Distinct Cell Death Mechanisms Research Articles

Cell Death Mechanism of Organometallic Ruthenium(II) and Iridium(III) Arene Complexes on HepG2 and Vero Cells.

Due to side effects and toxicity associated with platinum-derived metal-based drugs, extensive research has been conducted on ruthenium (Ru) complexes. We aim to synthesize a highly oil soluble Ru(II)-p-cymene complex (Ru1) with an aliphatic chain group, a bimetallic Ru(II)-p-cymene complex (Ru2) with N,S,S triple-coordination and a bimetallic Ir(III)-pentamethylcyclopentadienyl complex (Ir1) with S,S double-coordination. Subsequently, we investigate the effects of these complexes on Vero and HepG2 cell lines, focusing on cell death mechanisms. Characterization of the complexes is performed through nuclear magnetic resonance spectroscopy (1H and 13C NMR) and Fourier-transform infrared spectroscopy. The effective doses are determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay, applying different doses of the complexes to the two cell lines for 24 and 48 h, respectively. Immunoreactivities of Bax, Bcl2, caspase-3, RIP3, and RIPK1 are analyzed using the indirect immunoperoxidase technique. Notably, all the complexes (Ru1, Ru2, and Ir1) exhibit distinct cell death mechanisms, showing greater effectiveness than cisplatin. This study reveals the diverse mechanisms of action of Ru and Ir complexes based on different ligands. To the best of our knowledge, this study represents the first investigation of a novel RAED-type complex (Ru1) and unexpected bimetallic complexes (Ru2 and Ir1).

Benzimidazole carbamate induces cytotoxicity in breast cancer cells via two distinct cell death mechanisms

Metastatic breast cancer (mBC) is responsible for >90% of breast cancer-related deaths. Microtubule-targeting agents (MTAs) are the front-line treatment for mBC. However, the effectiveness of MTAs is frequently limited by the primary or acquired resistance. Furthermore, recurrent mBC derived from cancer cells that survived MTA treatment are typically more chemoresistant. The overall response rates for the second- and third-line MTAs in mBC patients previously treated with MTAs are 12–35%. Thus, there is an ongoing search for novel MTAs with a distinct mode of action that can circumvent chemoresistance mechanisms. Our results show that methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate (BCar), a microtubule-disrupting anthelmintic that binds to the colchicine binding site separate from the binding sites of clinically used MTAs, has the potential to treat MTA-resistant mBC. We have comprehensively evaluated the cellular effects of BCar in a panel of human breast cancer (BC) cell lines and normal breast cells. BCar effects on the clonogenic survival, cell cycle, apoptosis, autophagy, senescence, and mitotic catastrophe were measured. Approximately 25% of BCs harbor mutant p53. For this reason, the p53 status was included as a variable. The results show that BC cells are >10x more sensitive to BCar than normal mammary epithelial cells (HME). p53-mutant BC cells are significantly more sensitive to BCar treatment than p53 wild-type BC cells. Furthermore, BCar appears to kill BC cells primarily via either p53-dependent apoptosis or p53-independent mitotic catastrophe. When compared to docetaxel and vincristine, two clinical MTAs, BCar is fairly innocuous in HME cells, providing a much wider therapeutic window than docetaxel and vincristine. Together, the results strongly support the notion that BCar-based therapeutics may serve as a new line of MTAs for mBC treatment.

Ablative Dose Radiation Induces Distinct Waves of Cell Death Dependent on Cell Cycle Phase via DNA Repair Pathway Choice

