Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide
This research paper examines how a novel ruthenium(II) complex containing 4-carboxy N-ethylbenzamide (Ru(II)-CNEB) activates the p53-mediated glycolytic inhibition-oxidative stress-apoptosis pathway in Dalton's lymphoma (DL) cells in vivo. The study finds that Ru(II)-CNEB decreases expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2/PFKFB3), a key regulator of glycolysis in DL cells. It also activates superoxide dismutase but decreases catalase and glutathione peroxidase, imposing oxidative stress in DL cells. This is consistent with enhanced p
Brian Covello: Diabetes Research ProposalBrian Covello
Brian Covello's diabetes research proposal. Type 2 diabetes mellitus consists of an array of dysfunctions characterized by hyperglycemia and resulting from the combination of resistance to insulin action, inadequate insulin secretion, and excessive or inappropriate glucagon secretion.
Essential update: FDA approves subcutaneous albiglutide for management of DM2
The FDA has approved once-weekly injectable albiglutide (Tanzeum), a glucagonlike peptide 1 (GLP-1) receptor agonist, along with diet and exercise for the treatment of type 2 diabetes.[1, 2] This agent may be used either as monotherapy or in combination with metformin, glimepiride, pioglitazone, or insulin.
Albiglutide should not be used for the following[1, 2] :
Patients with type 1 diabetes
Patients with diabetic ketoacidosis
First-line therapy in patients who can’t be managed with diet and exercise
Patients who have a personal or family history of medullary thyroid carcinoma (MTC)
Patients who have multiple endocrine neoplasia syndrome type 2
The most common adverse reactions associated with albiglutide were nausea/diarrhea and injection-site reactions.
There will be a boxed warning on albiglutide’s labeling about thyroid C-cell tumors being observed in rodent studies with this class of drugs; it is currently unknown whether albiglutide causes these tumors in humans, including MTC.[1, 2] Moreover, the FDA is also requiring a number of postmarketing studies, including a pediatric trial; an MTC case registry (≥15 y); and a cardiovascular (CV)-outcomes trial in patients with a baseline high risk of CV disease.
Signs and symptoms
Many patients with type 2 diabetes are asymptomatic. Clinical manifestations include the following:
Classic symptoms: Polyuria, polydipsia, polyphagia, and weight loss
Blurred vision
Lower-extremity paresthesias
Yeast infections (eg, balanitis in men)
See Presentation for more detail.
Diagnosis
Diagnostic criteria by the American Diabetes Association (ADA) include the following[3] :
A fasting plasma glucose (FPG) level of 126 mg/dL (7.0 mmol/L) or higher, or
A 2-hour plasma glucose level of 200 mg/dL (11.1 mmol/L) or higher during a 75-g oral glucose tolerance test (OGTT), or
A random plasma glucose of 200 mg/dL (11.1 mmol/L) or higher in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis
Whether a hemoglobin A1c (HbA1c) level of 6.5% or higher should be a primary diagnostic criterion or an optional criterion remains a point of controversy.
Indications for diabetes screening in asymptomatic adults includes the following[4, 5] :
Sustained blood pressure >135/80 mm Hg
Overweight and 1 or more other risk factors for diabetes (eg, first-degree relative with diabetes, BP >140/90 mm Hg, and HDL < 35 mg/dL and/or triglyceride level >250 mg/dL)
ADA recommends screening at age 45 years in the absence of the above criteria
See Workup for more detail.
Brian Covello: Diabetes Research ProposalBrian Covello
Brian Covello's diabetes research proposal. Type 2 diabetes mellitus consists of an array of dysfunctions characterized by hyperglycemia and resulting from the combination of resistance to insulin action, inadequate insulin secretion, and excessive or inappropriate glucagon secretion.
Essential update: FDA approves subcutaneous albiglutide for management of DM2
The FDA has approved once-weekly injectable albiglutide (Tanzeum), a glucagonlike peptide 1 (GLP-1) receptor agonist, along with diet and exercise for the treatment of type 2 diabetes.[1, 2] This agent may be used either as monotherapy or in combination with metformin, glimepiride, pioglitazone, or insulin.
Albiglutide should not be used for the following[1, 2] :
Patients with type 1 diabetes
Patients with diabetic ketoacidosis
First-line therapy in patients who can’t be managed with diet and exercise
Patients who have a personal or family history of medullary thyroid carcinoma (MTC)
Patients who have multiple endocrine neoplasia syndrome type 2
The most common adverse reactions associated with albiglutide were nausea/diarrhea and injection-site reactions.
There will be a boxed warning on albiglutide’s labeling about thyroid C-cell tumors being observed in rodent studies with this class of drugs; it is currently unknown whether albiglutide causes these tumors in humans, including MTC.[1, 2] Moreover, the FDA is also requiring a number of postmarketing studies, including a pediatric trial; an MTC case registry (≥15 y); and a cardiovascular (CV)-outcomes trial in patients with a baseline high risk of CV disease.
Signs and symptoms
Many patients with type 2 diabetes are asymptomatic. Clinical manifestations include the following:
Classic symptoms: Polyuria, polydipsia, polyphagia, and weight loss
Blurred vision
Lower-extremity paresthesias
Yeast infections (eg, balanitis in men)
See Presentation for more detail.
Diagnosis
Diagnostic criteria by the American Diabetes Association (ADA) include the following[3] :
A fasting plasma glucose (FPG) level of 126 mg/dL (7.0 mmol/L) or higher, or
A 2-hour plasma glucose level of 200 mg/dL (11.1 mmol/L) or higher during a 75-g oral glucose tolerance test (OGTT), or
A random plasma glucose of 200 mg/dL (11.1 mmol/L) or higher in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis
Whether a hemoglobin A1c (HbA1c) level of 6.5% or higher should be a primary diagnostic criterion or an optional criterion remains a point of controversy.
Indications for diabetes screening in asymptomatic adults includes the following[4, 5] :
Sustained blood pressure >135/80 mm Hg
Overweight and 1 or more other risk factors for diabetes (eg, first-degree relative with diabetes, BP >140/90 mm Hg, and HDL < 35 mg/dL and/or triglyceride level >250 mg/dL)
ADA recommends screening at age 45 years in the absence of the above criteria
See Workup for more detail.
Drug-Drug interactions (DDI) is a serious clinical issue. An important mechanism underlying of DDI, is
induction or inhibition of drug metabolizing enzymes (DMEs) and transporters that mediate metabolism, cellular uptake and efflux of xenobiotics. DDI cannot be avoided in many cases, as they belong to routine medical practice.
