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ABSTRACT: PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancers with a 50% five-year survival rate, which has remained unchanged for the past three decades. One of the major reasons for the aggressiveness of this cancer is that HNSCCs readily metastasize to cervical lymph nodes that are abundant in the head and neck region. Hence, discovering new molecules controlling the metastatic process as well as understanding their regulation at the molecular level are essential for effective therapeutic strategies. EXPERIMENTAL DESIGN: Rab25 expression level was analyzed in HNSCC tissue microarray. We used a combination of intravital microscopy in live animals and immunofluorescence in an in vitro invasion assay, to study role of Rab25 in tumor cells migration and invasion. RESULTS: In this study, we identified the small GTPase Rab25 as a key regulator of HNSCC metastasis. We observed that Rab25 is downregulated in HNSCC patients. Next, we determined that re-expression of Rab25 in a metastatic cell line is sufficient to block invasion in a 3D collagen matrix and metastasis to cervical lymph nodes in a mouse model for oral cancer. Specifically, Rab25 affects the organization of F-actin at the cell surface, rather than cell proliferation, apoptosis or tumor angiogenesis. CONCLUSIONS: These findings suggest that Rab25 plays an important role in tumor migration and metastasis, and that understanding its function may lead to the development of new strategies to prevent metastasis in oral cancer patients.
Clinical Cancer Research 01/2013; · 7.74 Impact Factor
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Vyomesh Patel,
Daniel Martin,
Ruchika Malhotra,
Christina A Marsh,
Colleen L Doçi,
Timothy D Veenstra,
Cherie-Ann O Nathan,
Uttam K Sinha,
Bhuvanesh Singh,
Alfredo A Molinolo,
James F Rusling,
J Silvio Gutkind
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ABSTRACT: OBJECTIVES: The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC. MATERIALS AND METHODS: DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates. RESULTS: We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microfluidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes. CONCLUSION: We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients.
Oral Oncology 09/2012; · 2.86 Impact Factor
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ABSTRACT: The integrity of the epidermis and mucosal epithelia is highly dependent on resident self-renewing stem cells, which makes them vulnerable to physical and chemical insults compromising the repopulating capacity of the epithelial stem cell compartment. This is frequently the case in cancer patients receiving radiation or chemotherapy, many of whom develop mucositis, a debilitating condition involving painful and deep mucosal ulcerations. Here, we show that inhibiting the mammalian target of rapamycin (mTOR) with rapamycin increases the clonogenic capacity of primary human oral keratinocytes and their resident self-renewing cells by preventing stem cell senescence. This protective effect of rapamycin is mediated by the increase in expression of mitochondrial superoxide dismutase (MnSOD), and the consequent inhibition of ROS formation and oxidative stress. mTOR inhibition also protects from the loss of proliferative basal epithelial stem cells upon ionizing radiation in vivo, thereby preserving the integrity of the oral mucosa and protecting from radiation-induced mucositis.
Cell stem cell 09/2012; 11(3):401-14. · 23.56 Impact Factor
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ABSTRACT: Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg·mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and ~100,000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.
Analytical Chemistry 06/2012; 84(14):6249-55. · 5.86 Impact Factor
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ABSTRACT: The expression of heme oxygenase-1 (HO-1) was shown to be increased in multiple tumors compared with their surrounding healthy tissues and was also observed to be up-regulated in oral squamous cell carcinomas (OSCC). However, conflicting results were obtained and little information is available regarding HO-1 significance in head and neck squamous cell carcinoma (HNSCC). Therefore, the aim of the present study was to perform a wide screening of HO-1 expression in a large collection of human primary HNSCCs and to correlate the results with clinical and pathological parameters. For this purpose, we investigated the expression of this protein by immunohistochemistry (IHC) in tissue microarrays (TMAs) of HNSCC and in an independent cohort of paraffin-embedded tumor specimens. HO-1 expression was further validated by real-time qPCR performed on selected laser capture-microdissected (LCM) oral tissue samples. Both the number of HO-1-positive samples and HO-1 immunoreactivity in the cancerous tissues were significantly higher than those in the non-tumor tissues. These results were confirmed at the mRNA level. Interestingly, HO-1 localization was observed in the nucleus, and the rate of nuclear HO-1 in HNSCC was higher than that in non-malignant tissues. Nuclear HO-1 was observed in HNSCC cell lines and increased even further following hemin treatment. Analysis of HO-1 expression and sub-cellular localization in a mouse model of squamous cell carcinoma (SCC) and in human HNSCC revealed that nuclear HO-1 increases with tumor progression. Taken together, these results demonstrate that HO-1 is up-regulated in HNSCC and that nuclear localization of HO-1 is associated with malignant progression in this tumor type.
