ملخص موضوع البحث:

Cancer is a major public health burden in both developed and developing countries. Cancer can affect blood causing leukemia or affect tissues such as breast, prostate gland and various tissues in the body and causes tissue cancer. Chemotherapy can lower the number of healthy white blood cells, red blood cells and platelets. Some people may not be able to tolerate one particular drug, but do quite well on another. A lot depends on finding the right dose that will be effective against the cancer but still be tolerable for each person. While offering the chemotherapy treatment using different types of drugs depending upon the type of the cancer, sometimes the cancer cells may resist the treatment offered. This is termed as chemo drug resistance. Chemotherapy resistance and sensitivity studies are performed in the developed countries as routine tests to choice the appropriate chemotherapy drug, but in Gaza Strip these studies have never been performed.

In this study we will first propagate breast and prostate cancer cell lines in vitro and then investigate the cytotoxicity and anti- proliferative activity of chemical drugs against breast and prostate carcinoma cultured cell lines, using techniques such as trypan blue stain DNA degradation and MTT assay. Plants have a long history of use in the treatment of cancer, and this field of research is useful because plants have fewer side effects than chemotherapy agents and may be more effective in the treatment of some cases. Therefore, the cytotoxic effects of some chosen medicinal plants will be evaluated against breast prostate cancer  cell lines and the chemosensitivity of the chemotherapy drugs that are routinely used in the treatment of breast and prostate cancer patients will be assessed and the synergistic effect of medicinal plants and chemotherapy drugs will be evaluted. Then the same techniques will be assessed in a primary cell culture using patient’s cancerous tissues or biopsies after propagated in vitro.

Bone marrow cancer is the fifth most common type of cancer in Gaza strip after trachea, bronchus and lung cancers, colo-rectal and anus cancer, breast cancer and Brain and other nervous system cancer (Ministry of Health, 2006). Leukemia is a form of bone marrow cancer in which large numbers of abnormal, poorly functioning white blood cells are produced. There are two major kinds of leukemia, chronic and acute. Acute leukemia is a rapidly progressing disease involving the proliferation and accumulation of immature red, white blood cells or platelets (i.e., blasts and other very early cells). Accordingly, these cells cannot carry out their normal functions. Although leukemia collectively affects approximately 10 times more adults than children, it is the most common type of cancer among children, with ALL accounting for approximately 78% of all childhood leukemia. The most common type of leukemia in adults is AML, followed by CLL, CML and ALL (Rodgers and Young, 2005). We will first propagate Aacute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML) cell  lines in vitro and then investigate the cytotoxicity and anti- proliferative activity of chemical drugs against ALL and AML using techniques such as trypan blue stain DNA degradation and MTT assay. The cytotoxic effects of some chosen medicinal plants will be evaluated against ALL and AML cell lines, the chemosensitivity of the chemotherapy drugs that are routinely used in the treatment of ALL and AML patients will be assessed and the synergistic effect of medicinal plants and chemotherapy drugs will be evaluated. The effect of a combination of the resistant drugs with each plant and both plants will also be assessed.

     أهداف البحث:

Overall objective

·        To facilitate early and effective treatment of breast, prostate, acute lymphocytic and acute mylocytic leukemia patients using primary cell culture, specific cell lines, chemotherapy drug resistant technique and to test the synergistic effect of medicinal plants and chemotherapy drugs against cancerous cell lines and tissues.

Specific objectives

1-      To cultivate breast and prostate cancer cell lines in the lab and use them for research.

2-      To establish primary cell culture from human breast and prostate cancer tissue, in vitro.

3-      To test the antiproliferative effect of some selected medicinal plants against breast and prostate cancer cell line and patients cancerous tissues.

4-      To investigate the synergetic effect of some selected medicinal plants extract against breast and prostate cancer cell lines and patients cancerous tissues.

5-      To investigate the synergetic effect of some selected medicinal plants extract and chemotherapy drugs against breast, prostate cancer cell lines and patients cancerous tissues.

6-      To optimize chemosensitivity test of the chemotherapy drugs that are used in the treatment of breast and prostate cancer.

7-      To test a patient's own ALL or AML cells in the laboratory to chemotherapy drugs that may be used to treat the patient's leukemia.

8-      To measure the apoptotic and the anti-proliferative effect of selected medicinal plant extracts against patients own ALL or AML cells in the laboratory.

9-      To make a comparison between the chemotherapy drugs and medicinal plants extracts according to their effect on patient's own ALL or AML cells in the laboratory.

