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Anticancer drug screening
 

Anticancer drug screening

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Anticancer drug screening Anticancer drug screening Presentation Transcript

  • Anticancer Drug Screening ‘ Cancer’: a fatal disease of uncontrolled proliferation of genetically altered cells. In all known cases, cancer cells are derived from the repeated divisions of a mutant cell. Some of these mutations may be due to the effects of carcinogens, such as tobacco smoke, radiation, chemicals or infectious agents. A few other cancer-promoting mutations may be acquired through errors in DNA replication. Genetic alterations that render a normal cell cancerous usually arise in two classes of genes termed the oncogenes and tumors suppressors. Activate the cancer promoting oncogenes and/or inactivate the tumor suppressor genes. Cancer
    • An abnormal growth of tissue resulting from uncontrolled, progressive proliferation generates a mass of cells is called tumor.
    • Sometimes, such a mass can be self-limiting which does not invade neighboring tissue and hence pose no danger to life- called benign tumors .
    • If a tumor continues to grow at its original site, breaches the basement membrane and gets into the circulatory system, whereby these cancer cells migrate to new sites in the body and initiate secondary tumors. This process is called metastasis, and such tumors are called malignant . Thus cancer always refers to malignant neoplasms, whereas tumors can be either benign or malignant.
    • Cancer of almost all tissues is known today. The cancers of the epithelium are called Carcinomas. Those originating in the cells of connective tissues, like bones, cartilages, blood vessels or muscle are called Sarcomas. Cancers that start in blood forming tissues such as bone marrow are called Leukemia, and those that begin in cells of the immune system are termed as Lymphoma and Myeloma. Cancers of the central nervous system that arise in the tissues of the brain and spinal cord include Gliomas and Astrocytomas.
    • Chemotherapy, surgery and radiotherapy have remained the main attempts of cancer treatment. Chemotherapy includes use of cytotoxic drugs to attack the cancer cells, most often exploiting the property of rapid proliferation of the cancer cells.
    • Usually, a combination of all the three treatment regimes is used against any given cancer for maximum benefit.
  • Need for Novel Anticancer Agents
    • 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
  • In vitro cytotoxicity studies
    • Brine Shrimp Lethality Bioassay of compounds
    • Brine shrimp (artemia) eggs – hatched in a hatching chamber containing sea water- hatched Nauplli (10 nos) pippeted in to a vial containing suitable dilutions of the compound – vials were maintained under illumination- after 24 hrs survivors were counted.
    • 2. Potato disc assay ( Agrobacterium tumefaciens ) 
    • 3. Trypan blue exclusion assay on murine cell lines.
    • Based on the principle that the living cell membrane has the ability to prevent the entry of the dye. Hence they remain unstained and can be easily distinguished from the dead cells, which take the dye.
    • 4. Cytotoxicity assays on panel of human cancer cell lines
    • MTT-assay
    • SRB- assay
    • MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] is taken up by the viable cells and reduced to formazan by the mitochondrial enzyme succinate dehydrogenase. Formazan which is formed, is a purple coloured water-insoluble product that is largely impermeable to the cell membranes, thus resulting in its accumulation within the healthy cells, which is solubilized by adding dimethyl sulphoxide (DMSO). The optical density (OD) of purple coloured solution was read using a conventional ELISA plate reader at 540 nm (maximum absorbance).
    • SRB is a bright pink aminoxanthene dye with two sulfonic groups. Under mild acidic conditions, SRB binds to protein basic amino acid residues in trichloroacetic acid (TCA) fixed cells to provide a sensitive index of cellular protein content that is linear over a cell density range.
    • Hoechst 33342 Staining for Scoring Apoptosis
    • Induction of apoptosis is one of the important criteria for anticancer activity. Different morphological changes will occur during apoptosis like nuclear condensation, DNA fragmentation, membrane blebbing etc. In order to observe the alteration or morphological changes in the nucleus, specific fluorescent dyes which re-emit visible light upon absorbing ultraviolet light are used.