<h3>Purpose/Objective(s)</h3> Ablative Intensity Radiotherapy (ART; also called stereotactic ablative radiotherapy) enables treatment in a limited number of high-dose radiation fractions. ART leads to unprecedented cure rates, thought to be driven in part through immune responses. However, the mechanism(s) of ART-induced cancer cell death remain poorly characterized. We hypothesized that ART induces distinct mechanisms of cell death which drive tumor immunogenicity. <h3>Materials/Methods</h3> A range of cell lines including immortalized normal and cancer cell lines were subjected to single fraction doses of radiotherapy ranging from 2-20Gy <i>in vitro</i>. Outcomes were measured using clonogenic assays, long-duration live cell imaging, and biochemical assays. The Fluorescence Ubiquitin Cell Cycle Indicator system was used to monitor phase of the cell cycle during irradiation. Key DNA repair pathways were inhibited using genetic and/or pharmacological inhibition. <h3>Results</h3> Single doses of >=8Gy (hereafter ‘ART') induce a characteristic shift in timing and mechanism of cell death. As expected, ART induces senescence in cells with intact TP53. ART induces two distinct waves of cancer cell death in cells with inactive TP53. The first wave of death occurs exclusively during mitosis in cells irradiated in the S/G2 cell cycle phases. Mitotic death is signaled via BAX/BAK-dependent intrinsic apoptosis and driven by WAPL-dependent cohesion fatigue. The second cell death wave occurs days after ART, specifically within the cells that were irradiated in G1 phase. This latter cell death wave follows at least one cell division with chromosome segregation errors and is contemporaneous with a proinflammatory response, autophagy, and STAT-1 dependent extrinsic apoptosis. Strikingly, this cell-cycle dependence is absent with doses of 2-4Gy RT. Altering DNA repair pathways changes the spectrum of cell death outcomes. Specifically, inhibiting non-homologous end joining (NHEJ) promotes mitotic catastrophe of G1 phase irradiated cells; inhibiting canonical homology directed repair reduces the amount of mitotic death in S/G2 irradiated cells; and inhibiting single strand annealing significantly delays entry into mitotic catastrophe while increasing lethality. <h3>Conclusion</h3> ART lethality results from distinct mechanisms governed by cell cycle-dependent DNA repair pathways active at the time of irradiation. Our data suggest that NHEJ promotes immune activation and ‘toxic' HR drives mitotic catastrophe. Further, mode of ART lethality is tuneable through pharmacological manipulation, indicating the potential to enhance cancer cell death and potentiate immunotherapy effectiveness for patient benefit.

Contribution of Myocyte Apoptosis to Myocardial Injury in an &amp;lt;i&amp;gt;in Vivo&amp;lt;/i&amp;gt; Rabbit Preparation of Ischemia-Reperfusion

Objective: Exposure of the heart to repeated, brief episodes of coronary occlusion/reperfusion prevents lethal myocyte injury. Necrosis and apoptosis, two seemingly distinct mechanisms of cell death caused by ischemia could contribute independently to progressive loss of myocardium. Studies suggest that ischemic conditioning (IC) lessens myocyte injury by decreasing apoptosis. The goal of this study was to examine cell death in rabbit hearts subject to ischemia-reperfusion injury without (nIC) or with pretreatment by IC. Methods: In the control study, anesthetized, male rabbits (n = 4/group) underwent 30-min regional coronary occlusion (CO) and either 3, 6 or 24h reperfusion (REP). In the IC study, rabbits were pretreated by IC (2 cycles of 5-min CO and 10-min REP) before 30-min CO and subsequent REP. Additional groups were evaluated with 60, or 120-min CO followed by up to 96 h REP. Agarose electrophoresis was used to detect DNA laddering and poly (ADP-ribose) polymerase (PARP; chromatin bound nuclear DNA repair enzyme) was assessed in myocardial biopsies. Results: Genomic DNA from nIC and IC hearts showed no oligonucleosomal fragmentation. In addition, we did not detect any cleavage of PARP; however, myocardial PARP levels decreased when CO and REP durations were prolonged. Conclusion:Absence of genomic DNA fragmentation or PARP cleavage in an in vivo preparation of ischemia-reperfusion injury does not support the view that apoptosis contributes markedly to post-ischemic tissue necrosis.

Auranofin and Cold Atmospheric Plasma Synergize to Trigger Distinct Cell Death Mechanisms and Immunogenic Responses in Glioblastoma.

