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Based on this discovery, JHGC offers innovative methods by which IGF-I production is increased in the hair follicle. Sensory neurons that transmit the pain and heat sensation to the brain perceive the stimulatory information in the gastrointestinal tract and the skin, thereby releasing CGRP (calcitonin gene-related peptide) in the hair follicle. A powerful hair losing hormone called "DHT = dihydrotestosterone" is formed from testosterone by the action of an enzyme called "5?-reductase" in hair follicles.
Finasteride that inhibits DHT formation has recently been used to treat AGA, and, thus, it has become a hot topic.
Only JHGC provides hair care treatments based on the IGF-1 hair growth theory in the world. Green tea has been proven highly beneficial in the treatment of acne, comparable to a 4% benzoyl peroxide treatment without the same, negative side effects.
The EGCG component of green tea helps to counteract this by interfering with the IGF-I signals which encourage sebum production. According to Pubmed “The inflammatory stage of acne vulgaris is usually of greatest concern to the patient.
The EGCG in green tea is well known for its antioxidant properties and so may be beneficial in the treatment of acne to help reduce inflammation and stop pro-inflammatory responses.
Green tea has been seen to have three primary roles in the treatment of acne, namely, decreasing sebum production, acting as an anti-inflammatory and preventing the abnormal colonization of P. Some theorists believe that paranoid persons had parents who were distant, rigid, demanding, and perfectionistic, engendering rage, a sense of exaggerated self-importance, and mistrust in the individual. Science, Technology and Medicine open access publisher.Publish, read and share novel research. New Biomarkers for Cervical Cancer – Perspectives from the IGF SystemMartha Lucia Serrano1, Adriana Umana-Perez, Diana J. Sanchez-Gomez[1] Hormone Laboratory, Department of Chemistry, Faculty of Science, Universidad Nacional de Colombia, Bogota, Colombia1. 2000 Insulin Like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. A study of biomarkers in cervical carcinoma and clinical correlation of the novel biomarker MN. 1994 A functional insulina-like growth factor receptor I is required for the mitogenic and transforming activities of the epidermal growth factor receptor. IGF-II regulates metastatic properties of choriocarcinoma cells through the activation of the insulin receptor. The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. The serum assay of tumour markers in the prognostic evaluation, treatment monitoring and follow-up of patients with cervical cancer: a review of the literature.
Body fluid proteomics for biomarker discovery: lessons from the past hold the key to success in the future. Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: expression at the mRNA and protein level. Expression of the type 1 insulin-like growth factor receptor is up-regulated in primary prostate cancer and commonly persists in metastatic disease. 2008 Clinical implications of insulin-like growth factor I system in early stage cervical cancer. How do I remove a giant comedonal cyst (2x2 cm) from the interscapular area and leave minimal scarring in a keloid-forming patient? The incidence of obesity, type 2 diabetes mellitus (DM-2), and cancer has increased significantly in the past decade, having a huge impact on the healthcare system. What remains unknown is whether the association between DM and cancer is simply due to common risk factors for the two diseases (Figure 2). One of the main goals of diabetic therapy in addition to vascular risk reduction is effective glucose control to decrease microvascular complications.
Human studies14-16 have shown a relationship specifically between insulin glargine and cancer risk but are criticized for ignoring major confounding variables, having short follow-up times, and for including subjects with pre-existing cancer (Figure 4).5,7,17,18 Larger prospective trials that control for common confounding variables and have sufficient follow-up are required to more accurately study the specific link between all forms of insulin and the development of malignancy. Current observational evidence suggests an association between DM, DM therapy and cancer risk.
Hemkens LG, Grouven U, Bender R et al (2009) Risk of malignancies in patients with diabetes treated with human insulin or insulin analogues: a cohort study. Colhoun HM, SDRN Epidemiology Group Use of insulin glargine and cancer incidence in Scotland: a study from the Scottish Diabetes Research Network Epidemiology. However, it has not long been established how to increase IGF-I production without adverse effects. These methods will contribute to the restoration of normal hair growth and improvement of hair loss. The growth factor is also produced in the hair follicle and promotes hair growth by proliferating keratinocytes, thereby improving alopecia. Dermal papilla cells increase IGF-I production by receiving CGRP, and IGF-I thus formed activates keratinocytes to increase its proliferation.
DHT inhibits IGF-I production by inhibiting sensory neuron stimulation, thereby inducing hair loss. Since DHT inhibits IGF-I production, inhibition of DHT formation by finasteride or the other scalp care and treatments that increase IGF-I production may synergistically increase IGF-I production, thereby promoting hair growth; the former means release of brake of hair growth and the latter stepping on accelerator of hair growth. In the course of conducting research focusing on hematology primarily, he found a new way to increase the insulin-like growth factor which has the hair growth effect, and completed "IGF-1 hair growth theory" with isoflavone and capsaicin. Acnes bacteria and so studies have been done to show whether the antibacterial and antimicrobial effects of green tea may be beneficial against this particular form of bacteria as well.
First, a number of topical applications have been created in which green tea extracts can be used externally on the skin to treat acne.
Resolve conflicts, coping with anxiety without the use of threats or assaultive behavior.
Protein-protein interaction network representing the associations between the IGF system and identified membrane proteins expressed by the cervical cancer HeLa cell line.
IntroductionThe insulin-like growth factor (IGF) family is organized in a complex regulatory network at the cellular and sub-cellular levels. In: Human Proteome Organization Annual World Congress 2010, Sydney, Australia, October 2010. These include obesity, low levels of physical activity, high-fat and high-calorie diet, smoking and alcohol use.2 We have to remember that although the observational epidemiological evidence supports a link between DM and cancer, this relationship isn’t scientifically strong enough to be considered causal. Between 40% and 80% of type 2 diabetics will require insulin therapy.1 Observational studies have shown an increased cancer risk in diabetic patients on any type of insulin,11-13 but it’s the basal insulin analogue, glargine, that’s been most frequently linked to the development of cancer.
At this point it would be premature to definitively say that insulin or glargine insulin causes or promotes cancer. But observational research alone, without higher levels of evidence such as randomized-controlled trials and meta-analyses, leaves us unable to draw any solid conclusions. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin. Insulin glargine use and short-term incidence of malignancies—a population-based follow-up study in Sweden.
Prebtani, MD, FRCPC, is Associate Professor of Medicine and Program Director of the Endocrinology & Metabolism Residency Training Program at McMaster University in Hamilton, ON. Consequently, hair growth is promoted by sensory neuron stimulation in the gastrointestinal tract and scalp.
JHGC provides these synergistic treatments based on the above-mentioned theory established by extensive research.
This discovery is applied to the development of treatment methods that has demonstrated various effects of anti-aging effect such as rejuvenation of skin besides hair restoration. Including green tea in your diet is another solution and may help treat the acne from the inside out.
Most of these effects are due to the presence of EGCG, a strong antioxidant present in green tea. A fragile ego results in severely impaired self-esteem, a sense of loss of control, fear, and severe anxiety. In the human the IGF system has a key physiological role in the development of the organism and maintenance of normal cellular function during foetal and postnatal life.
Discovering the extent of these associations could impact our ability to modify this risk in our diabetic patients. Many relationships in medicine, such as that between hip fractures and older age, show us that association doesn’t mean causation. Furthermore, the known benefits of good glucose control with insulin, when indicated, outweigh the risks. These studies could potentially influence our cancer screening practices in diabetic patients, knowing that they’re likely a higher risk group.8 The absolute — not relative — risk of developing cancer needs to be considered in the context of the well-known morbidity in patients with poorly controlled DM. Type 2 diabetes mellitus and the risk of malignancy: is there a strategy to identify a subphenotype of patients with increased susceptibility to endogenous and exogenous hyperinsulinism. Preclinical and clinical evidence from prostate, breast, pancreatic, and colorectal research. No evidence of increased risk of malignancies in patients with diabetes treated with insulin detemir: a meta-analysis. He is also Director of the Internal Medicine International Health Program at McMaster University. Okajima found that stimulation of sensory neuron safely increases IGF-I production in the body. Therefore, promotion of IGF-I production by the safe method is critical for maintenance of normal hair growth and also for restoration of hair growing power in patients with alopecia. And the current level of evidence shouldn’t prevent appropriate glucose management in this patient population, with insulin if needed. IGF-I and IGF-II share a 62% homology in amino acid sequence and there is a 40% homology between the IGFs and proinsulin (Furstenberger & Senn, 2002). IGF-IR is a member of the family of the tyrosine kinase growth factor receptors and is highly homologous at the amino acid sequence level to the IR. The mature membrane receptor is a tetramer made of two ?-chains and two ?-chains, with several disulfide bridges (LeRoith et al., 1995).


The extracellular ?-subunits form the ligand binding domain and several lines of evidence suggest that the binding sites for IGF-I and IGF-II may be distinct (Samani et al., 2007). IGF-I and IGF-II bind to IGF-IR with high affinity, however, ligand affinities may vary with cell type and experimental conditions. IGF-II can also bind to the insulin receptor isoform A (IR-A) with an affinity similar to that of insulin. IR-A is expressed in certain tumours and has a more mitogenic effect than the IR-B isoform, the latter having a more metabolic function (Pandini et al., 2002). Early studies carried out with purified hybrid receptors indicate that these receptors mostly bind IGF-I and that they bind insulin with a much lower affinity (Belfiore et al., 2009). IGF-II can also bind to a second receptor, IGF-IIR, which is a multifunctional single transmembrane glycoprotein, identical to the cation-independent mannose 6-phosphate receptor.
It is composed of a large extracytoplasmic domain and a short cytoplasmic tail that lacks intrinsic cytoplasmic activity (El-Shewy & Luttrell, 2009).
The IGF-IIR receptors recycle continuosly between two cellular pools and at steady state most of the receptor localize in endosomes and the remainder (approx. All IGFBPs inhibit IGF action by sequestering IGFs and some IGFBPs (IGFBP-1, -3, -5) can also potentiate IGF action. The IGF signalling system network is tightly controlled under normal physiological conditions and alterations that disrupt the delicate balance of the system can trigger a number of molecular events that can lead to malignancy. Many studies have involved the IGF system in carcinogenesis and tumour progression of different cell types (LeRoith & Roberts, 2003). It appears that abnormal IGF-IR activation can result in oncogene activation leading to increased cellular proliferation and malignancy.
Compared to other types of cancer, like breast, colon, prostate, and lung, little research has been done on the relationship between the IGF family and cervical neoplasias, and by now, much remains to be learned. In this review, we will discuss some of the studies focused on the role of the components of the IGF system in the progression of cervical cancer and its potential utility as a diagnostic biomarker. The IGF system and cervical cancerCervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide, accounting for 9% (529,800) of the total new cancer cases and 8% (275,100) of the total cancer deaths among females in 2008. Worldwide, the highest incidence rates are in Eastern, Western and Southern Africa, as well as South-Central Asia and South America (Jemal et al., 2011). In Colombia, cervical cancer is one of the most common causes of cancer mortality among women (Ferlay, 2010). Although HPV infections are among the most frequent sexually transmitted diseases, infections are usually self-limited and revert spontaneously, with only a small group of women developing cervical cancer (Woodman et al., 2001). The evolution of infection to LSIL, HSIL and cancer is dependent on several factors, many of which remain to be identified.
Despite intensive investigation, the tumor biology of this disease is still largely unknown. Although prognostic factors such as pelvic lymph node metastasis affects the outcome of cervical cancer, the variability in progression-free and overall survival (OS) among patients with similar clinical and pathological characteristics, makes it difficult to predict the outcome reliably (Huang et al., 2008). Current research strives to determine why certain HPV-positive women develop cervical cancer while others do not (Schaffer et al., 2007). Serum levels of IGFs as cervical cancer biomarkersIGF-I is a potent mitogenic growth factor that plays a critical role during embryogenesis and development in human and animal species. Together with Growth Hormone (GH) constitute an axis that regulate postnatal growth and development in an endocrine, paracrine and autocrine mode of action. IGF-I is produced by numerous adult organs, with major contribution of liver to overall circulating IGF-I levels.
