Induction Of Apoptotic Cell Death Research Articles

Acquired Resistance to Shiga Toxin-Induced Apoptosis by Loss of CD77 Expression in Human Myelogenous Leukemia Cell Line, THP-1.

Shiga toxin (Stx) is a main virulence factor of Enterohemorrhagic Escherichia coli (EHEC) that causes diarrhea and hemorrhagic colitis and occasionally fatal systemic complications. Stx induces rapid apoptotic cell death in some cells, such as human myelogenous leukemia THP-1 cells expressing CD77, a receptor for Stx internalization, and the induction of apoptotic cell death is thought to be crucial for the fatal systemic complications. Therefore, in order to suppress the fatal toxicity, it is important to understand the mechanism how cells can escape from apoptotic cell death in the presence of Stx. In this study, we isolated resistant clones to Stx-induced apoptosis from highly sensitive THP-1 cells by continuous exposure with lethal dose of Stx. All of the ten resistant clones lost the expression of CD77 as a consequence of the reduction in CD77 synthase mRNA expression. These results suggest that downregulation of CD77 or CD77 synthase expression could be a novel approach to suppress the fatal toxicity of Stx in EHEC infected patient.

Benzyl Isothiocyanate Induces Apoptotic Cell Death Through Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

Gefitinib is used to treat patients with lung cancer, but in some patients, the disease becomes gefitinib-resistant. Benzyl isothiocyanate (BITC), found in cruciferous vegetables, has shown anticancer activity in many human cancer cell lines. However, the effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells in vitro have not been investigated. The effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells were investigated in vitro. Flow cytometric assay was used for determining the total viable cell number, apoptotic cell death, the production of reactive oxygen species (ROS) and Ca2+, mitochondriaI membrane potential (Ψm) and caspase-3, -8 and -9 activities. Furthermore, 4', 6-diamidino-2-phenylindole staining was used to examine chromatin condensation in NCI-H460 and NCI-H460/G cells. BITC reduced total viable cell number via the induction of apoptotic cell death, that was also confirmed by annexin V/propidium iodide double staining assay. BITC increased ROS and Ca2+ production, reduced Ψm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting assay also showed that BITC increased expression of cleaved caspase-3 and -9, cytochrome c, BCL2-associated X protein, endonuclease G, poly (ADP-ribose) polymerase, growth arrest and DNA-damage protein 153, caspase-7 and activating transcription factor 6 alpha, but reduced apoptosis-inducing factor and caspase-9, BH3-interacting domain death agonist, calpain 1, glucose-regulated protein 78 and inositol requiring enzyme 1 alpha in NCI-H460/G cells. BITC-induced apoptotic cell death appears to occur via caspase- and mitochondria-dependent pathways in both cell lines.

DNA/Protein Binding, Molecular Docking and Cytotoxicity Studies of Piperazinyl‐Moiety‐Based Copper(II) Complexes

AbstractCopper(II) complexes {[Cu(HL)(ClO4)(H2O)](ClO4)⋅3H2O} (1), {[Cu(HL)(m‐phth)]⋅5H2O} (2) and {[Cu(HL)(NCS)]2(ClO4)2⋅2H2O} (3) (HL=2‐{[2‐(1‐piperazinyl)ethylimino]methyl}phenol; m‐phth=1,3‐benzenedicarboxylate] have been synthesized and characterized by structural determination and spectroscopic studies. The mononuclear square pyramidal complex 1 resulted from the reaction of HL with copper perchlorate hexahydrate. Then mononuclear square planar complex 2 and dinuclear thiocyanato bridged complex 3 were obtained by reacting 1 with disodium 1,3‐benzenedicarboxylate (Na2(m‐phth)) and potassium thiocyanate, respectively. The interactions of 1–3 with CT‐DNA / serum albumins were investigated by UV‐visible absorption and fluorescence spectroscopy. The intrinsic binding constants of 1, 2 and 3 with CT‐DNA were calculated as 1.44 (±0.13) × 105, 4.86 (±0.11) × 105 and 4.51 (±0.16) × 105 L mol−1, respectively. Study of the interactions of 1–3 with human serum albumin (HSA) / bovine serum albumin (BSA) showed that all the complexes could quench intrinsic fluorescence of HSA and BSA through a static quenching process. Molecular docking technique was utilised to confirm the mode of interaction of complexes with CT‐DNA / serum albumin. Anticancer activities of the complexes have been tested using human breast cancer cell lines MCF7 and MBA‐MB‐231. Among the complexes studied 3 shows the higher cytotoxic activity and growth inhibition of cancer cells via induction of apoptotic cell death.

