catenin pathway led to a decreased expression of MMP7 and resistance to T-DM1. Conclusion: DKK1 and Wnt/β-catenin-dependent activation of MMP7 induces T-DM1 resistance and leads to the poor prognosis of gastric adenocarcinoma, which might be a novel potential therapeutical target for T-DM1 resistant gastric adenocarcinoma.

Effect of sunitinib and pazopanib on necrosis and autophagic cell death in cancer cells: Role of cathepsin B.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15513-e15513 ◽  
Author(s):  
Matteo Santoni ◽  
Consuelo Amantini ◽  
Maria Beatrice Morelli ◽  
Valerio Farfariello ◽  
Massimo Nabissi ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cathepsin B ◽  
Kinase Inhibitors ◽  
Autophagic Cell Death ◽  
Dependent Manner ◽  
Acridine Orange Staining

e15513 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on TKI-induced cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the anti-proliferative and cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrotic cell death was evaluated by Annexin-V/PI staining and FACS analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sunitinib and pazopanib markedly reduced at mM dose the viability of BC cells. Treatment for 24h with 20µM of sunitinib, by triggering “Incomplete autophagy”, induced necrosis of BC cells. In addition, sunitinib as a lysosomotropic agent, entered free within the lysosomes, where by increasing lysosomal pH and impairing cathepsin B activity, induced lysosomal-dependent necrosis. By contrast, treatment of BC cells for 72h with 20µM of pazopanib induced autophagic cell death, which was markedly reversed in a dose-dependent manner by the autophagic inhibitor 3-MA. The pazopanib-induced autophagic cell death was associated with increased procathepsin B cleavage and enhanced cathepsin B activity. Conclusions: Overall, our results show different cathepsin B-dependent cancer cell death mechanisms induced by sunitinib or pazopanib, providing the biological basis for novel molecularly targeted approaches.

scholarly journals ET-09 ACQUIRED MALIGNANT BEHAVIORS OF NPE6-PDT-SURVIVED GLIOBLASTOMA CELLS ARE SUPPRESSED BY USING MEK1/2 INHIBITOR TRAMETINIB

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii9-ii9
Author(s):  
Tatsuya Kobayashi ◽  
Yoshihiro Muragaki ◽  
Masamichi Takahashi ◽  
Kojiro Wada ◽  
Kentaro Mori ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Wave Length ◽  
Boyden Chamber ◽  
Concomitant Treatment ◽  
Dependent Manner ◽  
Molecular Signaling ◽  
Talaporfin Sodium ◽  
Human Gbm

Abstract INTRODUCTION In this study, we tried to investigate alteration of oncogenic properties and their molecular regulatory mechanism of talaporfin sodium (NPe6)-mediated photodynamic therapy (NPe6-PDT)-survived glioblastoma (GBM) cells. METHODS As the in-vitro NPe6-PDT model, human GBM cell lines (T98G, U87MG, U343), and patient derived GBM stem cells (GSY03, GSC23, MGG152) were pretreated with 0-30ug/ml NPe6 for 4 hours followed by laser irradiation (wave length 664 nm, laser-power 33 mW/cm2, total amount of irradiation 10 J/cm2) using a semiconductor laser irradiator (Panasonic Healthcare Co., Ltd., Tokyo, Japan). Cell death after PDT was evaluated by vital dye exclusion assay using Hoechst3342 and propidium iodide or CellTiter-Glo. Survived cells after NPe6-PDT (PDT-R cells) were repropagated, and alteration of intracellular molecular signaling or migration/invasion capability were analyzed by immunoblotting or Boyden chamber assay. RESULTS In both human GBM cell lines and patient derived GBM cells, cellular viability after NPe6-PDT was decreased with dose-dependent manner of pretreated NPe6. PDT-R cells showed not only resistance against NPe6-PDT-induced cell death but also higher invasiveness and migration capability compared with pre-PDT treated cells (PDT-Con cells), and immunoblot analysis demonstrated upregulation of ERK1/2 phosphorylation in PDT-R cells in comparison with PDT-Con cells. Furthermore, these acquired malignant behavior of PDT-R cells were repressed by concomitant use of MEK1/2 inhibitor Trametinib with NPe6-PDT. CONCLUSION We discovered PDT-R cells demonstrated higher malignant phenotypes via ERK1/2-dependent machinery compared with parent pre-PDT-treated cells. It was also suggested concomitant treatment with MEK1/2 inhibitor during PDT therapy in GBM cases would contribute to better outcome.