Phosphatidylinositol 4-Kinase Enzymes: Functional Roles and Potential for Dru...IOSRJPBS
The two types of phosphatidylinositol 4-kinases (PI-4Ks) synthesize phosphatidylinositol 4-phosphate (PI-4P), a member of the phosphoinositide family. Phosphoinositides (PIPs) are synthesized from phosphatidylinositol (PI), a lipid containing the myo-inositol head group. PI can be phosphorylated at positions 3, 4, and 5 of the inositol ring which allows for seven different PIPs. Indeed, all of these enzymes have been identified in the cell. For instance, one prominent function of PIPs is to serve as membrane markers typically in concert with organelle specific proteins. PI(4,5)P2 is the main lipid determinant of the plasma membrane and PI3P and PI(3,5)P2 of the early and late endosomes. PI-4P is the main lipid determinant of the Golgi and trans-Golgi network (TGN) but, additionally, helps to define the specific character of the plasma membrane. This article reviews the recent developments in research on these enzymes and their potential for drug target.
Presentation given by Dr. Wayne Danter at the 16th International p53 Workshop in Stockholm Sweden on June 16, 2014. This presentation provides an overview of Critical Outcome Technologies' lead cancer drug candidate, COTI-2, and outlines its p53-dependent mechanism of action.
Drug-Drug interactions (DDI) is a serious clinical issue. An important mechanism underlying of DDI, is
induction or inhibition of drug metabolizing enzymes (DMEs) and transporters that mediate metabolism, cellular uptake and efflux of xenobiotics. DDI cannot be avoided in many cases, as they belong to routine medical practice.
Phosphatidylinositol 4-Kinase Enzymes: Functional Roles and Potential for Dru...IOSRJPBS
The two types of phosphatidylinositol 4-kinases (PI-4Ks) synthesize phosphatidylinositol 4-phosphate (PI-4P), a member of the phosphoinositide family. Phosphoinositides (PIPs) are synthesized from phosphatidylinositol (PI), a lipid containing the myo-inositol head group. PI can be phosphorylated at positions 3, 4, and 5 of the inositol ring which allows for seven different PIPs. Indeed, all of these enzymes have been identified in the cell. For instance, one prominent function of PIPs is to serve as membrane markers typically in concert with organelle specific proteins. PI(4,5)P2 is the main lipid determinant of the plasma membrane and PI3P and PI(3,5)P2 of the early and late endosomes. PI-4P is the main lipid determinant of the Golgi and trans-Golgi network (TGN) but, additionally, helps to define the specific character of the plasma membrane. This article reviews the recent developments in research on these enzymes and their potential for drug target.
Presentation given by Dr. Wayne Danter at the 16th International p53 Workshop in Stockholm Sweden on June 16, 2014. This presentation provides an overview of Critical Outcome Technologies' lead cancer drug candidate, COTI-2, and outlines its p53-dependent mechanism of action.
P53 Tumor Suppressor Gene: Understanding P53 Based Dietary Anti Cancer Thera...Sheldon Stein
The P53 tumor suppressor gene which has been dubbed both the “Guardian of the Genome” (Lane 1992) and Science “Molecule of the Year”, is directly involved in the initiation of apoptosis and programmed cell death, to prevent an accumulation of abnormal cells. However apoptosis evasion is a characteristic feature of human cancers that promote tumor formation and progression (1). Presently, P53 is known to play a key role in practically all types of human cancers, and the mutation or loss of P53 gene function, can be identified in more than 50% of all human cancer cases worldwide.
This paper was uploaded on behalf of Professor Serge Jurasunas of Lisbon Portugal, www.sergejurasunas.com
The paper goes on to explain the role of the P53 gene and its relationship to Cancer and Apoptosis. It then elaborates on the importance of dietary agents can have a beneficial impact in cancer treatment, and provides a number of case studies. He addresses the importance of the P53 gene and DNA repair, as well as his use of Molecular Markers testing.
Professor Jurasunas believes:
We urgently need to put into clinical practice what we have discovered and learned. Targeting P53 and other genes remain one of the greatest challenges in the treatment of cancer. We have been working now for over 8 years with molecular markers as a diagnostic, prognosis, and follow up to treatment, selected the appropriate bioactive dietary compounds or anticancer agents, exceeding 1000 cases, blood tests, and successes. This may be an incentive for more doctors to venture into this new direction in order to achieve more beneficial results with their patient treatment, especially in cases where we can verify the ones who would be refractory to chemotherapy and have a poor response. It is always best to first check through patient testing, to determine whether or not chemotherapy would be beneficial.
GPCRs are the most dynamic and most abundant all the receptors. The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals. GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction. GPCRs interact with a number of ligands ranging from photons, ions, amino acids, odorants, pheromones, eicosanoids, neurotransmitters, peptides, proteins, and hormones.
Nevertheless, for the majority of GPCRs, the identity of their natural ligands is still unknown, hence remain orphan receptors.
The simple dogma that underpins much of our current understanding of GPCRs, namely,
one GPCR gene− one GPCR protein− one functional GPCR− one G protein −one response
is showing distinct signs of wear.
Similar to Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...JohnJulie1
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...AnonIshanvi
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids...
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...daranisaha
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids...
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules,
and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory
tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to
traffic small hydrophobic molecules such as lipids and retinoids. Due to its ability to sequester
iron-containing bacterial siderophores, LCN2 plays an essential part of the innate immunity as
well as the regulation of the cellular iron metabolism
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...EditorSara
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids...
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...ClinicsofOncology
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...NainaAnon
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids...
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...semualkaira
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids...
Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...semualkaira
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids..
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 Induced PI3K/Akt/mTor Pathway in the Regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive
cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC
directly related to the disease agressivity. Télomérase, is expressed
by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by
IL-6 is activated in the HCC. Our aim is to investigate the effect
of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/
PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Effectiveness of Resveratrol on Metastasis: A Reviewiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Similar to Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide (20)
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.
