Transcript Cancer—An Overview of the Disease

Cancer—An Overview of the Disease
• American Cancer Society slide show
• Cancer accounts for 6 million deaths per year
(>500 K in US alone)
• Causes are linked to genetic factors,
environmental factors, diet and lifestyle
(smoking, alcohol consumption, stress, lack of
exercise, etc.)
• Incidence: Breast / prostate > lung > colon >
urinary > lymphoma
• Mortality: lung > breast/prostate > colon >
pancreas
Steps in cancer initiation and
development
• Cancer generally begins with a mutation or alteration of
genetic material (DNA) in a cell (initiation)
• Cell begins to proliferate at an abnormally high rate
forming a group of “hyperplastic” cells
• Some of these cells further mutate and become
abnormal in appearance and function, or “dysplastic”
(promotion)
• Further mutations lead to formation of a tumor
• Certain tumors may under certain conditions invade
neighboring tissues to form more tumors, a process
called “metastasis”
• How this occurs is reviewed in “Hallmarks of Cancer”,
Hanahan & Weinberg, Cell (2000) 100: 57-70.
Why do cancer cells compete
against normal cells and survive?
• Normal cells have a limited life span (50 - 70 divisions)
• However cancer cells are capable of reproducing almost
infinitely - tumor cell lines are referred to as “immortal”
• Normal cells adhere to each other and to the ECM or
extracellular matrix, which is the protein-based material
filling the space between cells.
• Tumor cells develop their own abnormal signaling
pathways that override signals that control normal tissue
functions and maintain contact inhibition
• Result is uncontrolled proliferation of non-functional cells
• Tumors then form and eventually develop their own
blood supply through angiogenesis (VEGF signaling)
Why do cancer cells compete
against normal cells and survive?
• “Malignant” cells evade apoptosis and senescence –
becoming “immortalized.”
• They do not adhere properly and may begin to invade
and migrate through the ECM to other tissues.
• These malignant cells are able to then spread to other
sites in the body by traveling through bloodstream or
lymphatic system - metastasis.
• Examples: Colorectal cancer cells may migrate to the
liver; melanoma cells may migrate to lungs; prostate
cancer to bladder, bone, lymph nodes; breast cancer to
the lymph nodes
Some basic cancer types:
• Carcinoma = cancer originating in epithelial cells
(most common)
• Adenocarcinoma = originates in glandular tissue
• Sarcoma = cancer of connective tissues
• Glioma
= cancer occurring in brain cells
(non-neuronal)
• Lymphoma = originates in the lymphatic/immune cells
• Leukemia = cancer of the white blood cells or bone marrow
• Melanoma = originates in pigment-producing cells
Which cancers are most deadly* and what are
the risk factors?
• <20% survival (5 yr): lung (smoking)
pancreatic (diet, heredity, smoking)
• 40 - 60% survival:
kidney (mostly males, smoking, obesity)
ovarian (female: age, heredity)
late-stage melanoma (sun exposure, heredity)
• 60 – 80% survival: uterine (hormonal factors, reproductive history)
leukemia (genetic, viral, environmental)
colon (obesity, heredity, GI infections)
bladder (race, smoking, environmental)
• >80% survival:
prostate (male: age, obesity, race)
breast (female: age, genetics, obesity,
reproductive history)
skin, non-metastatic melanoma (sun exposure)
*This can be evaluated based on the five-year survival rate of individuals
diagnosed with the disease at any stage.
Many cancers diagnosed at an early stage have improved survival rates.