Droxinostat sensitizes human colon cancer cells to apoptotic cell death via induction of oxidative stress

Upregulation of histone acetylation plays a critical role in the dysregulation of transcription. It alters the structure of chromatin, which leads to the onset of cancer. Histone deacetylase inhibitors may therefore be a promising way to limit cancer progression. In this study, we examined the effects of droxinostat on the growth of HT-29 colon cancer cells. Our results show that droxinostat effectively inhibited cell growth and colony-forming ability by inducing cellular apoptosis and ROS production in HT-29 cells. Notably, the apoptotic inhibitor Z-VAD-FMK significantly decreased the levels of cellular apoptosis and the antioxidant γ-tocotrienol (GT3) significantly decreased ROS production induced by droxinostat treatment. Z-VAD-FMK and GT3 also partially reversed the negative growth effects of droxinstat on HT-29 cells. GT3 treatment decreased cellular apoptosis and increased colony-forming ability upon droxinostat administration. Z-VAD-FMK treatment also partially decreased droxinostat-induced ROS production. Our findings suggest that the effects of droxinostat on colon cancer cells are mediated by the induction of oxidative stress and apoptotic cell death.

Upregulation and activation of p53 by erastin‑induced reactive oxygen species contribute to cytotoxic and cytostatic effects in A549 lung cancer cells.

The tumour‑suppressor protein p53 is a key regulator of multiple cellular processes and exerts its tumour‑suppressor function by inducing apoptotic cell death. However, emerging evidence indicates that p53 is also involved in inducing ferroptosis, which is a unique iron‑dependent form of non‑apoptotic cell death triggered by the RAS‑selective lethal small molecule erastin. Previous studies have shown that erastin exposure induces increased ROS accumulation and oxidative stress. In the present study, we incubated A549 cells with erastin and detected ROS accumulation. Semi‑quantitative western blotting was performed to analyse the effect of the induced ROS on p53 activity. To determine how ROS activate p53, NAC, an ROS scavenger, and KU‑55933, an ATM kinase inhibitor, were employed to co‑incubate with erastin, followed by western blot analysis. Either p53 or SLC7A11 siRNA was introduced into A549 cells to silence the target‑gene expression, followed by ROS detection to illustrate the regulatory role of ROS‑activated p53 on its target gene SLC7A11. AnnexinV‑FITC/PI staining was performed to detect the induction of apoptotic cell death by erastin exposure. To further assess the effects of erastin treatment on cellular proliferation, EdU staining and cell cycle flow cytometric analysis were performed. Erastin exposure upregulated and activated p53 and thus, transcriptionally activated its downstream target genes, including p21 and Bax, in lung cancer A549 cells dependent on erastin‑induced ROS. Subsequently, activated p53 by erastin treatment suppressed SLC7A11 and induced ROS accumulation, indicating the potential feedback loop between p53 and erastin‑induced ROS. By employing the caspase inhibitor Z‑VAD‑FMK, it was revealed that erastin‑induced p53 contributed to both ferroptotic and apoptotic cell death and inhibited cell proliferation via arresting the cell cycle at G1phase. Collectively, these results indicated that p53 may contribute to the cytotoxic and cytostatic effects associated with establishing a feedback loop with ROS induced by erastin.

