Research Into The Cancer Biology Biology Essay

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Research into the cancer biology has taken a step further ahead with the identification of putative cancer stem cells in the tumor masses which are responsible for resistance to conventional therapy. The project is carried out to characterize the cancer stem cells CT26 from colon cancer and identify various resistance patterns to doxorubicin.

The aims of this project are:

1-To study the behaviour and growth characteristics of CT26 cells in various media such as DMEM, RPMI and soft agar medium.

2. To study the effect of various concentrations of doxorubicin on the growth of CT26 cells.

3. To culture Doxorubicin resistant cells in different growth media in order to characterize their growth requirements

4. To isolate and characterize CT26 cells using stem cell markers such as CD44,CD133 ,PML,MHC-1,P36 and SOX-2 with the help of PCR and RT-PCR.

This will be done by using the following techniques:

1-Cell culture technique.

2- Soft agar Assay

3-Proliferation Assay for drug resistance.

4- Characterization of stem cell markers by PCR and RT-PCR

2.0 Introduction

2.1 Background

A cancer may be defined as a group of diseases characterized by the uncontrolled growth of abnormal cells which have the ability to spread throughout the body or body parts (Bender and Bender, 2005). There are more than 100 types of cancer and can affect any part of the body (WHO, 2006). In 2004, cancer caused 7.4 million deaths all over the world which account for 13% of deaths globally (WHO, 2006). In the United Kingdom (UK) cancer is among the major mortalities resulting in a quarter of all deaths ( In 2007, almost 298,000 people were diagnosed with cancer in the UK which comes around 489 cases for every 100,000 people ( In the United States, it is the second most common cause of death and is responsible for one in every four deaths (USCS, 2010). According to estimates (USCS, 2010) in 2010, the total annual cost of cancer comes around $263.8 billion and hence signifies the enormous impact it can have on economy.

The etiology of cancer is complex and multifactorial. Many factors ccontribute towards development of cancer such as ultraviolet rays, diet, certain chemicals, drugs, viruses and hormones etc. An understanding of the biology of tumour cells is crucial to formulate various ways of treatment. Despite a number of treatment options devised to manage and treat cancer, researchers have met with only little success with early detection and prevention being the mainstay of dealing with cancer.

2.2 The "Cancer Stem Cell" Concept

The concept of "cancer stem cells" emerged in 1980s which led the scientists to believe that this might be partially responsible for achieving success in cancer treatment (Lin et al, 2008). However as a result of lack of specific markers for their isolation and less advanced technology, not much advancement could be made at that time. Later, Dick and colleagues isolated putative cancer stem cells (CSCs) from acute myelogenous leukemia and led to a series studies for isolation of these cells from various forms of cancer.

Cancer stem cells have been defined as cells within the tumor growth with a tumor which have the potential of initiating a tumor (Sagar, 2007). These cells have the property of self-renewal and form multiple forms of cancer cell lines that form the tumor (Clarke et al, 2006). Experimentally these cells can be identified by their capacity to continuously replicate the generation of growing tumor. Hence, other terms such as "tumor initiating cell" and "tumorigenic cells" have also been used to describe these CSCs (Clarke et al, 2006). This small subset of stem cells have the ability to both divide and increase the population of various types of non-tumorigenic differentiated cells in the tumor cells pool which constitute the bulk of tumor. Moreover, these cells have a slow rate of division and proliferation (Pérez-Caro and Sánchez-García, 2006) (Figure 1).

Figure 1: The cancer stem cell may develop as a result of mutation in specific stem cells, early stem cell progenitors or differentiated cells. Numerous factors in the host microenvironment may also be involved (Source: Bjerkvig et al, 2005)

2.3 Difference between a Cancer Stem Cell and Normal Stem Cell

A normal stem cell has the properties of self-renewal, strict control on cell division and ability to differentiate. However, the cancer stem cells do not have the ability to have control on cell numbers hence causing unchecked increases in these differentiated non-tumorigenic cell populations (Sagar et al, 2007). Thus a cancer stem cell works in a similar way for the growth and spread of tumor but it is not subject to the intrinsic and extrinsic controls to which normal stem cells are subject to (Pérez-Caro, M. and Sánchez-García, 2006).

2.4 Resistance to Current Chemotherapeutic Drugs

If the cancer stem cells theory proves to be true, then it would provide answers to many problems such as lack of response to therapy, resistance to therapy and recurrence (Sagar et la, 2007). Keeping the cancer stem cell hypothesis in view, if such tumor is treated with chemotherapeutic drugs targeting the rapidly dividing cells which will be cancer stem cells in this case and these CSCs are resistant to these drugs then the therapy will prove ineffective and will be followed by relapses (Clarke et al,, 2006) (Figure 2).