Genes that play a role in cells
turning cancerous
• Genes involved in cellular carcinogenesis
act to either promote or inhibit cancers and
the body strives to balance these actions
• These may be general to all cells or
specific for certain types of cells
• Regulation of such genes is the target of
many anti-cancer agents
Tumor Promotors
• Oncogenes encourage cancerous growth
• When mutated or over-expressed, oncogenes
cause excessive proliferation
• Example: Ras family of oncogenes controls
signal transduction – allows for growth signals in
absence of normal stimuli
• Ras is found mutated in about 25% of cancers
• Overproduction of growth factors (e.g. PDGF,
VEGF) leading to proliferation can occur as a
result of abnormal celllular signalling processes
Biological, chemical and environmental
tumor promoters
• “Carcinogens” include chemical or biological toxins such
as cigarette smoke, aflatoxins, benzopyrene, H. pylori,
HPV, bisphenol-A
• Environmental carcinogens – excessive exposure to UV
light, abnormal levels of radiation/radioactivity
• At the molecular level, mechanisms of action are varied
and in some cases unclear
–
–
–
–
DNA mutation
Oxidative stress – for example exposure to ROS
Pro-inflammatory processes
Up-regulation or down-regulation of the expression of
genes/proteins linked to proliferation
Tumor Suppressors and Inhibitors
• Tumor suppressor genes evolved to inhibit out of control
proliferation
• However, when inactivated, they fail to block cell division
• Examples: p53 tumor suppressor gene regulates cell
cycle, DNA repair, initiates apoptosis – if it is mutated,
apoptosis may not occur
• BRCA1 and BRCA2 are needed for normal DNA repair
processes, however in some families BRCA are found
mutated, leading to early-onset breast, ovarian cancers
• Some viruses also work by disabling tumor suppressor
genes – human papilloma virus (HPV) is a risk factor for
cervical cancer
Some mechanisms cells use to suppress
carcinogenesis
• apoptosis—a programmed cellular death
controlled by caspase family of enzymes and
regulated by many genes
• senescence – cell stops dividing in response to
shortened chromosome ends (telomeres)
• detection of DNA mutations and strand breaks,
and activation of DNA repair (nucleotide
excision, DNA ligation, etc.)
• expression of TNF-a = tumor necrosis factor
• expression of detoxification enzymes such as
quinone reductase
Cancer treatments
• Surgical removal of tumor—useful in isolated tumors but
may miss metastasized cancer cells
– Side effects: may cause organ damage, introduce infection
• Radiation (X-rays or gamma rays) – causes targeted
necrosis or apoptosis of cancer cells but may miss
metastasized cells.
– Side effects: weakened immune system, some tissue damage
• Chemotherapy – administration of highly toxic
substances kills tumor cells systemically and may be
more effective in catching metastasized cells
– Side effects: many, as the result of toxicity to normal tissue...
nausea, hair loss, fatigue, weakened immune system
Common classes of chemotherapeutic
agents by function:
• alkylating agents –these bind to DNA, disrupting gene
structure & function or bind to enzymes to inactivate
them (synthetic drugs)
• topoisomerase inhibitors—inhibit DNA replication in
rapidly dividing cells (lignans)
• antimitotics –inhibit cell division by blocking normal
microtubule function (taxol)
• antibiotics & anthracyclines – some can block DNA
replication & protein synthesis (doxorubicin)
Common classes of chemotherapeutic
agents by function:
• immunomodulators – stimulate the immune system to
inhibit proliferation
• enzymes -- proteases, tyrosinase inhibitors interfere
with proliferating cells
• inducers or inhibitors of enzymes involved in
proliferation: quinone reductase (QR) inducers, ornithine
decarboxylase (ODC) inhibitors
• hormones – work with endocrine system to inhibit
specific cancers
• Some targets being developed include monoclonal
antibody/vaccines and inhibitors of angiogenesis
(formation of blood vessels)
Why are many natural products anti-cancer agents?
(a theory expressed in Kintzios and Barberaki, 2004)
• Most natural anticancer agents are secondary
metabolites
• Most secondary metabolites are produced for
one of two reasons:
– Protection of the plant/organism from pathogens
– Growth regulation
• Both properties may make a compound cytotoxic
or capable of modulating tumor development
Some classes of natural products with
documented anticancer activity
• Flavonoids – cytotoxic, reduce oxidative stress
• Lignans -- cytotoxic, particularly to leukemia, skin, liver
• Other phenolics, stilbenes -- protein kinase inhibition, cell
membrane structure, and radical scavenging
• Isoprenoids -- cytotoxic to leukemia, prostate, other
cancers, anti-inflammatory properties
• Alkaloids: Indole, pyridine and piperidine alkaloids are
often cytotoxic, particularly to leukemia cell lines
• Aldehydes -- some are tyrosinase inhibitors
• Proteins and peptides – may induce apoptosis, inhibit
enzymes, block cell receptors
• Polysaccharides -- may stimulate the immune system
Anticancer bioassays:
Methods of evaluating compounds for
anticancer activity using in vitro (tissue
culture) model systems
Measurement of cytotoxicity / growth inhibition:
• Evaluates relative effectiveness of extracts and
compounds in inhibiting growth and proliferation
of specific types of tumor cells
• Most methods use dyes to quantify number of
live cells present after treatment.