Targeting the redox balance of malignant cells via the delivery of high oxidative stress unlocks a potential therapeutic strategy against glioblastoma (GBM). We investigated a novel reactive oxygen species (ROS)-inducing combination treatment strategy, by increasing exogenous ROS via cold atmospheric plasma and inhibiting the endogenous protective antioxidant system via auranofin (AF), a thioredoxin reductase 1 (TrxR) inhibitor. The sequential combination treatment of AF and cold atmospheric plasma-treated PBS (pPBS), or AF and direct plasma application, resulted in a synergistic response in 2D and 3D GBM cell cultures, respectively. Differences in the baseline protein levels related to the antioxidant systems explained the cell-line-dependent sensitivity towards the combination treatment. The highest decrease of TrxR activity and GSH levels was observed after combination treatment of AF and pPBS when compared to AF and pPBS monotherapies. This combination also led to the highest accumulation of intracellular ROS. We confirmed a ROS-mediated response to the combination of AF and pPBS, which was able to induce distinct cell death mechanisms. On the one hand, an increase in caspase-3/7 activity, with an increase in the proportion of annexin V positive cells, indicates the induction of apoptosis in the GBM cells. On the other hand, lipid peroxidation and inhibition of cell death through an iron chelator suggest the involvement of ferroptosis in the GBM cell lines. Both cell death mechanisms induced by the combination of AF and pPBS resulted in a significant increase in danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation, indicating a potential increase in immunogenicity, although the phagocytotic capacity of dendritic cells was inhibited by AF. In vivo, sequential combination treatment of AF and cold atmospheric plasma both reduced tumor growth kinetics and prolonged survival in GBM-bearing mice. Thus, our study provides a novel therapeutic strategy for GBM to enhance the efficacy of oxidative stress-inducing therapy through a combination of AF and cold atmospheric plasma.

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI–H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation.TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI–H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA.Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).

Abstract A59: Biologic rationale for radiation-induced release of circulating tumor DNA

Abstract Purpose: Rapid release of circulating tumor DNA (ctDNA) during cytotoxic therapy could provide an early indication of treatment efficacy. However, the biologic basis for treatment-induced ctDNA release is poorly understood. To study this phenomenon, we evaluated ctDNA release in head and neck squamous cell carcinoma (HNSCC) treated with radiotherapy (RT) or chemoradiotherapy (CRT). We sought to characterize the kinetics and biologic underpinnings of this process. Methods: 33 HPV+ HNSCC patients treated with RT (n=17) or CRT (n=16) had blood collected at baseline, after the 4th fraction of RT (early-RT), mid-RT, and 3 months post-RT. ctDNA was quantified using digital PCR for HPV-16. Nine HNSCC cell lines (3 HPV+, 6 HPV-) were used for in vitro ctDNA kinetic analysis. Apoptosis was measured by luminescent DEVD cleavage assay, triggered by staurosporine and blocked by z-vad-fmk. Senescence was measured by SA-β-galactosidase activity, triggered by HrasG12V-transduction of fibroblast cell lines and blocked by navitoclax. Three HNSCC cell lines (2 HPV+, 1 HPV-) were grown as xenografts in NRG mice for in vivo analysis. Endpoint xenograft tumors were assessed for necrotic area and for markers of apoptosis (cleaved-caspase 3) and senescence (p21) by immunohistochemistry. ctDNA was quantified from media or mouse plasma by quantitative PCR using human-specific LINE-1 primers. Results: Of 30 patients with detectable HPV-16 ctDNA (90.9%), we observed variable kinetic patterns of release. We grouped patients into three categories: pattern 1, peak at early RT (12, 40%); pattern 2, peak at mid RT (7, 23.3%); pattern 3, no peak (11, 36.7%). Based on these findings, we characterized the mechanisms dictating RT-induced ctDNA release in preclinical models of HNSCC. Following RT, HNSCC cell lines exhibited variable kinetics of ctDNA release in vitro and in vivo with peak release observed after 72-96 hours. RT-induced ctDNA release was not associated with intrinsic radiosensitivity, HPV status, or propensity to undergo apoptosis. Although caspase inhibition resulted in a near-complete reduction in RT-induced caspase activity (-84%±8%), a comparatively minor reduction in ctDNA release (-29%±9%) was observed. There was an inverse association between RT-induced senescence and ctDNA release. HrasG12V-triggered senescence reduced ctDNA release, while navitoclax treatment reversed this effect. HNSCC models with greater RT-induced ctDNA release displayed more necrosis and less senescence in endpoint xenograft tumors. Conclusion: Dynamic changes in HPV-16 ctDNA are observed during treatment, with variable patterns of release. Preclinical HNSCC models demonstrate RT-induced spikes within 3-4 days of treatment, revealing a complex interplay between distinct mechanisms of cell death and ctDNA release. Necrosis and senescence are the major determinants of RT-induced ctDNA release, while apoptosis has a comparatively minor impact. This study will inform future work on implementing ctDNA as a response biomarker. Citation Format: Ariana Rostami, John N. Waldron, Scott V. Bratman. Biologic rationale for radiation-induced release of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A59.

