Intrinsic Apoptotic Signaling Research Articles

HnRNP-L mediates bladder cancer progression by inhibiting apoptotic signaling and enhancing MAPK signaling pathways.

Heterogeneous nuclear ribonucleoprotein L (hnRNP-L) is a promoter of various kinds of cancers, but its actions in bladder cancer (BC) are unclear. In this study, we investigated the function and the underlying mechanism of hnRNP-L in bladder carcinogenesis. Our results demonstrated that enhanced hnRNP-L expression in BC tissues was associated with poor overall survival of BC patients. Depletion of hnRNP-L significantly suppressed cell proliferation in vitro and inhibited xenograft tumor growth in vivo. Furthermore, downregulation of hnRNP-L resulted in G1-phase cell cycle arrest and enhanced apoptosis accompanied by inhibition of EMT and cell migration. All these cellular changes were reversed by ectopic expression of hnRNP-L. Deletion of hnRNP-L resulted in decreased expression of Bcl-2, enhanced expression of caspases-3, -6 and -9 and inhibition of the MAPK signaling pathway. These findings demonstrate that hnRNP-L contributes to poor prognosis and tumor progression of BC by inhibiting the intrinsic apoptotic signaling and enhancing MAPK signaling pathways.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells.

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

HnRNP-L promotes prostate cancer progression by enhancing cell cycling and inhibiting apoptosis.

Expression of the RNA-binding protein HnRNP-L was previously shown to associate with tumorigenesis in liver and lung cancer. In this study, we examined the role of HnRNP-L in prostate cancer (Pca). We found that HnRNP-L is overexpressed in prostate tissue samples from 160 PC patients compared with tissue samples from 32 donors with cancers other than Pca. Moreover, HnRNP-L positively correlated with aggressive tumor characteristics. HnRNP-L knockdown inhibited cell proliferation and promoted cell apoptosis of Pca cell lines in vitro, and suppressed tumor growth when the cells were subcutaneously implanted in an athymic mouse model. Conversely, overexpression of HnRNP-L promoted cell proliferation and tumor growth while prohibiting cell apoptosis. HnRNP-L promoted cell proliferation and tumor growth in Pca in part by interacting with endogenous p53 mRNA, which was closely associated with cyclin p21. In addition, HnRNP-L affected cell apoptosis by directly binding the classical apoptosis protein BCL-2. These observations suggest HnRNP-L is an important regulatory factor that exerts pro-proliferation and anti-apoptosis effects in Pca through actions affecting the cell cycle and intrinsic apoptotic signaling. Thus HnRNP-L could potentially serve as a valuable molecular biomarker or therapeutic target in the treatment of Pca.

Nicotine plus a high-fat diet triggers cardiomyocyte apoptosis.

Cigarette smoking is an important risk factor for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75mg/kg body weight [BW]) or saline for 16weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for 16weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P < 0.05) attenuated the HFD-induced decrease in fibroblast growth factor 21 (FGF21) and silent information regulator 1 (SIRT1). We conclude that nicotine, when combined with HFD, triggers CM apoptosis through the generation of oxidative stress and inactivation of AMPK together with the activation of caspase-2-mediated intrinsic apoptotic signaling independently of FGF21 and SIRT1.

Manipulation of pH-Sensitive interactions between podophyllotoxin-chitosan for enhanced controlled drug release

Podophyllotoxin (PPT) offers a broad-spectrum of anticancer activities, but little has been reported for its controlled release. This work shows that by manipulating molecular interactions between PPT and Chitosan, efficient nanoscale capsulation of PPT can be realized. The drug encapsulation efficiency is as high as 52%, with a final particle drug loading in the order of 10% (wt/wt). It further demonstrates that changes in pH can also significantly affect the rate of drug release from the Chitosan nanoparticles. Upon contact with cancer cells, chitosan nanoparticles enable efficient internalization and drug release. In vitro evaluations with HepG-2 and MCF-7 cells indicate that the chitosan nanoparticle carriers can improve drug efficacy in comparison to free PPT, most likely by regulating the intrinsic apoptotic signaling pathway to induce apoptosis. Overall, PPT chitosan nanoparticles promise a safe and efficient drug delivery system for PPT.

