Anticancer effects on TACC3 by treatment of paclitaxel in HPV-18 positive cervical carcinoma cells.

Previously, we used proteome analysis to identify transforming acidic coiled coil (TACC) 3 as a protein that is down-regulated upon paclitaxel treatment in cervical cancer cells. TACC3 mRNA and protein levels decreased after paclitaxel treatment in a time- and dose-dependent manner, and the transactivation of TACC3 promoter was dramatically diminished by paclitaxel. Importantly, paclitaxel treatment and knockdown of TACC3 by siRNA led to a synergistic enhancement of significant G2/M phase arrest and apoptosis in HeLa cells. In contrast to TACC3-deficient cells, paclitaxel treatment of mTACC3-overexpressing cells failed to induce G2/M phase arrest, cell growth inhibition, and apoptotic cell death. We studied the associated gene in mTACC overexpressed cells using microarray. From these results, numerous genes have been identified as being associated with tumor progression (Ppia, TMSB10, Annexin A2, rab31, prostaglandin E2-EP2, UHRF1), chemoresistance (Akt, Plk-1, MAP kinase) and metastasis (MMP9, PECAM-1) in mTACC3 overexpressed HeLa cells. Thus, TACC3 is thought to be the critical molecule in mediating the anticancer mechanisms of paclitaxel in p53 inactivated cells by inducing G2/M arrest and apoptosis. And our data suggested that the overexpression of TACC3 may be associated with the mechanisms of chemoresistance, tumor progression, cell proliferation and metastasis.

Novel interaction between HPV E6 and BARD1 (BRCA1-associated ring domain 1) and its biologic roles.

Human papillomaviruses (HPVs), which are associated with the majority of cervical cancers, encode a transforming protein, E6, which interacts with the p53 tumor suppressor protein. There is a wide effort focused on searching for the target of the involvement of p53-independent HPV 16 E6-interacting proteins. We identified Breast Cancer 1 Gene (BRCA1)-associated ring domain protein 1 (BARD1) as a binding partner of E6 and investigated its biological function in cervical cancer cells. In vivo co-immunoprecipitation assay was performed to determine whether E6-BARD1 interaction occurred. We then used a degradation assay to determine whether E6-mediated inactivation of BARD1 transactivation function was associated with BARD1 degradation. A mutation assay revealed the site of interaction of E6 with BARD1. The effect of BARD1 on p53 transcriptional activity was tested using BARD1 knockdown and overexpression systems. BARD1 was not degraded by E6, and, instead, formed a physical complex with E6. Moreover, the mutations of the metal motif zinc-finger region decreased the ability of E6 to interact with BARD1. Transient transfection of BARD1 increased the p53-mediated activation of p21(WAF1) promoter despite the presence of E6. Additionally, the existence of BARD1 inactivated the expression of E6 in cervical cancer cells. These findings suggest that BARD1 may regulate the transcriptional activities of p53 as tumor suppressors.

Role of proteomics in translational research in cervical cancer.

Cervical cancer is one of the leading causes of cancer morbidity and mortality in women worldwide. More than 98% of cases are related to a human papillomavirus (HPV) infection. Infection with specific subtypes of HPV has been strongly implicated in cervical carcinogenesis. The identification and functional verification of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information for understanding cervical carcinogenesis and the development of cervical cancer-specific markers. In addition, proteomic profiling of altered proteins by anticancer drugs on cervical cancer cells may contribute to providing the fundamental resources for investigation of disease-specific target proteins, elucidation of the novel mechanisms of action and development of new drugs. The advent of proteomics has provided the hope of discovering novel biological markers for use in the screening, early diagnosis and prediction of response to therapy. This review describes the studies where profiles of protein expression in cervical cancer have been generated.

Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells.

Proteomic analyses can efficiently detect the variation of protein in high throughput screening. Using 2DE/MALDI-TOF-MS and SELDI-TOF-MS, we tried to search several cellular proteins that are responsive to ursolic acid (UA) in HeLa cervical carcinoma cells. Compared to control, UA-treated HeLa cells unfolded 25 proteins in significant changes by 2DE/MALDI-TOF-MS, most of which were involved in apoptosis. SELDI-TOF-MS with two types of protein chips profiled and analyzed proteomic features after administration of UA. Interestingly, eight polypeptide peaks can be detected. Further identification and characterization of these proteins may build the molecular basis of UA-induced apoptosis and provide insight into the anti-proliferative mechanism in cervical carcinoma cells.

Genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines.

cDNA microarray and proteomics studies were performed to analyze the genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines. Among 1024 known genes and ESTs tested by cDNA microarray, we found 50 upregulated and 35 downregulated genes in RC10.1 HPV-16 E6 transfected human colon adenocarcinoma cells compared to RKO cells, and 27 upregulated and 43 downregulated genes in A549E6 HPV-16 E6 transfected human lung adenocarcinoma cells compared to A549 cells. Employing two dimensional gel electrophoresis and MALDI-TOF-MS, the global pattern of protein expressions in RC10.1 human colon adenocarcinoma and A549E6 human lung adenocarcinoma cell lines stably expressing the HPV 16-E6 gene were compared with those of RKO and A549 cell lines to generate a differential protein expression catalog. We found 13 upregulated and 13 downregulated proteins in RC10.1 (E6-expressing RKO) cells compared to RKO cells and 12 upregulated and 14 downregulated proteins in A549E6 (E6-expressing A549) cells compared to A549 cells. The identified genes and proteins were classified into several groups according to the subcellular function. Expressing pattern of three genes and proteins (CDK5, Bak, and I-TRAF) were matched in both analyses of cDNA microarray and proteomics. These powerful approaches using cDNA microarray and proteomics could provide in-depth information on the impact of HPV-16 E6-related genes and proteins. Differential gene and protein expression patterns by transfection of HPV-16 E6 will provide the nucleus of valuable resource for investigation of the biochemical basis of cervical carcinogenesis. Further understanding of this data base may provide valuable resources for developing novel diagnostic markers and therapeutic targets of cervical cancer.

Proteomic analysis of antiproliferative effects by treatment of 5-fluorouracil in cervical cancer cells.

The global effects of 5-fluorouracil (FU) on cervical carcinoma cells were analyzed using an efficient proteomic method. More than 50 proteins showed a significant change in 5-FU-treated cervical carcinoma cells compared to control cells. Among them, 34 proteins have been identified by employing two-dimensional gel electrophoresis and MALDI-TOF-MS using peptide mass fingerprinting. In results, 22 proteins were upregulated (CIDE-B [cell death-inducing DFFA-like effector B], caspase-3, caspase-8, Apo-1/CD95 (Fas), etc.) and 12 proteins were downregulated (mitotic checkpoint protein BUB3, myc proto-oncogene protein [c-myc], src substrate cortactin, transforming protein p21A, etc.) by 5-FU treatment in HeLa cervical carcinoma cells as determined by spot volume (P <0.05). Our experiments showed that 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of caspase-9 and caspase-3 as well as the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with an Apo-1/CD95 (Fas)-dependent fashion. In addition, we could observe reduction of HPV-18 E6/E7 gene expression and activation of p53, pRb, and p21waf1 proteins by 5-FU treatment in HeLa cervical carcinoma cells. In conclusion, we suggest that 5-FU suppresses the growth of cervical cancer cells not only by antiproliferative effect but also antiviral regulation. Our findings may offer new insights into the mechanism of anticancer effect affected by 5-FU treatment in cervical cancer cells and its mode of action.

Exploring Rak tyrosine kinase function in breast cancer.

Protein tyrosine kinases have been implicated in the regulation of many cellular events such as cellular proliferation, differentiation and development. Deregulation of protein tyrosine kinase activity has been shown to result in human cancer. The majority of the protein tyrosine kinases studied to date localize to the cell membrane, where they function as components of signal transduction pathways. However, small group of nuclear tyrosine kinases has been identified that includes Rak. Our recent investigations demonstrated that Rak functions as a potent tumor suppressor by regulating PTEN protein stability and function. Rak also effectively suppresses phenotypes associated with in vitro transformation in breast cancer cells and tumorigenicity in vivo. Moreover, depletion of Rak is sufficient to induce tumorigenicity in mammary epithelial cells. However, the mechanisms by which Rak and its substrates function in cancer remain largely unexplored, leaving many potential therapeutic targets yet undiscovered. Therefore, fully elucidating the biological functions of Rak may contribute to effective therapeutic approaches for Rak-defective cancers.

BRIT1/MCPH1 links chromatin remodelling to DNA damage response.

To detect and repair damaged DNA, DNA-damage-response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions. ATP-dependent chromatin remodelling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA-damage-response protein that is mutated in human primary microcephaly. Here we report a previously unknown function of BRIT1 as a regulator of the ATP-dependent chromatin remodelling complex SWI-SNF in DNA repair. After damage to DNA, BRIT1 increases its interaction with SWI-SNF through ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and the reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired cell survival after DNA damage. Our findings therefore identify BRIT1 as a key molecule that links chromatin remodelling with response to DNA damage in the control of DNA repair, and its dysfunction contributes to human disease.

Rak functions as a tumor suppressor by regulating PTEN protein stability and function.

Expression of the PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrate that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr336. Knockdown of Rak enhanced the binding of PTEN to its E3 ligase NEDD4-1 and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

Biomarkers in cervical cancer.