Experimental and Molecular Pathology 05/2012; 93(2):237-45. · 2.42 Impact Factor
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ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. The recent identification of the mTOR complex 1 (mTORC1) signaling pathway as a highly prevalent molecular signature underlying HNSCC pathogenesis has provided the foundation to search for novel therapeutic approaches to prevent and treat HNSCC. Here, we asked whether metformin, the most widely used medication for the treatment of type II diabetes, which acts in part by stimulating the AMP-activated protein kinase (AMPK) signaling pathway thereby reducing mTORC1 activity, may lower the risk of HNSCC development. Indeed, we show that metformin reduces the growth of HNSCC cells and diminishes their mTORC1 activity by both AMPK-dependent and -independent mechanisms. We also optimized an oral-specific carcinogenesis mouse model that results in the accumulation of multiple oral premalignant lesions at the end of the carcinogen exposure, some of which then spontaneously progress into HNSCC. Using this mouse model, we observed that metformin specifically inhibits mTORC1 in the basal proliferating epithelial layer of oral premalignant lesions. Remarkably, metformin prevented the development of HNSCC by reducing significantly the size and number of carcinogen-induced oral tumoral lesions and by preventing their spontaneous conversion to squamous cell carcinomas. Collectively, our data underscore the potential clinical benefits of using metformin as a targeted chemopreventive agent in the control of HNSCC development and progression.
Cancer Prevention Research 04/2012; 5(4):562-73. · 4.91 Impact Factor
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Chan Eng Chong,
Kue Peng Lim,
Chai Phei Gan,
Christina A Marsh,
Rosnah Binti Zain,
Mannil Thomas Abraham,
Stephen S Prime,
Soo-Hwang Teo,
J Silvio Gutkind, Vyomesh Patel,
Sok Ching Cheong
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ABSTRACT: MAGE proteins have been shown to be good targets for cancer immunotherapy. We demonstrate that MAGED4B is over-expressed in more than 50% of Oral Squamous Cell Carcinoma (OSCC) tissues and the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines that over-express MAGED4B promote migration in vitro, exhibit an increase in cell growth both in vitro and in vivo, and are more resistant to apoptosis compared to control cells. Our data suggest that MAGED4B over-expression is a driver in oral carcinogenesis and argues strongly that this protein may represent a potential therapeutic target in OSCC.
Cancer letters 03/2012; 321(1):18-26. · 4.86 Impact Factor
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ABSTRACT: The incidence of head and neck squamous cell carcinomas (HNSCC) associated with human papillomavirus (HPV) infection has increased over the past decades in the United States. We aimed at examining the global impact of HPV-associated HNSCC and whether the established key role of mTOR activation in HNSCC is also observed in HPV(+) HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients.
An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16(INK4A), a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV(-) and HPV(+) HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays.
Approximately 20% of all HNSCC lesions could be classified as HPV(+), irrespective of their country of origin. mTOR pathway activation was observed in most HPV(+) HNSCC and CCSCC lesions and cell lines. The preclinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV(+) HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs and indicate that the activation of the mTOR pathway is a widespread event in both HPV(-) and HPV-associated HNSCC and CCSCC lesions.
The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies.
Clinical Cancer Research 03/2012; 18(9):2558-68. · 7.74 Impact Factor
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Dat T Vo,
Kotb Abdelmohsen,
Jennifer L Martindale,
Mei Qiao,
Kumiko Tominaga,
Tarea L Burton,
Jonathan A L Gelfond,
Andrew J Brenner, Vyomesh Patel,
Daniel Trageser,
Björn Scheffler,
Myriam Gorospe,
Luiz O F Penalva
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ABSTRACT: Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein (RBP) that has profound implications in cellular processes such as stem cell maintenance, nervous system development, and tumorigenesis. Msi1 is highly expressed in many cancers, including glioblastoma, whereas in normal tissues, its expression is restricted to stem cells. Unfortunately, the factors that modulate Msi1 expression and trigger high levels in tumors are largely unknown. The Msi1 mRNA has a long 3' untranslated region (UTR) containing several AU- and U-rich sequences. This type of sequence motif is often targeted by HuR, another important RBP known to be highly expressed in tumor tissue such as glioblastoma and to regulate a variety of cancer-related genes. In this report, we show an interaction between HuR and the Msi1 3'-UTR, resulting in a positive regulation of Msi1 expression. We show that HuR increased MSI1 mRNA stability and promoted its translation. We also present evidence that expression of HuR and Msi1 correlate positively in clinical glioblastoma samples. Finally, we show that inhibition of cell proliferation, increased apoptosis, and changes in cell-cycle profile as a result of silencing HuR are partially rescued when Msi1 is ectopically expressed. In summary, our results suggest that HuR is an important regulator of Msi1 in glioblastoma and that this regulation has important biological consequences during gliomagenesis.