Gonadal steroids block the calpain-1-dependent intrinsic pathway of apoptosis in an experimental rat stroke model

Objectives: Apoptosis plays an important role in the progression of the ischemic penumbra after reperfusion. Estrogen and progesterone have neuroprotective effects against ischemic brain damage, however the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In this study, we investigated the possible regulatory effects of a combined steroid treatment on extrinsic and intrinsic apoptotic signaling pathways after cerebral ischemia.Methods: Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAO) using an intraluminal filament technique for 1 h followed by 23 h reperfusion. Estrogen and progesterone were immediately injected after tMCAO subcutaneously. Sensorimotor functional tests and the infarct volume were evaluated 24 h after ischemia. Protein expression of calpain-1 and Fas receptor (FasR), key members of intrinsic and extrinsic apoptosis, were determined in the penumbra region of the ischemic brain using western blot analysis, immunohistochemistry, and TUNEL staining.Results: Neurological deficits and infarct volume were significantly reduced following hormone therapy. Calpain-1 up-regulation and caspase-3 activation were apparent 24 h after ischemia in the peri-infarct area of the cerebral cortex. Steroid hormone treatment reduced infarct pathology and attenuated the induction of both proteases. FasR protein levels were not affected by ischemia and hormone application.Conclusion: We conclude that a combined steroid treatment inhibits ischemia-induced neuronal apoptosis through the regulation of intrinsic pathways.

Marine guanidine alkaloids crambescidins inhibit tumor growth and activate intrinsic apoptotic signaling inducing tumor regression in a colorectal carcinoma zebrafish xenograft model.

The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo.Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.

Curcumin analog EF24 induces apoptosis via ROS-dependent mitochondrial dysfunction in human colorectal cancer cells.

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H2O2 and pretreatment with an ROS scavenger, NAC. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

Oxidative stresses-mediated apoptotic effects of ginsenoside Rb1 on pre- and post-implantation mouse embryosin vitroandin vivo

Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 μgmL-1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25-100 μgmL-1 ginsenoside Rb1 was associated with increased resorption of post-implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mgkg-1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses-triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre- and post-implantation development of mouse embryos, both in vitro and in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990-2003, 2017.

Mitochondrial ROS and the Effectors of the Intrinsic Apoptotic Pathway in Aging Cells: The Discerning Killers!

It has become clear that mitochondrial reactive oxygen species (mtROS) are not simply villains and mitochondria the hapless targets of their attacks. Rather, it appears that mitochondrial dysfunction itself and the signaling function of mtROS can have positive effects on lifespan, helping to extend longevity. If events in the mitochondria can lead to better cellular homeostasis and better survival of the organism in ways beyond providing ATP and biosynthetic products, we can conjecture that they act on other cellular components through appropriate signaling pathways. We describe recent advances in a variety of species which promoted our understanding of how changes of mtROS generation are part of a system of signaling pathways that emanate from the mitochondria to impact organism lifespan through global changes, including in transcriptional patterns. In unraveling this, many old players in cellular homeostasis were encountered. Among these, maybe most strikingly, is the intrinsic apoptotic signaling pathway, which is the conduit by which at least one class of mtROS exercise their actions in the nematode Caenorhabditis elegans. This is a pathway that normally contributes to organismal homeostasis by killing defective or otherwise unwanted cells, and whose various compounds have also been implicated in other cellular processes. However, it was a surprise that that appropriate activation of a cell killing pathway can in fact prolong the lifespan of the organism. In the soma of adult C. elegans, all cells are post-mitotic, like many of our neurons and possibly some of our immune cells. These cells cannot simply be killed and replaced when showing signs of dysfunction. Thus, we speculate that it is the ability of the apoptotic pathway to pull together information about the functional and structural integrity of different cellular compartments that is the key property for why this pathway is used to decide when to boost defensive and repair processes in irreplaceable cells. When this process is artificially stimulated in mutants with elevated mtROS generation or with drug treatments it leads to lifespan prolongations beyond the normal lifespan of the organism.

Nanomedicine-based paclitaxel induced apoptotic signaling pathways in A562 leukemia cancer cells

