Romaine E. Saxton

Gangliosides of human melanoma.

Human melanoma synthesizes a large quantity of gangliosides, glycosphingolipids containing sialic acid. The authors previously have demonstrated that the ganglioside profile differs among individual melanomas and is widely heterogenous. In the current study, a retrospective study was performed to compare the relationship between the quantity of five major gangliosides of human melanoma (GM3, GM2, GD3, GD2, and alkali‐labile ganglioside) and nine clinical factors (sex, age, site, stage, tumor size, pigmentation, histopathologic type of primary tumor, chemosensitivity, and prognosis). Melanoma specimens studied were obtained from patients of our clinic and included 52 biopsy specimens and 28 cultured cell lines. Analysis of melanoma biopsy specimens have shown a differential ganglioside expression among different sites of tumor, pigmentation, and histopathologic types. Results of cultured melanoma cell lines differed from those of biopsy specimens, but ganglioside expression also differed among the site of tumor, tumor size, histopathologic types, and chemosensitivity. GM3 positively correlated with a good prognosis in both biopsy and cultured melanomas.

Mass spectrometry (LC-MS/MS) site-mapping of N-glycosylated membrane proteins for breast cancer biomarkers

Cancer cell membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, cellular secretion or cell lysis, and represent promising candidates for interrogation. Because many known disease biomarkers are both glycoproteins and membrane bound, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins from breast cancer cell membrane fractions using the LTQ Orbitrap mass spectrometer. Our initial goal was to select membrane proteins from breast cancer cell lines and then to use the hydrazide method to identify the N-linked proteome as a prelude to evaluation of plasma/serum proteins from cancer patients. A combination of steps facilitated identification of the glycopeptides and also defined the glycosylation sites. In MCF-7, MDA-MB-453 and MDA-MB-468 cell membrane fractions, use of the hydrazide method facilitated an initial enrichment and site mapping of 27 N-linked glycosylation sites in 25 different proteins. However, only three N-linked glycosylated proteins, galectin-3 binding protein, lysosome associated membrane glycoprotein 1, and oxygen regulated protein, were identified in all three breast cancer cell lines. In addition, MCF-7 cells shared an additional 3 proteins with MDA-MB-453. Interestingly, the hydrazide method isolated a number of other N-linked glycoproteins also known to be involved in breast cancer, including epidermal growth factor receptor (EGFR), CD44, and the breast cancer 1, and early onset isoform 1 (BRCA1) biomarker. Analyzing the N-glycoproteins from membranes of breast cancer cell lines highlights the usefulness of the procedure for generating a practical set of potential biomarkers.

Metastatic head and neck malignancy treated using MRI guided interstitial laser phototherapy: An initial case report

Interstitial laser phototherapy (ILP) guided by magnetic resonance imaging (MRI) may become an attractive adjunctive modality for the treatment of deep and surgically inaccessible tumors of the head and neck when accurate methods of laser dosimetry and “real‐time” monitoring techniques with the MRI are introduced. We recently demonstrated in ex vivo and in vivo models, a linear relationship between levels of laser energies, thermal profiles, MR signal intensity changes, and histopathological tissue damage. Results of treatment in a patient with an unresectable large right jugulodigastric metastatic squamous carcinoma using new approach of MRI guided ILP are now reported. The patient complained of significant right‐sided neck pain and headaches secondary to a rapidly growing metastatic lymphadenopathy which recurred after previous surgery, radiation, and chemotherapy. Two treatment sessions were used at an interval of 2 weeks. Each treatment was performed in the MRI suite under heavy sedation. Using a 600‐μm bare fiber of the Nd:YAG laser implanted interstitially under MR guidance, the metastatic node was treated at three sites. T1‐ and T2‐weighted images were performed prior to, immediately after, 24 and 48 hours, and 4, 5, 7, 9, 16, and 25 days post‐treatment. Successful relief of pain and growth arrest of this node was observed after the second treatment and at the 3‐month follow‐up. These results demonstrate that this technique of ILP guided by MRI may be feasible in humans, and will become clinically practical when appropriate methods of dosimetry and instrumentation are developed.

Selective cyclooxygenase-2 inhibitor rofecoxib (Vioxx) induces expression of cell cycle arrest genes and slows tumor growth in human pancreatic cancer

Recent studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in pancreatic adenocarcinoma and may play a critical role in this rapidly progressing form of cancer. A human pancreatic adenocarcinoma cell line, Mia PaCa-2, was incubated for 18 hours with 5µ mol/L of rofecoxib (Vioxx), a selective COX-2 inhibitor. Total RNA was isolated and gene expression analyzed by DNA microarray chips. In a separate experiment, athymic mice were orthotopically injected with 7.5 x 105 Mia PaCa-2 cells through a minilaparotomy. After 1 month, laparotomy was repeated to measure tumor size, and mice were randomized to receive reformulated rodent chow containing either 12.5 mg/kg/day of rofecoxib or no drug for 21 days. Tumor growth was assessed by comparing volume before and after treatment. In vitro, rofecoxib decreased gene expression of cyclin D1/PRAD1, a key component of cell cycle progression, while increasing expression of several cell cycle arrest genes, including p21/WAF1, p33/ING, GADD34, and GADD45 (Pµ0.05). In vivo, tumor growth was significantly reduced in treated vs. control mice (Pµ0.05). No systemic toxicity was observed in mice receiving rofecoxib. These data suggest that rofecoxib slows the growth of human pancreatic cancer through changes in gene expression that favor cell cycle arrest.