Molecular Cancer Research 01/2012; 10(1):143-55. · 4.29 Impact Factor
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Vyomesh Patel,
Christina A Marsh,
Robert T Dorsam,
Constantinos M Mikelis,
Andrius Masedunskas,
Panomwat Amornphimoltham,
Cherie-Ann O Nathan,
Cherie Ann Nathan,
Bhuvanesh Singh,
Roberto Weigert,
Alfredo A Molinolo,
J Silvio Gutkind
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ABSTRACT: Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogues, as part of a molecular-targeted metastasis preventive strategy for the treatment of patients with HNSCC.
Cancer Research 11/2011; 71(22):7103-12. · 7.86 Impact Factor
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ABSTRACT: A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL⁻¹ levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL⁻¹ for PSA and 0.30 pg mL⁻¹ for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.
Biosensors & bioelectronics 07/2011; 26(11):4477-83. · 5.43 Impact Factor
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Angewandte Chemie International Edition 06/2011; 50(34):7915-8. · 13.45 Impact Factor
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ABSTRACT: Current first-line treatments for most cancers feature a short-list of highly potent and often target-blind interventions, including chemotherapy, radiation, and surgical excision. These treatments wreak considerable havoc upon non-cancerous tissue and organs, resulting in deleterious and sometimes fatal side effects for the patient. In response, this past decade has witnessed the robust emergence of nanoparticles and, more relevantly, nanoparticle drug delivery systems (DDS), widely touted as the panacea of cancer therapeutics. While not a cure, nanoparticle DDS can successfully negotiate the clinical payoff between drug dosage and side effects by encompassing target-specific drug delivery strategies. The expanding library of nanoparticles includes lipoproteins, liposomes, dendrimers, polymers, metal and metal oxide nano-spheres and -rods, and carbon nanotubes, so do the modes of delivery. Importantly, however, the pharmaco-dynamics and -kinetics of these nano-complexes remain an urgent issue and a serious bottleneck in the transition from bench to bedside. This review addresses the rise of nanoparticle DDS platforms for cancer and explores concepts of gene/drug delivery and cytotoxicity in pre-clinical and clinical contexts.
Pharmaceutics. 03/2011; 3(1):34-52.
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ABSTRACT: The human oral squamous epithelium plays an important role in maintaining a barrier function against mechanical, physical, and pathological injury. However, the self-renewing cells residing on the basement membrane of the epithelium can give rise to oral squamous cell carcinomas (OSCC), now the sixth most common cancer in the developed world, which is still associated with poor prognosis. This is due, in part, to the limited availability of well-defined culture systems for studying oral epithelial cell biology, which could advance our understanding of the molecular basis of OSCC. Here, we describe methods to successfully isolate large cultures of human oral epithelial cells and fibroblasts from small pieces of donor tissues for use in techniques such as three-dimensional cultures and animal grafts to validate genes suspected of playing a role in OSCC development and progression. Finally, the use of isolated oral epithelial cells in generating iPS cells is discussed which holds promise in the field of oral regenerative medicine.
Advances in experimental medicine and biology 01/2011; 720:27-38. · 1.09 Impact Factor
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ABSTRACT: To study the distribution and clearance of polyethylene glycol (PEG)-ylated single-walled carbon nanotube (SWCNTs) as drug delivery vehicles for the anticancer drug cisplatin in mice.
PEG layers were attached to SWCNTs and dispersed in aqueous media and characterized using dynamic light scattering, scanning transmission electron microscopy and Raman spectroscopy. Cytotoxicity was assessed in vitro using Annexin-V assay, and the distribution and clearance pathways in mice were studied by histological staining and Raman spectroscopy. Efficacy of PEG-SWCNT-cisplatin for tumor growth inhibition was studied in mice.
