The target population of this study is newly diagnosed ALL and AML children, breast and prostate newly diagnosed cases and specific ALL, AML, breast and prostate cell lines.
Aim1: To cultivate breast and prostate cancer cell lines in the lab and use them for research.
Aim2: To establish primary cell culture from human breast and prostate cancer tissue, in vitro.
1- The tumor specimen from breast and prostate cancer tissue will be removed at the excision operation and transferred immediately to the laboratory in Hanks' balanced salt solution.
2- Tissue samples will be minced and then dissociated overnight at 4 °C with 0.1% collagenase or trypsine in phenol red-free DMEM/F12 medium (1 g tissue/ml) supplemented with 10% fetal bovine serum (FBS) and an antibiotic-antimycotic (100 unit/ml penicillin G sodium, 100 Ìg/ml streptomycin sulfate and 0.25 Ìg/ml amphotericin B
3- Cell culture: The digested mixture will be centrifuged at 200 xg for 5 min at 25 °C. The cell pellet was re-suspended and allowed to settle by gravity. The sedimented cells, mostly epithelial cells, will be washed three times with phenol red-free DMEM/F12 medium and allowed again to settle by gravity. The sedimented epithelial cells will be resuspended in phenol red-free low calcium DMEM/F12 (0.04 mM CaCl2 ) supplemented with Chelex-100 treated FBS (10%).
4- Cell treatments: All experiments will be performed on primary cultured normal human breast epithelial cells not propagated beyond the third passage. Breast epithelial cells will be allowed to grow to 80% confluence, before being passaged using 0.5% trypsin – 5.3 mM EDTA with a 1/4 splitting ratio. The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not.
Aim3: To optimize chemosensitivity test of the chemotherapy drugs that are used in the treatment of breast and prostate cancer.
The cytotoxicity of drugs on cancer cells will be determined by the MTT assay. Cells {3000/well} will be plated in 100 mL of medium /well in 96-wellplate then incubated overnight. After incubation overnight, chemical drugs will be added in various concentrations 20, 40, 80,160 and 320 mg/ml, then the morphology of cells will be we mentored and test the cytotoxicity of cells by the MTT assay.
Experimental Design for Aim5, 6, 7, 8, 9, 10:
1- In the first (preparatory) experiments, the extracts will be prepared from the desired plant parts.
2- The working extract concentrations will be then determined by testing an array of extract dilutions on one cell type.
3- The working extract concentrations will be tested for each plant against each of the three cell lines in terms of cellular proliferation.
4- Also the used chemotherapy drugs against the leukemic cells in Gaza Strip will be tested alone, in combination with each plant and in combination with both plants against the three cell lines in terms of cellular proliferation.
5- The effects of the same extracts working concentration and the chemotherapy drugs will be further analyzed by viability assay to determine the type of cellular effect.
6- Observation of the morphological changes will be carried out in parallel to the viability assays.
7- The data will be statistically analyzed and comparison of the results from different methods will be done and reported. Also comparison between the effects of the plants and the chemotherapy drugs will be done.
Cytotoxicity Assay
1- Cells will be seeded into 96-well microplates (1×105 cells well?1) for 24 h and then treated with different concentrations of plant extracts as well as the chemotherapy drugs for 48 and 96 h. Cytotoxicity on the three cell lines will be assayed by means of commercially purchased kit that evaluates the lactate dehydrogenase (LDH) activity released into the culture medium by lysed cells. The product was measured colorimetrically using an ELISA microplate reader.
2- The percentage viability of cells corresponds to (100?% of cytotoxicity).
Evaluation of Viability and Proliferation
A commercially purchased colorimetric kit will be used to determine the proliferation activity of cells. Cells will be plated at a density of 1×105 cells well?1 in a 96-well cell culture plate and treated with plant ethanol extracts for either 2 or 4 days. The method is based on the ability of the mitochondria of metabolically active cells to convert tetrazolium salt into a dark red formazan product which is measured colorimetrically using an ELISA microplate reader. The intensity of the dye is proportional to the number of metabolically active cells.
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