An overview of the toxic effect of potential human carcinogen Microcystin-LR ...rkkoiri
The worldwide occurrence of cyanobacterial blooms due to water eutrophication evokes
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So far among these toxins, Microcystin-LR (MC-LR) is the most toxic and the most
frequently encountered toxin produced by the cyanobacteria in the contaminated aquatic
environment. Microcystin-LR is a potential carcinogen for animals and humans, and the
International Agency for Research on Cancer has classified Microcystin-LR as a possible
human carcinogen. After liver, testis has been considered as one of the most important target
organs of Microcystin-LR toxicity. Microcystin-LR crosses the blood–testis barrier and
interferes with DNA damage repair pathway and also increases expression of the protooncogenes,
genes involved in the response to DNA damage, cell cycle arrest, and apoptosis
in testis. Toxicity of MC-LR disrupts the motility and morphology of sperm and also affects
the hormone levels of male reproductive system. MC-LR treated mice exhibit oxidative
stress in testis through the alteration of antioxidant enzyme activity and also affect the
histopathology of male reproductive system. In the present review, an attempt has been
made to comprehensively address the impact of MC-LR toxicity on testis.
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
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MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
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Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
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Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
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Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide
2. apoptotic pattern of DNA cleavage which was consistent with the
decreased DL cell viability and increased life span of the tumor
bearing mice [11]. Importantly, since, this complex increased
certain apoptotic markers and also inhibited M4-LDH (LDH-5) in
those DL cells, it was argued that this complex, instead of targeting
DNA directly, is able to activate glycolytic inhibition-apoptosis
pathway in a tumor cell in vivo [11]. This necessitated further
studies on characterizing regulatory factors of this pathway as
target of Ru (II)-CNEB in the DL cells in vivo.
Switching over to aerobic glycolysis, known as ‘Warburg effect’,
is considered to be a common trait of most of the growing tumors
[12] and therefore, inhibition of enhanced tumor glycolysis is now
advocated as one of the therapeutic strategies in cancer therapy
[13,14]. In this regard, targeting regulatory enzymes of glycolytic
pathway assumes special importance for the novel anticancer
agents. Phosphofructokinase1 (PFK1) catalyzes committed step of
glycolysis. PFK2 domain of D-fructose-6-phosphate-2-kinase/fruc-
tose-2,6-bisphosphatase (PFK2/FBPase2) synthesizes fructose-2,6-
bisphosphate (FBP) to activate PFK1 and thereby, it acts as main
glycolytic regulator under a variety of pathological challenges
including tumor progression [15]. Cancer cells express C type PFK1
which is more sensitive to FBP [16] and also over express a cata-
lytically more efficient inducible form of PFK2 (iPFK2: PFKFB3 gene)
[17]. Since, iPFK2 repression has been reported to inhibit tumor cell
growth in vitro [13], this glycolytic activator could be considered as
relevant therpautic target for the novel anticancer agents. Similarly,
to sustain enhanced glycolysis, tumor cells adapt to produce lactate
from pyruvate by activating LDH-5. Reports also suggest that over
expression of LDH-5 gene (LDH-A) is associated with tumor growth
[12,18] and thus, advocating repression of LDH-5 gene as another
mechanism to define therapeutic target for a novel anticancer
agent.
So far up stream regulation of the cell bioenergetics is con-
cerned, p53, a tumor suppressor protein, has been found to
modulate overall balance between glycolysis and mito-OXPHOS
[19]. The loss of normal p53 has been reported to be critically
associated with tumor progression, particularly in leukemia and
lymphoma [20]. Similarly, p53 over expression has been demon-
strated to have prognostic significance in lymphoma [21]. Recently,
p53 has been reported to regulate tumor cell energy metabolism
[22] via down regulating expression of glucose transporters GLUT1
and GLUT4 [23] and also by activating the TIGAR gene which re-
presses iPFK2/PFKFB3 [24]. Thus, when tumor cells switch over to
glycolytic phenotype, it is accompanied with declined p53
expression [25]. Similarly, inhibition of glycolysis has been shown
to activate p53 [26].
It has been suggested that ROS are down stream mediators of
p53 dependent apoptosis [27], wherein, Bcl-2/Bax ratio acts as
main determinant of this pathway [28]. Low level of p53 induces
expression of antioxidant enzymes responsible to maintain ROS
level within a permissive range which otherwise can cause DNA
damage and genomic instability in the cells [29]. Higher level of p53
protein, on the other hand, is known to enhance the expression of
pro-oxidant and proapoptotic factors [30].
Tumor cells, in addition to acquiring glycolytic phenotype, also
modulate their oxygen metabolism in many ways [12,31]. Super-
oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and
glutathione reductase (GR) constitute main antioxidant defense
mechanism in most of the cells. The oxygen free redicals (O2
.-
),
produced during mitochondrial oxidative phosphorylation, are
dismutated to hydrogen peroxide (H2O2) by SOD followed by con-
version of H2O2 into water by catalase and GPx [32]. As SOD is the
committed enzyme of this pathway responsible to produce H2O2, it
appears to be the most relevant target for therapeutic intervention
in the tumor cells. In the recent past, both SOD isoforms (CueZn-
SOD: cytosolic; SOD1 and Mn-SOD: mitochondrial; SOD2) have
been found to be implicated in tumerogenesis [33].
Some earlier reports have described that reduced levels of SOD1
SOD2 facilitate tumerogenesis [34,35] via maintaining low level
of H2O2 in the cancerous cells and accordingly, increased SOD ac-
tivity and in turn, higher level of H2O2 may contribute for tumor
suppression [36,37]. However, effectiveness of this SOD mediated
mechanism depends on the status of the two down stream en-
zymes; catalase and GPx in the cancerous cells. Indeed, SOD and
catalase double transfectant (SOCAT3) cells and cells with over
activated GPx1 have shown protection from oxidative cell damage
[38,39]. Thus, to characterize oxidative mechanism based anti-
cancer potential of a novel anticancer agent, it is important to study
comparative profile of the three antioxidant enzymes involved in
H2O2 metabolism in the cancer cells.
In the present article, we have investigated whether Ru(II)-CNEB
is able to activate p53 mediated glycolytic inhibition-oxidative
stress-apoptosis pathway in the DL cells in vivo.
2. Materials and methods
2.1. Chemicals
Ruthenium(II)-complex containing 4-carboxy N-ethyl-
benzamide as ligand, Ru(II)-CNEB; [Ru(CNEB-H)2(bpy)2] 2PF6$0.5
NH4PF6, whose structural details have already been described
previously [10,11], was used in the present study. Antibodies
against p53, caspase 9, Bcl-2, Bax, SOD1, SOD2 PFK 2 were
purchased from Santa Cruz and b-actin was purchased from
SigmaeAldrich Co., USA. HRP- conjugated anti rabbit/goat/mouse
IgG were obtained from Genei. 20,70-dichlorofluorescin diacetate
and ECL super signal western pico kit were purchased from Fluka
and Pierce respectively. Lactate estimation kit was purchased
from Biorex Diagnostics, Ltd, UK. Other general chemicals and
reagents were obtained from Merck or SRL unless otherwise
specified.
