DMSO treatment of mice bearing Dalton's lymphoma resulted in 3 key effects:
1) Increased expression of tumor necrosis factor (TNF) and p53 proteins in the lymphoma cells, activating the TNF-p53 mediated apoptotic pathway.
2) Decreased expression of anti-apoptotic Bcl-2 and increased expression of pro-apoptotic Bax in the lymphoma cells, reducing the protective Bcl-2/Bax ratio.
3) Activation of caspase 9 and cleavage of PARP-1 in the lymphoma cells, consistent with induction of apoptosis through the mitochondrial pathway.
Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pa...rkkoiri
There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg
effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is
an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor
progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel
anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered
to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the
DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic
glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to
modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that
Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2:
PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase
(the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the
two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the
enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings
suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor
supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.
Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pa...rkkoiri
There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg
effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is
an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor
progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel
anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered
to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the
DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic
glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to
modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that
Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2:
PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase
(the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the
two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the
enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings
suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor
supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.
Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin-sensitive G-protein-coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG-mediated apoptosis in colon cancer cells.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
NAD-Glycohydrolase Depletes Intracellular NAD+ and Inhibits Acidification of Autophagosomes to Enhance Multiplication of Group A Streptococcus in Endothelial Cells
Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin-sensitive G-protein-coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG-mediated apoptosis in colon cancer cells.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
NAD-Glycohydrolase Depletes Intracellular NAD+ and Inhibits Acidification of Autophagosomes to Enhance Multiplication of Group A Streptococcus in Endothelial Cells
Targetting cancer with Ru(III/II)-phosphodiesterase inhibitor adducts: A nove...rkkoiri
Lack of specificity and normal tissue toxicity are the two major limitations faced with most of the anticancer
agents in current use. Due to effective biodistribution and multimodal cellular actions, during
recent past, ruthenium complexes have drawn much attention as next generation anticancer agents. This
is because metal center of ruthenium (Ru) effectively binds with the serum transferrin and due to higher
concentration of transferrin receptors on the tumor cells, much of the circulating Ru-transferrin complexes
are delivered preferentially to the tumor site. This enables Ru-complexes to become tumor cell
specific and to execute their anticancer activities in a somewhat targeted manner. Also, there are evidences
to suggest that inhibition of phosphodiesterases leads to increased cyclic guanosine monophosphate
(cGMP) level, which in turn can evoke cell cycle arrest and can induce apoptosis in the tumor
cells. In addition, phosphodiesterase inhibition led increased cGMP level may act as a potent vasodilator
and thus, it is likely to enhance blood flow to the growing tumors in vivo, and thereby it can further facilitate
delivery of the drugs/compounds to the tumor site.
Therefore, it is hypothesized that tagging PDE inhibitors (PDEis) with Ru-complexes could be a relevant
strategy to deliver Ru-complexes-PDEi adduct preferentially to the tumor site. The Ru-complex tagged
entry of PDEi is speculated to initially enable the tumor cells to become a preferential recipient of such
adducts followed by induction of antitumor activities shown by both, the Ru-complex & the PDEi, resulting
into enhanced antitumor activities with a possibility of minimum normal tissue toxicity due to
administration of such complexes.
Seminario basado en el artículo "Structural determination of group A Streptococcal surface
dehydrogenase and characterization of its interaction with
urokinase-type plasminogen activator receptor"
CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1a...Enrique Moreno Gonzalez
Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
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2 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx
cell death mechanisms in the tumor cells in vivo. In this paper, transferred to nitrocellulose membrane followed by detection of
we have investigated whether administration of a pharmaceutical different proteins against 1:1000 times diluted protein specific
dose of DMSO to DL bearing mice was able to induce pro-apoptotic polyclonal antibodies. Protein bands were detected by ECL kit. As
mechanisms and to modulate expressions of PFKFB3 and LDH-A in loading control, -actin was probed similarly using monoclonal
the DL cells without affecting normal lymphocytes. anti- -actin-peroxidase antibody (1:10,000). Protein bands were
quantified using gel densitometry software AlphaImager 2200.
2. Materials and methods
2.6. Analysis of LDH isozymes by non-denaturing polyacrylamide
2.1. Induction of Dalton’s lymphoma (DL) in mice gel electrophoresis (PAGE)
Inbred AKR strain mice of 16–18 weeks age weighing 24–26 g, Non-denaturing PAGE analysis of LDH employs substrate speci-
used for this experiment, were maintained at laboratory condi- ficity based detection of all LDH isozymes distinctly in the same gel,
tions and subjected to various treatments as per the guidelines and and it is considered highly relevant for interpreting LDH isozyme
approval from institutional animal ethical committee. based alterations at cellular level [14,18].
