Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

<p>Abstract</p> <p>Background</p> <p>Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine.</p> <p>Methods</p> <p>GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition.</p> <p>Results</p> <p>GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-X_L, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic.</p> <p>Conclusion</p> <p>GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be context or cell line dependent, but could also be explained on the basis that although NF-kappaB is an important mediator of pancreatic cancer cell survival, it plays a minor role in gemcitabine resistance. Further work is needed to understand the mechanisms of this effect, including the potential for rational combination of GSK3 inhibitors with other targeted agents for the treatment of pancreatic cancer.</p

The global burden of adolescent and young adult cancer in 2019 : a systematic analysis for the Global Burden of Disease Study 2019

Background In estimating the global burden of cancer, adolescents and young adults with cancer are often overlooked, despite being a distinct subgroup with unique epidemiology, clinical care needs, and societal impact. Comprehensive estimates of the global cancer burden in adolescents and young adults (aged 15-39 years) are lacking. To address this gap, we analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, with a focus on the outcome of disability-adjusted life-years (DALYs), to inform global cancer control measures in adolescents and young adults. Methods Using the GBD 2019 methodology, international mortality data were collected from vital registration systems, verbal autopsies, and population-based cancer registry inputs modelled with mortality-to-incidence ratios (MIRs). Incidence was computed with mortality estimates and corresponding MIRs. Prevalence estimates were calculated using modelled survival and multiplied by disability weights to obtain years lived with disability (YLDs). Years of life lost (YLLs) were calculated as age-specific cancer deaths multiplied by the standard life expectancy at the age of death. The main outcome was DALYs (the sum of YLLs and YLDs). Estimates were presented globally and by Socio-demographic Index (SDI) quintiles (countries ranked and divided into five equal SDI groups), and all estimates were presented with corresponding 95% uncertainty intervals (UIs). For this analysis, we used the age range of 15-39 years to define adolescents and young adults. Findings There were 1.19 million (95% UI 1.11-1.28) incident cancer cases and 396 000 (370 000-425 000) deaths due to cancer among people aged 15-39 years worldwide in 2019. The highest age-standardised incidence rates occurred in high SDI (59.6 [54.5-65.7] per 100 000 person-years) and high-middle SDI countries (53.2 [48.8-57.9] per 100 000 person-years), while the highest age-standardised mortality rates were in low-middle SDI (14.2 [12.9-15.6] per 100 000 person-years) and middle SDI (13.6 [12.6-14.8] per 100 000 person-years) countries. In 2019, adolescent and young adult cancers contributed 23.5 million (21.9-25.2) DALYs to the global burden of disease, of which 2.7% (1.9-3.6) came from YLDs and 97.3% (96.4-98.1) from YLLs. Cancer was the fourth leading cause of death and tenth leading cause of DALYs in adolescents and young adults globally. Interpretation Adolescent and young adult cancers contributed substantially to the overall adolescent and young adult disease burden globally in 2019. These results provide new insights into the distribution and magnitude of the adolescent and young adult cancer burden around the world. With notable differences observed across SDI settings, these estimates can inform global and country-level cancer control efforts. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd.Peer reviewe

GSK-3 Inhibition: A Novel Approach to Sensitization of Chemo-resistant Pancreatic Cancer Cells

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.Ph

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be context or cell line dependent, but could also be explained on the basis that although NF-kappaB is an important mediator of pancreatic cancer cell survival, it plays a minor role in gemcitabine resistance. Further work is needed to understand the mechanisms of this effect, including the potential for rational combination of GSK3 inhibitors with other targeted agents for the treatment of pancreatic cancer

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to TRAIL-Induced Apoptosis

<div><p>Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.</p> </div

TRAIL sensitization by AR18 is associated with loss of ΔΨm and increased ROI generation.

<p>PANC-1 and BxPC-3 cells were untreated or incubated with AR-18 (25 µM), TRAIL (10 ng/mL), or their combination for 24 h, and then analyzed by flow cytometry. Dot density plots of propidium iodide (PI) uptake vs ΔΨm show that loss of ΔΨm precedes PI uptake. TRAIL induced loss of ΔΨm in BxPC-3 but not in PANC-1, consistent with their greater sensitivity seen using the SRB assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041102#pone-0041102-g001" target="_blank">Figure 1</a>). Combined treatment with TRAIL and AR18 clearly sensitized both cell lines to loss of ΔΨm, and this was accompanied by increased ROI generation and loss of out membrane integrity.</p

Genetic knockdown of GSK-3 renders cells sensitive to TRAIL-induced apoptosis.

<p>PANC-1 cells were pre-treated using AR-18 or siRNA against both isoforms of GSK-3, and then exposed to TRAIL. Immunoblotting confirms successful knockdown of GSK3α and β. This resulted in increased cleavage of PARP and caspase-3 following exposure to TRAIL. Scr: Scrambled non-specific siRNA.</p

GSK-3 inhibition sensitizes TRAIL-resistant pancreatic cancer cells to apoptosis.

<p>PANC-1 (A) and BxPC-3 (B) were treated with TRAIL after 24 h pre-exposure to AR-18 at the indicated concentrations, and cell proliferation measured by SRB assay. Each bar graph signifies mean from three separate experiments with six replicates. error bars  =  ± SEM.</p

GSK-3 inhibition enhances TRAIL sensitization through PARP and caspase cleavage.

<p>(A) BxPC-3 and PANC-1 cells were treated with AR-18, in combination with or without TRAIL for 24 h and analysed by for PARP cleavage. (B) BxPC-3 (left) and PANC-1 (right) cells were treated with AR-18 (25 uM), TRAIL (10 ng/mL), z-VAD-fmk 50 µM, or the combinations indicated and SRB assay after 22 h incubation. Each bar graph signifies mean from three experiments with six replicates. error bars  =  ± SEM. *p<0.005, **p<0.0001.</p

Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes