This study examined the effects of the flavonoid quercetin on cancer cell viability and cell cycle progression when administered alone or in combination with the microtubule-targeting drugs taxol and nocodazole. The researchers found that while quercetin induced cancer cell death in a dose-dependent manner, lower doses of quercetin protected cancer cells from the G2/M cell cycle arrest induced by taxol and nocodazole. Specifically, quercetin delayed cell cycle progression, inhibited the accumulation of cyclin B1 at the microtubule organizing center, and partially restored viability of drug-treated cells for up to 72 hours. However, long-term exposure to quercetin still suppressed cancer cell proliferation and
Propolis with its active component CAPE (Caffeic Acid Phenethyl Ester) stops breast cancer cell growth. These results of CAPE are present in the naturopathic formulation
of propolis, a widely available natural substance with an extended safety record, making it a naturally-occurring and readily available epigenetic agent with great potential in breast cancer and oncology in general. The ability to link the biological effects of a naturopathic remedy to the pharmacologic effects seen with an exciting class of drugs in the treatment of cancer opens the door to a host of new therapeutic opportunities for patients.
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Propolis with its active component CAPE (Caffeic Acid Phenethyl Ester) stops breast cancer cell growth. These results of CAPE are present in the naturopathic formulation
of propolis, a widely available natural substance with an extended safety record, making it a naturally-occurring and readily available epigenetic agent with great potential in breast cancer and oncology in general. The ability to link the biological effects of a naturopathic remedy to the pharmacologic effects seen with an exciting class of drugs in the treatment of cancer opens the door to a host of new therapeutic opportunities for patients.
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Content Cytotoxicity Studies of Colorectal Carcinoma Cells Using Printed Impe...journalBEEI
Monitoring the effectiveness of drugs on cancer cells is crucial for chemotherapeutics studies. In-vitro cell-based biosensors can be used as an alternative for characteristic studies of cells’ response to drugs. Cell-based sensors provide real-time measurements and require smaller sample volumes compared to conventional T-flask measurement methods. This paper presents a biosensor that detects in real-time, impedance variations of human colon cancer, HCT-116 cells when treated with anti-cancer agent, 5-Fluorouracil (5-FU). Two different extracellular matrix (ECM); polyaniline and gelatin were tested and evaluated in terms of attachment quality. Polyaniline was found to provide the best attachment for HCT-116 cells and was used for cytotoxicity studies. Cytokinetic behavior indicated that 5-FU inhibited HCT-116 cells at IC50 of 6.8 µg/mL. Trypan blue exclusion method for testing cell viability was used to validate the impedance measurements, where the cancer cell concentrations were reduced to ~35% when treated with 2.5 µg/mL, and 50% when treated with 6.8 µg/mL. The results generated by the microfabricated impedance biosensor are comparable to the Trypan blue method since both gave similar cell growth trend. It can be concluded that the impedance biosensor has potential to be used as an alternative method in drug testing applications.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Growth inhibitory effects of vitamin k2 (menaquinones mk4) on bladder carcin...NAAR Journal
Ubiad1 is a tumor suppressor gene ubiquitously expressed in normal
human tissues and its protein harbors a prenyltransferase functional
domain conserved throughout the evolution. The ability of human
UBIAD1 enzyme to synthesize menaquinone-4 (MK-4) rescues
mitochondrial dysfunction in Drosophila and effectively initiates
apoptosis in various types of tumors through a caspase-dependent
pathway. The objective is to study menaquinone-4-induced apoptosis
signaling pathway and to investigate its anti-tumor activity in human
prostate (PC-3, LNCaP) and bladder (T24, J82) tumor cell lines, an
approach that has not been undertaken yet. Cell viability of cancerous cells
pre-treated with MK-4 showed a significant decrease in dose- and timedependent manner. Moreover, flow cytometry detected apoptotic cells
after treatment with the vitamin. Taken together, these observations
suggest that MK-4 could inhibit cell growth by promoting apoptosis in
prostate and bladder carcinoma cells via different-mediated signal
pathways, making it a potential therapeutic molecule for the prevention
and the cure of cancers.
Functional p53 is required for rapid restoration of daunorubicin-induced lesi...Enrique Moreno Gonzalez
The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
Content Cytotoxicity Studies of Colorectal Carcinoma Cells Using Printed Impe...journalBEEI
Monitoring the effectiveness of drugs on cancer cells is crucial for chemotherapeutics studies. In-vitro cell-based biosensors can be used as an alternative for characteristic studies of cells’ response to drugs. Cell-based sensors provide real-time measurements and require smaller sample volumes compared to conventional T-flask measurement methods. This paper presents a biosensor that detects in real-time, impedance variations of human colon cancer, HCT-116 cells when treated with anti-cancer agent, 5-Fluorouracil (5-FU). Two different extracellular matrix (ECM); polyaniline and gelatin were tested and evaluated in terms of attachment quality. Polyaniline was found to provide the best attachment for HCT-116 cells and was used for cytotoxicity studies. Cytokinetic behavior indicated that 5-FU inhibited HCT-116 cells at IC50 of 6.8 µg/mL. Trypan blue exclusion method for testing cell viability was used to validate the impedance measurements, where the cancer cell concentrations were reduced to ~35% when treated with 2.5 µg/mL, and 50% when treated with 6.8 µg/mL. The results generated by the microfabricated impedance biosensor are comparable to the Trypan blue method since both gave similar cell growth trend. It can be concluded that the impedance biosensor has potential to be used as an alternative method in drug testing applications.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Growth inhibitory effects of vitamin k2 (menaquinones mk4) on bladder carcin...NAAR Journal
Ubiad1 is a tumor suppressor gene ubiquitously expressed in normal
human tissues and its protein harbors a prenyltransferase functional
domain conserved throughout the evolution. The ability of human
UBIAD1 enzyme to synthesize menaquinone-4 (MK-4) rescues
mitochondrial dysfunction in Drosophila and effectively initiates
apoptosis in various types of tumors through a caspase-dependent
pathway. The objective is to study menaquinone-4-induced apoptosis
signaling pathway and to investigate its anti-tumor activity in human
prostate (PC-3, LNCaP) and bladder (T24, J82) tumor cell lines, an
approach that has not been undertaken yet. Cell viability of cancerous cells
pre-treated with MK-4 showed a significant decrease in dose- and timedependent manner. Moreover, flow cytometry detected apoptotic cells
after treatment with the vitamin. Taken together, these observations
suggest that MK-4 could inhibit cell growth by promoting apoptosis in
prostate and bladder carcinoma cells via different-mediated signal
pathways, making it a potential therapeutic molecule for the prevention
and the cure of cancers.
Functional p53 is required for rapid restoration of daunorubicin-induced lesi...Enrique Moreno Gonzalez
The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
Tras dirigir una fracasada sublevación contra la dictadura de Aramburu encabezada por un grupo de oficiales y suboficiales peronistas y sindicalistas de la resistencia, la vida del hombre más buscado en ese momento tuvo un giro increíble: el general Valle concurrió al velorio de su compañero, el coronel Cortínez. Allí se enteró de que el gobierno había lanzado un comunicado anunciando que, si se entregaba, se terminarían los fusilamientos de sus compañeros.
Моніторинг сприйняття реформ у суспільстві квітень 2016Kantar Ukraine
Спільний проект TNS та Національної ради реформ «Моніторинг сприйняття прогресу реформ» був ініційований проектним офісом Національної ради реформ у вересні 2015 року з метою більш повного відображення прогресу реформ. Відповідно до концепції проекту вимірювання громадської думки по загальних запитаннях довіри до органів влади, поінформованості та помітності прогресу реформ здійснюється щомісячно. Крім того, кожного місяця відбувається більш глибоке опитування респондентів по трьох з вісімнадцяти пріоритетних реформах.
У квітневому дослідженні, окрім загального блоку сприйняття реформ, розкриваються актуальні питання сприйняття антикорупційної реформи, децентралізації та реформи системи охорони здоров’я.
Venezuela. entre el estado de excepción oficialista y la amenaza de golpe de ...IADERE
La amenazante sanción del decreto de excepción constituye quizás, el develamiento de una enorme parodia. Apoyado en un discurso barnizado de un léxico “socialista”, que busca hacer ver continuidad con el gobierno de Chávez allí donde solo hay ruptura con él, el presidente Nicolás Maduro acaba de dar otro paso en el camino de la liquidación del proceso bolivariano tal como lo conocimos en estas dos décadas.
Indo-American Journal of Agricultural and Veterinary Sciences .It sounds like the journal you're referring to has a broad scope covering various aspects of Agricultural Sciences and Veterinary Medicine. The topics listed indicate a comprehensive range of fields within these discipline and submitting manuscripts to this journal can explore research and review articles of the journalism research.
The Indo-American Journal of Agricultural and Veterinary Sciences appears to be a scholarly journal focused on publishing research within the fields of agriculture and veterinary sciences of the journals public.
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
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
Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Ca...Jolene1981
ABSTRACT: The use of mushrooms contributes to human nutrition by providing low lipid content of lipids and high dietary fiber content, as well as significant content of other biologically active compounds such as polysaccharides, minerals, vitamins, and polyphenolic antioxidants. This study aimed to determine the content of polyphenols and polysaccharides, as well as the cytotoxic and antioxidative properties of several medicinal mushroom preparations. The content of total phenols and flavonoids of preparations of blended mushroom extracts (Lentifom, Super Polyporin, Agarikon, Agarikon Plus, Agarikon.1, and Mykoprotect.1) was evaluated quantitatively by using ultraviolet–visible spectroscopy spectrophotometric methods. The antioxidant capacity of the preparations was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and ferric reducing/antioxidant power assays. The content of water-soluble polysaccharides was determined using a specific gravimetric method, based on ethanol precipitation. To determine cytotoxic effects of single and blended mushroom extracts, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red assays were conducted using human small cell lung cancer, lung adenocarcinoma, colon cancer, and brain astrocytoma cancer cells. The obtained results suggest that due to the significant content of beneficial polyphenolic antioxidants and soluble polysaccharides, use of these mushroom preparations is beneficial in maintaining good health, as well as in the prevention and adjuvant biotherapy of various human pathological aberrations. These results reveal that these extracts exhibit different cytotoxic effects on tumor cells originating from different tissues. In addition, the comparison of investigated blended mushroom extracts with three well-known commercial mushroom products derived from single mushroom species or single mushroom compounds shows that blended mushroom extracts exhibit significantly stronger cytotoxic effects on human tumor cell lines.
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Multi-source connectivity as the driver of solar wind variability in the heli...
The flavonoid quercetin transientyly inhibits the activity of taxol and nocodazole through interference with the cell cycle (2010)
1. The flavonoid quercetin transiently inhibits the activity of taxol
and nocodazole through interference with the cell cycle
Temesgen Samuel*, Khalda Fadlalla, Timothy Turner, and Teshome E. Yehualaeshet
Pathobiology Department Tuskegee University, College of Veterinary Medicine, Nursing and
Allied Health Tuskegee, AL 36088
Abstract
Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of
quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination
with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death
in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and
nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-
induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-
targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup
of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not
inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells
by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by
quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was
critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-
term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the
interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-
targeting drugs to arrest cells at G2/M.
Keywords
quercetin; cell cycle; taxol; nocodazole; drug-diet interaction; flavonoid
Introduction
Consumption of foods of plant origin, especially fruits, vegetables and whole grains is
associated with a reduced risk of different types of cancer, including those of the lung, oral
cavity, esophagus, stomach, prostate and colon (Gonzalez, Pera et al. 2006; Kirsh, Peters et
al. 2007; Lunet, Valbuena et al. 2007; Millen, Subar et al. 2007). Dietary compounds are
being intensively studied for their chemopreventive, chemotherapeutic, or adjuvant potential
in cancer management. Dietary polyphenolic compounds, in particular, have attracted much
attention because of their abundance and due to well documented bioactivity that includes
their antioxidant effects.
Quercetin is one of the most abundant dietary flavonoids. Quercetin and its derivatives
constitute about 99% of the flavonoids in apple peel (He and Liu 2008), and it is also one of
(c) ‘Copyright Holder’, 2010
*
Corresponding author Phone: 334-724-4547 tsamuel@tuskegee.edu.
fadlallak@tuskegee.edu tyehuala@tuskegee.edu turner@tuskegee.edu
The authors have no conflict of interest to disclose.
NIH Public Access
Author Manuscript
Nutr Cancer. Author manuscript; available in PMC 2011 November 1.