Forskolin Sensitizes Human Acute Myeloid Leukemia Cells to H3K27me2/3 Demethylases GSKJ4 Inhibitor via Protein Kinase A.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring very often in older adults, with poor prognosis depending on both rapid disease progression and drug resistance occurrence. Therefore, new therapeutic approaches are demanded. Epigenetic marks play a relevant role in AML. GSKJ4 is a novel inhibitor of the histone demethylases JMJD3 and UTX. To note GSKJ4 has been recently shown to act as a potent small molecule inhibitor of the proliferation in many cancer cell types. On the other hand, forskolin, a natural cAMP raising compound, used for a long time in traditional medicine and considered safe also in recent studies, is emerging as a very interesting molecule for possible use in cancer therapy. Here, we investigate the effects of forskolin on the sensitivity of human leukemia U937 cells to GSKJ4 through flow cytometry-based assays (cell-cycle progression and cell death), cell number counting, and immunoblotting experiments. We provide evidence that forskolin markedly potentiates GSKJ4-induced antiproliferative effects by apoptotic cell death induction, accompanied by a dramatic BCL2 protein down-regulation as well as caspase 3 activation and PARP protein cleavage. Comparable effects are observed with the phosphodiesterase inhibitor IBMX and 8-Br-cAMP analogous, but not by using 8-pCPT-2′-O-Me-cAMP Epac activator. Moreover, the forskolin-induced enhancement of sensitivity to GSKJ4 is counteracted by pre-treatment with Protein Kinase A (PKA) inhibitors. Altogether, our data strongly suggest that forskolin sensitizes U937 cells to GSKJ4 inhibitor via a cAMP/PKA-mediated mechanism. Our findings provide initial evidence of anticancer activity induced by forskolin/GSKJ4 combination in leukemia cells and underline the potential for use of forskolin and GSKJ4 in the development of innovative and effective therapeutic approaches for AML treatment.

Abstract 4375: High hydrostatic pressure modified potato patatin induces apoptosis like cell death in human colorectal adenocarcinoma (HT-29) cell line

Abstract The current study assessed the anticancer activity of HHP-induced potato patatin on human colorectal adenocarcinoma (HT 29) cell line in vitro. Cell proliferation, apoptosis, necrosis and autophagy were investigated. HHP treatment inhibited HT-29 cell line proliferation in a dose a dependent manner. IC50 values showed antiproliferative activity (59.1%) at 4 mg mL-1 for HHP-induced patatin at 450 MPa. Flow cytometric analysis indicated that HHP-induced patatin at 450 MPa was the potent inducer of in vitro apoptotic cell death and showed significant (p<0.05) accumulation in S-phase followed by patatin pressurized at 550 MPa. Surface charge of patatin decreased with the increase in pressurization up to 350 MPa level, while at 450 MPa zeta potential value increased to (-13 mV) and (-11 mV) respectively. Overall, results indicated that 450 MPa patatin is a strong inhibitor of HT-29 and a potent inducer of apoptotic cell death. Thus, HHP treatment is an effective method for structural modification of patatin which ultimately enhance the anticancer potential against colorectal cancer cells. Citation Format: Rizwan Elahi, Tai Hua MU. High hydrostatic pressure modified potato patatin induces apoptosis like cell death in human colorectal adenocarcinoma (HT-29) cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4375.