After puberty, circulating IGF-I Figure 2.Schematic representation of cervical cancer and its precursor lesions. The proliferative and anti-apoptotic effects of IGF-I observed in animal and cell cultures, made it an obvious risk factor candidate in cancer development. In attempt to find a reliable serum biomarker to predict occurrence, progression or prognosis of human cancer, scientists have investigated the correlation of serum IGF-I or IGFBP-3, the most abundant IGFBP in circulation, to the prevalence of a variety of cancers. We conducted a case-control study on Colombian women with SIL and cervical cancer, and our findings illustrated an inverse association between cervical cancer risk and IGF-I circulating levels.
Similar results were obtained in a study conducted in patients with diagnosed pre-cancer lesions where levels of IGF-I were inversely associated with risk of high-grade cervical intraepithelial neoplasia (Schaffer et al, 2007).
A recent prospective study reported a possible influence of the IGF axis on the natural history of oncogenic HPV and the development of cervical neoplasia (Harris et al., 2008).
We conducted a case-control study with in a prospective populational-based cohort of 2200 women, followed-up during 10 years. Results adjusted by age, menarche, smoking, parity and hormonal contraceptives, showed that high IGF-I serum levels were associated with persistence (lower vs. In early-stage cervical cancer patients, lower IGF-I levels seemed to be associated with worse overall survival rate, but was not of an independent value, and there was no relationship between IGFBP-3 levels and survival (Huang et al., 2008). It is unclear why increased serum IGF-I levels may have a protective effect on development of cervical cancer, whereas in breast and prostate cancer it has the opposite effect. One of the theories is that the natural history to cervical cancer, where infection with HPV plays a crucial role, differs from sex hormone-related cancers (Schaffer et al., 2007). Larger prospective investigations are indicated to better clarify these associations, including any potential HPV type-specific differences in the effects of IGFs. Aberrant expression of IGFs in cancerSeveral lines of evidence support an important role for the IGF system in tumor tissue.
IGF-I expression rarely occurs in tumoral cells, but can be produced by stromal cells surrounding the tumor. A study identified the loss of imprinting (LOI) of the gene, frequently observed in the colonic mucosa of colorectal carcinoma patients, as a risk factor for developing colorectal carcinoma (Cui et al., 2003). There are two IR isoforms formed by alternative splicing of exon 11, IR-A which lacks exon 11 and IR-B which contains exon 11. After in vitro and in vivo studies demonstrating that insulin may also play a significant and independent role in tumorigenesis, insulin is now receiving more attention in this regard (Gallagher & LeRoith, 2011).
In the past few decades, accumulating evidence has established that insulin receptors (IRs) are usually abnormally expressed in cancer cells, where they mediate both the metabolic and nonmetabolic effects of insulin. As it was mentioned earlier, many tumors overexpress IGF-II, which also signals through the IR-A isoform. Both IGF-I and IGF-II stimulated JEG-3 choriocarcinoma cell invasion, although they signal through different receptors: IGF-I through IGF-IR, and IGF-II through IR-A. In JEG-3 cells, which predominantly express IR-A, IGF-II stimulated cell invasion more potently than insulin (Diaz et al., 2007).
Tissue expression of IGFs as risk or prognosis biomarkers in cervical cancerIn a recent study the expression levels and activated status of IGF-IR were measured by immunohistochemistry in formalin-fixed and paraffin-embedded specimens. IGF-IR levels and phosphorylation status were significantly high in cervical intraepithelial neoplasia (CIN III) and invasive cancer specimens (Kuramoto et al., 2008). An interesting retrospective analysis of patients with early-stage cervical cancer evaluated IGF-IR expression levels by immunoflorescent stain. Authors found that high-grade expression of IGF-IR, is an independent predictor of cervical cancer death and recurrence, and when combined with elevated squamous cervical cancer antigen (SCC Ag) serum level, could further help identify the subgroup of patients at higher death risk.