Different effects of sunitinib, sorafenib, and pazopanib on inducing cancer cell death: The role of autophagy.

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 270-270 ◽  
Author(s):  
Matteo Santoni ◽  
Consuelo Amantini ◽  
Maria Beatrice Morelli ◽  
Valerio Farfariello ◽  
Massimo Nabissi ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Cathepsin B ◽  
Kinase Inhibitors ◽  
Annexin V ◽  
Standard Of Care ◽  
Ros Generation ◽  
Dependent Manner ◽  
Acridine Orange Staining

270 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on the ability of TKIs to induce cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrosis and apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activity was evaluated by ELISA. Finally, by mRNA estraction and RT-PCR array the pazopanib-induced gene profile expression was evaluated. Results: We found that treatment of 5637 and J82 BC cells with the three TKI agents markedly reduced cell viability. Treatment for 24 h with sunitinib and sorafenib at 20 µM dose, triggers an incomplete autophagy of BC cells. In addition, inhibition of autophagy induced by sunitinib and sorafenib triggers cell death of BC cells. Thus, sunitinib by imparing the cathepsin B activity induces lysosomal-dependent necrosis. Similarly, sorafenib by defective lysosomial degradation triggers ROS- and mitochondrial-dependent apoptosis. As regard to pazopanib, we first demonstrate that treatment of BC cells for 72 hrs (20 µM) induces autophagic Type II cell death, which was markedly reversed in a dose-dependent manner by 3MA and chloroquine autophagic inhibitors. Finally, pazopanib upregulates the mRNA expression of α-glucosidase (GAA) and TP73 belonging to the p53 tumor suppressor genes. Conclusions: Overall, our results showing different TKI-induced cell death mechanisms provide the rationale for the sequential use of these agents and the biological basis for novel molecularly targeted approaches.

scholarly journals Fisetin Induced Cell Death in Human Ovarian Cancer Cell Lines via ZBP1 Mediated Necroptosis.

2021 ◽  
Author(s):  
Yaxian Liu ◽  
Wenhong Cao ◽  
Yanhui Zhao ◽  
Lijuan Shan ◽  
Shuhai Lan
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Annexin V ◽  
Dependent Manner ◽  
Apoptotic Process ◽  
Ovarian Cancer Cell Lines ◽  
And Migration

Abstract Background: Ovarian cancer leads to severe female mortality among all reproductive cancers. Fisetin, a natural flavonoid, exerts pharmacological characteristics on inhibiting cancer growth from various origins. Although multiple mechanisms involving in regulating cell death, there is still unclear if and how fisetin exhibits anti-cancer effect on ovarian cancer. The presented study aimed to evaluate cell apoptotic and necroptotic processes occurring in ovarian carcinoma (OC) cell lines induced by fisetin Methods: Cell growth was evaluated by MTT assay in both OC cell lines treated with or without fisetin. Annexin V/Propidium iodide staining followed by flow cytometry were used to characterize fisetin induced cell death. The apoptotic process was suppressed by z-VAD intervention then cell necroptosis was assessed by introducing ZBP1 knockdown OC cell lines coupled with fisetin intervention. The expression of necroptosis-related mediators and migration capability of respective cells were evaluated by western blotting and in vitro cell invasion assay. Result: Fisetin successfully reduced cell growth on both OC cell lines in a dose-dependent manner. Both apoptosis and necroptosis were induced by fisetin. Suppression on cell apoptotic process failed to enhance proliferation of fisetin treated cells. The induced cell death as well as robust expression of necroptotic markers RIP3 and MLKL were alleviated by knocking down the expression of ZBP1 protein in both OC cell lines.Conclusion: The present study demonstrated in vitro evidence supporting that both apoptosis and necroptosis were involved in fisetin induced OC cell death, while ZBP1 regulates necroptotic process via RIP3/MLKL pathway.

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