10-   To investigate the synergetic effect of selected medicinal plant extracts and resistant chemotherd drugs against patients own ALL or AML cells in the laboratory.

    خلفية مشروع البحث:

Leukemia is a disease resulting from the neoplastic proliferation of hemopoietic or lymphoid cells. It results from a mutation in a single stem cell, the progeny of which form a clone of leukemic cells. Often there is a series of genetic alterations rather than a single event. Acute leukemia is a fatal disease if untreated, with a median survival of 3 months or less. The therapy for acute leukemias, both AML and ALL, is separated into 2 phases: the induction phase and the post-remission phase. Induction chemotherapy usually is given over approximately 1 week, with the intent of eradicating all blasts from the bone marrow and peripheral blood. Post-remission therapy is initiated approximately 4 to 6 weeks after induction chemotherapy has ended, once the patient has recovered from the side effects of therapy and their blood counts have begun to normalize. While offering the chemotherapy treatment using different types of drugs depending upon the type of the cancer, sometimes the cancer cells may resist the treatment offered. This is termed as chemo drug resistance. During recent years several mechanisms have been described. In general decreased transport of drug into the cell,  defective  intracellular metabolism of the  drug to its active compound, increased  drug inactivation, enhanced cellular repair mechanisms, altered target molecules,  and multidrug resistance (MDR) by increased drug efflux represent  know mechanisms  of  chemo resistance. For centuries, plants have been used as remedies and treatments of diseases. The Middle Eastern Mediterranean region is rich in plant species; there are about 2,600 species of which many are considered to have medicinal effects. However, there is relatively limited research on medicinal plants in this region.

Cancer is a heterogeneous illness which can originate from many different organs of the human body. However, the most frequent cancer types in the world are lung, prostate, stomach, colorectal, and esophagus in men; and breast, lung, stomach, colorectal and cervical in women. There are two main types of breast cancer: Ductal carcinoma which starts in the tubes (ducts) that move milk from the breast to the nipple and Lobular carcinoma starts in the parts of the breast, called lobules that produce milk. In rare cases, breast cancer can start in other areas of the breast. Breast cancer may be invasive or noninvasive. Many breast cancers are sensitive to the hormone estrogen. This means that estrogen causes the breast cancer tumor to grow. Such cancers have estrogen receptors on the surface of their cells. They are called estrogen receptor-positive cancer or ER-positive cancer. Prostatic adenocarcinoma is the most frequently diagnosed malignancy and second leading cause of cancer death amongst men. Localized prostate cancer can be definitively treated by surgical resection or through radiation therapy. Androgen ablation triggers cell death or cell cycle arrest of prostate cancer cells, and hence remains the primary course of treatment for all patients with metastatic disease. The taxol is proven to be effective against breast and ovarian cancers, which was initially discovered in bark of yew trees. Of the 121 prescription drugs in use today for cancer treatment, 90 are derived from plants. Over the past few years, a number of medicinal plants have been investigated for their quenching activity of specific reactive oxygen species (ROS), such as the hydroxyl radical, superoxide anion, and lipid peroxides. It was reported in literature that selenium (Se) may reduce the incidence of cancer and associated mortality in humans. The search for new sources of biologically active compounds is important for the discovery of new drugs for the treatment of cancer.

  منهجية البحث:

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.

   مساهمة البحث في معالجة قضايا المجتمع أو تطوير وتنمية المعرفة:

Cancer is the leading cause of death worldwide and finding a cure for this disease is always an important objective for human.  One approach to discover novel lead compounds against cancer is the consideration of ancient ethno-medicinal knowledge and the investigation of locally available natural resources. The assessment of the cytotoxicity effect of the medicinal herbs on prostate and breast cancer, ALL and AML, may aid in the introduction of the herbal constituents in the treatment protocols of cancer patients, and may minimize the side effects of the chemotherapeutic agents and maximize the therapeutic effect of such protocols. Also herbal constituents are available and cheap in comparison to the chemotherapeutic agents. A chemosensitivity assay may help in choosing the best drug or drugs for the cancer being treated. This information can affect clinical decision making and can lead to the avoidance of ineffective and potentially harmful chemotherapeutic agents.To my knowledge this study will be the first to deal with the cell culture drug resistance testing and the cytotoxic effect of the medicinal herbs on breast and prostate cancer, leukemic cells in Gaza Strip. Drug resistance testing can affect clinical decision leading to the avoidance of ineffective and potentially harmful chemotherapeutic agents. The proper use of these drugs will both help individual patients and save the health care system a pile of money, a true win-win situation