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The prostate is a small gland located around the urethra and involve in the secretion of seminal fluid but it is known to cause problems more than beneficial function. Prostate cancer is slow growing, possibly fetal disease usually found in men over 60 years old. In UK, 34,000 men are diagnosed with prostate cancer each year. The most important risk factor to get prostate cancer is the age in addition to other factors including race, genetics, hormones and environment. The incidence is higher in some ethnic group than others. For example, black African-American men are most likely to develop prostate cancer than white men. The environmental factors include high fat diet and caloric intake, geographic location, and a sedentary lifestyle (Cancer research UK, 2010).
The prostate tumour can be either primary tumour is when the tumor has started growing inside the prostate itself or the metastasis tumour is when the cancer cells have metastasized from the prostate and spread to other part of the body. The most common places for the spreading of prostate cancer is the bones such as the spine, pelvis, thigh bone and ribs but other places can be affected as well like bone marrow, lymph nodes and in rare cases lungs,brain and liver (Ingrid et al., 2005).
The early prostate cancer is usually without any symptoms but the prostate gland often gets larger due to a non-cancerous condition called benign prostatic hyperplasia or hypertrophy (BPH). The symptoms of benign enlargement of prostate gland and malignant tumours are difficulty and more frequently passing urine associated with pain and sometimes haematuria (Ozden, et al., 2007).
The symptoms of metastasized prostate cancer are the same as mentioned above but in addition the symptoms depend which part of the body is affected and causing additional symptoms. The most common symptom is bone pain, especially vertebrae, pelvis, and ribs. If the spine bones are affected the symptoms are weakness and tingling or numbness in the legs, due to the pressure on the spinal cord by the tumour (Ingrid et al., 2005).
|The development of metastases requires that a cancer cell must go through a sequence of steps involving complex interactions between tumor cells and the host. Cells from primary tumors must separate, invade the stromal tissue, and then penetrate vessels by which they start spreading to reach secondary sites (LaTulippe et al., 2002).
The diagnosis of prostate cancer has improved too much with many new approaches involves genetic and molecular techniques but the widely used method of diagnosis is Prostate-specific antigen (PSA) which is a protein produced by the prostate and high levels are associated with prostate cancer. This test has poor tumour specificity |and some cases with prostate cancer will have a normal PSA. Another limitation of PSA is the difficulty in differentiation between prostate cancer and benign prostate disorders in slightly elevated PSA value. Another physical examination includes Digital rectal examination (DRE) of the prostate, via the rectum, to recognize any enlargement and should be used along PSA to increase the probability of detecting prostate cancer. Also, another diagnostic aid is the Trans Rectal Ultra Sound (TRUS) which is used for the visualization of the prostate malignant lesions and as a guide for accurate prostate biopsy. The biopsy is another diagnostic tool if there is susceptibility of cancer by initial tests but sometimes even if there is cancer in the prostate may not be found by biopsy.
As shown from the above diagnosis methods, there have some limitation which may miss out some cases of prostate cancer. This has lead to investigate in finding e new, more reliable tests and biological markers such as kallikrein, hK1 and hK2, Insulin-like growth factor-1 (IGF-1) and IGF bindingprotein- 3 (IGFBP-3). These markers are playing a role in the regulation and proliferation of prostate cancer.
The future for diagnostic tools is by the latest technology, gene chip (DNA microarrays) and proteomics which have opened up a new diagnostic approach to accelerate the identification of new molecular diagnostic targets. Gene chip analysis allows the pattern of gene expression in tumours to be profiled and monitoring thousands of genes at the same time. This technique can be used for the comparison of the level and the types of genes expressed in tumours and in normal tissue, and to follow changes in prostate cancer cells during the progression of the disease. The Proteomics technique involves the analysis of the slight changes in proteins in tissue, serum or urine and to identify changes in tumours against normal tissue samples, and to differentiate between prostate cancer and benign conditions.
