1

SCREENING OF ANTI CANCER AGENTS

2

INTRODUCTION• CANCER: Uncontrolled proliferation of

genetically altered cells.• Derived from the repeated divisions of a mutant

cell. • Due to the effects of carcinogens, such as

tobacco smoke, radiation, chemicals or infectious agents.

• Cancer-promoting mutations may be acquired through errors in DNA replication.

• Activate the cancer promoting oncogenes and/or inactivate the tumor suppressor genes.

3

•Cancer chemotherapy is a rather young discipline.

•It has been pursued with scientific vigour and multinational collaborations only since the mid-20th century.

•Although 92 approved anticancer drugs are available today for the treatment of more than 200 different tumor entities, effective therapies for most of these tumors are lacking.

•Out of the 92 registered drugs, 17 are considered by oncologists to be more broadly applicable and 12 additional agents are perceived as having certain advantages in some clinical settings

4

•They are mostly cytotoxic in nature and act by a very limited number of molecular mechanisms.

•Thus, the need for novel drugs to treat malignant disease requiring systemic therapy is still pressing.

• A preselection, called the screening process, is therefore required.

•The aim of screening efforts is to identify products that will produce antitumor effects matching the activity criteria used to define which compounds can progress to the next stage in the preclinical development program.

5

NEED FOR NOVEL ANTI-CANCER DRUG

• Development of multidrug resistance in patients.

• Long-term treatment with cancer drugs is also associated with severe side effects.

• Cytotoxic drugs have the potential to be very harmful to the body unless they are very specific to cancer cells.

•New drugs that will be more selective for cancer cells

6

ANTI- TUMOUR MODELS

INVITRO METHODS INVIVO METHODS

7

In-vitro methods

8

Advantages: •Reduce the usage of animals. •Less time consuming, cost effective & easy to manage •Able to process a larger number of compounds quickly with minimum quantity. •Range of concentrations used are comparable to that expected for in vivo studies. Disadvantages: •Difficulty in Maintaining of cultures. •Show Negative results for the compounds which gets activated after body metabolism and vice versa. Impossible to ascertain the Pharmacokinetics. 

Cell Viability

• Functional assay

• DNA labeling assay• Morphological assay • Reproductive assay

• Membrane integrity assay

9

10

The major criteria employed in viability assayCategory of

viability assay Assays Principles

Membrane integrity assay

-Exclusion dyes-Fluorescent dyes-LDH leakage

The determination of membrane integrity via dye exclusion from live cells

Functional assay

-MTT, XTT assay-Crystal violet/ Acid phosphatase(AP) assay-Alamar Blue oxidation- reduction assay - Neutral red assay -[3H]-thymidin/ BrdU incorporation

Examining metabolic components that are

necessary for cell growth

DNA labeling as-

say

-Fluorescent conjugatesSimultaneous cell selection and viabilityassay

Morphological as-say -Microscopic observation Determination of morphological change

Reproductive as-

say

-Colony formation assay Determination of growth rate

11

MEMBRANE INTEGRITY ASSAY

Possibly the simplest assay for cell death is measurement of plasma membrane integrity. This can be assessed in two ways: The ability of a cell to prevent a fluorescent dye from entering it and the ability of a cell to retain a fluorescent dye within it. As a cell dies it's plasma membrane becomes permeable allowing fluorescent dyes present outside the cell to enter it and fluoresce. The most common dyes used for this purpose are dyes that label nucleic acids.

12

The most commonly used dyes are DAPI, propidium iodide, 7AAD, and ToPro-3.