PEG-SWCNTs were efficiently dispersed in aqueous media compared with controls, and did not induce apoptosis in vitro. Hematoxylin and eosin staining, and Raman bands for SWCNTs in tissues from several vital organs from mice injected intravenously with nanotube bioconjugates revealed that control SWCNTs were lodged in lung tissue as large aggregates compared with the PEG-SWCNTs, which showed little or no accumulation. Characteristic SWCNT Raman bands in feces revealed the presence of bilary or renal excretion routes. Attachment of cisplatin on bioconjugates was visualized with Z-contrast scanning transmission electron microscopy. PEG-SWCNT-cisplatin with the attached targeting ligand EGF successfully inhibited growth of head and neck tumor xenografts in mice.
PEG-SWCNTs, as opposed to control SWCNTs, form more highly dispersed delivery vehicles that, when loaded with both cisplatin and EGF, inhibit growth of squamous cell tumors.
Nanomedicine 12/2010; 5(10):1535-46. · 5.05 Impact Factor
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ABSTRACT: This critical review evaluates progress toward viable point-of-care protein biomarker measurements for cancer detection and diagnostics. The ability to measure panels of specific, selective cancer biomarker proteins in physicians' surgeries and clinics has the potential to revolutionize cancer detection, monitoring, and therapy. The dream envisions reliable, cheap, automated, technically undemanding devices that can analyze a patient's serum or saliva in a clinical setting, allowing on-the-spot diagnosis. Existing commercial products for protein assays are reliable in laboratory settings, but have limitations for point-of-care applications. A number of ultrasensitive immunosensors and some arrays have been developed, many based on nanotechnology. Multilabel detection coupled with high capture molecule density in immunosensors and arrays seems to be capable of detecting a wide range of protein concentrations with sensitivity ranging into the sub pg mL(-1) level. Multilabel arrays can be designed to detect both high and ultralow abundance proteins in the same sample. However, only a few of the newer ultrasensitive methods have been evaluated with real patient samples, which is key to establishing clinical sensitivity and selectivity.
The Analyst 10/2010; 135(10):2496-511. · 4.23 Impact Factor
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ABSTRACT: Over the past several years, we have been interested in understanding the mechanisms by which mammary carcinomas acquire hormone independence. We demonstrated that carcinoma associated fibroblasts participate in the ligand-independent activation of progesterone receptors inducing tumor growth. In this study, we used DNA microarrays to compare the gene expression profiles of tumors from the MPA mouse breast cancer model, one hormone-dependent (C4-HD) and one hormone-independent (C4-HI), using whole tumor samples or laser-captured purified stromal and epithelial cells obtained from the same tumors. The expression of selected genes was validated by immunohistochemistry and immunofluorescence assays. We identified 413 genes specifically expressed in tumor stroma. Eighty-five percent of these genes were upregulated, whereas the remaining 15% were downregulated in C4-HI relative to their expression in the C4-HD tumor stroma. Several matrix metallopeptidases were overexpressed in the C4-HI tumor microenvironment. On the other hand, 1100 genes were specifically expressed in the tumor parenchyma. Among them, the 29% were upregulated, whereas the remaining 71% were downregulated in C4-HI relative to C4-HD tumor epithelium. Steap, Pdgfc, Runx2, Cxcl9, and Sdf2 were among the genes with high expression in the C4-HI tumor parenchyma. Interestingly, Fgf2 was one of the few genes upregulated by MPA in C4-HD tumors, confirming its pivotal role in regulating tumor growth in this model. In conclusion, we demonstrate herein a gene expression profile that distinguishes both the epithelial and the stromal cells in mammary tumors with different hormone dependence, supporting the hypothesis that the tumor-associated stroma may contribute to hormone-independent tumor growth.