In prostate cancer, the grading refers to the appearance of the cancer cells of the biopsy| sample under the microscope. The Gleason system is the most commonly used which looks at the pattern of cancer cells within the prostate and the biopsy samples which are graded to get the Gleason score of between 2 and 10. The lower the Gleason score, the lower the grade of the cancer. The stage of a cancer is a term used in describing the tumour size and whether it has spread beyond its original area of the body. This is helpful in deciding the most suitable treatment (Watson & Schalken, 2004).
2) Describe and evaluate the gene expression methodologies:
In this article, there are two gene expression methods have been used; the main one is microarrays for the analysis of gene expression and the second on is the quantitative reverse transcriptase-PCR technique for the validation of microarray-based expression analysis for some of the expressed genes.
Microarray is a high-throughput and multipurpose technology used to study the expression of many genes at once. It involves placing thousands of gene sequences in known locations on a microscope glass slide called a gene chip. This help in the study of the active genes and inactive genes in different cell types and for thousands of genes are at any time (Churchill, 2002).
Microarray technology helped in understanding more about many different diseases, including heart disease, infectious diseases, mental illness, and cancer. For example, microarray technology helped in classifying the types of cancers based on the patterns of gene activity in the tumor cells which eventually will help in finding the suitable and effective treatment. In addition, the microarray is used for the analysis of mutations and in gene expression studies.
There main two types of microarray are Affymetrix DNA chips and spotted microarrays. The research article used the Affymetrix DNA chips by using oligonucleotide arrays with 63,175 probe sets. This oligonucleotide array format is a complicated platform of microarray technology which is known also as Gene Chips (Abhilash, et al., 2009).
The principle of the microarrays technique is based on the available database of over 40,000 of gene sequences and the activation of gene which will produce copy of the gene segments resulting in messenger RNA (mRNA). The mRNA produced by the cell is complementary, and then will bind to the original part of the DNA strand from which it was copied.
To find out which genes are turned on and which are turned off in a given cell, messenger RNA present in that cell must be collected first. Then mRNA is labeled using a reverse transcriptase enzyme (RT) that generates a complementary cDNA to the mRNA. During that process fluorescent nucleotides are attached to the cDNA. Then tumor and the normal samples are labeled with different fluorescent dyes. Next, labeled cDNAs to be replaced onto a DNA microarray slide. The labeled cDNAs that represent mRNAs in the cell will then hybridize to their synthetic complementary DNAs attached on the microarray slide, leaving its fluorescent tag. Â Finally by using a special scanner the fluorescent intensity for each spot or areas on the microarray slide is measured.
The very active gene produces many molecules of messenger RNA, thus, more labeled cDNAs, which hybridize to the DNA on the microarray slide and produce a very bright fluorescent area. The less active genes produce fewer mRNAs, and as a result less labeled cDNAs, which results in dimmer fluorescent spots. If there is no fluorescence, it means that none of the messenger molecules have hybridized to the DNA, which indicate inactive gene (Gundogdu & Elmi, 2010).
Fig1. Microarray technology Cited from (www.genome.gov)
In this research article, the used of microarray technique has some advantages comparing with other gene expression techniques. These advantages are speed, high specificity and reproducibility. The Disadvantage of the microarray is the variation between individuals in gene expression, expensive and the analysis or the interpretation of the result is quite difficult.
To expand more about what the research article has performed in this technique, the samples after being stained with H&E stain then examined to cut apart cells of interest, the RNA was extracted from the tissue by using quanidinium isothiocyanate-based buffer which act to provide pure and quality intact RNA by suppress Rnase activity (Piercenet, 2010).
The RNA integrity was evaluated by denaturing agarose gel to check the overall quality and yield. Then, T7-promotertagged oligo(dT) primer used for the Complementary DNA synthesis from total RNA. After that is the hyperidization step which is used to set of known DNA specimens arrayed on Test2 arrays (Affymetrix, Santa Clara, CA). Then, Streptavidin phycoerythrin used to detect DNA on a microarray as it is used in applications that require either high sensitivity or simultaneous multicolor detection. The addition of Anti-Streptavidin has been optimally labeled to provide maximum fluorescence colouration. Gene expression was carried out using Affymetrix U95 human gene arrays with 63,175 probes.