In the present study, we have synthesized an amphiphilic pH-sensitive structure of poly(ethylene glycol) methyl ether-b-(poly lactic acid-co-poly(b-amino esters)) (MPEG-b-(PLA-co-PAE)) to load paclitaxel to increase the therapeutic efficacy in leukemia. The micelles exhibit excellent drug-loading capacities for paclitaxel (PTX) and exhibited a typical pH-responsive drug release pattern. The release of PTX from the micelles was significantly accelerated by decreasing pH from 7.4 to 5.0 which just fitted the pathological process. The most important advantage of this design is that the polymeric micelles provide an effective approach for rapid transport of cargo into the cytosol, which significantly increases the antitumor efficacy of PTX against K562 cancer cells. Paclitaxel-loaded polymer micelles (PTX-M) showed significantly higher cytotoxic effect than that of free PTX. The PTX-M exhibited a superior apoptosis effect in cancer cells compared to that of free PTX at all time points. We have showed that the PTX-M activated upstream of apoptosis signaling and inhibited the anti-apoptotic factors. The PTX-M remarkably increased the upregulation of Bax, caspase-3, caspase-9, and PARP-1 expression and downregulated the Bcl-2 expression in K562 cancer cells. The results show that PTX-M induced cell apoptosis through intrinsic apoptotic signaling pathway. Importantly, PTX had a remarkably prolonged plasma circulation time after administration of PTX-M. Overall, this novel cancer specific, pH-responsive, and potentially in vivo stable unimolecular micelles may provide a very promising approach for targeted cancer therapy in the effective treatment of Leukemia.

Abstract LB-354: Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array

Abstract Colorectal cancer (CRC) is the third and second most commonly diagnosed cancer in males and females, and the second most common cause of cancer-related deaths in the developed world. Identifying the importance of epidermal growth factor (EGF) signalling pathways for the survival of CRC cells (Van Cutsem, Kohne et al. 2011) resulted in development of therapies that target EGF-receptors (EGFR). Late stage CRC patients are mainly treated with monoclonal anti-EGFR antibodies such as cetuximab. Although anti-EGFR therapies have significantly improved survival in CRC patients (Van Cutsem, Kohne et al. 2009), they are not effective in patients with activating KRAS, BRAF, NRAS and PI3KCA mutations (De Roock, Claes et al. 2010). Nevertheless, between 50-60% of patients will not benefit from the additional anti-EGFR treatment, even when these have a quadruple wild-type status (De Roock, De Vriendt et al. 2011), suggesting that novel biomarkers and targeted therapies are required. In this study, we investigate potential biomarkers for cetuximab response in a panel of 93 quadruple wild-type, liver metastatic CRC samples from patient derived xenografts (PDX) with known responses to cetuximab. Matching PDXs were used to measure changes in tumour volumes post cetuximab treatment (Bertotti, Papp et al. 2015) and were later categorised as regressing, progressing or stabilised. Protein expression levels were measured in matched PDXs, using a reverse phase protein array (RPPA) with a panel of 72 antibodies targeting key cancer related proteins as previously described). Statistical analysis of measured values showed significant correlation between high protein expression levels, such as Chk-1, PARP, HIAP-2 (cIAP-1), and PDX progression post cetuximab treatment. These proteins were significantly expressed lower in both regressing and stable PDXs. Interestingly, we found a high cross correlation between expression levels of these proteins across all the samples, giving them a great potential to act as predictive biomarkers for cetuximab response. Bioinformatics pathway enrichment of these proteins showed a significant enrichment of intrinsic apoptotic and cell cycle signalling pathways. These results have also been investigated in publically available patient data with cetuximab response and together will be used to construct a deterministic mathematical cell cycle model that will help to predict the outcome of cetuximab therapy in future. Citation Format: Naser Monsefi, Robert O’Byrne, Steven Carberry, Eugenia R. Zanella, Ana Barat, Mattia Cremona, Clare Morgan, Bryan T. Hennessy, Andrea Bertotti, Livio Trusolino, Jochen H.M. Prehn. Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-354.

Suppression of human 8-oxoguanine DNA glycosylase (OGG1) augments ultrasound-induced apoptosis in cervical cancer cells

PurposeHuman 8-oxoguanine DNA glycosylase (OGG1) is a major base excision repair enzyme, and it was reported to suppress the activation of intrinsic apoptotic signaling pathway in response to oxidative stress. In this study, our aim was to investigate the effects of OGG1 downregulation on ultrasound-induced apoptosis in cervical cancer cells. MethodsOGG1 expression was silenced by shRNA in the cervical cancer SW756 and CaSki cells. Cell viability was evaluated by MTT assay after OGG1 knockdown following ultrasound treatment. Ultrasound-induced apoptosis was measured by Annexin V-FITC/propidium iodide. Intracellular reactive oxygen species (ROS) production and Ca2+ concentration were detected using a fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and a green fluorescent dye fluo-4AM, respectively. Western blotting was used to analyze the expression of Bcl-2, Bax, cleaved caspase-3, and nuclear factor-κB p65 (NF-κB p65). ResultsThe results indicated that OGG1 knockdown did not suppress cell proliferation, but significantly augmented ultrasound-induced inhibitory effects on the cell viability, and increased ultrasound-induced early apoptosis and late apoptosis and necrosis in the SW756 and CaSki cells when exposure to ultrasound (1MHz) at 1.5W/cm2 for 30 and 60s. OGG1 knockdown significantly increased intracellular ROS production and Ca2+ concentration after incubation of 6, 24, and 48h post-ultrasound treatment. The downregulation of Bcl-2 protein and the upregulation of Bax, cleaved caspase-3, and NF-κB p65 protein levels were observed in the shRNA-OGG1 cells and mock-shRNA cells, but no significant change of these protein levels was found between of them. ConclusionsThese results indicate that downregulation of OGG1 expression can augment ultrasound-induced apoptosis in cervical cancer cells, which suggests that OGG1 suppression might provide a new insight for ultrasound-induced therapeutic effects on cervical cancer treatment.