• SRB assay, Trypan Blue, MTT
• Disadvantage: doesn’t address mechanism
NCI Sulforhodamine B (SRB) method
(Skehan, et al, Journal of the National Cancer Institute, 82: 1107-1112; 1990)
48 h assay method to determine cytotoxicity
• Tumor cells are incubated with test extracts at various concentrations
for 48 hours at 37oC.
• A solution of 0.4% sulforhodamine B dye in 1% acetic acid is added.
The dye binds to the live cells.
• The cells are washed to remove excess dye so the remaining dye is a
function of adherent cell mass
• Bound sulforhodamine B is then solubilized in basic solution
• Dye concentration is quantified by measuring absorbance at 564 nm
• The amount of remaining cells in treated samples is compared to
untreated (control) and % inhibition of growth is determined
• A dose-response curve is generated to obtain GI50 values
(concentration inhibiting 50% of growth)
• Advantages: Can be used to evaluate cytotoxicity in a variety of tumor
cell lines, and it provides quantitative cytotoxicity data
Other dye-based methods
• Trypan blue staining method: only dead cells absorb
dye, can determine dead vs. live cell count.
• MTT assay: The yellow tetrazolium salt MTT is reduced
in metabolically active cells to a purple colored formazan
product by an enzyme produced in mitochondria
(succinate dehydrogenase)
• SYTOX green: Dye binds to DNA and becomes
fluorescent at 538 nm (excitation at 485 nm), then cells
are quantified by determining the amount of dye present
before and after lysing cells.
Apoptosis assays
Methods have been developed to determine whether
cytotoxicity is due to apoptosis (programmed cell death)
that determine changes in DNA fragmentation, morphology,
or activity or expression of enzymes controlling apoptosis
• Fluorescent TUNEL assay: detects DNA fragmentation
by fluorescent labeling – effective for determining % of
cell population undergoing apoptosis
• We used this assay to determine how cranberry
phytochemicals affect apoptosis rates in MCF-7 breast
cancer cells vs. MCF-10A normal cells, also colon
cancer cell lines HCT116 and HT-29
• The DNA fragmentation characteristic of apoptosis is
visualized by the fluorescein-labeling of free DNA ends
(apoptotic cells appear green). Non-apoptotic cell nuclei
are counterstained with DAPI (appears blue)
Mechanistic apoptosis assays
• Caspase family of enzymes controls apoptosis;
measurement of caspase enzyme activity can be done
via commercial kits:
– Cells are incubated with test compound
– Cells are homogenized to release caspase-3 for example into
the supernatant
– Incubation with caspase substrate produces a cleavage product
that can be quantified by fluorescence (460 nm)
• Effects on the expression of genes coding for tumor
suppressors (such as p53) can be measured by PCR
• Expression of apoptosis-regulating proteins (such as
Bax, Bcl-2) can be measured by Western blotting
Measurement of the expression of proteins
associated with invasion and metastasis
• Matrix metalloproteinases (MMPs) control invasion,
migration and metastasis by degrading the ECM
• Cranberry extracts inhibit expression of MMPs by
prostate tumor cells (R. Hurta, UPEI & C. Neto, UMD)
• Tumor cells (DU-145) are grown up in the Hurta lab and
then incubated with extracts from our lab
• Activity of matrix metalloproteinase enzymes is
evaluated at various timepoints by gel electrophoresis
(zymography) – MMP activity is identified as zones of
clearing in the gel due to their gelatinase behavior.