2.2. Induction of Dalton's lymphoma (DL) in mice
Inbred AKR strain mice of 16e18 weeks age weighing 24e26 g
were used for the experiments. Mice were maintained at standard
laboratory conditions with the supply of food and water ad libitum.
This work was approved by the institutional animal ethical com-
mittee (Dan/2006-07/962). Dalton's lymphoma was induced by
transplantation of 1 Â 107
viable tumor cells (assayed by trypan
blue method) i.p. per mice. Development of DL was confirmed by
abnormal belly swelling and increased body weight which became
visible on 10e12th post transplantation day. The DL bearing mice
survived up to 18 ± 2 days.
2.3. Treatment schedule
The DL mice were randomly divided into 2 groups with 10 mice
in each. The first group DL mice were treated with Ru(II)-CNEB
complex (10 mg/kg bw/day, ip), and the second group, designated
as DL control, were similarly injected with equal volume of Krebs
Ringer Buffer (KRB). As DL becomes visible on day 10e11 and DL
bearing mice survived up to 18e20 days post DL transplantation,
the treatments with the compounds were started from day 11 of
tumor transplantation and continued up to day 17th. The normal
control group mice were also treated simultaneously with KRB. To
study biochemical/molecular parameters, 3-4 mice from each
group were sacrificed on day 18th and tumor ascites pooled from 3
to 4 DL mice from each group were centrifuged at 2000 Â g at 4 C
to collect DL cells. The DL cell extract was prepared using lysis
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 53
3. buffer (20 mM Tris-Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 25 mM Na2 pyrophosphate and 1 mM PMSF). The
cell lysates were centrifuged at 20,000 Â g for 30 min and super-
natant obtained were used for biochemical and molecular studies.
Protein concentrations in the extracts were measured following the
method of Lowry et al. [40].
2.4. Western blotting
For western blot analysis of different proteins, DL cell extracts
containing 60 mg proteins, were subjected to 10% SDS-PAGE. As
described previously [11], proteins were transferred to nitrocellu-
lose membrane followed by detection of p53, caspase 9, Bcl-2, Bax,
iPFK2, SOD1 and SOD2 against specific polyclonal antibodies
(1:1000). Proteins on membrane were detected by ECL west pico
kit. As loading control, b-actin was probed similarly using mono-
clonal anti-b-actin-peroxidase antibody (1:10,000). Protein bands
were quantified using gel densitometry software AlphaImager
2200.
2.5. Assay of the antioxidant enzymes
Catalase activity was measured following an earlier method [41]
with some modifications. Briefly, 1 ml reaction mixture consisted of
0.05 M phosphate buffer (pH 7.0) and 0.003% H2O2. The reaction
was started by the addition of suitably diluted cell extract and
decrease in absorbance at 240 nm was recorded for 10 min. Unit of
the enzyme was defined as mmol of H2O2 depleted/min. The activity
was expressed as units/mg protein.
Glutathione peroxidase (GPx) activity was determined as
described earlier [42] with slight modifications. Briefly, cell ex-
tracts were added to the assay buffer consisting of 100 mM Tris-
Cl (pH 7.2), 3 mM EDTA, 1 mM sodium azide, 0.25 mM H2O2,
0.5 mM NADPH, 0.17 mM GSH, and 1 unit GR. The change in
absorbance per minute at 340 nm was recorded for 10 min and
enzyme activity was expressed as mmole NADPH oxidized/min/
mg protein.
2.6. Analysis of SOD and Gpx by non-denaturing PAGE
The active levels of superoxide dismutase (SOD) and glutathione
peroxidase (GPx) was determined using non-denaturing PAGE of
the cell extracts following the method recently reported from our
lab [42,43]. The cell extract containing 60 mg protein were loaded in
each lane of 10% non-denaturing PAGE. After electrophoresis at
4 ± 2 C, the gels were subjected to substrate specific staining of
different antioxidant enzymes.
The staining mixture for SOD consisted of 2.5 mM NBT, 28 mM
riboflavin, and 28 mM TEMED. Gels were incubated for 20 min in
the dark, followed by exposing them to a fluorescent light till
achromatic SOD bands developed against dark blue background.
GPx specific staining mixture was composed of 50 mM TriseCl
buffer (pH 7.9), 3 mM GSH, 0.004% H2O2, 1.2 mM NBT and 1.6 mM
PMS. Achromatic bands corresponding to GPx activity appeared
against a violeteblue background.
The intensities of all the bands were quantified by gel densi-
tometry using AlphaImager 2200 gel documentation software.
Specificity of the PAGE bands of different enzymes were confirmed
by obtaining clear negative results when similarly run gels were
treated in the absence of the enzyme specific substrates. In each
case, PAGE was performed at least 3 times and mean ± SD of
densitometry values of the bands, as percentage of the control lane,
have been presented in the result with one representative gel
photograph.
2.7. Biochemical estimations
2.7.1. Lactate
The concentration of lactate was determined by measuring
lactate oxidation by lactate oxidase as per the manufacturer's in-
structions given in the lactate assay kit obtained from Biorex Di-
agnostics Ltd, UK.
2.7.2. H2O2
Following the method described earlier [42]; intracellular H2O2
was determined by measuring H2O2 dependent oxidation of DCFH-
DA into DCF. Briefly, 100 ml of cell extracts were incubated with
10 mM DCFH-DA at 37 C for 30 min in dark. Using excitation at
504 nm and emission at 529 nm, intensity of DCF fluorescence was
measured. H2O2 concentration in the extract was determined by
using a calibration plot made against different H2O2 concentrations
(5e100 mM).
2.7.3. Total glutathione (GSH þ GSSG)
Following the method described earlier [42], DL cell extracts
were precipitated with 5% sulfosalicylic acid in the ratio of 1:2 and
centrifuged. The supernatant collected was neutralized. In a 96 well
micro plate, 50 ml neutralized supernatant was incubated with
100 ml of the reagent containing 0.30 mM NADPH, 0.22 mM DTNB
and 1.6 units/ml GR prepared in 100 mM phosphate buffer (pH 7.4)
containing 1 mM EDTA. The absorbance was recorded at 412 nm for
10 min using Micro Scan MS5608A (ECIL) micro plate reader. Total
glutathione content was expressed in terms of nmol/mg protein.
2.8. Semi-quantitative RT-PCR
Total RNA was isolated from DL cells using TRI reagent
following the manufacturer's protocol. DNA-free™ (Ambion) was
used to remove any contaminating DNA from the RNA preparation
following the manufacturer's protocol. Briefly, reaction mixture
consisting of DNase I, 10Â DNase I buffer and RNA sample was
incubated at 37 C for 20 min. Thereafter, DNase I inactivation
reagent slurry was added and incubated for 2 more min at room
temperature and centrifuged. The upper phase was collected as
DNA free RNA solution. From this RNA isolate, 2 mg RNA was
subjected for reverse transcription using 200 U of reverse tran-
scriptase and 200 ng random hexamer to make ss-cDNA (Revert
Aid First strand cDNA synthesis kit, MBI fermentas). The PCR re-
action mixture contained 1Â Taq polymerase buffer, 0.2 mM each
of the four dNTPs, 1.0 U of Taq polymerase, and 10 pmol of the
specific primer. The mouse gene-specific primers used were: Bcl-2
(forward 50-TAC CGT CGT GAC TTC GCA GAG-30; reverse 50-GGC
AGG CTG AGC AGG GTC TT-30); Bax (forward 50-CGG CGA ATT GGA
GAT GAA CTG-30; reverse 50-GCA AAG TAG AAG AGG GCA ACC-30);
PFKFB3 (forward 50-GGC AAG ATT GGG GGC GAC TC-30; reverse 50-
GGC TCC AGG CGT TGG ACA AG-30); LDH A (forward 50-ATG CAC
CCG CCT AAG GTT CTT-30; reverse 50-TGC CTA CGA GGT GAT CAA
GCT-30); SOD2 (forward 50-GCA CAT TAA CGC GCA GAT CA-30;
reverse 50-AGC CTC CAG CAA CTC TCC TT-30) and b actin (forward
50-ATC GTG GGC CGC TCT AGG CAC C-30; reverse 50-CTC TTT GAT
GTC ACG ATT TC-30). Linearity of PCR amplifications was checked
for each gene using various cycles (20, 24, 28, 32, 36 and 40 cycles)
vs densitometric value of the PCR product of the corresponding
gene. Accordingly, the optimal number of cycles from the linear
phase and other conditions were chosen for amplification for each
gene as; b actin, 30 cycles of 30 s at 94 C, 30 s at 52 C, and 30 s at
72 C; Bcl-2 LDH A, 34 cycles of 45 s at 94 C, 45 s at 55 C, and
1 min at 72 C; Bax, 30 cycles of 30 s at 94 C; 60 s at 56 C, and
1 min at 72 C; PFKFB3, 31 cycles of 60 s at 95 C, 60 s at 50 C, and
1 min at 72 C; SOD2, 27 cycles. The amplification products,
R.K. Koiri et al. / Biochimie 110 (2015) 52e6154
4. analyzed by 1e2% agarose gel electrophoresis, were visualized by
ethidium bromide staining and were found to be; Bcl 2 (350 bp),
Bax (160 bp), PFKFB3 (329 bp), LDH A (103 bp), SOD2 (241 bp) and
b actin (543 bp). Amplification of b actin served as a control. Three
RT-PCR repeats from 3 RNA isolates for each gene were performed.
The expression levels were measured by densitometry using
AlphaImager 2200.
2.9. Statistical analysis
Experimental data were expressed as mean ± SD and Student's t
test was applied for determining the level of significance between
control and experimental groups and value of p 0.05 was
considered significant.
3. Results
3.1. Ru(II)-CNEB mediated decline of lactate level in the DL cells
in vivo
Lactate serves as an alternate metabolic fuel for the high energy
demanding tumor cells. Therefore, overproduction of lactate is
considered one of the hallmarks of the tumor growth. According to
Fig. 1b, as compared to the DL cells from untreated group, there is a
significant decline (p 0.001) in lactate level in case of the DL cells
from Ru(II)-CNEB treated DL mice. To ascertain, whether decline in
lactate level is due to decline in the expression of M4-LDH, the level
of LDH-A mRNA was measured in the DL cells from the untreated
the treated group DL mice. According to Fig. 1a, the level of LDH-A
Fig. 1. Effect of Ru(II)-CNEB (Rc) on the expression of LDH-A and iPFK2 lactate production in the DL cells in vivo. (a) shows representative RT-PCR photograph for LDH-A with the
ratio of LDH-A/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b) shows lactate level represent as mean ± SD where n ¼ 4. (c) shows representative RT-PCR
photograph for PFKFB3 with the ratio of PFKFB3/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. In (d), a representative western blot photograph, with 60 mg
protein in each lane, presented with the relative densitometric values of iPFK2/b actin as mean ± SD from three western blot repeats. *p 0.05; ***p 0.001 (untreated vs. treated
DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 55
5. mRNA in the DL cells from Ru(II)-CNEB treated DL mice is found to
be unchanged as compared to the untreated counterpart.
3.2. Downregulation of iPFK2/PFKFB3 in the DL cells due to the
treatment with Ru(II)-CNEB
Over expression of iPFK2/PFKFB3 is associated with tumor
growth. Recently, p53 has been reported to regulate tumor cell
energy metabolism indirectly by activation of the TIGAR gene
which declines iPFK2/PFKFB3 induced fructose-2,6-bisphosphate
production in the tumor cells. As illustrated in Fig. 1c and d, the
levels of PFKFB3 mRNA and its protein product declined signifi-
cantly (p 0.05e0.001) in the DL cells from Ru(II)-CNEB treated DL
mice than that from the untreated counterpart.
3.3. Ru(II)-CNEB increases p53 level in the DL cells in vivo
Restoration of p53 level is an important strategy of anticancer
chemotherapy. Based on immunoblot analysis, level of p53 was
observed to be ~5 times higher in the DL cells (p 0.001) from the
Ru(II)-CNEB treated DL mice than that from the control group mice
(Fig. 2).
3.4. Enhanced expressions of SOD1 SOD2 in the DL cells from
Ru(II)-CNEB treated DL mice
Oxidative stress and mitochondrial dysfunction are known to
initiate final steps of apoptosis. Infact ROS has been suggested to act
as a down stream mediator of p53 dependent apoptosis. SOD is the
first commited enzyme that neutralizes superoxide anion free
radical (O2
À
) based oxidative stress in the cells. According to Fig. 3a,
the level of active SOD2 SOD1, both shows significant increments
in the DL cells from Ru(II)-CNEB treated DL mice. To confirm
whether activity increment is contributed due to the similar in-
crease in the expression of these antioxidant enzymes, RT-PCR and
western blot analysis was performed. Fig. 3bed illustrate that
Ru(II)-CNEB is able to increase the expression of both, SOD1 and
SOD2 significantly (p 0.05e0.001) in the DL cells in vivo.
3.5. Ru(II)-CNEB mediated in vivo modulation of pro-oxiative
factors in the DL cells
The two antioxidant enzymes, catalase GPx, down stream to
SOD, show significant decline in their activity (p 0.05) in the DL
cells from Ru(II)-CNEB treated DL mice (Fig. 4aec). However,
treatment with Ru(II)-CNEB caused a significant decrease in the
activity of GPx1, with a concomitant increase in the activity of GPx2
(Fig. 4c). Such a reciprocal change between SODs and catalase
GPx is likely to allow accumulation of H2O2 at cellular level. Fig. 5a
shows that indeed there is a marked increase (~6 times; p 0.001)
in H2O2 level in the DL cells from Ru(II)-CNEB treated DL mice than
those from the untreated groups. Level of glutathione is another
marker of oxidative stress in the cells. According to Fig. 5b, as
compared to the untreated DL group, the DL cells from Ru(II)-CNEB
treated DL mice show a significant decline (p 0.05) in total
glutathione (GSH þ GSSG) level.
3.6. Ru(II)-CNEB mediated in vivo modulation of pro-apoptotic
factors in the DL cells
Bcl2/Bax ratio serves as one of the critical determinants of
apoptotic induction at cellular level. As compared to the untreated
DL mice, treatment with Ru(II)-CNEB caused a significant decline
(p 0.001) in the level of Bcl2 protein in the DL cells with a
concomitant increase in the Bax protein (Fig 6b). Such reciprocal
changes in Bcl2 vs Bax resulted into a significantly declined Bcl2/
Bax ratio (~3.5 times; p 0.01) in the DL cells from Ru(II)-CNEB
treated DL mice (Fig. 6b). The RT-PCR results (Fig 6a) also demon-
strate a significant decline in Bcl2/Bax mRNA level, however, mainly
due to a significant decrease in Bcl2 mRNA level in the DL cells from
the treated rats.
Caspase 9 cleavage is an important event of the mitochondrial
pathway of apoptosis and that of PARP-1 cleavage associates with
DNA fragmentation in the apoptotic cells. As shown in Fig. 6c,
treatment with Ru(II)-CNEB is able to produce cleaved products of
caspase 9. Full length PARP-1 is a 116 kDa protein, which, upon
activation, is cleaved into a 89 kDa C-terminal a 24 kDa N-ter-
minal protein fragments. Fig 6d illustrates ~1.5 times increase in the
levels of the cleaved PARP-1 fragments with a proportionate
decline in its full length PARP-1. This was consistent with the DNA
fragmentation pattern of the DL cells reported previously [11].
4. Discussion
The two main findings reported in our previous paper [11]; one,
Ru(II)-CNEB could inhibit M4-LDH and second, it induced release of
mitochondrial cytochrome c in the DL cells in vivo, hinted towards
induction of metabolic derangement led apoptosis in the DL cells
due to the treatment with this compound. This tempted us to
explore the regulatory aspects of such cellular effects produced by
Ru(II)-CNEB. Since, decline in M4-LDH activity in the DL cells, due to
the treatment with an anti-DL agent, could be attributed to the
reduced expression of this enzyme [28], first we checked the mRNA
level of LDH-A (M4-LDH) in the DL cells from Ru(II)-CNEB treated
DL mice. The RT-PCR result (Fig. 1a) shows no change in the level of
LDH-A mRNA in the DL cells due to the treatment with Ru(II)-CNEB.
Thus, suggesting that Ru(II)-CNEB does not affect expression of M4-
LDH. This implies that decline in M4-LDH activity, observed pre-
viously due to the treatment with Ru(II)-CNEB [11], is a conse-
quence of inhibiting catalytic efficiency of this LDH isozyme by the
compound at protein level. Indeed, this complex has already been
demonstrated to interact with and inhibit M4-LDH non-
competitively [10].
β actin
DL DL+Rc
p53
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
DL DL+Rc
Densitometricvalue
(p53/βactin)
***
Fig. 2. Treatment with Ru(II)-CNEB (Rc) increased the level of p53 in the DL cells
in vivo. A representative western blot photograph is presented with the relative
densitometric values of p53/b actin as mean ± SD from three western blot repeats.
***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e6156
6. Overproduction of lactate is considered as one of the hallmarks
of the tumor growth [44]. It has been reported that tumor cells
survival is greatly supported by excess of lactate produced by the
tumor cells [45]. Also, blockage of tumor M4-LDH, responsible to
synthesize lactate, has been found to suppress this additional route
of metabolic supplementation and thereby renders tumor cells
susceptible to death [45]. Therefore, keeping aside the mechanism
by which Ru(II)-CNEB decreases M4-LDH activity, the resultant
decline in lactate production could be of high therapeutic relevance
for this compound. Indeed, Ru(II)-CNEB, earlier found to inhibit
M4-LDH [11], is also able to significantly decline lactate level in the
DL cells (Fig. 1b), and thus, advocating M4-LDH as a therapeutic
target for this compound at least in case of the DL cells in vivo. Such
a mechanism gets support from inhibition of tumor cell prolifera-
tion in vitro due to the treatment with certain N-hydroxyindole-
based inhibitors of M4-LDH [46].
Moreover, M4-LDH catalyzed rapid production of lactate is
mainly dependent on the adequate supply of pyruvate as a conse-
quence of enhanced glycolysis in the tumor cells [18,47]. The
committed step of glycolysis is catalyzed by PFK1 which is evident
to be the target of multimodal regulation under a variety of path-
ophysiological conditions including tumor development [16]. PFK2,
Fig. 3. Effect of Ru(II)-CNEB (Rc) on (a) active levels of SOD2 SOD1, (b) on the expression of SOD1 and (c d) on the expression of SOD 2 in the DL cells from untreated and treated
DL mice. (a) gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the relative densitometric values for the respective SOD bands from
3 PAGE repeats. (c) shows a representative RT-PCR photograph with the densitometric values of SOD2/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b and d) shows
representative western blot photographs, with 60 mg protein in each lane, presented with the densitometric values of SOD1/b actin and SOD2/b actin respectively as mean ± SD from
3 western blot repeats. *p 0.05; **p 0.01; ***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 57
7. the kinase domain of a bi-functional enzyme (PFK2/FBPase2),
synthesizes FBP, the most effective allosteric activator of PFK1, and
thereby, considered as a master regulator of the glycolytic pathway.