Quantitative Reverse Transcriptase-PCR was performed using the Light Cycler thermal cycler system in which 50-100 ng of total RNA was used followed by steps of reverse transcription, denaturation, annealing and extension. The use of this technique was for the validation of the microarray by selecting some differentially expressed genes for transcript levels measurement. Quantitative Reverse Transcriptase-PCR is a sensitive technique for mRNA detection and quantification (Genome, 2010).
3) Critically review the contribution of this article to the current understanding of the relevant human disease:
The research conducted by (LaTulippe et al., 2002) is ''Comprehensive Gene Expression Analysis of Prostate Cancer Reveals Distinct Transcriptional Programs Associated with Metastatic Disease" which was done by performing an analysis and identification of the different gene expression in primary and metastatic prostate cancers. It is important to have more understanding about the prostate metastasis stage which has a clinical manifestation of spreading to other part of the body and especially to the bones (Schmitz, 2009).
This research aimed to identify the genes, gene expression profile, and biological pathways that can lead to metastasis which will be of important value in improving tumor classification and treatment.
The research article in a prospective aspect has started the analysis first by identifying the different expressions of genes between all the samples of primary and metastatic tumors which have shown a very strong possibility to have different expression profiles. Then the Functional features including the molecular and biological function of expressed genes was evaluated in which different expressed genes are reflecting different biological functions between the primary and metastasis stages.
The study article reveals that there is an expression value for more than 3,000 tumor-intrinsic genes differed by at least 3-fold between primary and metastatic tumors. Also, the gene expression in prostate cancer metastasis stage is expressed by many genes involved in cell cycle regulation, DNA replication and repair, or mitosis including many genes, such as RFC5, TOP2A, RFC4 and MAD2L1. Also, genes involved in signaling and signal transduction, cell adhesion and migration or extracellular matrix including HMMR. In addition, it has been found that metastasis involved genes that playing a role in transcriptional regulation, signaling, signal transduction, cell structure, RNA splicing and motility. This finding is of great help as indicates that many of these gens expressed are important for the cell activity, proliferation, microenvironment interaction, synthesis, function of gene product. Theses cell activities can be aimed for the suitable diagnosis and proper treatment of prostate tumour.
One of the important finding in this research is the overexpressions of genes that are playing a role in cell functional pathways. The MYBL2 activates CDC2 gene expression in proliferating fibroblasts and CDC that induce cell entry into mitosis. The identification of these genes will eventually help in diagnosis and treatment. This has contributed to some extant to the new research finding (BioMed, 2008) in the treatment by chemopreventative and chemotherapeutic which involve NFÎºB that control DNA transcription (Brasier, 2006).
In addition, the well differentiated and recognized expressed genes in the prostate cancer have a great contribution to the gene therapy trials. By using a viral delivery vector (adenovirus), the gene product will act on immune system stimulation and destroying the tumor cells. This approach requires time to be achieved with the most effective and safest treatment for the prostate cancer (Freytag et al., 2007).
The research article make to some extent more perceptive on the different genes involved in the progression of prostate cancer and in connection with other researches done for the DNA methylation and silences of gene (Yegnasubramanian et al., 2004; Xiang & Dolan, 2008). The understanding of these approaches of gene expression and silencing in prostate metastasis, suitable and more effective treatments can be obtained to activate them and will be helpful in preventing the development and progression of the prostate cancer. For instance, DNA methylation plays a role during development by regulating gene expression during the prostate cancer and DNA is released from the cancer cell which can be investigated for the presence of methylated genes. This can be used for the diagnosis and screening of prostate cancer (Das et al., 2006).
