Katrina Briggs

Glutathione S-transferase genotypes as risk factors for head and neck cancer

BACKGROUND Several enzymatic systems, including glutathione S-transferases, are involved in the metabolism of environmental agents. The absence of glutathione S-transferases mu (GSTM1) and theta (GSTT1) results in decreased detoxification of carcinogens, for example, chemicals in cigarette smoke. These metabolic deficiencies may predispose individuals to the development of smoking-related tumors, such as cancers of the lung, head and neck, and bladder. METHODS The glutathione S-transferase genotypes of 186 previously untreated patients with squamous cell carcinoma of the head and neck and 42 healthy controls were determined with polymerase chain reaction (PCR) methodologies. Lymphocytes separated from heparinized peripheral blood or whole blood extracts served as sources of genomic DNA. The presence or absence of the gene-specific PCR products revealed the positive or negative genotypes, respectively. RESULTS The absence of the GSTM1 genotype conferred an odds ratio of 2.37, and the 95% confidence interval (CI) was 1.20 to 4.67. The absence of the GSTT1 gene conferred an odds ratio of 1.47 (CI 0.71 to 3.02). In the population of 42 patients and their matched 42 controls, the absence of the GSTM1 and GSTT1 genotypes conferred odds ratios of 3.10 (CI 1.24 to 7.75) and 2.18 (CI 0.91 to 5.23), respectively. CONCLUSIONS Despite the small study size, our preliminary data suggest that genetically determined factors of carcinogen metabolism may be associated with increased risk for head and neck cancer.

Stability of antibody-conjugated gold nanoparticles in the endolysosomal nanoenvironment: implications for noninvasive radiofrequency-based cancer therapy.

UNLABELLED The use of noninvasive radiofrequency (RF) electric fields as an energy source for thermal activation of nanoparticles within cancer cells could be a valuable addition to the emerging field of nano-mediated cancer therapies. Based on investigations of cell death through hyperthermia, and offering the ability for total-body penetration by RF fields, this technique is thought to complement and possibly outperform existing nano-heat treatments that utilize alternative heat production via optical or magnetic stimuli. However, it remains a challenge to understand fully the complex RF-nanoparticle-intracellular interactions before full system optimization can be engineered. Herein we have shown that liver cancer cells can selectively internalize antibody-conjugated gold nanoparticles (AuNPs) through receptor-mediated endocytosis, with the nanoparticles predominantly accumulating and aggregating within cytoplasmic endolysosomes. After exposure to an external RF field, nonaggregated AuNPs absorbed and dissipated energy as heat, causing thermal damage to the targeted cancer cells. We also observed that RF absorption and heat dissipation is dependent on solubility of AuNPs in the colloid, which is pH dependent. Furthermore, by modulating endolysosomal pH it is possible to prevent intracellular AuNP aggregation and enhance thermal cytotoxicity in hepatocellular cancer cells. FROM THE CLINICAL EDITOR Gold nanoparticles absorb energy from RF fields and can exert hyperthermic effects leading to cell death. Combining this known effect with antibody-based targeting of the nanoparticles, selective cancer specific hyperthermia induced cell death therapies can be designed, as demonstrated in this article.

Resetting the histone code at CDKN2A in HNSCC by inhibition of DNA methylation

Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent cancer in the US. Several genetic and epigenetic alterations are associated with HNSCC tumorigenesis, including inactivation of CDKN2A, which encodes the p16 tumor suppressor, in cell lines and primary tumors by DNA methylation. Reactivation of tumor suppressor genes by DNA-demethylating agents and histone deacetylase (HDAC) inhibitors shows therapeutic promise for other cancers. Therefore, we investigated the ability of these agents to reactivate p16 in Tu159 HNSCC cells. Treatment of cells with 5-aza-2′deoxycytidine (5-aza-dC) increases CDKN2A expression and slightly increases histone H3 acetylation at this gene. No reactivation of CDKN2A is observed upon treatment with the HDAC inhibitor trichostatin A (TSA), but synergistic reactivation of CDKN2A is observed upon sequential treatment of Tu159 cells with both 5-aza-dC and TSA. Silencing of CDKN2A in Tu159 cells is correlated with increased methylation of histone H3 at lysine 9 and decreased methylation at lysine 4 relative to the upstream p15 gene promoter. Interestingly, global levels of H3-K9 methylation are decreased upon treatment with 5-aza-dC. Together these data indicate that DNA methylation is a dominant epigenetic mark for silencing of CDKN2A in Tu159 tumor cells. Moreover, changes in DNA methylation can reset the histone code by impacting multiple H3 modifications.