Colocalisation of IGF-I and IGF-IR in the cancerous tissues, and the lack of correlation between circulating IGF-I or IGFBP-3, and IGF-IR overexpression in cervical cancer tissue, give support to a paracrine or autocrine function of the IGF system in early-stage cervical cancer, with the corresponding adverse IGF-I stimulation of IGF-IR signaling (Huang et al., 2008). The remaining cellular material after preparation of routine Pap smear can be used for the search of new biomarkers at both the mRNA and protein levels. The use of cervical scrapes instead of biopsies for biomarker studies has several advantages, since this is a noninvasive procedure, the material obtained is not affected by stroma or other contaminants, as is the case in the analysis of tissue homogenates and can be the base for molecular epidemiology studies. In a study on cervical scrapes from SIL and cancer patients, we found no difference in IGF-II mRNA levels between cancer and normal cells, whereas at the protein level, IGF-II expression was reduced in cancer cervical scrapes (Serrano et al., 2007, 2010a). High levels of IGF-II protein, but not mRNA were found in pheocromocytomas (Haselbacher et al., 1987). These evidences indicate that regulation of IGF-II expression involves transcriptional mechanisms, as genomic imprinting, but also post-transcriptional regulation, that can occur through malignant progression. A member of the IGF-II mRNA binding protein family (IMP), IMP-3, has been reported to be an activator of IGF-II mRNA translation and therefore may play a critical role in IGF-II-dependent cellular proliferation (Liao et al., 2005). IMPs were detected in various cancers; increased levels were found in ovarian cancer and correlated with prognosis.
There are no studies about IMP in cervical cancer.Few studies have examined the expression of hybrid receptors in cervical cancer cells.
In a recent study, we found that IR-A, IR-B and hybrid receptors coexist with IGF-IR in human papillomavirus-positive cervical cancer cells. Tyrosine phosphorylation of the receptors and activation of the MAPK and PI3-K pathways were observed upon ligand binding, which may explain the anti-apoptotic effect mediated by IGF-I in these cells (Serrano et al., 2008).
The role of IGF in radioresistance in cervical cancerThe crucial role of IGF-IR in promoting resistance to cancer treatments, such as radiation and chemotherapy, is well documented for several cancer types (Samani, et al., 2007).
IGF-IR down-regulation by antisense nucleotides or its inhibition by tyrosine kinase inhibitors increases cancer cell sensitivity to both radiation and chemotherapy in a variety of malignancies. Some studies indicate an important role of IGF-IR in several pathways involved in the induction of radioresistance (Belfiore et al., 2009). In a study with advanced cervical cancer patients (CIN I-III), we found that tumor tissues co-expressing IGF-IR and IGF-II, showed an increase (4.6-fold) in the risk of developing resistance to radiotherapy. The increased expression of the glycolitic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in these cancer tissues, demonstrated the metabolic shift to glycolisis under hypoxic conditions (Moreno-Acosta et al., 2010). Biomarkers for cervical cancerBiomarkers used for screening in cancer, usually consist of biomolecules that are released from tumors or precancerous lesions into the bloodstream, urine, or other means, they should have the property to survive the metabolic degradation to be measurable (Ahlquist, 2010). Molecular changes that occur in the development of tumors may take several years; some biomarkers can be used to detect early stage disease. However, biomarkers are usually found at relatively low level compared to other biomolecules; research and validation as possible diagnostic tests depend critically on the ability to achieve measurements with high precision and sensitivity. Several biologic markers or indexes have been studied as potential tools to determine the prognosis and biological behaviour of various types of gynaecological neoplasias. This fact prompted investigators to develop and test antibodies in a widespread range of neoplasic lesions. Because HeLa is a cultured human uterine cervix carcinoma cell line, these authors concluded that MN-p had great use as a tumour marker in the evaluation of cervical carcinoma and could predict progression in precursor cervical intraepithelial neoplasic (CIN) lesions. Ki67, a nuclear proliferation associated antigen is expressed in the growth and synthesis phases of the cell cycle (G1, S, G2, and mitosis) but not in the resting phase, G0.