Resistance have been shown against a number of chemoptherapeutic drugs such as Gemcitabine, 5-fluorouracil (5-FU), bevacizumab and doxorubicin.

A recent study by Zheng et al (2010) showed that thyroid cancer were resistant to doxorubicin and the tumor continued to progress despite therapy with this drug. These thyroid cells were then characterized by FACS for their content of cancer stem cells, their in vitro sphere-forming capacity and their expression of multidrug resistance transporters of the ABC gene family which may confer drug resistance to the cells. The cells were treated with doxorubicin and then analyzed. It was shown that treatment with doxorubicin killed the non-side (stem cell) population of cancer cells derived from anaplastic thyroid carcinoma cells. As a result, there was an advantage to stem cells and they overgrew in the culture.

Figure 2:

The traditional therapies may shrink the size of the tumour. On the other hand, if the treatment is directed against the cancer stem cells, they are more effective in eradicating the tumour (Source: Sagar et al, 2007)

2.5 Origin of Cancer Stem Cells

The mechanisms and origin of these cancer stem cells have yet to be elucidated although various theories have been put forward. It is agreed that some mutations is the basis of transformation into a cancer stem cells. However it is not clear whether the cancer stem cells are derived mutations in the self-renewing normal stem cells transforming them into cancer stem cell or the normal progenitor differentiated cells have acquired the ability of acting as a stem cell by acquiring self-renewal capacity by mutations (Lin et al, 2008).

2.6 Isolation of Cancer Stem Cells

One problem which arose is that was difficult to recognize these cells with tumor initiative potential as they are very few in number with plating efficiency ranging from 1/1,000-5,000 to 1/1,000,000 (Lin et al, 2008). However, identification of stem cell markers led the researchers to isolate these cells from various other tumors such as CD 44, CD 133, Sca1, and Thy1. It must be kept in mind that these markers are not universally present on all cancer stem cells and vary widely from one organ to another (Clarke et al, 2006).

The initial evidence for the presence of these cancer stem cells came from haematological tumors but they have been now isolated from a variety of solid tumors such as breast, colon, prostate, lungs, pancreas, head and neck, kidney, liver and brain tumors. (Lin et al, 2008).

2.7 Cancer Stem Cell Essays

The cancer stem cells in all organs share the ability of self-renewal and differentiation however the type of stem cell varies significantly from one organ to the next. One common error made while attempting to isolate stem cells is the tendency to apply the stem cell findings from organ and apply them to the other organ. As a result, the properties used for recognition and characterization of stem cells from one organ may differ in the other organ. Same is true for cancer stem cells belonging to different tumors and organ types. The common key features of all stem cells are self-renewal and tumor propagation. For analysis of these cells a serial transplantation in animal models is the gold standard experimental studies. In these studies, the cells are xenografted into a non-obese diabetic mouse with severe combined immunodeficiency syndrome that are then analysed at various time intervals for tumor formation (Clarke et al,2006).

This analysis for tumor transformation is done by checking for the presence of various cell surface markers. Various studies suggest the presence of cell surface markers on surface of tumor cells. For example CD 133 was initially used with success for isolation of stem cells in brain tumors but it was revealed that this is present on normal stem cells and other tumors as well such as colon cancer. The definitive characterization of these stem cell markers in various tumors is essential as they will be the mainstay of any future therapies intended to target CSCs (LaBarge and Bissell, 2008). Other markers like CD 44 have also been studied and Liu and colleagues (2007) have suggested that there is a positive expression of CD44 in breast cancer stem cells as compared to the normal breast cells and this showed a significant association between the presence of this gene and the overall survival rate.

Other stemness markers have also been studied such as SCA 1 have been identified in characterization of stem cells in mammary tumors of BLAB-neu T transgenic mice (Grange et al, 2008).

The studies for identification and characterization of these makers commonly employ flow cytometry technique to recognize these cell surface markers either isolated from the mouse models or maintained in a tissue cell line. The methods employs use of cells which have been tagged by antibodies and use of FACS (Grange et al, 2008).

Other technique that can be used for this purpose is PCR and RT-PCR (Haraguchi et al, 2005).

2.9 PCR and RT-PCR

The characterization of SCSs can also be carried out by PCR and RT-PCr effectively (Haraguchi et al, 2005).

PCR is an in vitro procedure or method which helps amplify definite target DNA sequences found within a source of DNA. Generally PCR is developed to allow selective amplification of a particular target DNA sequence/s within a heterogeneous set of DNA sequences. As the newly synthesised DNA strands act as templates for more DNA synthesis in the following cycles PCR acts as a chain reaction.

(Strachan and Read, 1999). Major advantages of the PCR technique as a cloning method are its rapidity, sensitivity and robustness.