An Orthotopic Model of Papillary Thyroid Carcinoma in Athymic Nude Mice

OBJECTIVE To develop a reproducible orthotopic model of papillary thyroid carcinoma for the BRAF(V600E) mutation (GenBank NM004333) and an RET/PTC rearrangement (GenBank M31213) that recapitulates the clinical picture in humans. DESIGN In vitro and in vivo study. SETTING Department of Head and Neck Surgery, M. D. Anderson Cancer Center. SUBJECTS Eight- to 12-week-old athymic female nude mice. INTERVENTIONS Either BRAF-mutated or RET/PTC1-rearranged papillary thyroid carcinoma cells were injected into the thyroid glands of athymic female nude mice. The mice were euthanized when the tumor burden exceeded 1.0 cm or when they exhibited significant morbidity. MAIN OUTCOME MEASURES Tumorigenicity, extent of tumor invasion and metastasis, cell invasion and migration, and median survival. RESULTS All the BRAF-mutated cell lines and 1 selected RET/PTC1-rearranged cell line were 100% tumorigenic in mice. These mouse tumor models exhibited a wide range of biological potential, including laryngeal invasion, lymph node metastasis, and pulmonary metastasis, thus reflecting the clinical spectrum of papillary carcinoma. CONCLUSIONS An orthotopic model of papillary thyroid carcinoma was successfully established in nude mice using BRAF-mutated and RET/PTC1-rearranged cell lines. These models mimic the human disease and will thus be useful for evaluating the clinical potential of novel targeted therapies.

Headpin: A Serpin with Endogenous and Exogenous Suppression of Angiogenesis

Headpin is a novel serine proteinase inhibitor (serpin) with constitutive mRNA expression in histologically normal oral mucosa but with lost or down-regulated expression in head and neck squamous cell carcinoma. Several serpin family members are similarly lost in multiple cancer types and hold tumor suppressor functions including the inhibition of angiogenesis. However, the functional significance for the loss of headpin expression in cancer is not known. Using immunohistochemical analysis of invasive squamous cell carcinoma and matched normal squamous mucosa of patient specimens, headpin expression was lost or down-regulated in the vast majority of tumor specimens. We investigated the functions of exogenous recombinant headpin and endogenously expressed headpin related to angiogenesis. In a rat corneal assay of neovascularization, recombinant headpin protein blocked in vivo angiogenesis mediated by interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF). In assays of cellular events in angiogenesis, headpin blocked the invasion, migration, and tube formation of endothelial cells. In light of our findings of nuclear subcellular localization of headpin, we investigated the expression and secretion of angiogenic factors and found reduced mRNA, protein, and promoter activities of IL-8 and VEGF. Finally, using a murine flank tumor model, headpin expression reduced growth and microvessel density in tumors derived from headpin-expressing UMSCC1 cells relative to those from vector control cells. These findings of nuclear regulatory functions of a serpin in the inhibition of angiogenesis bring new understanding to the cellular and molecular mechanisms of serpins. Therefore, this novel serpin targets diverse mechanisms against tumor angiogenesis on which to base therapeutic strategies.

Autophagy and enhanced chemosensitivity in experimental pancreatic cancers induced by noninvasive radiofrequency field treatment

Patients with pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options and poor response to the standard gemcitabine (GCB)‐based chemotherapy. In the current study, the authors investigated the feasibility of noninvasive short‐wave radiofrequency (RF) electric fields to improve the cytotoxic effect of GCB on PDAC cells and determined its mechanism of action.