Breast Cancer Research and Treatment 10/2010; 129(1):49-67. · 4.43 Impact Factor
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ABSTRACT: Squamous cell carcinomas of head and neck (HNSCC) are associated with immune, inflammatory, and angiogenic responses involving interleukin-6 (IL-6). This article reports an ultrasensitive electrochemical immunosensor for human IL-6 and proof-of-concept studies of IL-6 detection in HNSCC cells. Single wall carbon nanotube (SWNT) forests with attached capture antibodies (Ab(1)) for IL-6 were used in an electrochemical sandwich immunoassay protocol using enzyme label horseradish peroxidase (HRP) to measure very low (<or=30 pg mL(-1)) and elevated levels of IL-6. Two levels of multienzyme labeling were used to measure a broad concentration range of IL-6 in a representative panel of HNSCC cells. Secondary antibodies (Ab(2)) attached to carboxylated multiwall carbon nanotubes with 106 HRP labels per 100 nm gave the highest sensitivity of 19.3 nA mL (pg IL-6)(-1) cm(-2) and the best detection limit (DL) of 0.5 pg mL(-1) (25 fM) for IL-6 in 10 microL of calf serum. For more concentrated samples, biotinylated Ab(2) bound to streptavidin-HRP to provide 14-16 labels per antigen was used. These immunosensors accurately measured secreted IL-6 in a wide range of HNSCC cells demonstrated by excellent correlations with standard enzyme-linked immunosorbent assays (ELISA), suggesting that SWNT immunosensors combined with multilabel detection have excellent promise for detecting IL-6 in research and clinical applications.
Analytical Chemistry 03/2010; 82(8):3118-23. · 5.86 Impact Factor
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ABSTRACT: Previous work from our laboratory has demonstrated overexpression of chemokines in head and neck cancer and the utility of targeting these proteins for tumor therapy in a preclinical model. However, the mechanisms involved are unexplored. Through gene expression analysis, we found that expression of vascular endothelial growth factor (VEGF-C) was elevated in HN12 cells expressing high levels of CXCL5. In the present study, we have investigated the contribution of VEGF-C to tumor cell growth and motility. RNAi-mediated knockdown of VEGF-C expression in HN12 cells, which express high levels of CXCL5, resulted in a decrease in proliferation. Conversely, forced expression of VEGF-C in HN4 tumor cells with low endogenous CXCL5 levels increased cell growth. Suppression of VEGF-C inhibited migration of HN12 cells. Similarly, HN4 cells showed reduced migration towards conditioned media collected from HN12 cells with VEGF-C knockdown compared to controls, while HN4/VEGF-C conditioned media stimulated cell migration. Moreover, tumor growth in vivo was markedly reduced when VEGF-C expression was blocked by shRNA. Finally, determination of VEGF-C expression in squamous carcinoma cell lines revealed universal overexpression compared to normal keratinocytes. These findings support a role for VEGF-C in head and neck squamous cell carcinogenesis.
Oral Oncology 03/2010; 46(4):e19-24. · 2.86 Impact Factor
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ABSTRACT: Previous studies from our laboratory have indicated that overexpression of the epidermal growth factor receptor pathway substrate 8 (EPS8) enhances cell proliferation, migration and tumorigenicity in vivo, although the mechanisms involved remain unexplored. A microarray screen to search for potential mediators of EPS8 identified upregulation of multiple cell cycle-related targets such as the transcription factor FOXM1 and several of its reported downstream mediators, including cdc20, cyclin B1, cyclin A, aurora-B kinase and cdc25C in cells with elevated EPS8, as well as matrix metalloproteinase-9, which we reported previously to be upregulated by EPS8-dependent mechanisms. Cells engineered to overexpress FOXM1 showed increased proliferation, similar to EPS8-overexpressing cells. Conversely, targeted knockdown of FOXM1 in EPS8-overexpressing cells reduced proliferation. Cotransfection of EPS8 with a FOXM1-luciferase reporter plasmid into 293-T- or SVpgC2a-immortalized buccal keratinocytes demonstrated that EPS8 enhances FOXM1 promoter activity, whereas chromatin immunoprecipitation assays revealed elevated levels of acetylated histone H3 associated with the FOXM1 promoter in cells expressing high levels of EPS8. Treatment of EPS8-overexpressing cells with inhibitors of phosphoinositide 3-OH kinase or AKT reduced expression of FOXM1 and aurora-B kinase, a transcriptional target of FOXM1. Overexpression of EPS8 induced expression of the chemokine ligands CXCL5 and CXCL12 in a FOXM1-dependent manner, which was blocked by LY294002 or a dominant-negative form of AKT. Additionally, overexpression of FOXM1 enhanced cell migration, whereas targeted knockdown of CXCL5 or inhibition of AKT reduced migration of EPS8-expressing cells. These data suggest that EPS8 enhances cell proliferation and migration in part by deregulating FOXM1 activity and inducing CXC-chemokine expression, mediated by PI3K- and AKT-dependent mechanisms.
Carcinogenesis 03/2010; 31(6):1132-41. · 5.70 Impact Factor
