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.

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.

Effect of sorafenib on cathepsin B-dependent BID-mediated apoptosis in cancer cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15515-e15515
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Maria Beatrice Morelli ◽  
Valerio Farfariello ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Cathepsin B ◽  
Tyrosine Phosphatase ◽  
Ros Generation ◽  
Tyrosine Phosphatases ◽  
Multikinase Inhibitor ◽  
Acridine Orange Staining ◽  
Induced Apoptosis

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib 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. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric 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 sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

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 Anticancer Activity of Cynomorium coccineum

Cancers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 354 ◽  
Author(s):  
Mouna Sdiri ◽  
Xiangmin Li ◽  
William Du ◽  
Safia El-Bok ◽  
Yi-Zhen Xie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cycle Progression ◽  
Cycle Progression

The extensive applications of Cynomorium species and their rich bioactive secondary metabolites have inspired many pharmacological investigations. Previous research has been conducted to examine the biological activities and numerous interesting pharmaceutical activities have been reported. However, the antitumor activities of these species are unclear. To understand the potential anticancer activity, we screened Cynomorium coccineum and Cynomorium songaricum using three different extracts of each species. In this study, the selected extracts were evaluated for their ability to decrease survival rates of five different cancer cell lines. We compared the cytotoxicity of the three different extracts to the anticancer drug vinblastine and one of the most well-known medicinal mushrooms Amaurederma rude. We found that the water and alcohol extracts of C. coccineum at the very low concentrations possessed very high capacity in decreasing the cancer cells viability with a potential inhibition of tumorigenesis. Based on these primitive data, we subsequently tested the ethanol and the water extracts of C. coccineum, respectively in in vitro and in vivo assays. Cell cycle progression and induction of programmed cell death were investigated at both biological and molecular levels to understand the mechanism of the antitumor inhibitory action of the C. coccineum. The in vitro experiments showed that the treated cancer cells formed fewer and smaller colonies than the untreated cells. Cell cycle progression was inhibited, and the ethanol extract of C. coccineum at a low concentration induced accumulation of cells in the G1 phase. We also found that the C. coccineum’s extracts suppressed viability of two murine cancer cell lines. In the in vivo experiments, we injected mice with murine cancer cell line B16, followed by peritoneal injection of the water extract. The treatment prolonged mouse survival significantly. The tumors grew at a slower rate than the control. Down-regulation of c-myc expression appeared to be associated with these effects. Further investigation showed that treatment with C. coccineum induced the overexpression of the tumor suppressor Foxo3 and other molecules involved in inducing autophagy. These results showed that the C. coccineum extract exerts its antiproliferative activity through the induction of cell death pathway. Thus, the Cynomorium plants appear to be a promising source of new antineoplastic compounds.

scholarly journals Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Betty Yuen Kwan Law ◽  
Francesco Michelangeli ◽  
Yuan Qing Qu ◽  
Su-Wei Xu ◽  
Yu Han ◽  
...  
Keyword(s):  
Cell Death ◽  
Cancer Therapy ◽  
Cancer Cells ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Receptor Activation ◽  
Autophagic Cell Death ◽  
Dependent Manner ◽  
Computational Docking ◽  
Calcium Dependent

AbstractResistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells via new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from Nelumbo nucifera, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells, via calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.

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.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Atorvastatin to inhibit pancreatic cancer cells proliferation and invasion via suppression neurotrophin receptor signaling.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15725-e15725
Author(s):  
Shengjun Ji
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Medical Center ◽  
Neurotrophin Receptor ◽  
Dependent Manner ◽  
Receptor Signaling ◽  
Pancreatic Cancer Cells ◽  
Trk A

e15725 Background: Pancreatic cancer is an uncommon but fatal malignant tumor, only 20% of which show any significant response to chemo-radiotherapy. Tumor metabolism research revealed that pancreatic cancer cells highly dependent on cholesterol uptake. So, in the present study, we have determined the effectiveness of atorvastatin, one drug originally used to lower cholesterol, against pancreatic carcinogenesis in various pancreatic cancer cell lines. Methods: To investigate atorvastatin effects in pancreatic cancer cells, two cell lines (PANC-1 and SW1990) were treated with different atorvastatin doses. The anti-proliferative, apoptotic and anti-invasive properties of atorvastatin were evaluated using MTS, cytoplasmic histone-DNA fragmentation and matrigel invasive assays, respectively. And western blot was used to evaluate the neurotrophin receptor signaling of NGF/Trk A, BDNF/TrkB and NT3/Trk C. Results: Atorvastatin suppressed pancreatic cancer cells proliferation, clone formation and invasion in a dose-dependent manner. The dose of atorvastatin at 10^-5mmol/L would be able to achieve obvious anti-tumor effect in vitro. Cell-cycle analysis demonstrated that cells were arrested in the S phase. Western blot showed increased protein expression of cleaved caspase-3, caspase-9, p21, p-chk2 and decrease in protein level of NGF/Trk A and NT3/Trk C. Conclusions: The current results demonstrated the anti-tumor effects of atorvastatin on pancreatic cancer cells, providing initial evidence towards its potential therapeutic use. Acknowledgment:This study was supported by Suzhou Cancer Clinical Medical Center Szzx201506.

scholarly journals Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide

2016 ◽  
Vol 84 (7) ◽  
pp. 2042-2050 ◽  
Author(s):  
Nobuo Okahashi ◽  
Masanobu Nakata ◽  
Hirotaka Kuwata ◽  
Shigetada Kawabata
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Death ◽  
Cathepsin B ◽  
Opportunistic Infections ◽  
Specific Inhibitor ◽  
Fluorescent Labeling ◽  
Dependent Manner ◽  
Content Type ◽  
Acridine Orange Staining ◽  
Streptococcus Oralis

Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced byS. oralisis sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2is a cytotoxin. In the present study, we investigated whether streptococcal H2O2impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death.S. oralisinfection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2also induced cell death. Infection with either a mutant lackingspxB, which encodes pyruvate oxidase responsible for H2O2production, orStreptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection withS. oralisor exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection withS. oralisor exposure to H2O2. The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

scholarly journals PKM2 Knockdown Induces Autophagic Cell Death via the AKT/mTOR Pathway in Human Prostate Cancer Cells

2019 ◽  
Author(s):  
Prasanta Dey ◽  
Amit Kundu ◽  
Richa Sachan ◽  
Jaehyun Park ◽  
Mee Young Ahn ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Cancer Metabolism ◽  
Aerobic Glycolysis ◽  
Autophagic Cell Death ◽  
Prostate Cancer Cells ◽  
Glucose Transporter 1 ◽  
Acridine Orange Staining ◽  
Cancer Tissues

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis and is highly expressed in various cancer tissues. Although high PKM2 expression is observed in prostate cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. PKM2 expression was silenced using various PKM2 small interfering RNAs (siRNAs) and then we measured PKM2-related cellular pathways associated with autophagy. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange staining and immunoblotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. To the best of our knowledge, this is the first study to show that PKM2 inhibition alters cancer cell metabolism and induces autophagy. Thus, the present study provides a strategy for the development of PKM2-targeted novel anticancer drugs for the treatment of prostate cancer.

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