Engineering of caveolae-specific self-micellizing anticancer lipid nanoparticles to enhance the chemotherapeutic efficacy of oxaliplatin in colorectal cancer cells

Novel nanomaterials for the intracellular transport of therapeutic cargos have been actively sought to effectively breach cell-membrane barriers. In this study we developed novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells (CRCs). We demonstrated that NPs with special affinity to caveolae could be designed and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. We demonstrated a remarkable uptake of OL-loaded SMAL NPs (SMAL-OL) in HCT116 and HT-29 cells via the caveolae-mediated endocytosis (CvME) pathway. The higher accumulation of SMAL-OL in the intracellular environment resulted in a significantly elevated anticancer effect compared to that of free OL. Cell cycle analysis proved G2/M phase arrest, along with substantial presence of cells in the sub-G1 phase. An immunoblot analysis indicated an upregulation of pro-apoptotic markers (Bax; caspase-3; caspase-9; and PARP1) and downregulation of Bcl-xl and the PI3K/AKT/mTOR complex, indicating a possible intrinsic apoptotic signaling pathway. Overall, the ability of SMAL NPs to confer preferential specificity towards the cell surface domain could offer an exciting means of targeted delivery without the need for receptor-ligand-type strategies. Statement of SignificanceIn this work, we developed a novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells. We demonstrated that NPs with special affinity to caveolae could be realized and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. In addition, oxaliplatin-loaded SMAL were efficiently endocytosed by the cancer cells and represent a significant breakthrough as an effective drug delivery system with promising potential in cancer therapy. We believe this work holds promising potential for the development of next generation of multifunctional nanocarriers for an exciting means of targeted delivery without the need for receptor-ligand-type strategies.

Accumulation of apoptosis-insensitive human bone marrow-mesenchymal stromal cells after long-term expansion.

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long-term-cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long-term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence-associated β-gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15-19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7-10 passages). This resistance occurred via caspase-9 and caspase-3 rather than via caspase-8. The senescent cells are gradually accumulated during long-term expansion. The oxidative stress-sensitive proteins ataxia-telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl-2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late-passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long-term-cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long-term expansion. Copyright © 2016 John Wiley & Sons, Ltd.

P62 modulates the intrinsic signaling of UVB-induced apoptosis

BackgroundUVB radiation is the main source of sunburn and skin cancers. Apoptosis eliminates photodamaged cells, and is thus important for preventing epidermal carcinogenesis. The cytoplasmic regulatory protein p62/A170/sequestosome 1 (p62) molecule is involved in a variety of cellular and signaling pathways. p62 is known to be and important in autophagy, but its role in UVB-induced apoptosis remains to be clarified. ObjectiveTo investigate the role of p62 against UVB-induced apoptotic changes, using mouse embryonic fibroblasts (MEFs) derived from p62 homozygous knockout (p62−/−) mice. Methodsp62−/− and wild-type (p62+/+) mice and MEFs were subjected to UVB irradiation, and the resultant apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blots. ResultsApoptosis was decreased in the p62−/− MEFs compared to p62+/+ MEFs in response to UVB treatment. Compared with p62+/+ MEFs, p62−/− MEFs expressed significantly more Bcl-2 and less Bax, and showed increased Src and Stat3 phosphorylation. Our results show that p62 regulates apoptotic pathways by modifying critical signaling intermediates such as Src and Stat3. Conclusionp62 reduces UVB-induced apoptosis by modulating intrinsic apoptotic signaling through Src phosphorylation.

