Cancer—An Overview of the Disease

1. 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

2. 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.

3. 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)

4. 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

5. 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

6. 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.

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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) 

14. 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)

15. 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

16. 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

17. Anticancer bioassays: Methods of evaluating compounds for anticancer activity using in vitro (tissue culture) model systems

18. 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

19. 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

20. 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.

21. 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)

23. 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

24. 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

25. 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)

26. 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.

27. 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

28. 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!

29. 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 dimericindole 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

30. 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)

31. Animal models of cancer

32. 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]

33. 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.