DL was induced by transplantation of 1 × 107 viable tumor cells LDH isozymes in various tissue extracts, in DL cell extracts and
(assayed by trypan blue method; 14) i.p. per mice. Development in cell free ascitic fluid were analyzed using 10% PAGE as described
of DL was confirmed by belly swelling and increased body weight earlier [18]. The extracts containing 60 g protein were loaded in
which became visible on 10–12th post transplantation day. The DL each lane and electrophoresed under non-denaturing conditions at
bearing mice survived up to 18 ± 2 days. 4 ◦ C. Gels were subjected to activity based detection and identifi-
cation of different LDH isozymes in the gel.
2.2. Experimental protocol
2.7. Semi-quantitative RT-PCR
DL bearing mice were randomly divided into two groups with
4–5 mice in each. The experimental group (DL + DMSO) mice were Total RNA was isolated from DL cells using TRI reagent fol-
treated with 200 l DMSO (∼7.5 g/kg b.w., i.p.) and those of DL con- lowing manufacturer’s protocol. After DNase I (DNA free-Ambion)
trol were similarly injected with equal volume of normal saline on digestion, reverse transcription of 2 g RNA was done using 200 U
post transplantation day 10. For biochemical and molecular studies, of reverse transcriptase and 200 ng random hexamer to make ss-
3–4 mice from each group were sacrificed on day 18th. cDNA (Revert Aid First strand cDNA synthesis kit, MBI fermentas).
The PCR reaction mixture contained 1× Taq polymerase buffer,
2.3. Collection and preparation of cell/tissue extracts 0.2 mM dNTPs, 1 U of Taq polymerase, and 10 pmol of specific
primer.
DL cells were collected by centrifuging tumor ascites pooled The mouse gene-specific primers used were: PFKFB3
from 3 to 4 DL mice from each group at 2000 × g at 4 ◦ C. Normal lym- (forward 5 -GGCAAGATTGGG GGCGACTC-3 ; reverse 5 -
phocytes were separated from heparin containing blood, collected GGCTCCAGGCGTTGGACAAG-3 ); LDH A (forward 5 -ATG CACC-
from severed neck of mice, using density gradient centrifugation at CGCCTAAGGTTCTT-3 ; reverse 5 -TGCCTACGAGGTGATCAAGCT-
400 × g for 30 min with histopaque-1077 reagent (Sigma diagnos- 3 ); Bcl-2 (forward 5 -TACCGTCGTGACTTCGCAGAG-3 ; reverse
tics protocol). 5 -GGCAGGCTGAGCAGGGT TT-3 ); Bax (forward 5 -CGGCGAAT-
The DL cell and normal lymphocyte extracts were prepared TGGAGATGAACTG-3 ; reverse 5 -GCAAAGTAGAAG AGGGCAACC-
using lysis buffer (20 mM Tris–Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 3 ); TNF˛ (forward 5 -ATGAGCACAGAAAGCATGATCC-3 ; reverse
1 mM EGTA, 1% triton X-100, 25 mM Na2 pyrophosphate and 1 mM 5 -GAAGATGATCTGAGTGTG-3 ) and ˇ Actin (forward 5 -ATCG-
PMSF). The cell lysates were centrifuged at 10,000 × g for 30 min TGGGCCGCTCTAGGCAC C-3 ; reverse 5 -CTCTTTGATGTCACGAT-
and supernatants obtained were used for biochemical and molec- TTC-3 ). PCR were run as: for -actin, 26 cycles; for LDH and Bcl-2,
ular studies. Liver and spleen extracts were prepared in a protease 31 cycles of 45 s at 94 ◦ C, 45 s at 55 ◦ C, and 1 min at 72 ◦ C. For Bax,
inhibitor containing extraction medium as described previously 31 cycles of 60 s at 56 ◦ C, and 1 min at 72 ◦ C; for PFKFB3, 30 cycles
[14,18]. of 60 s at 95 ◦ C, 60 s at 60 ◦ C, and 1 min at 72 ◦ C. Amplification
products were analyzed by 1–2% agarose gel electrophoresis and
2.4. DNA ladder study visualized by ethidium bromide staining. -Actin amplification
served as a control.
Total DNA from DL cells was isolated as described previously
[14]. Briefly, 5 × 106 DL cells were lysed in 1 ml lysis buffer for 2.8. Statistical analysis
1 h, added with 0.4 ml 5 M NaCl and after 5 min, centrifuged at
3000 × g for 30 min. The supernatants were treated with RNase Experimental data were expressed as mean ± SD and Student’s
(20 g/ml) for 15 min. DNA was precipitated by adding 2× (v/v) t-test was applied for determining the level of significance between
chilled ethanol. DNA collected after centrifugation was dissolved DL control vs DMSO treated DL groups.
in TAE buffer (40 mM Tris–acetate + 1 mM EDTA).