One of such method includes TRYPHAN BLUE DYE EXCLUSION ASSAY The trypan blue dye exclusion assay is the most commonly used and accepted method for the measurement of cell viability It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability Based on optimal image analysis, the technology allow precise cell-viability and cell-density determination The system performs automatic and reproducible measurements of human or animal suspension cell densities as well as standardized differentiation between viable and dead cells, based on the trypan blue dye exclusion method

13

14

Other dyes, such as CFDA and Indo-1, are readily taken up by live cells, and upon internalisation are cleaved into a fluorescent form that can no longer cross the membrane. The result is that live cells become loaded with the dye, but dead cells, that do not have the enzymes necessary for cleavage of the non-fluorescent pre-cursor, will not. As a cell dies, the membrane becomes permeable and the fluorescent dye leaves the cell resulting in a loss of fluorescence.Example includes:

Ethidium bromide (EtBr)/fluorescein diacetate(FDA) and propidium iodide (PI)

15

Intact cell –PI and FDA is added

Fluorescein in intact cells

Schematic illustration of the principle of PI/FDA cell viability assay

● FDA (Fluorescein diacetate)● PI (Propidium iodide)

Plasma membrane is damaged ; fluorescein leaks out PI enters and strains

nucleic acids

16

•A well-established feature of apoptosis is the externalisation of the lipid phosphatidyl serine (PS) from the inner to the outer plasma membrane.• Annexin-V (five) is a protein that specifically binds PS and fluorescent labelling of the annexin-V enables the flow cytometric detection of externalised PS, and hence apoptotic cells.•When used in conjunction with a live/dead cell discriminator that measures membrane integrity (such as PI, 7AAD, and DAPI), early apoptotic cells (annexin-V positive only) can be distinguished from late apoptotic/necrotic cells (annexin-V and PI/7AAD positive) •The early apoptotic phase can be quite rapid and can often be missed, making it appear that cells are either live or late apoptotic/necrotic.

AnnexinV

17

18

LDH ASSAY

Test principleThe assay is based on consideration that tumor cells possess high concentration of intracellular LDH and the cleavage of a tetrazolium salt when LDH is present in the culture supernatant.

19

.

Functional assays

Evaluate viability by examining the metabolic components that are necessary for cell growth, on the premise that cellular damage will inevitably result in the loss of ability to maintain and provide energy for metabolic function and growth

20

Microculture Tetrazolium Test(MTT Assay) • MTT assay a quantitative colorimetric assay for

measuring cellular growth, cell survival and cell Proliferation based on the ability of living cells.

• Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) a tetrazolium salt is reduced to purple formazan by mitochondrial dehydrogenase of living cells in which tetrazolium ring gets cleaved in mitochondria . 

21

Receipt of Tissues Tissue Shipment Dosing

Rinsing UVA Exposure and Post-Treatment Incubation

22

Transfer to MTT

Extraction in Isopropanol and Plate Reading

23

Compare with MTT assay and XTT assay

Culture cells in a MTP for a certain period of time (37 )℃

MTT assay XTT assay

Prepare labeling mixture

Incubate cells (0.5-4 h, 37 )℃

Add solubilizing solution(Isopropanol) and incubate

Measure absorbance using an ELISA reader

Add XTT labeling mixtureAdd MTT labeling reagent

Insoluble formazan Soluble formazan

24

Example: MTT and XTT

MTT XTT

Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002. Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for

quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73

25

Other functional assays are:

Crystal violet dye elution (CVDE) Acid phosphatase (AP) assay Alamar blue oxidation-reduction assay Neutral Red (NR) assay [3H]-thymidine and BrdU incorporation

26

DNA labeling assay Detection of DNA synthesis in proliferating cells relies on the incorporation of labeled DNA precursors into cellular DNA during the S phase of the cell cycle.

The labeled DNA precursors, usually pyrimidine deoxynucleosides, are added to cells during replication, and their incorporation into genomic DNA is quantified or visualized after incubation and sample staining.

The same labeled deoxynucleosides can be injected into experimental animals to assay cellular proliferation in specific organs and tissues.

27

[3H]-thymidine and BrdU incorporation

The most common deoxynucleosides used for assaying DNA replication are [3H]thymidine and 5-bromo-2-deoxyuridine (BrdU).

[3H]Thymidine incorporated into DNA is usually detected byautoradiography, where as detection of BrdU is accomplished immunologically, through specific anti-BrdU antibodies.