Biological Rationale for Radiation-induced Release of Circulating Tumor DNA

Rapid release of circulating tumor DNA (ctDNA) during cytotoxic therapy could provide an early indication of treatment efficacy. However, the biological basis for treatment-induced ctDNA release is poorly understood. To study this phenomenon, we selected head and neck squamous cell carcinoma (HNSCC) as an archetypal epithelial cancer treated with ionizing radiation (IR). We hypothesized that IR-induced ctDNA release would be correlated with intrinsic radiosensitivity, human papillomavirus (HPV) association, and propensity to undergo apoptosis. Nine HNSCC cell lines (3 HPV+, 6 HPV-) were grown in tissue culture for in vitro ctDNA kinetic analysis. Intrinsic radiosensitivity was determined by clonogenic assays. Apoptosis was measured by luminescent DEVD cleavage assay, triggered by staurosporine, and blocked by z-vad-fmk. Senescence was measured by SA-β-galactosidase activity, triggered in the fibroblast cell lines IMR90 and WI38 by transduction with oncogenic HrasG12V, and blocked by navitoclax. Three HNSCC cell lines (1 HPV+, 2 HPV-) were grown as subcutaneous xenografts in Nod-Rad-Gamma (NRG) mice for in vivo ctDNA kinetic analysis. Endpoint xenograft tumors were assessed for necrotic area (H&E) and for markers of apoptosis (cleaved-caspase 3), senescence (p21), vessel density (CD31), and hypoxia (CAIX) by immunohistochemistry. ctDNA was quantified from media or plasma by quantitative PCR using human-specific LINE-1 primers. Following IR, HNSCC cell lines exhibited variable magnitudes and kinetics of ctDNA release in vitro and in vivo with peak release observed after 72-96 hours. IR-induced ctDNA release was dose-dependent but not associated with intrinsic radiosensitivity, HPV status, or propensity to undergo apoptosis. Although caspase inhibition resulted in a near complete reduction in IR-induced caspase activity (-84%±8%), a comparatively minor reduction in ctDNA release (-29%±9%) was observed. There was an inverse association between IR-induced senescence and ctDNA release. HrasG12V-triggered senescence reduced ctDNA release, while navitoclax treatment reversed this effect. HNSCC models with greater IR-induced ctDNA release displayed more necrosis and less senescence in endpoint xenograft tumors; in contrast, neither vessel density nor hypoxia could account for differences in IR-induced ctDNA release between models. IR-induced ctDNA spikes are commonly observed within 2-3 days of treatment in preclinical HNSCC models. Our striking findings reveal the complex interplay between distinct mechanisms of cell death following IR exposure and their influence on ctDNA release. Necrosis and senescence are the major determinants of IR-induced ctDNA release, while apoptosis has a comparatively minor impact. This study will inform future work on implementing ctDNA as a response biomarker in HNSCC and other cancer types.

Effects of SAHA and RG7388 on p21WAF1/CIP1 and p27KIP1 mediated Pathways in Cancer Cells

Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21WAF1/CIP1 and p27Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Saturated Fatty Acids, MUFAs and PUFAs Regulate Ferroptosis

Apoptosis, necroptosis, autophagy, and ferroptosis are distinct mechanisms of cell death. In this issue of Cell Chemical Biology, Magtanong etal. (2019) demonstrated that exogenous monounsaturated fatty acids (MUFAs) induce a ferroptosis-resistant cell state by suppressing the accumulation of lipid peroxides and decreasing levels of oxidizable polyunsaturated fatty acids (PUFAs).