Real-time PCR has been introduced which provides PCR quantification. This permits the researcher to actually see or view the increase in the quantity of the DNA as it is amplified (Wong and Medrano, 2005).

Real-time PCR greatly helps simplify the amplicon identification through providing the means to monitor the accumulation of particular products continuously during the cycling process. The Real-time PCR is also beneficial as analysis through this method can be done without opening the tube which can then be discarded without the risk or danger of dissemination of PCR amplicons or other target molecules in the lab circumstances. This is also efficient, cost-effective and economical. Real-time PCR also covers certain drawbacks of standard PCR, such as, standard PCR formats depend on the end-point analysis. These are not quantitative because the final product yield is not mainly reliant on the concentration of the target sequence in the sample. The Real-time PCR triumphs over this demerit of the standard PCR.

Aims and Objectives

The aims of this project are

1-To study the behaviour and growth characteristics of CT26 cells in various media such as DMEM, RPMI and soft agar medium.

2. To study the effect of various concentrations of doxorubicin on the growth of CT26 cells.

3. To culture Doxorubicin resistant cells in different growth media in order to characterize their growth requirements

4. To isolate and characterize CT26 cells using stem cell markers such as CD44,CD133 ,PML,MHC-1,P36 and SOX-2 with the help of PCR and RT-PCR.

3.0 Experimental Approach

Carcinoma cell line CT-26 will be used to obtain multiple clones of cancer stem cells. These stem cells will be differentiated on the basis of surface markers on them such as CD44,CD133 ,PML,MHC-1,P36 and SOX-2. This characterization of cancer stem cells will be done by real time PCR.

3.1 Cell Culture and Maintenance

The carcinoma cell lines will be revived from storage vial in liquid media. In order to achieve this revival, DMEM media with 10% FCS and RPMI media with 10% FCS and NEEA will be used. Cells will be incubated for 48-96 hours at 37 ° C in an atmosphere of 5% CO2 and then it will be repeatedly sub-cultured to obtain a confluent growth in culture flasks. This confluent growth will be them washed with PBS and will be then trypsinized and incubated for a few minutes.

3.2 Soft Agar Assay

The cells will be then seeded in semi-solid agar medium with 0.4% agar in a 96 well plate. This will be done as the cancer cells have an ability to achieve independent growth in soft agar. Normal cells do not have this ability to grow in semisolid media in a three dimensional manner as do the cancerous or transformed cells. This is superior as drug testing is more effectively carried out in a three dimensional format as compared to monolayer cultures as 3D growth mimics the in vivo cellular environment.

After the agar has solidified, the seeded plates will be incubated for several days to allow cells to proliferate.

3.4 Proliferation Assay for Drug Resistance

Confluent growths obtained in the flasks mentioned above will be used for chemo resistance essay. The trypsinized cells will be counted to obtain about 106 cells. This cell count will be cultured in DMEM and RPMI respectively. Doxorubicin (an anticancer chemotherapeutic drug) will be added in different concentrations to observe the effect of these on growth of cells.

To achieve this, doxorubicin resistance comparative analysis will be done over a range of drug concentrations varying between 100ng/ml to 1000ng/ml. The behaviour and growth patterns of cells will be observed.

Populations of cells resistant to doxorubicin will be re-cultured through another passage. During this passage, various growth nutrients will be added to the different sets of media in which the doxorubicin cells will be cultures. The plates will be incubated again at 37° C in an atmosphere of 5% CO2 for 48-96 hours.

The steps of can be described as under:

Stage I:

CT-26 cells will be cultured under normal culture conditions for 72 Hrs in culture flask with seeding of 750000 cells.

Stage II:

The culture media for these cells will be DMEM media with 10% FCS containing 1000ng Doxorubicin/ml of the media and incubated for 4 hours.

Stage III:

The medium contents will be then drained off and fresh DMEM will be added. This will again be incubated for 48 hours to allow cells to revive themselves.

Stage IV

The doxorubicin resistant cell population will be divided in two parts after trypsinization.

Population I will be incubated under normal media and normal incubation environment for 96 hours.

Population II will be treated with various growth nutrition to observe the effect of these on growth of doxorubicin cells. Any viable cell population thus obtained will be re-cultured in normal medium and normal environmental conditions.

3.5 - RNA extraction and RT-PCR Analysis

The cells will be then analysed for the presence of CD44, CD133, MHC, PML.

Almost 104 cells will be collected in a microcentrifuge tube with 350  µl RLT buffer containing 1% 2-merceptoethanol. The total RNA from these cells will be then extracted using the RNA extraction kit and purity and concentration of the RNA will also be determined.

T7 linear amplification will be applied and this amplified RNA will be then transcribed to DNA to be analysed using PCR and real time PCR.

The results and data thus obtained will be analyzed by using statistical tests to obtain quantifiable and dependable results.