    • Cell Cycle Analysis by Flow Cytometry (propidium iodide staining)
  • Figure-1: Normal (Control) HeLa cells showing no signs of necrosis or apoptosis Figure-2: Effect of Doxorubicin on HeLa cells showing cell shrinkage, nuclear condensation Figure-3: Effect of compound - 6 on HeLa cells showing apoptosis and cell necrosis Figure-4: Effect of Com -4 on HeLa cells showing cell shrinkage and nuclear condensation
  • Cell Cycle Analysis Control R6 R3 R4 R5 DOX R6 R3 R4 R5 Phase Sub-G 0 G 0 -G1 S G 2 -M Phase Sub-G 0 G 0 -G 1 S G 2 -M % cells 1.15 74.27 6.34 18.65 % cells 2.22 16.62 4.01 76.76 Figure-7: Control HeLa cells showing normal cell cycle Figure-8: Doxorubicin showing prominent G 2 -M phase arrest
  • BE-7 R5 R2 R3 R4 BE-4 R6 R3 R4 R5 Phase Sub-G 0 G 0 -G 1 S G 2 -M Phase Sub-G 0 G 0 -G 1 S G 2- M % cells 27.87 44.46 9.73 17.91 % cells 0.62 88.41 1.04 9.72 Figure-9: Com-6 treated HeLa cells showing sub G 0 (apoptotic) arrest Figure-10: Com-4 treated HeLa cells showing prominent G 0 -G 1 phase arrest
  •  
  • In Vivo Study Goals: Animal Models
    • Efficacy: Proof of therapeutic principle
    • Toxicology: Toxicity profile
    • Practical Issues:
      • Animal pharmacokinetics and pharmacodynamics
      • Starting dose and schedule for clinical trials
  • Animal Models Proof of Principle
    • Animal screening is too expensive for routine use
    • Efficacy in animal models of specific disease states occurs after in vitro studies
    • Evaluation of therapeutic index
      • Toxicity versus efficacy
  • Ideal Animal Model
    • Validity
    • Selectivity
    • Predictability
    • Reproducibility
    “ There is no perfect tumor model”
  • Animal Models in Cancer
    • Spontaneous tumors
      • Carcinogen-induced (N-nitrosodiethyl amine)
      • Transgenic/gene knockout animals: p53, RB, etc
    • Transplanted tumors
      • Animal tumors: Lewis lung, S180 sarcoma, etc
      • Human tumor xenografts: human tumor lines implanted in immunodeficient mice (current NCI standard in vivo efficacy testing system)
      • Human tumors growing in vivo in implantable hollow fibers
  • IN VIVO ANTICANCER ACTIVITY
    • On EAC Cells by Liquid Tumour Model (Ehrlich Ascites Carcinoma) on (Swiss albino mice)
    • EXPERIMENTAL PROTOCOL
    • Induction 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.
    • The mice were weighed on the day of tumor inoculation and then for each three days. Cisplatin was injected on two alternative days 1 st and 3 rd day after tumor inoculation (intraperitoneally). The drugs were administered after 24 hours of tumor inoculation and were admistered till 9 th day intraperitoneally.
    • - On 15 th day blood was collected from the animal through the retroorbital plexus to determine the heamatological parameters and lipid profile
    • Preparation of the Drug
    • The solution of the compounds were prepared by suspending them in 4% acacia and administered intraperitoneally daily for a period of 9 days from the 2 nd day of tumor inoculation in the volume of 0.1ml/10g mouse.
    Group No. Treatment Dose Route 1 Control-S (2% gum acacia) Equivolume i.p 2 Cisplatin 3.5 mg/kg i.p 3 Control-A (2% gum acacia) Equivolume i.p 4 Compound-A 50 mg/kg i.p 5 Control-B Equivolume i.p 6 Compound-B 50 mg/kg i.p 7 Control-C Equivolume i.p 8 Compound-C 50 mg/kg i.p
    • Parameters monitored
    • 1) % Decrease in weight variation compared to control.
    • 2) Median survival time (MST) and percentage increase in lifespan (%ILS).
    • 3) Mean survival time (MEST) and percentage increase in lifespan (%ILS).
    • 4) Cell viability test. (% Survivors of malignant cells in ascitic fluid).