Cell Death Markers in Sepsis

Abstract Cell death mechanisms play an important role in the pathogenesis of sepsis. The goal of our study was to understand cell death regulatory mechanisms such as pyroptosis, necroptosis and apoptosis in sepsis. Our preliminary study design included patients diagnosed with sepsis that were treated in clinical settings. Patients diagnosed with sepsis (test) were compared with healthy individuals (control). Cell death-regulating markers were assessed in plasma, peripheral blood mononuclear cells (PBMCs) and PBMC cell lysate samples derived from patients (N =8) and healthy controls (N=8). Samples were analyzed by ELISA, flow cytometry and immunoblotting methods. Preliminary flow cytometry evaluation of PBMC samples from patients showed elevated levels of cell death markers when compared to samples from healthy individuals. Results from ELISA experiments showed both plasma and PBMC cell lysate samples from septic patients had an increase in a marker for necroptosis, in comparison with control samples (P &amp;lt; 0.05 and P &amp;lt; 0.1, respectively). Interestingly, test samples showed modulation in a marker for apoptosis and a marker for pyroptosis in comparison with controls (P &amp;lt; 0.05). Preliminary results suggest that there exists distinct cell death mechanisms with respect to the clinical outcomes observed in septic patients.

Correction: Modulation of Pathways Underlying Distinct Cell Death Mechanisms in Two Human Lung Cancer Cell Lines in Response to SN1 Methylating Agents Treatment.

[This corrects the article DOI: 10.1371/journal.pone.0160248.].

PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1-/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.

A novel real-time imaging technique to quantify mechanisms of cell death in neutrophils

Abstract Neutrophils have distinct mechanisms of cell death, including the formation of neutrophil extracellular traps (NETs- a process called NETosis). NETosis is characterized by reactive oxygen species generation, chromatin decondensation, membrane rupture and extrusion of a meshwork of chromatin bound to granule peptides. Objective, efficient techniques to quantify NETosis are required. We developed a new method to automatically quantify the percentage of neutrophils undergoing NETosis using the IncuCyte ZOOM™ (Essen BioScience, Inc.) platform to image neutrophils at 20× in a 96-well plate and a dual-dye system dependent on membrane integrity to stain DNA, collecting both phase and fluorescence images. DNA in healthy control neutrophils were stained with a cell membrane permeable NUCLEAR-ID Red DNA dye. Neutrophils were then incubated with Sytox, a membrane-impermeable DNA dye, used to assess cell death. Conditions were run in triplicate and 3 images per well were captured at 10-minute intervals. A processing definition was set and optimized to count all neutrophils (NUCLEAR-ID stained) and neutrophils undergoing cell death (Sytox stained) using fluorescence intensity and stained area size to identify cells undergoing NETosis. Changes in nuclear morphology, quantified by the processing definition, distinguished PMA, Ionomycin and SLE sera induced NETosis from staurosporine induced apoptosis. Findings were confirmed and correlated with the gold-standard of immunofluorescence microscopy. This novel real time assay distinguishes types of neutrophil cell death and quantifies NETosis in a rapid, accurate and reproducible way, significantly improving the study of neutrophils.

MTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells.

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN(-/-) LNCaP and androgen independent (AR-), PTEN(+/-) DU145 prostate cancer cells were exposed to purvalanol (20µM) and roscovitine (30µM) with or without rapamycin for 24h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (-) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR(+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.

Modulation of Pathways Underlying Distinct Cell Death Mechanisms in Two Human Lung Cancer Cell Lines in Response to SN1 Methylating Agents Treatment.