28

29

Morphological assay

Large-scale, morphological changes that occur at the cell surface, or in the cytoskeleton, can be followed and related to cell viability. Damage can be identified by large decreases in volume secondary to losses in protein and intracellular ions of due to altered permeability to sodium or potassium. Necrotic cells: nuclear swelling, chromatin flocculation, loss of nuclear basophilia Apoptotic cells: cell shrinkage, nuclear condansation, nuclear fragmentation

Example; Morphological feature (Human skin keratinocyte)

Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated human skin keratinocyte(B).

(A) (B)

30

Example; Morphological feature (Human skin fibroblasts)

Fig. Morphological feature of (A) normal human skin fibroblasts, and aging human skin fibroblasts(B).

(A) (B)

31

32

Reproductive Assay• Colony-forming Efficiency

Clonogenic Cell: Defined as a cell with the capacity for sustained proliferation Have undergone a minimum of 5-6 doublings to give rise to colonies containing at least 50 cells

Example; Rat keratinocytes

(A) (B)

(C) (D)

Colony forming Non-colony form-ing

48 hr af-ter

subcul-ture

6 days af-ter

subcul-ture

: colony , : Single cells33

34

35

Preclinical Toxicity Studies• Aimed at predicting

(a) Safe starting dose & dosage regimen for human clinical trials(P1)

(b) The toxicities of the compound, &(c) The likely severity and reversibility of drug toxicities.

• Regulatory requirement : Two acute preclinical toxicity studies1. Rodent (mice) - single- and multiple-dose lethality

studies.2. Non rodent (dogs) - single- and multiple-dose

confirmatory toxicity.

• Cytotoxic & non cyotoxic drugs

36

Tumor model

1. Carcinogen induced models

2. Viral infection models

3. Transplantation Models

4. Genetically Engineered Mouse Models

5. In vivo hollow fibre assay

37

CHEMICAL CARCINOGEN MODELDMBA induced mouse skin papillomas • Two stage experimental carcinogenesis

– Initiator – DMBA (dimethylbenz[a]anthracene),– Promotor – TPA. (12-O-tetradecanoyl-phorbol-13-

acetate)• Mice : Single dose – 2.5 µg of DMBA f/b 5 to 10 μg of

TPA in 0.2 ml of acetone twice weekly. • Papilloma begins to appear after 8 to 10 wks - Tumor

incidence & multiplicity of treatment group is compared with DMBA control group

38

RAT MAMMARY GLAND CA

Mouse skin papillomas

39

Cancer site

Cancer Type Species

Carcinogen

Colon Adenocarcinomas

Rat AOM (azoxymethane)

Prostate Adenocarcinomas

Rat MNU (methylnitrosourea)

Esophagus

Squamous cell carcinoma

Rat NMBA(N-nitroso-methylbenzylamine)

Breast Adenocarcinoma

Mice NMU (N-Nitroso-N-methylurea)

40

Viral infection models• Mouse Mammary Tumor Virus (MMTV) was the first

mouse virus, isolated at Jackson labs as the “non-chromosomal factor” that caused mammary tumors in the C3H strain of mice.

• Some viruses cause cancer via random integration in certain cells

• Some viruses carry cellular oncogenes – Abelson murine leukemia virus – Abl– Moloneymurine sarcoma virus – Raf

• Engineered viruses now used routinely in the laboratory to induce cancer.

41

Transplantation Models• Tumor cells or tissues (mouse or human)

transplanted into a host mouse.• Ectopic – Implanted into a different organ

than the original (typically subcutaneous or kidney capsule)

• Orthotopic – Implanted into the analogous organ of the original tumor.

• Advantages :– Typically cheap, fast & easy to use.– Not covered by patents

42

On EAC Cells by Liquid Tumour Model (Ehrlich Ascites Carcinoma) on (Swiss

albino mice)

EXPERIMENTAL PROTOCOLInduction of ascitic carcinoma - The ascitic tumor

bearing mice (donor) were used for the experiment 12 days after tumor transplantation. The ascitic fluid was drawn using an 18 gauge needle into a sterile syringe. A small amount of tumor fluid was tested for microbial contamination. Tumor viability was determined by tryphan blue exclusion test and cells were counted using haemocytometer. The ascitic fluid was suitably diluted with saline to get a concentration of 10 million cells/ml of tumor cell suspension. 250 µl of this fluid was injected in each mouse by i.p. route to obtain ascitic tumor.