Silencing thioredoxin induces liver cancer cell senescence under hypoxia.

Aim:  Although thioredoxin 1 (TXN) has pleiotropic cellular functions as a redox‐sensitive protein, very little is known about its role in tumor survival and growth under hypoxia. MHCC97H hepatocellular carcinoma cells have a high metastatic potential and high thioredoxin expression levels compared with their parent cell line, MHCC97. Thus, we used this cell line to explore the functional connections between TXN and hypoxia.

Abstract 2270: Non-invasive radiofrequency increases the anticancer effect of gemcitabine in treating pancreatic cancer via autophagy

Pancreatic cancer has a high mortality rate. Currently the main treatment for unresectable pancreatic adenocarcinoma is chemotherapy based on gemcitabine (GCB) use. Trials of GCB, combined with radiation therapy and other cytotoxic agents, did not produce a substantial improvement in patient response over GCB alone. This indicates that new therapeutic approaches should be developed for the treatment of this disease. In our laboratory we developed a new method based on the use of non-invasive radiofrequency (RF) field. Electromagnetic energy produced by 220 MHz was shown to have low tissue specific absorption rate and therefore, excellent whole body tissue penetration with documented safety in humans exposed to the RF field for several hours. The RF frequency of our device is lower, 13.56 MHz and generates mild hyperthermia. In the current study we used human Panc-1 tumor cells, known for their poor response to GCB, and studied the effect of RF on tumor cells with and without GCB. Combination of GCB with RF (GCB+RF) significantly inhibited proliferation of Panc-1 cells in vitro when compared with either treatment alone (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2270. doi:1538-7445.AM2012-2270

Abstract 3704: Gemtuzumab ozogamicin conjugated gold nanoparticles induce cell death in D1.1 leukemia cells when exposed to a radiofrequency field

Introduction: Acute T-cell leukemia cells may express the marker CD33. Gemtuzumab is a monoclonal antibody against CD33, used clinically in combination with ozogamicin, a cytotoxic antibiotic, for treatment of acute leukemias. It has been previously demonstrated that gold nanoparticles delivered intracellularly and exposed to nonionizing radiofrequency (RF) fields induce hyperthermic cytotoxicity. RF fields are not cytotoxic without the presence of nanoparticles. The purpose of this experiment is to investigate cell death in cells treated with gemtuzumab ozogamicin conjugated to gold nanoparticles after RF field exposure. Methods: D1.1 (acute T-cell leukemia) cells were plated in 60mm cell culture dishes. Two groups (n=3 in each group) of plated cells were treated for two hours with gemtuzumab ozogamicin conjugated to 10nm solid gold nanoparticles in media. One group of cells was then exposed to a radiofrequency field for a total generator energy of 1500 W-minutes. The control cells were treated with the same gold conjugate, but were not exposed to an RF field. Two days later, cell viability, apoptosis, and death was determined by flow cytometry. The groups were compared with Student9s t-test. Results: RF fields induced apoptosis in 58.7% ± 3.95% of treated cells, compared with 8.66% ± 0.56% of the controls (p=0.002). The viability after RF exposure was 13.9% ± 6.11% while gemtuzumab ozogamicin gold conjugate alone decreased viability to 74.2% ± 0.85% (p=0.003). The temperature of the media was kept below 40°C to avoid nonspecific cell injury. Conclusions: Multimodality therapy (chemotherapy and nanoparticle-mediated hyperthermia) induced significantly more death in acute T-cell leukemia cells than antibody-chemotherapy alone. Antibodies specific to molecular targets on cells permit targeting of the heat mediator (nanoparticles) while preventing non-specific delivery of both nanoparticles and chemotherapeutic agent. The use of non-invasive RF fields to specifically induce cell death by hyperthermia with nanoparticles may present new options for the treatment of leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3704.