For agarose gel electrophoresis, DNA samples were prepared in 3. Results
a loading solution (0.25% bromophenol blue, 0.25% xylene cyanol FF
and 30% glycerol) in the ratio of 1:5 and samples containing 10 g 3.1. DMSO induced apoptosis in the DL cells in vivo
DNA were electrophoresed on 1% agarose gel containing 0.5 g/ml
ethidium bromide in TAE buffer for 2–3 h. The DNA bands in gel In general, TNF mediated apoptosis implicates p53 induced
were observed under UV transilluminator. mitochondrial mechanism. We have compared expression levels of
the key partners of this pathway in the DL cells from DMSO treated
2.5. Western blotting DL mice with that from the untreated DL group. Based on RT-PCR
analysis, level of TNF mRNA was observed to be ∼2-times higher
DL cell extracts containing 60 g protein, were subjected with a significant increment in the level of p53 protein (p < 0.05)
to 10% SDS-PAGE. As described previously [14], proteins were in the DL cells from DMSO treated DL mice than that from the
Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto-
sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
doi:10.1016/j.leukres.2010.12.029
3. ARTICLE IN PRESS
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R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx 3
Fig. 1. DMSO caused increased expressions of TNF (A) and p53 (B) in the DL cells in vivo. (A) A representative RT-PCR photograph with the normalized values of TNF / actin
mRNA levels as mean ± SD from three RT-PCR repeats. (B) A representative western blot photograph with the normalized values of p53/ actin protein levels as mean ± SD
from three western blot repeats. * p < 0.05; *** p < 0.001.
untreated group (Fig. 1A and B). Furthermore, levels of Bcl-2 mRNA 9 and PARP-1 with proportionate decline of their respective pro-
and its protein were found to be declined markedly with the con- caspases were observed in the DL cells from DMSO treated DL mice
comitant increments in Bax expression resulting into a significant than that from the untreated group (Fig. 3A). Additionally, as com-
decline in Bcl-2/Bax ratio (Fig. 2A and B) in the DL cells from DMSO pared to a single intact genomic DNA band seen in case of DL cells
treated DL mice than that of the untreated counterpart. from untreated DL group, DNA from DL cells of DMSO treated group
Caspase 9 activation is associated with induction of mitochon- showed many DNA fragments in the range of ∼800–200 bp (Fig. 3B).
drial pathway of apoptosis and that of PARP-1 cleavage with DNA
fragmentation in the apoptotic cells. As prescribed in supplier’s 3.2. Down regulation of PFKFB3 and LDH-A by DMSO in the DL
manual, anti-caspase 9 and PARP-1 antibodies could detect pro- cells
caspases as well as their cleaved products; 46- and 35 kDa and
116 and 24 kDa for caspase 9 and PARP-1 respectively (Fig. 3A). Over expression of iPFK2 (PFKFB3) and LDH-A are associated
Accordingly, ∼2-times increases in the levels of cleaved caspase with tumor growth. As illustrated in Fig. 4A and B, the levels of
Fig. 2. DMSO caused declined expression of Bcl-2 with concomitantly increased expression of Bax in the DL cells in vivo. (A) Representative western blot photographs with
Bcl-2/Bax ratio as mean ± SD from three western blot repeats. (B) Representative RT-PCR photographs with the ratio of Bcl-2/Bax mRNA level as mean ± SD from three RT-PCR
repeats. ** p < 0.01.
Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto-
sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
doi:10.1016/j.leukres.2010.12.029
4. ARTICLE IN PRESS
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4 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx
Fig. 3. DMSO caused activations of caspase 9 and PARP 1 (A) and DNA fragmentation (B) in the DL cells in vivo. (A) Representative western blot photographs from three
western blot repeats for each protein; caspase 9 and PARP 1 and that of -actin as loading control. (B) Represents a representative (out of three repeats) ethidium bromide
stained DNA gel photograph.