    • 5) Haematological parameters
    • a. Total W.B.C. and differential leukocyte counts.
    • b. Total R.B.C. and Hemoglobin content.
    • 1. % Increase in weight as compared to day “o”weight
    • Upon weighing the animals on the day of inoculation and after once in 3 days in the post inoculation period the % increase in weight was calculated as follows
    • 2. Median survival time and increase in life span [%ILS]
    • Total number of days an animal survived from the day of tumor inoculation was counted. . Subsequently the Median and Mean survival times were calculated. The %ILS was calculated as follows.
    • Cell viability test
    • Tryphan blue exclusion test is based on the principle that the living cell membrane has the ability to prevent the entry of the dye. Hence they remain unstained and can be easily distinguished from the dead cells, which take the dye.
    • On day15 after tumor inoculation 0.1 ml of ascitic fluid was taped and subjected to Tryphan Blue Assay to assess the number of viable tumor cells. Equal volumes of EAC cell suspension and 0.1% Tryphan blue solution were mixed thoroughly. The diluted suspension was charged into Haemocytometer. The viable (unstained) were counted in WBC chamber under microscope and mean number of cells in 4 chambers were counted.
    • Total number of cells =Mean number of cells x dilution factor x10 4
    • Hematological parameters
    • In order to detect the influence of synthetic drugs on the hematological status of EAC bearing mice, comparison was made amongst four groups of mice for each extract on the fourteenth day after transplantation. These comprised:
    • Normal mice
    • Tumor bearing mice
    • Tumor bearing mice treated with Cisplatin
    • Tumor bearing mice treated with Compounds.
    • Blood was drawn from each mouse from retro orbital and the following parameters were evaluated:
    • White blood cell total count
    • Differential leukocyte count
    • Red blood cell total count
    • Biochemical parameters
    • Glutathione (GSH)
    • Glutathione –S-transferase
    • Lipid Profile
    • Total cholesterol
    • Triglycerides
  • 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 contamination
    • Tumor 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 10 6 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.
  • Parameters Monitored
    • Tumor volume
    • The diameter of developing tumor were measured using a vernier calipers at three days interval for one month and tumor volume was calculated using the formula
    • V=0.4 ab2
    • Where a and b represents the major and minor diameters respectively.
    • Tumor Weight
    • At the end of the fifth week animals were sacrificed under anesthesia tumor was excised and weighed
    • % Inhibition was calculated = 1-B/A X 100
    • A= average tumor weight of control group
    • B= average tumor weight of treated group
  • 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
  • Murine Xenograft Sites
    • Subcutaneous tumor (NCI method of choice) with IP drug administration
    • Intraperitoneal
    • Intracranial
    • Intrasplenic
    • Renal subcapsule
    • Site-specific (orthotopic) organ inoculation
  • Xenograft Study Endpoints
    • Toxicity Endpoints:
      • Drug related death
      • Net animal weight loss
    • Efficacy Endpoints
      • Tumor growth assay (corrected for tumor doubling time)
      • Treated/control survival ratio
      • Tumor weight change
  • Xenograft Tumor Weight Change
    • Tumor weight change ratio (used by the NCI in xenograft evaluation)
    • Defined as: treated/control x 100%
    • Tumor weight in mg = (a x b 2 )/2
      • a = tumor length
      • b = tumor width
    • T/C < 40-50% is considered significant
  • Xenograft Advantages
    • Many different human tumor cell lines transplantable
    • Wide representation of most human solid tumors
    • Allows for evaluation of therapeutic index
    • Good correlation with drug regimens active in human lung, colon, breast, and melanoma cancers
  • Xenograft 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
  • Other Animal Models
    • Orthotopic animal models: Tumor cell implantation in target organ
      • Metastatic disease models
    • Transgenic Animal Models
      • P53 or other tumor suppressor gene knockout animals
      • Endogenous tumor cell development
  • In Vivo Hollow Fiber 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 and SC in lab mice
    • After in vivo drug treatment, fibers are removed and analyzed in vitro
    • Antitumor (growth inhibitory) activity assessed
  • Thank you