The proportion of cells which express Ki67 (Ki67 index) is an excellent measure of neoplasm proliferation.
Production of specific antibodies against recombinant DNA Ki67 antigen (MIB-1) confirmed the definite use of MIB-I immunoreactivity, exhibiting close correlation with CIN grade and providing additional risk prediction beyond HPV type (Resnick et al., 1996).
In low-grade CIN, anti-MIB-1 staining is not detected in superficial levels of epithelium, whereas in high-grade CIN full thickness staining is present.
In contrast with MN-p expression, anti-MIB-1 shows consistent results by most investigators and exhibits definitive, important associations with clinical outcome and basic carcinogenesis hypotheses in the uterine cervix (Costa, 1996).For several years, it has been known that high vascularity is characteristic of grade 3 CIN and invasive lesions, and angiogenesis has been associated as indicator of prognosis. The onset of angiogenesis in human cervical cancer occurring during the premalignant dysplastic stage is unique. An association between lymphatic high microvessel density with vascular invasion and a lower global survival rate has been described.
There is a study demonstrating that microvessel density in carcinomas of the uterine cervix is a factor associated with poor prognosis (Tjalma et al., 1999). The authors showed that during the progression from noninvasive to microinvasive cervical carcinoma, the microvessel density increases significantly.
Once the tumour cells have passed the basal membrane, the tumour apparently switches to an angiogenic phenotype.
Measures of factor VIII, CD31, and vimentin, showed no association between microvessel count and lymph node invasion, depth of invasion, and histological differentiation. Nevertheless, these studies found an association between lymphatic vessel invasion and a larger number of newly formed vessels (Di Leo et al., 1998). Other studies have proposed anti-CD34 as a marker for evaluating angiogenesis in cervical cancer (Vieira et al., 2004, 2005).
They suggested that anti-CD34 antibody reactivity in cervical carcinoma is associated with pathoanatomical features, as microvessel density or invasion, indicative of poorer prognosis and greater risk of recurrence (Vieira et al., 2005).
Briefly, they showed lymphatic invasion in 40% of the cases, while vascular and perineural invasion was observed in 24% and 19% of the cases, in a population of 62 patients diagnosed with invasive cervical carcinoma, stages Ib and IIa.
They also observed that 55% of women whose carcinoma presented high microvessel density had an undifferentiated type of cancer, suggesting that the presence of lymphatic invasion may associate with higher microvessel density. In general, different markers for angiogenesis may or may not present a statistically significant association with different pathoanatomic features of cervical cancer.
These possible markers should be tested prospectively as a prognostic and predictive factor of the response to different therapies used in cervical carcinoma. On the other hand, a recent study explored the power of serum markers such as squamous cell carcinoma antigen (SCC), CYFRA 21-1, CA 125, immunosuppressive acidic protein (IAP) and vascular endothelial growth factor (VEGF) in patients with cervical cancer (Gadducci et al., 2008). Squamous cell carcinoma antigen comprises two similar proteins, SCC-1 and SCC-2, that posses protease inhibitory property: SCC-1 inhibits chymotrypsin and cathepsin L, while SCC-2 inhibits cathepsin G and mast cell chymase. SCC-1 and SCC-2 reside in the citosol of squamous cells, and their presence in the sera of patients with advanced squamous cell carcinomas is mainly due to a passive release rather than an active secretory process into the circulation.
Increased SCC levels have been observed in 28-88% of patients with squamous cell cervical cancer.
Elevated CA 125 levels are detectable in 20-75% of patients with cervical adenocarcinoma and have been associated with advanced tumour stage, large tumour size, high histological grade, lymph node involvement and status.