In general, tumor cells are evident to express a C-type PFK1 that
shows greater sensitivity for FBP activation [16] and also express a
catalytically more efficient isoform of PFK2 (iPFK2: PFKFB3) [13,17].
Importantly, iPFK2 is found to be over expressed as a universal trait
of most of the growing tumor cells including DL [28] and many
tumors of human origin as well [17]. It was interesting to observe
that this compound could significantly decline the expression of
iPFK2, both at mRNA and at protein levels (Fig. 1c and d). This
clearly suggested that Ru(II)-CNEB is able to downregulate the
synthesis of master regulator of the glycolytic pathway in the DL
cells in vivo. The argument is well supported by a report describing
correlation between PFKFB3 gene silencing by siRNA and apoptosis
in the Hela cells in culture [13]. Though information is scanty on
modulation of iPFK2 by synthetic compounds in tumor cells, a 3-(3-
pyridinyl)-1-(4-pyridinyl)-2-propen-1-one has been found to
inhibit iPFK2 resulting into decreased glucose uptake by the tumor
cells leading into tumor growth suppression in vivo [48]. In this
context, the findings of Fig. 1c and d are first of its kind to
demonstrate that Ru(II)-CNEB is able to repress iPFK2 in a tumor
cell in vivo and thereby speculated to render less production of
adequate glycolytic intermediates to sustain high glycolytic effi-
ciency of the DL cells in vivo.
So far tumor growth associated regulator of cell bioenergetics is
concerned, p53, a tumor suppressor protein, has been given much
emphasis as it has been reported to be involved in imposing War-
burg effect during tumor development [19,22]. It is now evident
that switching over to glycolytic phenotype by the tumor cells is
accompanied with the declined p53 level [25]. Similarly, the
enhanced level of p53 has been found to repress glucose trans-
porters GLUT1 and GLUT4 [23]. Also, increased p53 has been
demonstrated to decline the expression of iPFK2 via activating the
TIGAR gene [24] and consequently, it inhibits glycolysis [26]. We
have observed significant increase in p53 level (Fig. 2) vis a vis a
significantly declined iPFK2 expression (Fig.1c and d) in the DL cells
due to the treatment with Ru(II)-CNEB and thus suggesting an as-
sociation between Ru(II)-CNEB mediated enhanced p53 level and
declined activity of the committed step of glycolytic pathway in the
DL cells in vivo.
It is known that activation of aerobic glycolysis by the tumor cells
is a metabolic strategy to prevent production of ROS, an inevitable
outcome of the oxidative energy metabolism, and thereby to protect
tumor cells from ROS induced apoptosis [49]. In addition, tumor
cells are known to modulate main antioxidant enzymes to prevent
oxidative stress [34,35]. Therefore, deranging such enzymatic
mechanisms by a therapeutic agent is argued to be a relevant option
for driving tumor cells to undergo apoptosis [28,43].
SOD is the first and commited enzyme of antioxidant pathway
that neutralizes O2
À
into H2O2. It has been reported that reduced
levels of SOD1 and SOD2 maintain low level of H2O2 in the
cancerous cells to facilitate tumerogenesis [34,35]. Similarly,
increased SOD activity and in turn, higher level of H2O2 is specu-
lated to inhibit tumor progression [36,37]. According to Fig. 3a,
there is a significant increase in the activity of both the SOD iso-
forms; SOD2 SOD1, in the DL cells from Ru(II)-CNEB treated DL
mice. Since, the pattern of active levels of both of them coincided
with the similar increments in expression of both these enzymes
(Fig. 3bed), it is evident that Ru(II)-CNEB is able to enhance
expression of SOD2 and SOD1 to make overall increment in SOD
activity in the DL cells in vivo. Since reports are limited on metal
complex induced expression of antioxidant enzymes, it is a first
report wherein, a Ru(II)-complex is demonstrated to enhance ac-
tivity of SOD by overexpressing SOD2 and SOD1 proteins. Moreover,
keeping aside these explanations, the enhanced level of SODs is
likely to ultimately produce higher level of H2O2 in the DL cells due
to the treatment with Ru(II)-CNEB.
H2O2 produced by SODs is metabolized by GPx and Catalase and
activity of both these enzymes were observed to be declined
significantly in the DL cells from Ru(II)-CNEB treated DL mice
(Fig. 4aec). Such a reciprocal pattern between SOD vs catalase
GPx has been reported accountable for unusually increased level of
H2O2 in the DL cells due to the treatment with emodin [50]. Indeed,
0
1
2
3
4
5
6
DL DL+Rc
Catalaseactivity
(U/mgprotein)
*
a
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
DL DL+Rc
GPxactivity
(U/mgprotein)
*
b
0
10
20
30
40
50
60
DL DL+RcsdnabxPGfoyrtemotisneD
(Relativeintensity)
GPx1 GPx2
**
*
GPx1
DL DL+Rc
GPx2
GPx3
c
Fig. 4. Effect of Ru(II)-CNEB (Rc) on the activity of catalase (a) and GPx (b and c) in the DL cells in vivo. The values in (a and b) are mean ± SD, where n ¼ 4. (c) represents active level
of GPx in the DL cells from untreated and complex treated DL mice. The gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the
relative densitometric values for the respective GPx bands from 3 PAGE repeats. *p 0.05; **p 0.01 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e6158
8. H2O2 concentration was found to be remarkably increased (~6Â) in
the DL cells from Ru(II)-CNEB treated DL mice (Fig. 5a). Thus, it is
argued that Ru(II)-CNEB is able to modulate all the three enzymes
of antioxidant pathway; SOD, catalase and GPx, to maintain higher
level of H2O2 in the DL cells. Such a condition might drive the DL
cells to undergo apoptosis, as increase in intracellular H2O2 is
known to cause a significant drop in cytosolic pH [51] which is
considered accountable for translocation of Bax, a pro-apoptotic
factor, to mitochondria [49].
Though the mechanism by which Ru(II)-CNEB regulates
reciprocal changes in SODs vs catalase and GPx could be a matter
of further investigation, in the present context, however,
enhanced level of p53 protein (Fig. 2) could be considered as one
of the integrators of such enzymatic changes. This is because,
not only a close association between p53 dependent modulation
of ROS metabolism and cell apoptosis is on record [26,27] but
also, higher level of p53 protein has been found to enhance the
expression of pro-oxidant and proapoptotic factors [28,30].