Despite advances in the understanding of tumor biology in recent years, lung cancer remains the leading cause of cancer death worlwide. Non small cell lung cancer (NSCLC; adenocarcinoma, squamous carcinoma, and large cell carcinoma) is the most common type, representing more than 80% of lung cancer diagnoses. Clinical improvements following treatment with traditional chemotherapy have reached a plateau in patients with NSCLC and 5 year survival still remains at 15%. Moreover, the molecularly targeted therapies having already progressed to clinical trials have not proved successful [1,2]. Although NSCLC is very often extremely resistant to chemotherapeutic agents, chemotherapy remains an established treatment for advanced NSCLC. Platinum-based chemotherapy frequently combined with other agents and/or approaches remain the standard in the first-line treatment of patients with advanced NSCLC [3,4]. Methylating agents such as temozolomide, an oral SN1 methylating agent which crosses the blood brain barrier achieving effective concentrations in the Central Nervous System (CNS), have already been used combined with platinum-based regimen and whole brain radiotherapy as prophylaxis against brain metastasis in NSCLC and/or as second-line treatment in NSCLC patients with brain metastases [5–8]. Methylating agents of SN1 type, including temozolomide (TMZ), procarbazine, dacarbazine and streptozotocine, constitute a widely used class of anticancer drugs. These DNA damaging agents are highly cytotoxic, mutagenic, recombinogenic and clastogenic inducing about a dozen DNA methylation adducts [9]. Among them, O6-methylguanine (O6-meG), induced in small amounts (maximally 8% of total methylation products), is the most biologically significant lesion; it mispairs with thymine during DNA replication finally generating G to A transitions in the second round of replication. The DNA mismatches are recognized by the Methyl-directed Mismatch Repair (MMR) system that removes the mis-incorporated base opposite the O6-meG lesion. Repair resynthesis of DNA leads to reinsertion of thymine opposite O6-meG and re-initiation of futile mismatch repair. The DNA double-strand breaks (DSBs) resulting from this process are thought to initiate a series of events including prolonged G2 arrest leading to cell death [9]. A critical factor influencing the cellular response to methylating agents is O6-methylguanine- DNA methyltransferase (MGMT), the DNA repair protein that stoichiometrically and selectively removes methyl lesions from the O6 position of guanine and returns the DNA to its pre-lesioned state [10]. Pre-replicative repair by MGMT as well as post-replicative MMR determine the level of methylating agent-induced genotoxicity and cell death [11–13]. Chemotherapeutic agents inducing DNA damage, such as SN1 methylating agents and cisplatin, may activate cell death by apoptosis or necrosis. They could also induce autophagy, senescence or mitotic catastrophe, which may then be followed by apoptosis or necrosis [13–15]. The molecular basis underlying the decision- making process is currently the subject of intense investigation because a deeper understanding of how a given chemotherapy affects all of the signalling pathways involved in cell death is highly relevant in order to develop more effective therapeutics. Despite their use in combination therapies, the effect of SN1 methylating agents on human NSCLC has not been studied thoroughly. We thus investigated the mechanism of the cell death induced by a model SN1 methylating agent, N-methyl-N-nitrosourea (MNU) in two human NSCLC cell lines, A549 (p53wt) and H157 (p53null) [13] through a time course gene expression profiling study 24, 48 and 72 hours after treatment. The list of differentiated genes, biological processes and cellular pathways were identified using appropriate bioinformatics tools and the results were further validated through RT-PCR of selected genes. MNU induced cell death through distinct responses at the gene expression level in the above cell lines. Our results overall support the use of SN1 methylating agents in platinum-based combination regimen against advanced NSCLC.

Classical and Targeted Anticancer Drugs: An Appraisal of Mechanisms of Multidrug Resistance.

The mechanisms by which tumor cells resist the action of multiple anticancer drugs, often with widely different chemical structures, have been pursued for more than 30 years. The identification of P-glycoprotein (P-gp), a drug efflux transporter protein with affinity for multiple therapeutic drugs, provided an important potential mechanism and further work, which identified other members of ATP-binding cassette (ABC) family that act as drug transporters. Several observations, including results of clinical trials with pharmacological inhibitors of P-gp, have suggested that mechanisms other than efflux transporters should be considered as contributors to resistance, and in this review mechanisms of anticancer drug resistance are considered more broadly. Cells in human tumors exist is a state of continuous turnover, allowing ongoing selection and "survival of the fittest." Tumor cells die not only as a consequence of drug therapy but also by apoptosis induced by their microenvironment. Cell death can be mediated by host immune mechanisms and by nonimmune cells acting on so-called death receptors. The tumor cell proliferation rate is also important because it controls tumor regeneration. Resistance to therapy might therefore be considered to arise from a reduction of several distinct cell death mechanisms, as well as from an increased ability to regenerate. This review provides a perspective on these mechanisms, together with brief descriptions of some of the methods that can be used to investigate them in a clinical situation.