Published in final edited form as:
Nutr Cancer. 2010 November ; 62(8): 1025–1035. doi:10.1080/01635581.2010.492087.
NIH-PAAuthorManuscriptNIH-PAAuthorManuscriptNIH-PAAuthorManuscript
2. the major constituents in foods consumed in the United States (Harnly, Doherty et al. 2006;
Huang, Wang et al. 2007). Numerous in vitro and animal model studies using quercetin
alone or quercetin in combination with other bioactive compounds have shown the anti-
cancer activities of the compound (Gee, Hara et al. 2002; Huynh, Nguyen et al. 2003;
Nguyen, Tran et al. 2004; Ong, Tran et al. 2004; Zhang, Huang et al. 2004; Kim, Bang et al.
2005; Mertens-Talcott and Percival 2005; Granado-Serrano, Angeles Martin et al. 2008).
While much is known about the bioactivities and the major signaling pathways modulated
by quercetin (see ref in (Ramos 2008)), less is known about the potentials of the compound
as a complementary supplement once the cancer has established itself and therapy has been
implemented to treat the cancer. The benefits and dangers of the concomitant use of
antioxidants and chemotherapeutic agents has been controversial (Keith I. Block 2008). This
has especially been true for therapeutic agents that induce oxidative stress as the mechanism
of action, as antioxidants may also reduce the side effects of chemotherapeutic agents. A
definitive recommendation is still lacking as to whether or when antioxidants should at all
be used in the course of chemo- or radiation-therapy (Bairati, Meyer et al. 2005; Block,
Koch et al. 2007; Keith I. Block 2008; Lawenda, Kelly et al. 2008). Drug-diet interactions
among antioxidants and classes of drugs that act through non-oxidative mechanisms is not
well known.
We examined the effect of the co-treatment of cancer cells with the flavonoid quercetin and
two anti-microtubule drugs, namely taxol and nocodazole. We analyzed cells treated with
single agents or a drug-flavonoid combination. We hypothesized an additive or a synergistic
effect with this drug-flavonoid combination, but unexpectedly, quercetin protected cells
from the activity of these anti-microtubule drugs, and sustained the viability of the cells.
However, prolonged exposure of the cells to the highest protective dose of quercetin was
still able to prevent cell proliferation. Thus, we identify bimodal activity of the flavonoid
quercetin, a short term activity which is cytoprotective against chemotherapeutic drugs, and
a long term activity which is inhibitory to cancer cell growth.
Materials and Methods
Cell culture and treatments
The human colorectal cancer HCT116 cell lines (wild type and p53 null) were generous gifts
from Dr Bert Vogelstein (Johns Hopkins). The cells were maintained in McCoy's medium
(Lonza, Walkersville, MD) supplemented with 10% Fetal Bovine Serum (FBS) and
Penicillin/Streptomycin. Prostate cancer PPC1 cells (gift from Dr John C. Reed, Burnham
Institute) were grown in RPMI medium (Invitrogen, Carlsbad, CA), supplemented with 10%
FBS and Penicillin/Streptomycin. RKO colorectal cancer cells (ATCC, Manassas, VA) were
maintained in similarly supplemented DMEM. MCF7 cells were kindly provided by Dr
Leslie Wilson (UC Santa Barbara) and were maintained in DMEM. For most experimental
treatments, cells were seeded in 96-well, 6-well or 6-cm dishes, at approximate densities of
103, 104, or 105 cells per well, respectively. For experiments requiring longer than 48 hours,
the cell numbers for the entire experimental set up were reduced by half. All cell cultures
were incubated at 37°C and 5% CO2 in a humidified incubator. Cells were synchronized by
the double thymidine block method.. Exponentially growing cells were treated overnight
with 2mM thymidine in growth medium. The next morning, culture medium was removed
and the monolayer was washed 3 times with plain growth medium to remove thymidine. The
cells were allowed to grow in complete medium for 8 hours and were treated again
overnight with 2mM thymidine. The synchronized monolayer cells were washed again and
released into complete growth medium. Cell cycle was analyzed at different time points
after the release.
Samuel et al. Page 2
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3. Reagents
Quercetin (Q0125), nocodazole, and taxol were purchased from Sigma (St. Luis, MO). A
stock solution of 50mM quercetin was prepared in DMSO, aliquoted in single use portions,
and stored at −20°C. Unused portions of any thawed aliquots were discarded. Nocodazole
and taxol were also dissolved in DMSO as 5mM and 10mM stock solutions. Working
dilutions of the stock were prepared in culture medium. Polyclonal antibodies to cyclin-B1
(AHF0062) and CDK1 (AHZ0112) were purchased from Invitrogen. Monoclonal antibody
to α-tubulin (clone DM1A) was purchased from Sigma (St Louis, MO).
Colony formation and clonogenic assays
Colony formation assay was performed by seeding approximately 500 cells per well of a 12-
well dish. Depending on the cell types or experimental designs, cells were allowed to adhere
for up to 18 hours and then treated with quercetin, or they were directly seeded in culture
medium containing quercetin. Culture medium was changed every 48 hours until discreet
colonies were visible to the naked eye, after which they were stained with 10% crystal violet
in methanol, washed and air-dried. Clonogenic assay was performed as described (Franken,
Rodermond et al. 2006). The number of cells in colonies was counted microscopically
(200X magnification), whereas the number of established clonal colonies was counted using
a stereo microscope. Due to their small sizes, HCT116 cells were allowed to grow up to
about 130 cells per colony before staining with crystal violet.
Flow cytometry
Cells were harvested and prepared for flow cytometry as described, with some modifications
(Samuel, Okada et al. 2005). Cells were harvested by trypsinization using 0.25% trypsin
EDTA. Prior to trypsinization, floating or loose cells were harvested by gentle manual
rocking of the culture dishes and transferring the culture medium containing the cells into
centrifuge tubes. Trypsinized and loose cells were then combined and centrifuged. Pellets
were resuspended in 300μl phosphate buffered saline, fixed by the addition of 700μl 100%
ethanol while vortexing, and stored at –20°C for at least overnight. Fixed cells were
centrifuged, and stained in FACS staining solution (320 mg/ml RNase A, 0.4 mg/ml
propidium iodide) in PBS without calcium and magnesium for 30 minutes at 37°C. Stained
cells were filtered through a 70 microns pore sized filter and analyzed by flow cytometry
(FACScalibur® Beckton Dickinson and C6 Accuri® flow cytometers). Data was analyzed
and histograms were prepared using CellQuest and CFlow software.
MTT/MTS and BrdU incorporation assays
MTT reagent was obtained from American Type Culture Collection (ATCC), whereas the
MTS assay was performed using CellTiter 96® AQueous One Solution cell proliferation
assay kit from Promega® (Madison, WI, USA). The assays were performed on cells seeded
in triplicates in 96-well plates, according to the manufacturer's instructions. Absorbance was
recorded at 570nm (MTT) or 490nm (MTS) using Synergy HT multimode plate reader or
PowerWave XS2 (BioTek®, Winooski, VT). To account for absorbance of quercetin at
490nm, during each MTT or MTS experiment, separate wells were set where quercetin was
diluted in culture medium without cells. The average A490 readings from wells containing
quercetin in culture medium were subtracted from the readings of treated cells. To calculate
MTT viability index, absorbance readings from DMSO treated control wells were set at
100% and the relative A490 was calculated as a percentage of the control.
BrdU incorporation ELISA
BrdU incorporation was analyzed by Cell Proliferation BioTrak ELISA (GE Lifesciences)
according to the manufacturer's instructions. For this assay, about 5×103cells were seeded
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4. per well of 24-well plates. After allowed to adhere for about 12 hours, cells were serum
deprived for about 24 hours by culturing them in serum-free medium. Cells were then
released into serum containing culture medium, and after 3 hours treated with quercetin,
taxol, or quercetin and taxol. Five hours after the treatment, BrdU labeling reagent was
added to the culture medium to label those cells synthesizing DNA. Cells were labeled
overnight, fixed the next morning, and processed for BrdU ELISA as recommended.
Absorbance readings were taken at 405nm using PowerWave XS2 plate reader (BioTek®).
Cell monolayer immunocytochemistry
HCT116 cells were seeded in 4-well chamber slides and allowed to adhere for about 16
hours. Then, cells in each well were treated with control (DMSO), single agents (quercetin
or taxol or nocodazole), or a combination of quercetin and taxol or quercetin and
nocodazole. About six hours after treatment, the culture medium was removed and the cells
were fixed in 4% formaldehyde for 15 minutes at room temperature. The fixed cells were
washed with PBS and processed for immunocytochemical staining at the
immunohistochemistry lab of the College of Veterinary Medicine, Nursing and Allied
Health (CVMNAH), Tuskegee University. Cyclin-B1 primary antibody (Invitrogen
AHF0062) was used at 15mg/ml concentration. Peroxidase conjugated secondary antibody
(Envision+ Dual Link System, Dako®, Carpinteria, CA) and DAB+ Chromogen (Dako®)
were used for the detection. Mayer's Hematoxylin (Lillie's modification, Dako®) was used
as counter stain. Slides were mounted using Micromount mounting medium (Surgipath®
Richmond, IL) and cover slips.
Immunofluorescent staining and Microscopy
Images of unstained live cells and immunocytochemically stained cells were taken at 20X
and 40X magnification objectives using Leica or Olympus microscopes fitted with digital
image capture cameras (Digital Microscopy Lab, CVMNAH). Photographs saved in TIFF
format were directly imported to Microsoft PowerPoint and cropped or adjusted for
brightness, contrast, or grayscale conversion. MCF7 cells for immunofluorescent staining
were grown in 4-well chamber slides. Staining was performed as described (Samuel, Okada
et al. 2005). Confocal images were taken at the Carver Research Center at Minority
Institutions (RCMI, Tuskegee University) core-facility using Olympus DSU spinning disk
confocal microscope using 40X dry objective. Images were captured using Metamorph
Premium® software and further processed in Adobe Photoshop®.
Immunoblotting
Cell lysates were prepared in NP-40 lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl,
10% Glycerol and 0.2% NP-40 plus protease inhibitor cocktail) and protein concentrations
were determined using NanoVue spectrophotometer (GE Healthcare Life Sciences,
Piscataway, NJ). Samples containing equivalent protein concentrations were mixed with
Laemmli buffer, and boiled for 5 minutes. Proteins were resolved by SDS-PAGE,
transferred to PVDF membranes (GE Healthcare Life Sciences) and blocked in 5% non-fat
dry milk. Primary antibodies used were rabbit anti cyclin-B1 (Invitrogen) at 1:200, rabbit
anti-CDK1 (Invitrogen) at 1:500, β-actin (Cell Signaling) at 1:1000. Peroxidase conjugated
anti-rabbit and anti-mouse IgG secondary antibodies were purchased from GE Healthcare
Life Sciences and used at 1:5000 dilution. Chemiluminescent detections were done using
LumiSensor™ Chemiluminescent HRP Substrate (Genescript, Piscataway, NJ).
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5. Results
We examined the bioactivity of quercetin singly and in combination with two
chemotherapeutic drugs known to act via disruption of the microtubule dynamics, namely
taxol and nocodazole. Both drugs induce G2/M arrest as phenotype.
Dose dependent induction of apoptosis by quercetin
To examine the apoptosis-inducing activity of quercetin, we exposed human colorectal
tumor HCT116 cells to increasing doses of quercetin, and analyzed the cell cycle profile of
the cells at 24 and 48 hours post treatment. As shown in Fig. 1 A, quercetin induced
apoptosis, which was evident as increased sub-G1(s-G1) population, most significantly by
48 hours of exposure, accompanied by reduction in the G2/M population. We also examined
the proapoptotic effect of quercetin on an adherent PPC1 prostate cancer cell line. PPC1
cells were treated with 0 to 100μM quercetin in growth medium. As shown in Fig. 1B, by 48
hours of exposure to 25μM and 50μM quercetin, the sub-G1 population of PPC1 cells began
to increase. The increase in apoptosis was concurrent with the reduction in the proportion of
cells at G1as well as G2/M phases of the cell cycle. At the dose level of 100μM, over 40%
of the cells were in sub-G1 state (apoptotic), indicating that higher doses of quercetin are
cytotoxic. Similar results on the cell death inducing potential of quercetin have previously
been reported (Murtaza, Marra et al. 2006). From these data, the bioactivity of quercetin
appears to be similar in both colorectal and prostate cancer cells, though the latter seemed to
be more sensitive to the flavonoid.
Inhibition of microtubule-acting drugs by quercetin
Bioactive compounds with antioxidant properties have been suggested to antagonize the
activity of chemotherapeutic agents that induce oxidative stress (Lawenda, Kelly et al.
2008). However, it is not well known if flavonoids may enhance or inhibit the activities of
other classes of anti-cancer drugs. We investigated the bioactivity of quercetin in the
presence of microtubule-targeting chemotherapeutic drugs. Since quercetin alone induced
apoptosis in colon and prostate cancer cells, we hypothesized the cell cycle inhibitory
activity of the anti-microtubule drugs nocodazole and taxol would be enhanced by co-
treatment with quercetin. To test this, we first examined the effect of combination treatment
of nocodazole, a microtubule-destabilizing agent, and quercetin on HCT116 colon cancer
cells. Adherent wild type and p53-null HCT116 cells were treated with the carrier alone
(DMSO), with single agents (nocodazole 10μM, or quercetin 50μM), or with a combination
of both agents. Cell morphology was examined by microscopy, and cell cycle profile was
analyzed by flow cytometry. While HCT116 cells treated with nocodazole alone were
completely rounded as expected, surprisingly, cells treated with a combination of quercetin
and nocodazole were morphologically indistinguishable from the control cells (Fig. 2A-D).
HCT116 cells treated with quercetin alone did not show any major morphological alteration
within 24 hours. Additionally, flow cytometric analysis showed that while 10μM nocodazole
induced 70-90% G2/M accumulation of cells, co-treatment with 50μM quercetin completely
abolished the G2/M arrest induced by nocodazole in both wild type and p53-null cells (Fig.
2 G-I). Quercetin at 25μM dose showed moderate inhibition of nocodazole activity in wild
type cells within 24 hours. Within this time frame, lower doses of quercetin had neither
inhibitory nor enhancing effects on nocodazole activity (G2/M arrest). Additionally, to
assess the inhibitory effect of quercetin on another microtubule-targeting drug, we tested the
combination of taxol and quercetin on colon cancer cells. Unlike nocodazole, taxol prevents
cell cycle progression by stabilizing the microtubules. We performed similar single
(quercetin or taxol) and combination (quercetin and taxol) treatments of HCT116 cells with
the two agents. As with nocodazole, the cells treated with the combination of quercetin and
taxol were morphologically indistinguishable from control DMSO treated cells (Fig. 2E, F)
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6. and displayed cell cycle profile similar to the control cells (not shown). This suggested that
quercetin-treated cells may not have responded to the cell cycle effects of the microtubule
targeting drugs.
To further test that quercetin protected cells from taxol activity, we treated PPC1 prostate
cancer cells with taxol or with taxol and quercetin, and examined the cells by flow
cytometry. To this end, we treated the cells overnight with increasing doses of taxol
(0-400nM) with or without co-treatment with 50μM quercetin. The cell cycle profiles of
treated and untreated cells were analyzed by flow cytometry. As with HCT116 cells, co-
treatment of PPC1 prostate cancer cells with quercetin completely abolished the prominent
G2/M arrest induced by the drug taxol (Fig. 3A, B).
Since we found that quercetin blocked the cell cycle arrest induced by nocodazole and taxol,
we became interested in examining if the viability of cells treated with the microtubule
acting drugs would be restored by quercetin. To assess this, we performed MTT assay on
singly (quercetin or nocodazole) or doubly (quercetin and nocodazole) treated cells at 24,
48, and 72 hours after the treatments. The MTT viability index showed that quercetin alone
in doses above 50uM reduced the viability of both wild type and p53-null HCT116 cells
(Fig. 4A, B). However, doses of quercetin as low as 3.13μM attenuated the activity of
nocodazole, while nocodazole (10μM) alone reduced the viability of the treated cells (Fig.
4C, D). At 72 hours after treatment, the viability index of nocodazole treated HCT116 cells
was about 65%, whereas the viability index of cells treated with nocodazole plus 50μM
quercetin was comparable to that of the carrier treated control cells. Quercetin at 100μM
dose was less protective than 50μM, suggesting the cytotoxicity of quercetin at higher doses.
To assess if the viability of cells treated with quercetin and taxol was accompanied with cell
cycle progression, we performed BrdU incorporation assay as an indicator of cellular DNA
synthesis, and analyzed BrdU incorporation in singly or combination treated cells. As shown
in Fig. 4E, cells treated with the combination of taxol and quercetin incorporated BrdU to a
degree comparable to singly treated cells. Therefore, it appears that the sustained viability of
quercetin-taxol combination treated cells may not necessarily be accompanied by DNA
replication, but by steady state maintenance of viability.
To further test the effect of quercetin on cell cycle progression, we synchronized HCT116
cells at G1-S boundary by the double thymidine block method and released them into
culture medium containing 50μM quercetin. Progression of the released cells through the
cell cycle was assessed by flow cytometry of cells harvested at different time points after the
release. We found that cells released into quercetin medium showed marked delay in cell
cycle. By 9 hours after release, most cells in the control medium were in G1 phase of the
next cell cycle, whereas the majority of the cells in quercetin medium were still in S-G2
phase of the first cell cycle after the release (Fig. 4F).
Quercetin does not interfere with microtubule targeting of taxol and nocodazole
The inhibition of the activity of taxol and nocodazole by quercetin led us to speculate that
quercetin might interfere with the uptake, intracellular distribution, or microtubule targeting
of the two drugs. To rule out this possibility, we examined the α-tubulin architecture in
MCF7 cells treated with taxol or nocodazole in the presence or absence of quercetin. Similar
to HCT116 and PPC1 cells, treatment of MCF7 cells with taxol and nocodazole in the
presence of quercetin also resulted in absence of G2/M arrest of the cells. However, unlike
HCT116 and PPC1 cells, 50μM and 25μM quercetin were cytotoxic to MCF7 cells, whereas
12.5μM was protective against the G2/M arrest of cells (not shown). Confocal images of
cells immunostained for α-tubulin showed that in the presence of quercetin nocodazole and
taxol were still able to destabilize or stabilize microtubules, respectively (FIG. 5). Since the
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7. drugs target microtubule dynamics in the presence of quercetin, we conclude that the
absence of G2/M arrest of combination-treated cells is not due to lack of uptake or increased
efflux of the anti-microtubule drugs.
Taxol/nocodazole and quercetin combination treatment prevents accumulation of cyclin-
B1 at the microtubule organizing center (MTOC)
As shown above, cells treated with quercetin and taxol or quercetin and nocodazole did not
accumulate at the G2/M phase of the cell cycle. Since mitotic entry is regulated mainly by
the cell cycle dependent subcellular dynamics and stability of cyclin-B1 and its partner
CDK1 through the MTOC (Jackman, Lindon et al. 2003), we examined the localization of
these proteins in HCT116 cells treated singly with quercetin or taxol or nocodazole or by a
combination of quercetin and taxol or quercetin and nocodazole for 8 hours. Monolayers of
HCT116 cells grown in chamber slides were immunohistochemically stained using an
antibody against cyclin-B1. Interestingly, combination-treated cells showed weak to no
detectable accumulation of cyclin-B1 at the MTOC in contrast to those cells treated with
either the drugs or quercetin alone (Fig. 6A). This indicates that the lack of cell cycle arrest
by taxol and nocodazole in the presence of quercetin is accompanied by the absence of
proper mobilization of cyclin-B1-CDK complex to the MTOC to initiate mitosis. However,
since the cells did not accumulate in S-phase, combination treated cells could also be
blocked at other phases of the cell cycle. Indeed, as shown above (Fig. 4E), cells treated
with quercetin alone or quercetin-taxol combination did not incorporate BrdU more than
taxol treated cells, suggesting quercetin treatment may have stalled the progression of the
cell cycle also before the S-phase. The decrease in the levels of cyclin-B1 in combination-
treated cells was also confirmed by immunoblotting. While taxol-treated cells accumulated
cyclin-B1 as expected, taxol-quercetin treated cells had markedly low levels of cyclin-B1
(Figs. 6B)
Quercetin inhibits colony formation of both wild type and p53-null colorectal tumor cells
It is estimated that more than 50% of human cancers carry p53 protein mutations, almost all
of which have been cataloged (Magali Olivier 2002; Christophe Béroud 2003). As p53 is
also a key protein regulating the apoptotic and cell cycle signaling, we became interested to
examine if the anti-proliferative activity of quercetin would be dependent on the p53 status
of colon cancer cells.
To address this, we exposed wild type and the isogenic p53-null human colorectal tumor
HCT116 cells to varying concentrations of quercetin, and examined growth of the cells by
colony formation assay. Both wild type and p53-null cells were seeded in the presence of 0 –
100μM concentrations of quercetin under two different conditions. In one instance, the cells
were allowed to adhere for overnight before adding quercetin, and under the second
instance, the dissociated cells were seeded in the presence of quercetin. Growth medium was
replaced at 72 hours intervals with a fresh supplementation of quercetin at the same
concentration as the initial dose.
As shown in Fig. 7A-B, long term exposure to quercetin (50μM or more) inhibited colony
formation in both p53 positive and negative cells at a comparable dose, which suggests that
the long term cell proliferation inhibitory effect of quercetin probably does not require
cellular p53. Moreover, the same dose of quercetin (50μM) that abrogated the G2/M arrest
by taxol and nocodazole also inhibited colony formation by HCT116 cells. Additionally, we
observed that both wild type and p53-null cells were more sensitive to the activity of
quercetin when the cells were seeded in the presence of the flavonoid. While 50μM
quercetin was needed to inhibit colony formation of adherent HCT116 cells, 12.5μM
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8. quercetin was sufficient to achieve an even stronger inhibition of colony formation of both
wild type and mutant cells when they were treated before they adhered to the culture dishes.
To examine if quercetin provided long-term survival advantage to cancer cells exposed to
anti-microtubule drugs, we performed clonogenicity assays on wild type HCT116 cells
treated with only quercetin or a combination of quercetin and taxol. The numbers of clonal
colonies formed and the number of cells per colony were compared. As shown in Fig. 7C
and D, quercetin doses (25μM and 50μM) that interfered with taxol and nocodazole still
inhibited the clonogenicity of HCT116 cells. Moreover, the number of cells per colony was
lower in cells treated with 12.5μM or higher quercetin, compared to control cells, suggesting
that quercetin may have interfered with cell cycle progression and therefore limited the rate
of cell proliferation or survival. When we tested the clonogenicity of HCT116 cells treated
with 25μM quercetin and taxol (0.6nM to 5nM) combinations, we observed that quercetin
provided no clonogenicity advantage to cells. On the contrary, the combination of quercetin
with taxol consistently suppressed the clonogenic survival of treated cells, and sensitized the
cells to lower doses of taxol which did not inhibit clonogenic survival. Cells treated with
1.25nM and 0.6nM taxol retained clonogenicity, while combination of 25μM quercetin with
the same doses of taxol markedly inhibited clonogenic survival of the cells (Fig. 7E).
Discussion
We have found that quercetin, a ubiquitous flavonoid abundantly available in green
vegetables and fruits, has pleiotropic effects on cancer cell survival as a single agent and
when combined with conventional chemotherapeutic drugs that target the microtubules.
While we initially predicted that quercetin would enhance the activity of taxol or
nocodazole, we unexpectedly found that quercetin antagonized the G2/M arrest induced by
both drugs. We also found that even in the presence of quercetin the uptake of nocodazole or
taxol was not inhibited, as shown by the distinctive effects of the drugs on the microtubules.
The antagonistic activity of quercetin on taxol and nocodazole was accompanied by the
absence of recruitment of cyclin-B1 to the MTOC in combination-treated cells. Cyclin-B1
and CDK1 are partner proteins crucial for mitotic entry (Jackman, Lindon et al. 2003). At
the end of the S phase, cyclin-B1 protein level is elevated, cyclin-B1 – CDK complexes are
formed, and the CDK component is activated. Activated cyclin B1-CDK complex
phosphorylates substrate proteins, including those at the MTOC, to drive cells into mitosis.
We propose that quercetin's interference with the cell cycle progression inhibits the activity
of the two microtubule-acting drugs to arrest cells at G2/M.
Although we found that quercetin interfered with the mitotic arrest induced by microtubule-
targeting drugs, we did not find evidence to suggest that the cells continue to synthesize
DNA and proliferate when combination-treated. Indeed, quercetin by itself inhibited the
long-term growth and survival of cells at the same concentrations that interfered with anti-
microtubuledrugs. Though our in vitro observations are limited, our data suggest that the
continued presence of quercetin in the cellular environment may attenuate the activity of
microtubule acting agents in the short run. Since the viability of cells in the presence of
microtubule disrupting drugs was maintained even by low concentration of quercetin
(3.13μM or higher in our study), the co-administration of quercetin during treatment with
anti-microtubule agents such as paclitaxel may diminish drug activity. In vivo studies need
to be performed to elucidate the relevance of this interference. However, our clonogenic
assays suggest that long term administration of high doses of quercetin alone or even low
doses of quercetin in combination with taxol may not promote the clonogenic survival of
colorectal cancer cells.
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9. The current thought on the bioactivity of quercetin and other flavonoids is that these
compounds act by scavenging free radicals induced by endogenous and exogenous pro-
oxidants (Valko, Rhodes et al. 2006). These pro-oxidant agents include DNA damaging
chemotherapeutic drugs and irradiation. However, recent studies suggest that polyphenolic
compounds and antioxidants may antagonize diverse groups of chemotherapeutic drugs. Liu
et al. (Liu, Agrawal et al. 2008) showed that dietary flavonoids, especially quercetin, inhibit
bortezomib-induced apoptosis in malignant B-cell lines and primary chronic lymphocytic
leukemia (CLL) cells, by direct association with bortezomib. The authors also found that the
inhibitory effect of quercetin was abolished by boric acid, thereby restoring the apoptotic
effect of bortezomib on CLL cells. Similarly, Golden et al. (Golden, Lam et al. 2009) found
that green tea polyphenols blocked the activities of bortezomib and other boronic acid-based
proteasome inhibitors through direct interference. Our data adds taxol and nocodazole to the
list of drugs potentially antagonized by quercetin.
It is not clear, however, if the antioxidant properties of flavonoids explain all of such anti-
drug bioactivity. For example, a recent study on vitamin C -another antioxidant dietary
compound - showed that vitamin C significantly attenuated the activity of diverse classes of
chemotherapeutic compounds such as doxorubicin, cisplatin, vincristine, methotrexate, and
imatinib, independent of its anti-oxidant potential (Heaney, Gardner et al. 2008). The
chemotherapeutic compounds used in the study and found to be inhibited by vitamin C are
known to target cellular DNA, the cytoskeleton, or diverse cell signaling mechanisms.
These results and our data suggest that compounds such as quercetin, other polyphenols, and
vitamin C may have hitherto unknown bioactivities that may be independent of their
antioxidant properties. Competitive interference of polyphenols with bortezomib for
proteasome inhibition has been documented (Liu, Agrawal et al. 2008; Golden, Lam et al.
2009), but mechanisms of antagonism of polyphenols against other drugs remain unknown.
In the cases of taxol and nocodazole, the effects of quercetin do not appear to stem from the
inhibition of uptake of the drugs. Also, unlike bortezomib, the two drugs are not known to
directly target the proteasome, excluding the possibility of competitive proteasomal
inhibition. Therefore, it is possible that the cell cycle inhibitory effects of quercetin and the
resulting lack of cycling cells may explain the antagonistic effect of quercetin on taxol and
nocodazole.
We also observed that the bioactivity of quercetin varies with the adherence status of the
treated cells. In colony formation assay, non-adherent colon carcinoma cells were inhibited
by a dose of quercetin fourfold less than that required for the adherent cells. This
observation, together with lack of a major difference between p53 wild type and p53-null
HCT116 cells suggests that the adherence status rather than the p53 status renders tumor
cells more sensitive to the bioactivity of quercetin. Moreover, the observation that adherent
cell lines are also more sensitive to quercetin before they attach to surfaces suggests that the
mechanisms and pathways that support cell attachment may confer a degree of resistance to
the growth inhibitory effects of quercetin. This in turn may imply that cells may be more
sensitive to the actions of the flavonoid quercetin if they are detached from their anchor, as it
may occur during metastasis. However, this possible mechanism of action can't explain the
cancer preventive activities of flavonoids such as quercetin because metastatic events occur
during later stages of oncogenesis. The chemopreventive mechanisms of dietary levels of
quercetin and other flavonoids remain to be elucidated.
In conclusion, quercetin appears to have a bimodal bioactivity where it may provide a short-
term transient survival benefit to cells exposed to taxol and nocodazole, but has a long-term
anti-cell proliferative effect. The anti-proliferative effects appear to be strong especially
when the cells have lost their attachment to the growth matrix. Although quercetin
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10. attenuated the cell cycle effects of taxol and nocodazole in the short term, we observed
diminished survival and clonogenicity of cancer cells exposed to combinations of quercetin
and taxol, which suggests no long-lasting antagonistic effects. Further studies are needed to
examine the in vivo effects co-administration of quercetin or other flavonoids with
microtubule-acting drugs.
Acknowledgments
We thank Dr Tsegaye Habtemariam, Dr Cesar Fermin and Dr Frederick Tippett for research support; Mrs Tammie
Hughley for secretarial assistance; Dr John Williams for technical assistance at the Tuskegee University RCMI
imaging core facility; Dr John Heath, Dr Clayton Yates, Mrs Starlette Sharp, and Mrs Patricia Adams for various
technical supports and advise. We thank Dr Bert Vogelstein for HCT116 cells, Dr John Reed for PPC1 cells, and Dr
Leslie Wilson for MCF7 cells. We acknowledge the research training support by the TU/UAB/MSM partnership
U54 CA118948 to T.S. This research was supported by NIH/NCI/NIGMS grant 1SC2CA138178 (T.S.) and
partially by grant number S21 MD 000102 (T.E.Y).
Abbreviations
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium
BrdU bromodeoxyuridine
DAB diaminobenzidine
PVDF polyvinylidene fluoride
CDK1 cyclin dependent kinase 1
MTOC microtubule organizing center
DAPI 4′,6-diamidino-2-phenylindole
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13. Figure 1. The effect of quercetin on the cell cycle profile of HCT116 colorectal and PPC1
prostate cancer cells
A, HCT116 cells were treated with 10μM nocodazole, 100nM taxol, or with the indicated
concentrations of quercetin for 24 hours or 48 hours. Cells were harvested and analyzed by
flow cytometry. The proportions of cells in each phase of the cell cycle (sub-G1, G1, S, G2/
M) for each treatment are indicated in the table. B, PPC1 cells are treated with 0 to 100μM
quercetin (as shown) for 24 hours. Cells were harvested and analyzed by flow cytometry.
Histograms of the cell cycle profiles of the cells are shown on the upper panel. The lower
panel shows the proportion of cells in phases of the cell cycle (sub-G1, G1, S, G2/M) for
each dose of quercetin. Representative data from two independent experiments are shown.
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14. Figure 2. Quercetin blocks the activity of nocodazole and taxol
A-F, HCT116 cells were treated with carrier DMSO (A), 50μM quercetin alone (B), 10μM
nocodazole (C), 10μM nocodazole plus 50μM quercetin (D), 100nM taxol (E), or 100nM
taxol plus 50μM quercetin (F). Cells remained under treatment for 24 hours (A-D), or 16
hours (E, F), and phase contrast images were taken at 200X magnification. G-I, Quercetin
inhibits G2/M arrest in HCT116 cells. Wild type (G) and p53-null (H) HCT116 cells were
treated with DMSO, 50μM quercetin, 10μM nocodazole, or the indicated decreasing
concentrations of quercetin in the presence of 10μM nocodazole as shown. 50μM quercetin
effectively blocked the cell cycle effect of nocodazole on both cell types, while lower
concentration showed weaker or no inhibition. I. Tabular presentation of the data in G and
H.
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15. Figure 3. Quercetin inhibits the activity of taxol on PPC1 prostate cancer cells
PPC1 cells were treated with 0 - 400nM taxol as shown (A) or a combination of 0 – 400nM
taxol and 50μM quercetin (B), and incubated for 12 hours. Cells were harvested and
analyzed by flow cytometry. The histograms in upper panels show the cell cycle profiles of
the cells, and the lower panels (tables) show the numerical proportion of cells in each phase
of the cell cycle for each treatment in A and B. One of three independent experiments is
shown.
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16. Figure 4. Quercetin maintains the viability of colorectal cancer cells treated with nocodazole but
delays cell cycle progression
A-D, effect of quercetin or quercetin-nocodazole combination on the viability of HCT116
cells. Wild type or p53-null HCT116 cells were treated with quercetin alone (A, B) or with
combinations of 10μM nocodazole and increasing doses of quercetin (C, D) as shown. Cell
viability was measured after 24, 48, and 72 hours by MTT assay. Cell viability is plotted as
MTT index, relative to that of the control DMSO treated cells. E. BrdU uptake in wild type
HCT116 cells treated with DMSO, 100nM taxol, 50μM quercetin, or a combination of taxol
and quercetin was measured by BrdU incorporation ELISA. Relative BrdU uptake is shown
as a percentage of uptake by the control cells. The difference in BrdU incorporation between
taxol, quercetin, and combination treated cells was not significant. F. RKO colorectal cancer
cells were synchronized by double thymidine (2mM) block, and released into growth
medium containing DMSO (Contr.) or quercetin (Qctn). Aliquots of cells growing
asynchronously or at different time points (at release (t0), 2 hours, 4 hours or 9 hours) after
release from the block were analyzed by flow cytometry. Cell cycle profiles are shown as
histograms in the top panels, and the proportion of cells in G1, S, or G2 at the time points
are shown in the lower panels (tables). Cells exposed to quercetin medium showed
considerable delay (underlined values) in cell cycle progression compared to control cells.
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17. Figure 5. Quercetin does not interfere with microtubule targeting of taxol and nocodazole
MCF7 cells were treated for 16 hours (overnight) with carrier (DMSO), quercetin (QCTN,
10μM), taxol (TAX, 50nM), nocodazole (NOC, 10μM) or combinations of taxol and
quercetin (TAX + QCTN) or nocodazole and quercetin (NOC + QCTN) as shown. Cells
were then fixed and immunofluorescently stained for tubulin (upper row). DAPI was used as
a counterstain for nuclei (middle row). Merged images (tubulin and DAPI) are shown in the
bottom row. Confocal images were taken using a 40X dry objective.
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18. Figure 6. Treatment of HCT116 cells with a combination of quercetin and taxol disrupts the
localization of cyclin-B1 at the MTOC
A, HCT116 wild type cells grown in chamber slides were exposed to DMSO, 50μM
quercetin (Qctn), 100nM taxol (TAX), or 50μM quercetin and 100nM taxol combination
(TAX+Qctn). After 8 hours of treatment, cell monolayers were stained with anti cyclin-B1
antibody by immunocytochemistry. Arrows indicate the localization of cyclin-B1 at the
MTOC. B, HCT116 cells grown in 6 cm diameter dishes were treated with DMSO, 50μM
quercetin, 100nM taxol or a combination of 50μM quercetin and 100nM taxol for 8 hours.
Cell lysates were prepared as described in the Materials and Methods section. Cyclin-B1,
CDK1, and β-actin proteins were detected by immunoblotting. * Indicates a non specific
band.
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19. Figure 7. Continued exposure of HCT116 cells to quercetin inhibits colony formation
A. Wild type and p53-null HCT116 cells were seeded in 12-well cell culture dishes and
allowed to adhere to the plate for about 16 hours. Adherent cells were treated with the
indicated concentrations of quercetin and colony formation was examined over 8 days as
described under materials and methods. B. Wild type and p53-null HCT116 cells were
seeded in 12-well cell culture dishes in the presence of the indicated concentrations of
quercetin in culture medium. Colony formation was examined as described. C-E, Quercetin
does not provide lasting clonogenicity and survival advantage to HCT116 cells.
Clonogenicity of HCT116 cells exposed to 6.25μM -100μM quercetin was examined by
clonogenicity assay (Franken, Rodermond et al. 2006). The colonies that formed after the
treatments, and the number of cells per colony for each treatment are shown in C and D,
respectively, relative to the numbers from control cells. Doses of quercetin that antagonized
taxol or nocodazole still inhibited clonogenic survival of the cells. E. Clonogenic survival of
HCT116 cells treated with quercetin (25μM) or quercetin in combination with taxol (0.6nM
– 5nM). Clonogenicity of the cells is shown as the number of colonies that formed relative
to the control (DMSO) treatment. Quercetin in combination with taxol provided no
clonogenic advantage; on the contrary, combination treated cells had the poorest clonogenic
survival.
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