Fig. 5. Effect of Ru(II)-CNEB (Rc) on the level of H2O2 (a) and total glutathione (b) in the DL cells in vivo. (a) represents level of H2O2 in the DL cells. Values are mean ± SD and n ¼ 4.
(b) represents level of total glutathione in the DL cells in vivo. Values are mean ± SD, where n ¼ 3. *p 0.05; ***p 0.001 (untreated vs. treated DL groups).
Fig. 6. Treatment with Ru(II)-CNEB (Rc) caused a significant decline in the expression of Bcl2 with concomitantly increased expression of Bax (a and b), activation of caspase 9 (c)
and PARP1 cleavage (d) in the DL cells in vivo. a shows representative RT-PCR photographs with the b-actin normalized densitometric ratio of Bcl2/Bax mRNA where values
represent mean ± SD from 3 RT-PCR repeats. b shows representative western blot photographs with the b-actin normalized densitometric ratio of Bcl2/Bax where values represent
mean ± SD from three western blot repeats. ***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 59
9. Glutathione content is known to represent actual redox status of
the cells and its enhanced level in the cancer cells is directly
correlated with the tumor progression. Higher glutathione level, in
addition to providing multidrug and radiation resistance [52], has
been found to prevent apoptosis in the tumor cells [53]. Similarly,
depletion of glutathione level has been reported to sensitize tumor
cells to undergo apoptosis via release of cytochrome c [54]. The
anti-apoptotic role of Bcl-2 has also been linked with the gluta-
thione content in the tumor cells [55]. We have shown previously
that Ru(II)-CNEB causes release of mitochondrial cytochrome c [11]
and according to Fig. 6a and b, it is evident to decline Bcl2/Bax ratio
also. Importantly these cellular alterations are consistent with a
significant decline in total glutathione (GSH þ GSSG) level in the DL
cells (Fig. 5b) and thereby providing another biochemical mecha-
nism by which Ru(II)-CNEB could be able to induce apoptosis in the
DL cells.
One of the important aspects of p53 biochemistry is that it is
considered to be involved in regulating apoptosis in the tumor cells
[19,22,27,28]. Normally, p53 remains sequestered with Mdm2 in
the cytosol and thereby prevents cells to undergo apoptosis [56].
This implies that release of p53 from Mdm2 could be one of the
mechanisms to induce apoptosis in the cells. This may happen due
to many cellular changes including increased DNA damage caused
by the exogenous agents [57]. However, we observed that the
increasing concentration of Ru(II)-CNEB, when incubated in vitro at
37 C for 24 h with pBR322 plasmid DNA, it did not convert
supercoiled plasmid into the nicked circular DNA (Supplementary
data; Fig. S1) and thereby excluding direct nuclease activity of
this compound. Therefore, in the present context, as reported
earlier [28,56], abrrent ellular signaling could be argued account-
able for a significantly enhanced level of p53 in the DL cells from
Ru(II)-CNEB treated DL mice (Fig. 2). Moreover, keeping aside the
mechanism by which p53 level gets enhanced in the DL cells, it is
considered to act as a strong apoptotic inducer in multimodal ways.
The enhanced cytoplasmic p53 level has been reported to activate
Bax and its oligomerization [58] which in turn, induces cytochrome
c release from the mitochondria [59]. Bcl2 is an anti-apoptotic
factor and therefore, declined Bcl2/Bax ratio is considered
accountable for initiating intrinsic pathway of apoptosis [28]. In the
present context, Ru(II)-CNEB significantly declined the level of Bcl2
protein with a concomitant increase in Bax level (Fig. 6b). This
suggested that Ru(II)-CNEB is able to reciprocally modulate level of
both these factors resulting into a significant decline in Bcl2/Bax
ratio in the DL cells in vivo.
Caspase 9 activation is a hallmark of mitochondrial pathway of
apoptosis [60] and that of PARP-1 cleavage is associated with DNA
fragmentation in the cells undergoing apoptosis [61]. We observed
significant increase in the levels of cleaved caspase 9 and PARP-1 in
the DL cells from Ru(II)-CNEB treated DL mice (Fig. 6c and d).
Consistent with these observations, Ru(II)-CNEB mediated release of
mitochondrial cytochrome c, the main initiator of the intrinsic
pathway of apoptosis, in those DL cells, is already on record [11].
Thus, taking together, these findings strongly advocate for p53
mediated activation of Bcl2/Bax-cytochrome c release-caspase 9 led
intrinsic pathway of apoptosis in the DL cells due to treatment with
Ru(II)-CNEB in vivo. Though not much information is available on
induction of intrinsic pathway of apoptosis by metal complexes, a
Ru(II)-arene compound [Ru(g6-p-cymene)Cl2(pta)] has been
described to implicate this pathway for inducing apoptosis in the
Ehrlich ascite carcinoma [62]. Recently, another Ru(II)-complex
(Ru(II) b-carboline complex) has also been demonstrated to induce
apoptosis in the cancer cells by involving p53 [63]. In this context,
the findings of this paper are of special merit with regard to eluci-
dation of the biochemical mechanism by which a Ru(II)-complex can
induce p53 dependent apoptosis in the tumor cells in vivo.
In conclusion, there is an evolving concept of restricting tumor
growth by depriving tumor cells from adequate energy production
and by rendering them susceptible to oxidative stress. The present
article demonstrates that a Ru(II)-CNEB, characterized previously as
an anti tumor compound, is evident to activate p53 mediated
glycolytic inhibition-oxidative stress-apoptosis pathway in the DL
cells when administered in vivo. As such these findings provide a
biochemical mechanism which can be utilized for defining phar-
macological targets for the novel anticancer agents suitable for
in vivo applications.
Conflict of interest
The authors declare no conflict of interest with respect to this
article.
Acknowledgements
This work was financially supported by a project from Depart-
ment of Biotechnology (DBT), Govt. of India, (BT/PR5910/BRB/10/
406/2005) sanctioned jointly to LM and SKT at BHU. The contri-
bution of Dr. SK Dubey in synthesizing Ru(II)-CNEB in the lab of LM
is also acknowledged. RKK thanks CSIR, Govt. of India for awarding
Senior Research Fellowship during the tenure of this work at BHU.
The authors are thankful to UGC Centre of Advanced Study pro-
gramme to Department of Zoology and DBT-ISLS, BHU, for
providing facilities and assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biochi.2014.12.021.
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