Abstract 2325: Protein homeostasis adaptation to Hsp70 inhibition in cancer

Abstract Cancer cells experience acute stress conditions due to their increased proliferation rate and synthesis of misfolded proteins. However, by modulating the mechanisms that control protein-folding, cancer cells avoid cell death. Thus, it is perhaps not surprising that molecular chaperones, like Hsp70, are upregulated in cancer cells compared to their normal counterparts. These data suggest that these chaperones are potential targets for cancer therapy. Previous work in our lab demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that treatment of on RMS13 cell line with an Hsp70 inhibitor, known as MAL3-101, activates the PERK driven unfolded protein response that results in CHOP-dependent cell death (Sabinis et al., 2016). Nevertheless, the mechanism underlying how Hsp70 inhibition triggered apoptosis was poorly understood. By taking advantage of a MAL3-101-resistant cell line (RMS13-R), we have now determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying the increased demand on two protein degradation pathways upon inhibition of Hsp70. Specifically, autophagy related genes were upregulated, and increased conversion of LC3BI to LC3BII and accumulation of LC3BII was detected in RMS13-R cells. Further experiments demonstrated that autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components. Finally, we discovered that only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, which was apparent from an induction of apoptotic markers and cell death. These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, the unfolded protein response, and autophagy in rhabdomyosarcoma. Citation Format: Sara Sannino, Christopher J. Guerriero, Amit J. Sabnis, Trever G. Bivona, Jeffrey L. Brodsky. Protein homeostasis adaptation to Hsp70 inhibition in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2325.

Anti-cancer effects of Kaempferia parviflora on ovarian cancer SKOV3 cells

BackgroundKaempferia parviflora (KP) is an herb found in the north of Thailand and used as a folk medicine for improving vitality. Current reports have shown the anti-cancer activities of KP. However, the anti-cancer effects of KP on highly aggressive ovarian cancer have not been investigated. Therefore, we determined the effects of KP on cell proliferation, migration, and cell death in SKOV3 cells.MethodsOvarian cancer cell line, SKOV3 was used to investigate the anti-cancer effect of KP extract. Cell viability, cell proliferation, MMP activity, cell migration, and invasion were measured by MTT assay, cell counting, gelatin zymography, wound healing assay, and Transwell migration and invasion assays, respectively. Cell death was determined by trypan blue exclusion test, AnnexinV/PI with flow cytometry, and nuclear staining. The level of ERK and AKT phosphorylation, and caspase-3, caspase-7, caspase-9 was investigated by western blot analysis.ResultsKP extract was cytotoxic to SKOV3 cells when the concentration was increased, and this effect could still be observed even though EGF was present. Besides, the cell doubling time was significantly prolonged in the cells treated with KP. Moreover, KP strongly suppressed cell proliferation, cell migration and invasion. These consequences may be associated with the ability of KP in inhibiting the activity of MMP-2 and MMP-9 assayed by gelatin zymography. Moreover, KP at high concentrations could induce SKOV3 cell apoptosis demonstrated by AnnexinV/PI staining and flow cytometry. Consistently, nuclear labelling of cells treated with KP extract showed DNA fragmentation and deformity. The induction of caspase-3, caspase-7, and caspase-9 indicates that KP induces cell death through the intrinsic apoptotic pathway. The antitumor activities of KP might be regulated through PI3K/AKT and MAPK pathways since the phosphorylation of AKT and ERK1/2 was reduced.ConclusionsThe inhibitory effects of KP in cell proliferation, cell migration and invasion together with apoptotic cell death induction in SKOV3 cells suggest that KP has a potential to be a new candidate for ovarian cancer chemotherapeutic agent.

Rational Design of Cancer-Targeted Selenadiazole Derivative as Efficient Radiosensitizer for Precise Cancer Therapy.

Chemical drug design based on the biochemical characteristics of cancer cells has become an important strategy for discovery of targeted therapies for personalized cancer medicine. Herein, cancer targeting RGD peptide has been covalently conjugated to selenadiazole derivative (RGD-SeD) to improve its cancer selectivity. The RGD decoration significantly enhances the anticancer efficacy of RGD-SeD in αVβ3 integrin-overexpressing HepG2 liver cancer cells but not in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of RGD-SeD to integrin-overexpressing cancer cells. RGD-SeD strongly sensitizes HepG2 cells to clinically used X-ray radiotherapy through ROS overproduction, which triggers DNA damage-mediated apoptosis and G2/M cell cycle arrest. This X-ray-responsive DNA damage activates p53 signaling pathways by phosphorylation of ATM/ATR and γ-H2A.X. Furthermore, in a HepG2 nude mice xenograft model, the combined treatment of RGD-SeD and X-ray demonstrates potent in vivo antitumor efficacy via induction of apoptotic cell death but shows no toxicity on the functions of major organs. In summary, this study provides a strategy to design a selenium-based cancer targeting radiosensitizer for precise cancer therapy.

