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.
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).
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Autophagy has been researched in laboratories since the 1960'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).
3.4 The relationship between apoptosis and autophagy
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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).
3.5 Cervical cancer
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
Fig. 3 The stages are defined by how abnormal the cells appear, slight, moderate and high. The risk of cancer development increases with increasing CIN grade. CIN is relatively common, with 1.4 million low grades and 330,000 high grade cases diagnosed in the United States in 2006. Cervical lesions are treated depending on the degree of severity. CIN 1 lesions may be removed or closely monitored; CIN 2/3 lesions are usually surgically removed. In either case, careful follow-up screening is performed to ensure that there is no recurrence. Despite the high incidence of CIN, if these irregularities are treated, progression to cancer is very rare. The figure shows microscopic images of normal cervical tissue, CIN 1, CIN 2 and CIN 3.
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.
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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).
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).
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 tumour growth (Wang, 2007).
mTOR is a kinase which is the mammalian target of rapamycin. It has a role in regulating translation within the cell and cell growth. mTor kinase is also an important regulatory component that controls the initiation of autophagy. The inhibition of this molecule leads to the impeding of the cell cycle and autophagy induction. The connection between beclin -1 and mTOR is still uncertain as most observations were performed on in-vitro experiments. Results by Faisal, (2009) revealed that more normal breast tissue expressed the beclin-1 protein strongly in comparison with tumour tissue. However, after all the results were analysed the study concluded that there was no association identified between Beclin-1 protein expression and the patients clinicopathological parameters. The aberration in these findings may have been caused by a defective or unreliable antibodies used in the experiment. These study findings are contrary to those of many other studies which suggest that decreased expression of autophagy proteins may contribute to the growth or progression of breast and other human malignancies (Liang, 2009). 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.
Bcl-2 is the prototype of a family of bcl-2 proteins. Many of the bcl-2 family proteins can interact with beclin-1. Beclin-1 constitutively interacts with antiapoptotic proteins of the bcl-2 family, bcl-2 and bcl-XL, which may inhibit autophagy. However, bad and bik, proapoptotic proteins of the bcl-2 family, could induce autophagy by dissociation of beclin-1 from bcl-2 or bcl-XL.
A study by Won et al., 2010 studied the expression of Beclin-1 and bcl-2 in human breast cancer with the objective of finding a clinicopathological correlation between the proteins. The study showed a decreased expression of beclin-1 in breast carcinoma cells as compared to the normal ductal epithelium, which is in accordance with the group's earlier findings (Liang, 1999).
These results support the hypothesis that Beclin-1 might be related to tumour suppression in human breast cancer tissue. In the study by Liang et al a small number of cases were assessed. Several researchers favour the idea that autophagy represses tumour development as in the afore mentioned findings, whereas other investigators suggest that autophagy increases tumour development and protects tumour cells from cell death.
It was observed that beclin-1 expression was inversely correlated with bcl-2 expression in breast cancer tissue. Taking the previous findings of Pattingre (2005) into consideration, bcl-2 interacts with beclin-1; it is probable that bcl-2 may have a role in the regulation of beclin-1 and in autophagy. The findings of Won et al. (2010) are consistent with the latest reports which have shown that bcl-2 negatively regulates beclin-1-dependent autophagic cell death. Therefore, it may be assumed that bcl-2 has the ability to reduce autophagy as well as to block apoptosis.
Beclin-1 expression showed a statistically considerable correlation with pleomorphism and mitotic count. These interpretation may allow assumptions that beclin-1 expression in breast cancer could be allied to the destructive qualities of cancer cells, but a significant prognostic implication was not reached. Beclin-1 expression in breast cancer has not yet been assessed as a determinant of survival for breast cancer (Won, 2010).
Pattingre (2005) found that Bcl-2-mediated inhibition of Beclin 1-dependent autophagy is prevented by either mutations in Bcl-2 that obstruct the binding to Beclin 1 or by mutations which occur in Beclin 1 that obstruct binding to Bcl-2.
This observation presents the argument for an independent role of Bcl-2-Beclin 1 interactions in the inhibition of autophagy. The ability of Bcl-2 to restrain autophagy through its contact with Beclin 1 is of particular significance in relation to cancer. Bcl-2 was originally discovered in the context of its translocation which results in the development of human follicular lymphoma, and the deletion of one allele of beclin-1 disposes mice to developing spontaneous B cell lymphomas, which suggests that levels of Beclin 1-mediated autophagy influence the risk of malignancies arising from B lymphocytes (Qu, 2003).
Their findings also demonstrate an accelerated development of hepatitis B virus induced premalignant lesions. Their findings provide further evidence that beclin 1 is a haplo-insufficient tumour-suppressor gene and supply genetic information that autophagy is a novel mechanism of cell-growth control and tumour suppression.
4.1The role of autophagy in cancer therapeutics
Further research has more recently been directed at determining whether and how autophagy can inhibit or support cancer. Some types of cancer cells utilise autophagy as a means to survive in the hypoxic, nutrient-limiting, and metabolically stressful tumour microenvironment and therapeutically provoked cell stress or damage. A number of anti-neoplastic therapies, including radiation therapy, chemotherapy, arsenic trioxide, TNFα, IFNγ, imatinib, rapamycin, and anti estrogen hormonal therapy, have been detected to induce autophagy as a protective and prosurvival mechanism in human cancer cell lines. As mentioned earlier, the therapeutic value of these agents can be improved if autophagy is blocked. Also, hypoxia induces autophagy that can occur without Hypoxia-inducible Factor-1α (HIF1α), suggesting that autophagy plays a role in tumour cell survival by increasing resistance to hypoxic stress in the tumour microenvironment. These results sustain the hypothesis that these therapies induce a type of prosurvival autophagy, which increases the tumour cells' resistance to therapies, and that inhibiting autophagy may lead to greater cell death and the blocking of tumour growth (Dalby, 2010).
New data has suggested that equally and depending on the context, the induction of autophagy can provide therapeutic benefits to patients and that the design and production of modulators of autophagy may provide original therapeutic tools and may eventually lead to new therapeutic tactics in cancer care.
Failings in apoptosis can lead to more resistance to chemotherapy, radiotherapy and other and targeted therapies. Hence, the initiation of autophagic cell death may be an ideal avenue in those cancers that are resistant to apoptosis by anticancer therapies. Therefore, induction of autophagic cell death may serve as a new therapeutic tool to eradicate cancer cells with defective apoptosis, which is used for many advanced, drug-resistant and metastatic cancers. It has recently been demonstrated that the inhibition of some protein kinases or the targeting of proteins that are implicated in the suppression of autophagy for example Bcl-2 can trigger autophagic cell death without any other treatment (Dalby, 2010).
Autophagy is a greatly regulated process of degrading and reusing of cellular constituents, partaking in organelle turnover, and contributing in the bioenergetic management of starvation. Autophagy has a complex relationship with cancer development, often seeming contradictory.
Furthermore, it is becoming ever clearer that autophagy is implicated in cancer formation and survival, and it reacts to several forms of cancer treatments. The study of the yet unknown relationship between malfunctioning autophagy and other cancer-promoting functions may provide precious insight into the oncogenesis and may have momentous prognostic and curative implications for cancer patients. Beclin-1 levels have emerged as one of the significant factors that affect the stimulation of autophagy. additional study about the in depth mechanisms by which beclin 1 and autophagy operate in relation to tumor suppression and extra study of this pathway as a new target for cancer therapy are helpful and warrant a great deal of consideration
The four stages of Autophagy .
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.