The critical review will enable you to approach the practical research of your project with a broad understanding of current knowledge of your chosen subject. This will include historical background, reports of experiments, trials and current hypotheses. Investigating and analysing this information will help you to develop personal opinions on the subject and to justify your chosen line of research.
Beclin-1 expression in cervical neoplasia.
1.1 The research problem
A brief overview
This should be a brief section that outlines the subject of the review. You should assume that the reader has little knowledge of the topic.
Autophagy is a cellular catabolic process in which cytoplasm portions are sequestered into a double membrane autophagosome. These join with lysosomes and are subsequently degraded by the lysosomal proteases. On the initial discovery of the autophagic process, autophagy was outlined as a cell survival mechanaism under conditions of limited nutrients. Also, it has been described that autophagy partakes in a cell death pathway which is viewed as an alternative to apoptosis. It is understood that this mechanism is engaged once the cell reaches a point and cannot continue to survive without nutrients.
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The role of autophagy in relation to tumourigenesis is multi-faceted one. In some situations autophagy was deemed as an essential process in the tumour formation process. Conversely, cancer has been shown to develop with the loss of autophagic processes. The protein Beclin-1 is a progenitor of autophagy and has been profiled as a tumour suppressor. Autophagy is a complex process with numerous biological features (Eisenberg-Lerner, 2009).
Autophagy has been researched in laboratories since the 1960â€™s and it was the analysis of the autophagy process in yeast which provided knowledge of the molecular processs involved. Up until recently electron microscopy was used to analysis the process however this is a very time-consuming process and identification can be difficult.
(Autophagy may be outlined in basic steps, cell signalling, the sequestration of cytoplasm, autophagosome formation, lysosomal identification of autophagosome and the degradation step. The autophagosome and the lack of caspase involvement are the defining features of autophagy in comparison to the apoptotic process (Wang, 2003).)
Beclin 1 was initially identified as a protein which interacts with Bcl-2 and is structurally similar to yeast Atg6. After studying Beclin 1 in yeast, mice and plants, it was elucidated that it was present in a variety of species and had tumour suppressing capabilities. The Beclin-1 gene locus (17q21) was found to be frequently deleted in human cancers such as breast, ovarian and prostate proving the tumour suppressor role of Beclin 1. These findings were corroborated by studies on mice. The tumour suppressive function of Beclin 1 is related to positive autophagy regulation. As outlined in figure 1, Beclin 1 binds with the Vps34 complex which activated the complex and initiates autophagy (Eisenberg-Lerner, 2009).
Since autophagy is affected by many stimuli, it is understandable that a number of signalling pathways are involved in its regulation. One of the primary ways is through a molecule called mTOR which is an inhibitor of autophagy. The Atg1ortholog controls autophagy. As mentioned previously a kinase, Vps34 forms a complex with Beclin 1; many proteins are included in this complex including UVRAG and Ambra1. The latter named proteins control the kinase and resultantly control autophagy. Many of the Atg proteins are involved in autophagasome formation. Atg9 is one such protein that travels between the endosomes and golgi and is capable of recognising the membranes used in the creation of the of the vessel of the autophage. Rab7 GTPase is used in the lysosome/autophagosome fusion step. By identifying the molecules involved in bringing about autophagy, it is possible to augment the reaction by blocking autophagy at the chosen stage. It is worth noting; that the stage the mechanism is blocked will affect the outcome for the cell. While, much has been revealed about the molecular mechanism of autophagy in the past decade, there are still many unanswered questions regarding the molecular process (Thorburn, 2008).
Fig.1. Diagram demonstrating the molecular regulation of autophagy. Growth factor receptor signaling activates class I PI3K are activated by growth factor signalling, which in turn activates the downstream targets Akt and mTOR. This inhibits autophagy. Rapamycin, (which is an inhibitor of mTOR) acts to induces autophagy. The PTEN gene mutation promotes the activation of Akt. Ras has a twofold effect on autophagy; on the activation of Class I PI3K, autophagy is inhibited, but when it activates the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase cascade, autophagy is encouraged. A complex of Class III PI3K and beclin1 (a tumor suppressor gene) is required for the primary step in autophagy. The cell death-associated protein kinase (DAPK) and the death-associated related protein kinase 1 (DRP1) may also induce autophagy. Class III PI3K is essential for the formation of autophagic vesicles and vesicular transport to the lysosome (Lefranc, 2006).
3.3 The relationship between apoptosis and autophagy
Always on Time
Marked to Standard
Since autophagy and apoptosis are both involved in cell death and autophagy can prevent apoptosis, it would be expected that their mechanism are interlinked. It has been discovered that p53 which induces apoptosis can also do so for autophagy. In the same way that the PI3 kinase pathway blocks apoptosis has an identical effect on autophagy. Hence, the same essential signalling pathways are involved in both processes. The key autophagic protein Beclin-1 was found to interact with the well known apoptotic regulator protein Bcl-2 and other members of the Bcl family including Bcl-xL (see fig.2). The latter named protein can inhibit autophagy by binding with Beclin 1 which has an important influence on starvation generated autophagy. Some of the pathway regulators may be influenced by the cellular reaction location. Apoptotic Bcl-2 is active at the mitochondria and endoplasmic reticulum (ER), the Bcl-2 autophagy inhibitor functions at the ER and the apoptotic inhibitor occurs at the mitochondria. The Bcl-2 at the different locations can only affect their specific mechanism. Structural analysis demonstrates that the interference of the interaction between Beclin-1 BH3 domain and BcL-2 leads to an increase in autophagy. In this way, a BH3 domain interaction controls autophagy as opposed to apoptosis. Aside from these mentioned, there are several other Bcl mechanisms that link the two processes and that it may be the protein location that determines whether the regulate autophagy or apoptosis (Thorburn, 2008).