PFKFB3 mRNA and its protein product were declined significantly apoptotic proteins, is the most plausible mechanism [20–23]. We
(p < 0.01 and 0.05 respectively) in the DL cells from DMSO treated observed ∼2-times increment in the level of TNF mRNA with sig-
DL mice than that from the untreated counterpart. nificantly increased level of p53 protein in the DL cells from DMSO
For native PAGE analysis based identification of LDH isozymes, treated DL mice (Fig. 1). The TNF -p53 related apoptotic mecha-
the LDH band obtained from DL extracts was compared with a nism is suggested to involve mitochondrial pathway of apoptosis
standard pattern showing all the five LDH isozymes in the kidney [21,22] wherein, p53 induced alterations in Bcl-2/Bax ratio acts as a
extracts of normal mice (Fig. 4C(a)). Accordingly, an over activated determining factor [23]. Thus, significantly declined levels of Bcl-2
LDH-5 band was observed in the DL cell extracts of the untreated DL mRNA and its protein with concomitant increments in Bax mRNA
mice (Fig. 4C(b)), however, with a significant decline (p < 0.001) in and its protein (Fig. 2), in effect, represented a significantly reduced
the DL cell extracts from DMSO treated DL group. The RT-PCR result Bcl-2/Bax ratio in the DL cells from DMSO treated group. Keeping
(Fig. 4D) of the corresponding gene (LDH-A) further suggested a aside some fragmentary reports from in vitro studies on involve-
similar decline in LDH-A mRNA level (p < 0.05) in the DL cells from ment of Bcl-2/Bax in DMSO induced apoptosis [5], the present
DMSO treated group. finding is the first report to demonstrate up regulations of TNF and
p53 with concomitant decline in Bcl-2/Bax ratio and thereby, sug-
3.3. Regression of DL by DMSO and effect on normal tissues gesting induction of mitochondrial pathway of apoptosis by DMSO
in a non-Hodgkin’s lymphoma in vivo. In case of myeloid leukemia
Decline in ascitic volume and release of DL specific LDH-5 in U937 cells also, DMSO has been reported to induce death recep-
cell free ascitic fluid are considered good parameters to ascertain tor mediated apoptosis via depolarizing mitochondrial membrane
DL regression in vivo. DMSO caused significant decline in ascitic [19].
volume (p < 0.05) with concomitant release of LDH-5 in the cell free Caspase 9 activation is a hall mark of mitochondrial pathway of
ascitic fluid (Fig. 5A and B). This was consistent with a declining apoptosis [23] and that of PARP-1 cleavage is associated with DNA
trend in the body weight of DMSO treated DL mice also. Further, fragmentation in the cells undergoing apoptosis. We observed ∼2-
to ascertain DL cell specific effect of DMSO, the level of LDH-5, as times increments in the levels of cleaved caspase 9 and PARP-1 in
a susceptible parameter, was compared in normal lymphocytes, the DL cells from DMSO treated DL mice (Fig. 3A). Thus, together
spleen and liver from normal, DL bearing and DMSO treated DL with the production of DMSO dependent apoptotic pattern of DNA
mice. Fig. 5C–E suggests that DMSO did not alter the level of LDH-5 ladder in the DL cells (Fig. 3B), these results suggested induction of
in these tissues. mitochondrial pathway of apoptosis, consistent with up regulation
of TNF and p53, in the DL cells due to treatment with DMSO in vivo.
4. Discussion There could be many biochemical aberrations accountable for
apoptosis in the tumor cells. As tumor cells in vivo depend more
DMSO, as a vehicle for hydrophobic compounds, is considered on anaerobic energy production, inhibiting key glycolytic steps
non-toxic to the animals. In one such evaluation, a single dose in tumor cells is of current focus as a novel anticancer strategy
of 200 l (∼7.5 g/kg b.w.) DMSO was also found to be non-toxic [11,13,14,17]. PFK1 catalyzes committed step of glycolysis. FBP2 ,
to the normal mice (data of pilot experiments). However, when a metabolic activator of PFK1, is synthesized by PFK2. Tumor cells
administered to the DL bearing mice, it significantly declined ascitic express a C-type PFK1 with increased sensitivity for FBP2 activation
volume and caused LDH-5 release in the cell free ascitic fluid (Fig. 5A [15] and concordantly, a catalytically more efficient iPFK2: PFKFB3
and B). As reported earlier [14], these findings suggested DL cell gene [11,16]. We observed DMSO mediated decline in the levels of
death/regression in vivo by DMSO. Additionally, apoptotic pattern PFKFB3 mRNA and its protein (iPFK2) in the DL cells (Fig. 4A and
of DNA ladder observed in the DL cells from DMSO treated DL mice B) suggesting down regulation of iPFK2 by DMSO in these cells. As
(Fig. 3B) led us to investigate mechanistic aspects of DMSO induced decline of iPFK2 has been correlated with the regression of certain
apoptosis in the DL cells. tumors in vitro [11], it is argued that DMSO dependent down reg-
Although limited, but some in vitro studies suggest that DMSO ulation of this enzyme could be associated with DL cell apoptosis
primarily potentiates death receptor mediated apoptosis by involv- in vivo.