Fluorescent Nanodiamonds are Efficient, Easy-to-Use Cyto-Compatible Vehicles for Monitored Delivery of Non-Coding Regulatory RNAs.

MicroRNAs are short molecules of RNA regulating most cellular processes via the mechanism of RNA interference. Their dysregulation leads to a disease burden, making them important therapeutic targets. For the successful development of a therapeutic device, the uptake of a functionalized carrier by live cells and the sufficient release of effector therapeutic molecules are limiting factors. Here for the first time, the inhibition of oncogenic microRNA-21 in CT-26 colon cancer cells is achieved, using an advanced nanosystem consisting of fluorescent nanodiamond and antisense RNA. Stable nanocomplexes efficiently deliver antisense RNA into cell cytoplasm, encouraging further study of microRNA-21 function in target cells. Engaging the fluorescent nanoparticle enables monitoring of transfection and release of the antisense RNA load into cell cytoplasm. Importantly, the internalized antisense RNA effectively destroys target microRNA-21 in CT-26 cancer cells. The absence of oncogenic microRNA-21 liberates tumor suppressor genes Pdcd4 and Timp3 from silencing, and results in a decrease of cell invasion and migration, and in the induction of apoptotic cell death. This study uses a nanodiamond-based imaging and delivery system, and shows that the multidimensional performance of the presented device makes nanodiamond-based complexes promising therapeutic devices.

Simultaneous targeting of EGFR, HER2, and HER4 by afatinib overcomes intrinsic and acquired cetuximab resistance in head and neck squamous cell carcinoma cell lines.

The epidermal growth factor receptor (EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR‐targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti‐EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first‐generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines, independent of the HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G0/G1 cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.

P-28 - Idh2 deficiency exacerbates acrolein-induced lung injury through mitochondrial redox environment deterioration

Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of IDH2 in acrolein-induced lung injury using idh2 short hairpin RNA (shRNA)-transfected Lewis lung carcinoma (LLC) cells and idh2-deficient (idh2–/–) mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2–/– mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

Transformation of mouse T cells requires MYC and AKT activity in conjunction with inhibition of intrinsic apoptosis.

Peripheral T-cell lymphoma is an aggressive non-Hodgkin's lymphoma characterized by excessive proliferation of transformed mature T cells. The number and nature of genetic aberrations required and sufficient for transformation of normal T cells into lymphomas is unknown. Here, using a combinatorial in vitro-approach, we demonstrate that overexpression of MYC together with activated AKT in conditions of inhibition of intrinsic apoptosis rapidly resulted in transformation of mature mouse T cells with a frequency approaching 100%. Injection of transformed cells into mice resulted in rapid development of aggressive T cell lymphoma, characterized by spread to several organs, destruction of tissue architecture and rapid death of the animals. TcR-sequencing revealed a polyclonal repertoire of tumor cells indicating that co-expression of MYC, activated AKT and BCLXL is sufficient for tumor transformation and do not require acquisition of additional genetic events. When analyzing cells with inducible expression we found that proliferation of transformed T cells required sustained expression of both MYC and AKT. AKT exerted a dual function as it inhibited induction of, and promoted exit from, cellular quiescence and contributed to inhibion of apoptosis. Downregulation of AKT and/or MYC together with BCLXL resulted in rapid and complete elimination of cells through induction of apoptotic cell death.