The role of calcium sulfide in intrinsic and extrinsic apoptotic pathways in non‐small lung cell adenocarcinoma

Apoptosis is the process of programed cell death and it is characterized by metabolic changes and energy‐dependent mechanisms. It occurs normally in the development and regeneration of tissues, to insure proper functionality of systems, and it can also function as part of the cellular response against disease or cytotoxicagents. Still, a variety of internal and external stimuli trigger apoptotic cascades, which are categorized in two signaling pathways, extrinsic and intrinsic; they are based on the origin of the stimuli, and the mediators involved. Previous studies in our laboratory, showed that non‐small lung cell adenocarcinoma cell line (ATCC HCC827) treated with proprietary calcium sulfidenanoclusters (CaS) [3.8μM] showed increased apoptotic activity following 48 hours that become statistically significant at 72 hours (p&lt;0.05) without affecting non‐cancerous cell lines (MCR5) after 24‐hours. The objective of this study was to determine the expression of intrinsic (Bcl‐2 and caspase‐3) and extrinsic (caspase‐8 and Bax) apoptotic factors in HCC827 cells upon addition of a single dose of CaS nanoclusters evaluated at 24 hours intervals up to 72‐hours. We hypothesized that CaS nanoclusters will increase the intrinsic and extrinsic apoptotic signaling pathways. DMSO [1%] was used as a negative control and Etoposide[10μM] as a positive control. The flasks were randomly assigned to one of the treatments in triplicates. We used Western Blot, densitometry, and statistical analyses to evaluate changes in expression of the studied proteins. The preliminary data showed that: (1) at 72 hours, the expression of the pro‐apoptotic Bax increased and the anti‐apoptotic Bcl‐2 decreased; (2) at 24 and 48 hours the expression of full caspase‐8 and their respective cleaved variations increased, as well as the pro‐apoptotic caspase‐3 when HCC827 cells were treated with CaS nanoclusters (p&lt;0.05). These results suggest that CaS nanoclusters have apro‐apoptotic effect, involving both intrinsic and extrinsic pathways during the specified timelines. We are currently studying additional pro apoptotic mediators in the intrinsic (caspase‐9, ‐12) and extrinsic (caspase‐7) to elucidate the level of effect of CaS nanoclusters in these pathways.Support or Funding InformationThis work was supported by: UPRPRISE Program NIH Grant #R25GM096955

Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells.

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

MiRNA expression profiles in high-grade prostatic intraepithelial neoplasia.

45 Background: High-grade prostatic intraepithelial neoplasia (HGPIN) is widely believed to be a precursor of prostate cancer (PCa). However, little is known about the expression of miRNAs variations in HGPIN compared to normal tissues and PCa. Methods: The expression data of 1054 miRNAs from TCGA were applied to identify relevant miRNAs associated with tumor progression (i.e., miR-98-5p, miR-183-5p, 345-5p, miR-143 miR-210-3p and miR-378-3p). miRNA were isolated by miRNeasy FFPE kit (Qiagen, Hilden, Germany) from paraffin-embedded tissues (FFPE) of prostate specimens with PCa, HGPIN and normal tissues. Early-stage PCa was defined as PCa with pT2 tumor stage, Gleason score &lt;=7a (3+4) and PSA level &lt;10 ng/ml. Quantitative miRNA expression data were acquired and analyzed using a real-time TaqMan-based PCR with the ABI Prism 7900HT (Life Technologies, Darmstadt, Germany). ANOVA analysis were performed to evaluate the expression of miRNAs between HGPIN, Normal and PCa tissues. All statistical analysis was performed using SPSS (IBM, Armonk, USA). P values were adjusted using the false discovery rate for multiple comparisons. The small nuclear U6 RNA was used as an endogenous control. Results: The expression of miR-143-3p, miR-210-3p and miR-345-5p and miR-98-5-p were varied between normal tissue, HGPIN and early-stage PCa. Interestingly, decrease in expression of miR-143.-3p, miR-98-5p and miR-210-3p was associated with tumor development (Normal tissues &gt; HGPIN &gt; early-stage PCa) (FDR&lt;0.001). Furthermore, overexpression of miR-345-5p was observed in normal tissues compared to HGPIN and early-stage PCa, which both showed similar expression level of miR-345-5p. No significant differences in expression of miR-375, miR-183-5p and miR-378-3p were observed between HGPIN and PCa. These miRNAs were interacted with genes related to HIF-1 signaling pathway, p53 signaling pathway, androgen receptor signaling pathway, intrinsic apoptotic signaling pathway, RNA transcription, homologous recombination and non-homologous end-joining. Conclusions: HGPIN shows an altered expression of miRNAs interact with genes related to hypoxia, androgen receptor signaling pathway, cell cycle and epigenetic regulation.