CA 125 is detected in normal adult fallopian tube, endometrium, endocervix and peritoneum, and in vitro studies showed that CA 125 secretion by human mesothelial cell monolayers may be enhanced by the inflammatory cytokines interleukin-1 and tumour necrosis factor. However, the specificity of the antigen is not yet optimal, since elevated serum CA 125 can be found in benign gynaecological conditions, such as endometriosis and pelvic inflammatory disease, benign non-gynaecological conditions, such as hepatitis, pancreatitis, renal failure and pleural effusion and non-gynaecological malignancies, including lung cancer, pancreatic cancer and non-Hodgkin’s lymphoma.Serum levels of vascular endothelial factor (VEGF) are often elevated in patients with cervical cancer, and decrease significantly after successful treatment.
Proteomic studiesProteomics has emerged as a promising tool for unravelling protein signatures that are associated with a particular malignancy that can be useful biomarkers of the disease. Validated protein biomarkers could be useful in early detection of disease, monitoring disease progression or monitoring response to treatment.
Proteomic studies on body fluidsAfter exploring markers in tissue, several studies have focused on biomarker discovery in body fluids which would be advantageous for eventual clinical implementation. Cervical mucous or cervical vaginal fluid (CVF) is potentially an ideal sample to screen for biomarkers for early detection of cervical cancer.
As cervical mucous is produced in the microenvironment where cervical neoplasia arises, it is likely to include proteins produced by the lesion as well as by the host in response to the lesion. A recent study identified 151 new proteins that included proteins present in the lower female genital tract, such as HBD-2 and cathelicidin, two proteins that play an important role in the innate immunity of the cervicovagina (Zegels et al., 2009).
Another recent study on cervical mucous proteome indicated that plasma proteins were abundant in cervical mucous (Panicker et al., 2010).
The majority of proteins identified were categorized under metabolism and immune-response functional groups. In contrast to plasma samples, some authors discuss the advantages of cervical mucous as a potential source of concentrated biomarkers due to lesser dilution of the sample in CVF-vaginal washings (volume ±50 mL) in comparison to plasma volume (±3 L) (Good et al., 2007). In addition, altered biomarker expression patterns in plasma are often not very specific, as they may be associated with different pathologies, because plasma comes in contact with all organs of the body. In contrast, when using CVF samples, it is expected that expression patterns will directly correlate with gynaecological pathologies (Zegels et al., 2009). However, it should be taken into account that CVF is a body fluid that can be highly influenced by many biological factors including menstruation, age, infection, sexual intercourse, usage of contraceptives, pregnancy, etc. Moreover, conditions of CVF collection and the experimental proteomic strategy could affect the proteome results. Membrane proteomic studiesThe main potential of proteomics in cancer research is not only derived from individual experiments, but from comparative studies between different types and states of cancer. There is a need to integrate proteomics, genomics and metabolomics with the aim of achieving a functional and comprehensive interpretation of clinical and pathological data. Comparative proteomic studies have provided tools for establishing some molecular mechanisms of cancer progression, either from normal to neoplasic cells, or from cancer models with different malignant phenotypes.
Membrane proteomic studies constitute an analytical challenge due to its heterogeneous composition, dynamic physicochemical characteristics and hydrophobicity; however, 60% of therapeutic targets are directed against membrane proteins. In particular, cellular motility depends on the adaptive response to environment that extensively relies on key molecular elements of the plasma membrane and cytoskeleton, and therefore usually considered as tumour targets.
Functional assays showed that IGF-I and IGF-II stimulate migration and invasion in HeLa but not in C33-A cervical cancer cells.