Compound 21 (C21), a Selective Angiotensin Type 2 (AT2) Receptor Agonist Attenuates Bleomycin induced Alveolar Epithelial Cell Death

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by excessive deposition of collagen‐rich matrix, which progressively leads to respiratory failure and death. Patients with IPF have a reduced survival rate and the disease remains refractory to current therapies. Hence, identifying novel targets and developing effective drugs are of paramount importance. Accumulating evidence indicate that death of alveolar epithelial cells (AEC) is a key event for the initiation of IPF. Bleomycin, a well‐known fibrotic agent is a potent inducer of apoptotic cell death in AEC through synthesis of Angiotensin II (Ang II). While the apoptotic actions of Ang II on AEC are mediated via the angiotensin type 1 (AT1) receptor, the functional role of angiotensin type 2 (AT2) receptor in AEC has not yet been characterized. We hypothesized that stimulation of the angiotensin type 2 (AT2) receptor by a non‐peptide agonist, Compound 21 (C21) protects against bleomycin‐induced alveolar epithelial cell death.MethodsIn this study, we investigated the effects of C21, the selective AT2 receptor agonist against bleomycin‐induced death of human pulmonary alveolar epithelial cells (A549). The MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) assay was performed to determine cell viability of A549.ResultsWe first determined the concentration of bleomycin required for the induction of cell death in cultured A549 cells. A dose‐dependent induction of cell death was obtained with different concentration of bleomycin (μg/mL: % cell death; 1: 30%; 10:42% and 20:61%). Since we observed optimal cell death with 10 μg/mL concentration of bleomycin, we selected this dose for subsequent experiments. Next, we evaluated the cell‐protective effects of C21 at different drug doses (0.001, 0.005, 0.01, 0.05 and 0.1 μM). C21 treatment of bleomycin‐challenged A549 cells (10 μg/mL) produced a dose‐dependent decrease in cell death (μM: % cell death; 0.001:31%; 0.005:29%; 0.01:16%; 0.05:14% and 0.1:9%) as analyzed by the MTT assay. We also observed that treatment of control A549 cells with the above mentioned doses of C21 had little effect on cell proliferation or cell death.ConclusionOur results suggest that C21, the selective AT2 receptor agonist protects against bleomycin‐induced alveolar epithelial cell death. Further studies are warranted to elucidate the mechanisms underlying the cytoprotective effects of C21.Support or Funding InformationAmerican Heart Association Grant (SDG12080302) awarded to Vinayak ShenoyStart‐up Grant from California Health Sciences UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oral exposure to silver nanoparticles increases oxidative stress markers in the liver of male rats and deregulates the insulin signalling pathway and p53 and cleaved caspase 3 protein expression

The present study was aimed at assessing the impact of AgNPs on the liver of male rats orally exposed to 0, 50, 100 and 200 mg/kg/day of polyvinyl pyrrolidone coated AgNPs (PVP-AgNPs) for 90 days. The induction of apoptotic cell death -by measuring the protein levels of the active form of caspase 3- and the levels of the microtubule-associated protein 1A/1B-light chain (LC3) protein were measured as a marker of the induction of autophagy. PVP-AgNPs caused an increase of the activity of superoxide dismutase (SOD) and catalase (CAT) in the liver of male rats. However, the activity decreased after exposure to high amounts of PVP-AgNPs. Increased protein levels of IRS-1, AKT, GSK3β and mTOR proteins were observed in a dose-dependent manner. However, these proteins showed a decrease at 200 mg/kg/day. The same pattern was observed for the p53, p21 and cleaved caspase 3 protein levels. The current results suggest that the increase of ROS production by PVP-AgNPs stimulated SOD and CAT activity, as well as IRS-1, AKT, mTOR, p53, p21 and caspase 3 as protective mechanisms of cell survival and preserve DNA fidelity. However, cellular damage by excessive ROS production might induce the depletion of these survival mechanisms at 200 mg/kg/day.

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from Loranthus yadoriki

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.