Fig. 2. An outline of the apoptosis/autophagy relationship. The mutual inhibition of both processes is regulated by Bcl-2, which inhibits autophagy by interacting with Beclin 1 and could also inhibit apoptosis by blocking the activation of Bax (Zarzynska, 2008).
Cervical cancer is the second most common cancer among women worldwide and the most widespread female cancer in sizeable areas of the developing world where approximately 80% of new cases arise (Clifford, 2003). Widespread screening has dramatically reduced the incidence of cervical cancer but by contrast the prevalence of cervical intraepithlia neoplasia (CIN) has increased. The increase is most likely caused by improved detection methods (Kumar, 2003).
The vaginal portion of the cervix is covered by stratifies squamous epithelia and the endocervical canal is lined by a mucin secreting simple columnar epithelium. The junction between these cell types is positioned at the external os. Due to the influence of menstrual cycle hormones, an area of the cervix called the transformation zone arises. This is where columnar epithelium has been converted to squamous epithelium by a process called squamous metaplasia. The transformation zone is subjected to dysplastic changes caused by external factors. On removal of the causative factors, the dysplasia may regress or undergo neoplastic change with the occurrence of CINIII (Stevens, 2002). A lesser quantity of develop into invasive squamous cell carcinoma (SCC).
With CINI, the cells located in the lower third of the epithelium are compacted, have an increased mitotic activity and are hyperchromatic; the occasional koilocyte is also visible (see fig.3)(need to place arrow in photomicrograph). CINII is recognisable by basal cell proliferation occupying two thirds of the epithelium thickness, a greater variation in cell and nuclear size. The cells have a disordered orientation with normal and/or abnormal mitosis. CINIII, the dysplasia encompasses the entire epithelium and mitotic figures are widespread (Stevens, 2002).
CIN changes are confined to the epithelium (Kumar, 2003). CIS continue
Epidemiological studies have been carried out and it has been established that human papilloma-virus (HPV) infection is the fundamental causative agent of invasive cervical cancer. HPV DNA was identified in nearly all (99.7%) cases of cervical cancer in a study carried out in 22 countries by the International Agency for Research on Cancer.
The genital affected HPV virus types are all members of the Alphapapillomavirus genus (Brown, 2009).There are 40 different types of the HPV virus that may infect the genital tract and 14 of these are capable of progressing to invasive cancer. HPV types 16 and 18 together cause about 70 percent of cervical cancers; the other cancer causing types are phylogenetically related to types 16 and 18. The most significant type after HPV16 and 18 seem to be HPV45, preceded by types 31, 33, 58 and 52, their importance varying by geographical area (Clifford, 2003).
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HPV vaccines summon great hope of reducing the global burden of ICC. However, the vaccine must be multivalent since previous infection serves no protection to a new strain of HPV (Wheeler, 2009).
Autophagy literature review
The mechanism of autophagy was studied in order to produce an alternative tumour suppressing means, to overcome the resistance of many cancers to pro-apoptotic chemotherapy. A letter published in 2010 by Yang Sun et al. described how a study was carried out to determine if the expression of Beclin 1 had an influence over the chemosensitivity of cervical anti-cancer drugs. An MTT assay was performed, which works to assess the viability and the proliferation of cells under certain conditions. It is used to determine the cytotoxicity of would-be medicinal agents and chemicals, since those products would increase or inhibit cell proliferation.
The assay results confirmed that an increased Beclin 1 expression improved the cancer cells response to chemotherapy drugs, i.e supporting the apoptotic response.
It has been suggested that the autophagic capacity is lower in cancer cells than in normal counterparts. This suggests that the collapse of autophagy may trigger tumour development (Gozuacik, 2004). Heterozygous disruption of beclin 1 in mice resulted in the increased regularity of spontaneous carcinomas and lymphomas (Qu X, 2003)
A study was carried out to examine the role of Beclin 1 in HeLa cells to provide added insight into the relationship between autophagy and apoptosis. The methodology was based on the study of cell lines in which the Beclin 1 gene was silenced and over-expressed. The findings which showed that enforced expression of Beclin 1 promoted autophagy in cervical cancer and inhibited tumours. This signified that autophagy may be an essential mechanism for preventing the tumour growth (Wang, 2007).
A study by wang et al, 2006 found a lower expression of Beclin 1 in cervical SCC tissue, demonstrating the correlation between autophagy and cervical cancer.
Autophagy has a complex relationship with cancer development, often seeming contradictory. Possible future functions for the beclin 1 protein product include therapeutic application.
The four stages of AutophagyÂ
1) Induction: Following external/internal stimuli (e.g. nutrient depletion or ischemia) mTOR is inhibited, leading to induction of Autophagy. Key genes in yeast are Atg1 and Atg13, for which the mammalian homologues are yet to be identified.
2) Autophagosome formation: Cytosolic proteins and organelles are sequestered by a double membrane vesicle, the origin of which is uncertain, but may arise from the endoplasmic reticulum. Formation of this vesicle is co-ordinated by complexes of Atg proteins, in particular Atg5 and Atg12, that are conjugated enabling the recruitment of LC3 (Atg8). Beclin-1 forms a complex with Atg14.
3) Docking and fusion with the lysosome
4) Breakdown of the autophagic vesicle. The molecular mechanism behind the fusion with the lysosome and subsequent breakdown of the autophagic vesicle are poorly understood, although Lamp-2 is thought to play a key role.
This is a narrative account of the history of the topic, including any development of ideas that have been reported in scientific literature.