Cervical cancer, a potentially preventable disease, remains the second most common malignancy in women worldwide. Human papillomavirus (HPV) is the single most important etiological agent in cervical cancer, contributing to neoplastic progression through the action of viral oncoproteins, mainly E6 and E7. Cervical screening programs using Pap smear testing have dramatically improved cervical cancer incidence and reduced deaths, but cervical cancer still remains a global health burden. The biomarker discovery for accurate detection and diagnosis of cervical carcinoma and its malignant precursors (collectively referred to as high-grade cervical disease) represents one of the current challenges in clinical medicine and cytopathology.

The role of HPV E6 and E7 oncoproteins in HPV-associated cervical carcinogenesis.

Cervical cancer is one of the leading world causes of cancer morbidity and mortality in woman, with more than 98% related to a human papillomavirus (HPV) infection origin. Infection with specific subtypes of HPV has been strongly implicated in cervical carcinogenesis. The identification and functional verification of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis and the development of cervical cancer-specific markers. The advent of functional genomics and proteomics has provided hope of discovering novel biological markers for use in the screening, early diagnosis, prognostication and prediction of response to therapy. Herein, we review the studies where the profiles of host proteins associated with HPV E6 and E7 oncoproteins in cervical cancer were generated.

Proteomic analysis of anti-cancer effects by paclitaxel treatment in cervical cancer cells.

OBJECTIVES: Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used in the treatment of ovarian, lung and breast cancer. At present, there is little information regarding the anti-cancer mechanism of paclitaxel against cervical carcinoma cells. We thus tried to show the anti-cancer effect of paclitaxel on cervical carcinoma cell line carrying HPV by using a proteomic analysis and to investigate the mechanism of actions. METHODS: We treated paclitaxel to cervical carcinoma cells and then carried out MTT assay to observe the anti-proliferate activity. Using proteomics analysis including two-dimensional (2-DE) gel electrophoresis and MALDI-TOF-MS, we tried to find the anti-proliferate activity-related proteins. Among them, paclitaxel treatment suppressed the expression of the mitotic checkpoint protein BUB3. Functional proteomic analysis by small interfering RNA (siRNA) targeting was tried to illuminate a role of mitotic checkpoint protein BUB3 in cell cycle progression. RESULTS: The cytotoxicity effects of paclitaxel were determined in HPV-16 positive CaSki, HPV-18 positive HeLa and HPV-negative C33A cervical carcinoma cell lines. Using efficient proteomics methods including 2-DE/MALDI-TOF-MS, we identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells. Paclitaxel treatment elevated mainly apoptosis-related, immune response-related and cell cycle check point-related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Paclitaxel showed anti-proliferate activity through the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with a TRAIL-dependent fashion as well as the mitochondrial-mediated pathway involving down-regulation of bcl-2 by cytochrome c release. Furthermore, we found siRNA-induced BUB3 knock down on cell cycle progression blocked by cell cycle arrest after paclitaxel treatment. CONCLUSIONS: The proteome profiling technique provided a broad-base and effective approach for the identification of protein changes induced by paclitaxel and showed anti-proliferate activity through the membrane death receptor-mediated apoptotic pathway, the mitochondrial-mediated pathway. This study shows the power of proteomic profiling with functional analysis using RNAi technology for the discovery of novel molecular targets and a better understanding of the actions of paclitaxel at the molecular level in cervical carcinoma cells.

Proteome analysis of differential protein expression in cervical cancer cells after paclitaxel treatment.

PURPOSE: It is well known that infection with HPV (human papillomavirus) is the main cause of cervical cancer and certain types of HPV are recognized as carcinogens. At present, there is little information regarding the antineoplastic mechanism of paclitaxel against cervical carcinoma cells. We thus tried to analyze differential protein expression and antineoplastic mechanism-related proteins after paclitaxel treatment on cervical cancer cells by using a proteomic analysis and to investigate the mechanism of action. MATERIALS AND METHODS: Using proteomics analysis including 2-DE and MALDI-TOF-MS, we detected the antineoplastic mechanism-related proteins. Then, we performed western blot analysis for apoptosis- and transformation-related proteins to confirm expression patterns derived from proteome analysis after paclitaxel treatment. RESULTS: We identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells using proteomics methods. Paclitaxel treatment elevated mainly apoptosis, immune response and cell cycle check point-related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Also, in the HPV-associated cervical carcinoma cells, paclitaxel demonstrated anti-proliferative activity through the membrane death receptor-mediated apoptotic pathway and the mitochondrial-mediated pathway. CONCLUSION: Identification and characterization of functionally modulated proteins involved in anti-cancer regulatory events should lead to a better understanding of the long-term actions of paclitaxel at the molecular level and will contribute to the future development of novel therapeutic drug treatments based upon current therapies.