The positive aspect in the research is the results of gene expression obtained from oligonucleotide array has been validated by selecting some differentially expressed genes for the determination with quantitative reverse transcriptase-PCR technique and results were found to be the nearly the same. To a great extent, this validation confirms gene expression profiles in metastatic stage of prostate cancer. In addition study done by Dallas et al., 2005 indicated that, there is generally a strong correlation between QRT-PCR and microarray when identical transcripts are targeted by these two methods.
In this study, the association between the androgen receptor sensitivity and metastasis cancer was not taking into consideration during the selection of the samples with metastasis cancer. This can affect the accuracy of the gene expression that reflects the biology of the metastasis stage as the development of metastasis cancer has a great correlation with the androgen signaling and sensitivity. Therefore, the selection of samples should be correlated with previous history and if patient has undergone androgen ablation therapy and whether progress of disease has been shown at the time of androgen ablation (Chandran, et al., 2007).
In the other hand, the article stated that, there weren't many similar studies done earlier due to the low number of samples well preserved and suitable for the analysis during the metastasis stage. This is to some extent true however more samples would be better for more accuracy. Also, using oligonucleotide array with high number of probe set for the analysis has a positive aspect of the research as the use of microarray technology helps to great extend, the fast and more reliable analysis of thousands of genes expression, and more tissue samples (Foley et al. 2004).
Furthermore, in this research, the use of noncancerous prostate samples as controls of non expression which can help in the differentiation between the normal expression, primary and metastasis expression which is practically useful.
In addition, the possibility of contamination by the nonneoplastic prostate tissue from the primary tissue can lead to difference in the differentiation between the primary and metastatic tumors. To overcome this, the research article has an additional confirmation by comparing these results with the nonneoplastic prostate expression data. Then the difference of remaining gene expression expected to be related to tumor cells.
This article research has identified large number of expressed sequence tag (EST) clusters which were highly differentially expressed between primary and metastatic prostate cancers. These EST has been found to play important biological roles to in which by knowing the EST location, the corresponding gene function can be known (Shivashankar, et al 2006). Also, the ESTs identification has helped in the design of very accurate probes for microarray gene expression technique (GEN, 2005).
To the extent that this research is very helpful, the results and information of the research provide insights into the importance of prostate gene expression in the metastasis stage in prostate cancer. This information were used by the subsequent researchers in the progress of developing better diagnosis and prognosis indicators and development of treatments as well.
4) Discuss other approaches that the authors could have adopted to address the research question:
The article research used microarray which is quite expensive method despite the other advantages of being fast, more reliable and thousands of genes can be studied at the same time. However, Real-Time Reverse Transcription PCR (RT-PCR) could have been used by the research article which is also a sensitive and accurate method for the gene expression in prostate cancer but this method is impractical and time consuming since many genes are to be looked into. Also, in RT-PCR, RNA carryover and possible RNA contaminating can lead to false positives result. The research article could have used Serial Analysis of Gene Expression (SAGE) as a technique for the expression which is as same as microarray expensive but very direct and quantitative for measuring transcript abundance and built in with quality control. Even though this technique has some of the disadvantages which are time consuming, should be done with fully sequenced genomes and the 3' ends in some genes can be very polymorphic.
Therefore, the selection of microarray despite the disadvantages of being expensive for the gene expression analysis in prostate cancer was very good and proper choice which eventually leads to more accurate results.
In addition to the above mentioned gene expression techniques, other approaches could have been adopted by the research article, is the selection of samples which should put into consideration the variation in gene expression between human populations and ancestry. This can cause differences in the expression of gens and the susceptibility to diseases and treatment. In prostate cancer Caucasians and African-Americans have significant difference in some of the genes for example, EGFR expression (Zhang and Dolan, 2008). In addition, the history of the patient should be verified during the selection of samples. For example, there is a correlation between patient who has undergone androgen ablation therapy and the development of metastasis cancer (Chandran, et al., 2007).