Differential Action of Anti-CD20 Monoclonal Antibodies: Role in Induction of Cell Death

Purpose: To study and assess to the sensitivity of Burkitt’s lymphoma cells harboring differential levels of cell surface CD20 for anti-CD20 monoclonal Antibodies. Material and methods: Burkitt’s lymphoma cell line ‘Daudi’ was used during present study. Cells were exposed to single dose of γ-radiation (0.5 Gy) and thereafter incubated with rituximab (Rtx) or tositumomab (Tst) (0.5 μg/ ml concentration each). The changes in expression of RelA, Akt and Bax/Bcl-2 ratio was measured to assess sensitivity of cells in term of cell death. Results: During the present investigation, we found that cells incubated with anti-CD20 mAbs at +20 hrs post radiation exposure showed higher levels of cell death in term of Bax/Bcl-2 ratio. In addition, we also found the significant changes in expression of pro-survival signaling proteins such as RelA and Akt pathways. Moreover, we found that tositumomab is a potent inducer of apoptotic cell death. Conclusion: These findings suggested that the efficacy of anti-CD20 mAbs depends on the number of CD20 molecules expressed on cell surface and type of antibody used. It may provide new treatment options for selection of anti-CD20 mAbs even in aggressive B-cell lymphoma, which harbors low levels of CD20 or even resistant to current therapies in vivo.

Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model.

The aim of this study is to investigate the antitumor activity and possible molecular mechanism of Phenethyl isothiocyanate (PEITC) against Ehrlich ascites carcinoma in-vivo and in-vitro. In-vivo, ascetic fluid volume, body weight, serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined using Ehrlich ascites carcinoma (EAC) bearing mice. In-vitro, MTT assay was used. RT-PCR was used to investigate role of PEITC in apoptosis by analyzing the expression of Bax, caspase-9, and Bcl-2 genes. The effect of PEITC on caspase-9 enzyme activity was also tested. PEITC and/or Doxorubicin (Dox) treatment significantly suppressed EAC growth as compared to EAC/oil control mice. PEITC treatment showed a dose-dependent inhibition of EAC cells as indicated by MTT assay. We found that significant increase in MDA level and decrease in TAC caused by Dox treatment were significantly reduced by combination with PEITC treatment. Bax, caspase-9 genes' expression and caspase-9 enzymatic activity were significantly increased, while Bcl-2 gene expression was significantly decreased in PEITC treated mice. PEITC may act as a promising anticancer agent either alone or more effectively in combination with Dox through apoptotic cell death induction.

Upregulation of Mcl-1 inhibits JQ1-triggered anticancer activity in hepatocellular carcinoma cells

Bromodomains and extra-terminal (BET) proteins inhibitors are promising cancer therapeutic agents. However, tumor cells often develop resistance to BET inhibitors, greatly limiting their therapeutic potential. To study the mechanism underlying the resistance of BET inhibitors in hepatocellular carcinoma (HCC) cells, we herein investigated the impact of BET inhibitor JQ1 on the gene expression of Bcl-2 family members by RNA sequencing analysis, and found that acute treatment with JQ1 triggered upregulation of Mcl-1 in HCCLM3 and BEL7402 cell lines. This JQ1-triggered Mcl-1 upregulation was further confirmed by quantitative reverse transcription polymerase chain reaction and western blotting analysis, both at mRNA and protein levels. Inhibition of Mcl-1 by RNA interference dramatically enhanced JQ1-triggered caspase-3 activation, cleavage of poly (ADP-ribose) polymerase and apoptotic cell death induction in multiple HCC cell lines. Moreover, JQ1 in combination with cyclin-dependent kinase inhibitor flavopiridol at a subtoxic concentration that reduced expression of Mcl-1, triggered massive apoptotic cell death in HCCLM3 and BEL7402 cell lines. Together, these data suggest that Mcl-1 is a major contributor to BET inhibitor-resistance in HCC cells, and that combining drugs capable of down-regulating Mcl-1 may promote therapeutic potential in human HCC.