Pseudomonas aeruginosa PAO1 induces distinct cell death mechanisms in H9C2 cells and its differentiated form.

Bacterial infections in myocardium may lead to the myocardial damage, which may progress to dilated cardiomyopathy and cardiac arrest. Pseudomonas aeruginosa has been reported to cause myocarditis and other systemic infections especially in immunocompromised patients. To understand the cellular responses during the establishment of infection in myocardium, we challenged differentiated H9C2 cells with P. aeruginosa PAO1. We also did comparison studies with infected undifferentiated form of H9C2 cells. Invasion studies revealed that PAO1 can invade both forms of cells and is able to survive and replicate within the host. Internalization of PAO1 was confirmed by live cell imaging and flow cytometry analysis. Though invasion of the pathogen triggered an increased ROS production in the host cells at earlier post-infection periods, it was decreased at later post-infection periods. Invasion of PAO1 induced cell death through apoptosis in differentiated H9C2 cells. Significant decrease in cell size, formation of polarized mitochondria, and nuclear fragmentation were observed in the infected differentiated cells. On the contrary, cell death preceded by multinucleation was observed in infected undifferentiated H9C2 cells. Morphological markers such as multinuclei and micro nuclei were observed. Cell cycle arrest in G2/M phase corroborates that the undifferentiated H9C2 cells experienced cell death preceded by multinucleation.

The use of linear accelerator with RapidArc option for the purposes of stereotactic radiotherapy for lung cancer

In this introductory lecture of the Symposium, essential knowledge concerning biological mechanisms of post highradiation dose will be present. Emerging evidence suggests that distinct mechanisms of cell death, likely including a major contribution from vascular epithelial cell apoptosis (devascularizing effect), might play an important role in the success of SBRT in controlling tumors. Also “5Rs” of conventional fractionated RT will be discussed in aspect of SBRT, including new pathways of tumor clonogen repopulation, i.e. “phoenix rising”, as well as constructing SBRT cell survival curve attempts. However, studies taken toward maximizing this effect should also be designed with the cautionary awareness that tissue regenerative processes might also be triggered as a result.

Pazopanib and sunitinib trigger autophagic and non-autophagic death of bladder tumour cells

Background:Tyrosine kinase inhibitors (TKI) such as sunitinib and pazopanib display their efficacy in a variety of solid tumours. However, their use in therapy is limited by the lack of evidence about the ability to induce cell death in cancer cells. Our aim was to evaluate cytotoxic effects induced by sunitinib and pazopanib in 5637 and J82 bladder cancer cell lines.Methods:Cell viability was tested by MTT assay. Autophagy was evaluated by western blot using anti-LC3 and anti-p62 antibodies, acridine orange staining and FACS analysis. Oxygen radical generation and necrosis were determined by FACS analysis using DCFDA and PI staining. Cathepsin B activation was evaluated by western blot and fluorogenic Z-Arg-Arg-AMC peptide. Finally, gene expression was performed using RT–PCR Profiler array.Results:We found that sunitinib treatment for 24 h triggers incomplete autophagy, impairs cathepsin B activation and stimulates a lysosomal-dependent necrosis. By contrast, treatment for 48 h with pazopanib induces cathepsin B activation and autophagic cell death, markedly reversed by CA074-Me and 3-MA, cathepsin B and autophagic inhibitors, respectively. Finally, pazopanib upregulates the α-glucosidase and downregulates the TP73 mRNA expression.Conclusion:Our results showing distinct cell death mechanisms activated by different TKIs, provide the biological basis for novel molecularly targeted approaches.