43

The mice were weighed on the day of tumor inoculation and then for each three days. Cisplatin was injected on two alternative days 1st and 3rd day after tumor inoculation (intraperitoneally). The drugs were administered after 24 hours of tumor inoculation and were administered till 9th day intraperitoneally.- On 15th day blood was collected from the animal through the retro orbital plexus to determine the heamatological parameters and lipid profile.

Normal mouse Tumor bearing mouse

Solid tumor model using DLA cell lines

• The DLA(dalton’s lymphoma ascitic) bearing mouse was taken 15 days after tumor transplantation. The ascitic fluid was drawn using a 18 guage needle into a sterile syringe. A small amount was tested for microbial contaminationTumor viability was determined using trypan blue exclusion method and cells were counted using haemocytometer.

• The ascitic fluid was suitably diluted in phosphate buffer saline to get a concentration of 106 cells per ml of tumor cell suspension.

• Around 0.1ml of this solution was injected Subcutaneously to the right hind limb of the mice to produce solid tumor.

• Treatment was started 24 hours after tumor inoculation. Cisplatin was injected on two alternate days i.e. the 1st and 3rd day. Extracts were administered till 9th day intraperitonially.

45

Transplantation Models : Human Tumor Xenografts

• Athymic “nude”mice developed in 1960’s• Mutation in nu gene on chromosome 11• Phenotype: retarded growth, low fertility, no fur, immunocompromised

– Lack thymus gland, T-cell immunity

• First human tumor xenograft of colon adenocarcinoma by Rygaard & Poulson, 1969

46

Xenograft Study Endpoints• Toxicity Endpoints:

– Drug related death– Net animal weight loss

• Efficacy Endpoints:– Tumor weight change– Treated/control survival ratio– Tumor growth assay (corrected for tumor

doubling time)

47

Human Tumor XenograftsAdvantages• Many different human

tumor cell lines transplantable

• Wide representation of most human solid tumors

• Good correlation with drug regimens active in human lung, colon, breast, and melanoma cancers

Disadvantages• Brain tumors difficult to

model• Different biological

behavior,metastases rare– Survival not an ideal

endpoint: death from bulk of tumor, not invasion

• Shorter doubling times than original growth in human. Difficult to maintain animals due to infection risks

48

49

In Vivo Hollow Fibre Assay• In vivo screening tool implemented in

1995 by NCI• 12 human tumor cell lines (lung, breast,

colon, melanoma, ovary, and glioma• Cells suspended into hollow

polyvinylidene fluoride fibers implanted IP or SC in lab mice

• After in vivo drug treatment, fibers are removed and analyzed in vitro

• Antitumor (growth inhibitory) activity assessed

50

51

In Vivo Hollow Fibre Assay

Subcutaneous Hollow Fibre implants

52

• Currently, pharmaceutical firms spend a large amount of money

on the compound efficacy and cytotoxicity test.

• There is still a 78% failure rate for all drugs, which may be

devastating to developing companies.

• Effective compounds in vitro may be non-effective in vivo for

many reasons, including differences between in vitro and in vivo

target biology, interrelated biochemical mechanism,

metabolism, poor penetration into solid tissues, etc.

2-D and 3-D Cell-based Assays in Drug Screening

53

• Currently, almost all cell-based assays or biosensors are

developed in 2-D culture systems, although conventional 2-D

cultures usually suffer from contact inhibition and a loss of

native cell morphology and functionality.

• In comparison with 2-D cultures, 3-D cell models create a

more realistic representation of real human tissues, which is

critical to many important cell functions, including

morphogenesis, cell metabolism, gene expression,

differentiation and cell-cell interactions.

54

55

• In studying cytotoxicity and drug testing, maintaining cells in

their native functional state in a proper 3-D environment

would improve predictions and have the potential to reduce

clinical trial failures.

• Therefore, although designing 3-D models is much more

complicated than designing the 2-D counterparts, cell- and

tissue-based assays with a 3-D model are superior and are the

assays of choice for HTS (High-throughput screening) of drug

cytotoxicity.

56