ing different mechanisms in different tumor cells [19,20]. However, Over activation of LDH-5 is associated with tumor growth in vivo
TNF mediated apoptosis, involving p53 and p53 related pro- because, it preferentially converts pyruvate into lactate to facilitate
Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto-
sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
doi:10.1016/j.leukres.2010.12.029
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Fig. 4. DMSO declined the levels of iPFK2 (A) and its mRNA (B) and those of active LDH-5 (C) and LDH-A mRNA (D) in the DL cells in vivo. (A) A representative western
blot photograph and values of iPFK2/ actin as mean ± SD from three western blot repeats. (B and D) Representative RT-PCR photographs for PFKFB3 and LDH-A with the
ratio of PFKFB3/ actin and LDH-A/ actin mRNA respectively as mean ± SD from 3 RT-PCR repeats. (C) Representative photographs from three PAGE repeats obtained for
tissue specific standard LDH isozymes in the kidney extract (C a) and for the LDH-5 bands in the DL extracts (C b) with relative densitometry values as mean ± SD. * p < 0.05;
**
p < 0.01, *** p < 0.001.
anaerobic energy production in the tumor cells [10,17,24]. Thus, gene silencing by siRNA has been correlated with the depletion of
repression of LDH-5 can affect tumor cell bioenergetics severely. cellular energy substrates and induction of apoptosis in the HeLa
Like most of the tumors, DL cells also over activate LDH-5, whose cells [11]. Blockage of tumor LDH-A has also been reported to ren-
inactivation by a novel anticancer compound has been correlated der tumor cells susceptible to death [24]. Recently, we have also
with apoptosis of DL in vivo [14]. An intense band of active LDH-5 demonstrated a correlation between decline of LDH-5 and induc-
in DL cell extracts from untreated DL mice (Fig. 4C(b)) corroborated tion of apoptosis by a ruthenium complex in the DL cells in vivo
these earlier findings and accordingly, significantly declined level of [14]. In the present context, therefore, it is argued that as a con-
active LDH-5 in DL cells from DMSO treated mice suggested DMSO sequence of DMSO mediated decreased expressions of PFKFB3 and
led decline in the activity of this enzyme in the DL cells in vivo. As LDH-A (Fig. 4), DL cells might be deprived of adequate energy pro-
RT-PCR band for the corresponding gene (LDH-A) was also observed duction and concomitantly forced to undergo apoptosis. Moreover,
to be declined similarly in the DL cells from DMSO treated group induction of apoptosis due to inhibition of tumor glycolysis is a rel-
(Fig. 4D), it is argued that decline in LDH-5 activity resulted due atively newer concept [12,14] and therefore, need to be defined in
to DMSO mediated decreased expression of LDH-A in the tumor many tumor models. In this respect, this is a first report to describe
cells. declined expressions of tumor growth supportive glycolytic factors
As compared to in vitro conditions, tumor cells in vivo face consistent with the induction of TNF -p53-mitochondrial path-
greater hypoxia and therefore, they rely much on anaerobic gly- way of apoptosis by DMSO in a tumor cell in vivo. Indeed, TNF
colysis for their energy requirements [17]. Depletion of energy induced apoptosis in another non-Hodgkin’s lymphoma has been
substrates is considered as strong apoptotic signal [25]. PFKFB3 found associated with changes in the LDH isozymes [26].
Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto-
sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
doi:10.1016/j.leukres.2010.12.029
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6 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx
Fig. 5. DMSO declined ascitic volume (A) and enhanced LDH-5 release in the cell free ascitic fluid (B) without any adverse effects on active level of LDH-5 in the normal
lymphocytes (C), spleen (D) and liver (E) of DL mice. (A) Data as mean ± SD where n = 4. (B) A representative photograph with relative densitometry of LDH-5 bands as
mean ± SD from three PAGE repeats. (C–E) Representative photographs from three PAGE repeats obtained for normal lymphocytes, spleen and liver respectively. *p < 0.05.
Tumor specificity is the main limitation of anticancer agents. Fellowship to R.K.K. for working on this topic. The facilities pro-
LDH is a highly sensitive enzyme to undergo change during tumor vided due to UGC-CAS and DST-FIST programmes to Department of
growth and tissue toxicity [14,17,27]. We observed unaltered pat- Zoology, BHU, are also acknowledged.
tern of LDH-5 in normal lymphocytes, in spleen (lymphocyte
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Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto-
sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
doi:10.1016/j.leukres.2010.12.029