In order to make comparisons between proteomic maps, we obtained proteomic maps of the two cell lines by two–dimensional electrophoresis (2-DE), and used the PDQuest (Bio-Rad®) and Progenesis SameSpot (Nonlinear Dynamics Ltd) softwares, to perform between-gel spot comparisons (Fig. A total of 641 spots in C33-A and 493 spots in HeLa proteomes were identified, with 399 spots matching between the two cell lines. This means that the whole proteomic profiles of cervical adenocarcinoma and cervical carcinoma cells share 80% of similarity, suggesting that the differences in occurrence, prognosis, dissemination and recurrence after treatment, may be related to a limited number of proteins and their relative distribution and expression. Study of sub-cellular compartments could improve the identification of a higher number of proteins and consequently a more efficient detection of therapeutic targets. We obtained enriched protein membrane extracts from HeLa and C33-A cells by cell surface biotin labelling followed by avidin purification and separation by 2-DE to obtain the corresponding membrane proteomes (Fig.
We detected 351 spots in HeLa sub-proteome, out of which 207 spots (41%) were present in the whole proteome and 144 spots corresponded to new proteins not visualized in the total proteome. With respect to C33-A, 395 spots were found in the membrane proteome where 170 spots (43%) matched with the total proteome; additionally 255 new spots were detected.
Furthermore, comparison of membrane-enriched proteomes, showed a 58% match between the two cell lines with 272 common proteins, out of which, 86 proteins were found to be differentially expressed (>3-fold).
The invasive abilities of HeLa in opposition to C33-A cells, may be related to the observed differences in the membrane protein profile.In order to identify a higher number of proteins and overcome the limitations associated with the separation of membrane proteins by bi-dimensional electrophoresis, we conducted a multidimensional analysis of enriched membrane fractions. Proteins were separated by SDS-PAGE, each lane in the gel was cut and divided in equal fragments and digested with trypsin.
The peptides were separated by reversed-phase liquid chromatography (LC) and analyzed in a hybrid quadrupole mass spectrometer–time of flight (Q-TOF) instrument. Bioinformatics analysis was performed using Mascot Distiler® and Proteome Software Scaffold, which allowed the identification of 44 and 56 proteins in HeLa and C33-A fractions, respectively.
Results showed a differential or exclusive protein expression profile that correlated with cell phenotype.
We identified 33 proteins exclusively expressed in C33-A cells that corresponded to cell cycle regulation, metabolism, stress response and immune system evasion, more related to a proliferative non-invasive phenotype.
Meanwhile, 22 proteins exclusively expressed in HeLa cells, where mainly involved in cytoskeleton remodelling and associated with cell motility, according to the invasive phenotype.
The interaction network was built including all the previously identified proteins in the membrane proteome and the members of the IGF system. This analysis confirmed the interaction of some components of the membrane with members of the IGF family, and suggests a potential role in the promotion of the invasive phenotype display by these cells.
In particular, this analysis confirmed a direct interaction between IGFBP-3 and the CD44 - Moesin complex, associated with cell adhesion and tumor progression, meaning a promising target for cervical cancer invasion and metastasis control. In conclusion, our Figure 4.Protein-protein interaction network representing the associations between the IGF system and identified membrane proteins expressed by the cervical cancer HeLa cell line.
Connecting lines represent association.approach is unique as it considers the IGF receptors in a wider context within a protein network, and shows that the membrane is a rich reservoir for potential targets for cancer. ConclusionThe evidence reviewed above shows that the IGF system plays a central role in many aspects of the development and progression of cervical cancer. The effects of components of the IGF axis in cervical carcinogenesis share some similarities with those observed in other types of cancer, however, clear differences exist that reflect specificities, possibly due to associations with oncogenic and nononcogenic HPV.
The available data support dysregulation of the IGF system expression and signaling, that could promote the progression to a malignant phenotype. It is plausible to consider these molecules as promising targets for cervical cancer invasion and metastasis control and source of valuable biomarkers of the disease, as deduced from the proteomic approach of membrane targets.
Advances in molecular biology and high throughput technologies will improve our understanding of the disease and the search for reliable biomarkers that will facilitate screening, early detection, management, and individualized targeted therapy.5.
AcknowledgementsThis work was supported by the Research Council of the Universidad Nacional de Colombia (Grants No.
Domont, Universidade Federal de Rio de Janeiro, Brazil, for the helpful support with the proteomic studies.



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