• Ursolic acid hydroxycinnamate esters inhibit MMP-2 and
MMP-9 expression by 50-75% at 1 uM concentration
• Cranberry PACs also inhibit MMPs at 25 uM
DNA synthesis & repair mechanisms
• Some groups researching natural products
screen extracts for compounds that affect
DNA damage and repair:
• Topoisomerase II (etoposide)
• DNA polymerase b lyase –inhibition of the
lyase activity indicates DNA-damaging
agents (neolignans)
Camptothecin
• Camptotheca acuminata
(Nyssaceae)
• “xi shu” or “happy tree”
• Bark, wood & young
leaves used in traditional
Chinese medicine to treat
stomach, liver cancers &
leukemia
• Location: Southern
China, Tibet originally;
now cultivated in
provinces south of
Yangtze River.
Camptothecin
Mechanism: Topoisomerase II
inhibitor
• Camptothecin was so toxic that
semi-synthetic derivatives had
to be developed
• However camptothecin itself is
required as starting material
• topotecan (Hycamtin) —
ovarian cancers
• 9-nitro camptothecin
(Rubitecan)—pancreatic
cancer
Dr. Monroe E. Wall of USDA first
reported the anticancer properties
and isolated the quinoline alkaloid
camptothecin
Vincristine and vinblastine
• Vinca rosea or Catharanthus
roseus (Apocynaceae)
• aka “Rosy periwinkle”
• Traditional uses: cramps, skin
ailments, wasp stings, skin &
eye infections
• Location: originally from
Madagascar and tropical
Africa, they are now cultivated
in Caribbean & U.S.
• They even grow in MA in the
summer!
Vincristine and vinblastine
History: in the 1950’s, Dr. Charles
Beer identified alkaloids which
decreased WBC due to
destruction of bone marrow
• Vinblastine: Used to treat
Hodgkin’s disease,
lymphomas, testicular
• Vincristine: childhood leukemia
& Hodgkin’s
• Both are dimeric indole
alkaloids
• More recent use includes
diabetes treatment: extracts
of the leaves used in Jamaica
to make tea for diabetics
Mechanism of action:
Like taxol, vinblastine binds to
tubulins, disrupting mitosis
Viscum album (Loranthaceae)
aka “Mistletoe”
• Mechanism of action:
enhancement of immune
system
• Lectins from mistletoe boost
the production of interleukins
(IL-1, IL-6) and tumor necrosis
factor (TNF-a)
• Effective at very low
concentrations (pg/mL)
• Some cytotoxicity in vitro
(melanoma, leukemia,
lymphoma) but its major use is
as an adjuvant to other cancer
treatments
Location: Europe, Asia, N. Africa
Leaves and twigs of plant contain
viscotoxin, alkaloids and
lectins (a type of peptide with affinity
for sugars)
Animal models of cancer
More about senescence
(from Wikipedia)
Senescence, an irreversible state in which the cell no longer divides, is
a protective response to the shortening of the chromosome ends.
The telomeres are long regions of repetitive noncoding DNA that
cap chromosomes and undergo partial degradation each time a cell
undergoes division (see Hayflick limit).[12] In contrast, quiescence is
a reversible state of cellular dormancy that is unrelated to genome
damage (see cell cycle).
Senescence in cells may serve as a functional alternative to apoptosis
in cases where the physical presence of a cell for spatial reasons is
required by the organism,[13] which serves as a "last resort"
mechanism to prevent a cell with damaged DNA from replicating
inappropriately in the absence of pro-growth cellular signaling.
Unregulated cell division can lead to the formation of a tumor (see
cancer), which is potentially lethal to an organism. Therefore, the
induction of senescence and apoptosis is considered to be part of a
strategy of protection against cancer.[14]
Nature Protocols 1, - 1112 - 1116 (2006)
Published online: 17 August 2006 | doi:10.1038/nprot.2006.179
Subject Categories: Cell and tissue culture | Pharmacology and toxicology
Sulforhodamine B colorimetric assay for cytotoxicity screening
Vanicha Vichai1 & Kanyawim Kirtikara1
Abstract
The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular
protein content. The method described here has been optimized for the toxicity screening of compounds to
adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol)
trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1%
(vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510
nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is
comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested
within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore
an efficient and highly cost-effective method for screening.