Neutralizing Monoclonal Antibody to Periostin Inhibits Ovarian Tumor Growth and Metastasis

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutra-lizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein. Mol Cancer Ther; 10(8); 1500–8. ©2011 AACR.

Interstitial laser phototherapy assisted by magnetic resonance imaging: A new technique for monitoring laser-tissue interaction

The rapid technological advances of magnetic resonance imaging, laser fiberoptics, and compatible probes may allow treatment of deep and sometimes surgically unreach‐able tumors of the head and neck with minimal morbidity through interstitial laser phototherapy. In this study, a new application of magnetic resonance imaging was developed to monitor and quantify laser‐induced tissue damages. Pig skin was exposed to increased levels of argon laser (514.5 ran) at energy densitites between 62.5 and 375 J/cm2 as determined by an accurate and reproducible method of dosimetry. Thermal profiles were recorded using an infrared sensor and Tl‐ and T2‐weighted magnetic resonance images were taken; afterward, biopsies were performed to quantitate the level of tissue damage. Our results demonstrate that above a certain threshold of laser energy, the magnetic resonance imaging findings are temperature dependent. Appropriate development of a scale matching laser energies, temperature profiles, Tl‐ and T2‐weighted magnetic resonance images, and histological quantitation of tissue destruction will allow us to optimize the three‐dimensional control and monitoring of laser‐tissue interactions.

Imaging of human tumor xenografts in nude mice with radiolabeled monoclonal antibodies. Limitations of specificity due to nonspecific uptake of antibody

To determine if in vivo binding specificities of monoclonal antibodies to tumor nodules would reflect in vitro antibody specificities as determined by radioimmunoassay, two monoclonal antibodies were selected for imaging of human tumor xenografts in nude mice. By in vitro radioimmunoassay, antibody 436 binds to the M20 melanoma cell line, but not to the P3 carcinoma cell line; antibody 44 binds to P3 but not to M20. Both antibodies are IgG1 isotypes. Nude mice bearing an M20 melanoma in one flank and a P3 carcinoma in the other were injected intravenously with 5 to 25 μg of each antibody labeled with either I‐125 or I‐131; in separate animals the labels were reversed. Animals were imaged daily with a scintillation camera equipped with a pinhole collimator. On day 6 the animals were sacrificed, and binding of the antibodies to the tumors and normal tissue were compared. Antibody 436 had up to a two‐fold binding advantage in vivo for the melanoma, whereas antibody 44 had up to a two‐fold binding advantage for the carcinoma, thus confirming the in vitro specificities of each. However, imaging on day 4 and computer analysis of percent radioactivity in the tumors showed that tumor images were related directly to tumor size and relatively uninfluenced by antibody specificity. Thus, even though antibody specificity can be demonstrated by differential tissue counting, imaging of tumor deposits appears to involve a nonspecific phenomenon that is largely dependent upon tumor weight.

Hypericin: A New Laser Phototargeting Agent for Human Cancer Cells†‡

Laser activation of anthracycline‐related drugs combines chemotherapy with photoablation for improved treatment. Hypericin, a structurally related anthraquinone, was tested for laser activation and cytotoxicity in human cancer cells. Viability of P3 squamous cell carcinoma cells incubated with 1 to 20μg/mL hypericin was reduced by more than 95% after 1 minute exposure at 4°C to an argon laser (514 nm, 5 W), a KTP‐532 laser (532 nm, 5 W), or a 20‐A xenon lamp. Viability was reduced over 90% in six human carcinoma, sarcoma, and melanoma cell lines by this combined treatment, but only trace toxicity was seen after separate exposure to hypericin or light alone. These results show that hypericin is a sensitive agent for phototherapy of human cancer cells in vitro and indicate that this drug may be useful for tumor targeting via minimally invasive imaging‐guided laser fiberoptics.

Hypericin uptake in rabbits and nude mice transplanted with human squamous cell carcinomas: study of a new sensitizer for laser phototherapy.

Tissue uptake and biodistribution of hypericin was measured in rabbits and in nu/nu mice xenografted with P3 human squamous cell carcinoma to assess the value of this dye as an in vivo sensitizer for laser photoinactivation of solid tumors. Hypericin has absorption maxima at 545 and 590 nm with a fluorescence emission peak at 640 nm in ethanol. Dye uptake after intravenous injection was tested at 4 and 24 hours in rabbit tissues by ethanol extraction and quantitative fluorescence spectrophotometry. Maximum dye levels were seen at 4 hours in most vascular organs with lung having fivefold higher uptake than spleen followed by liver, blood, and kidney. Mice were examined after 2, 4, 6, 8, and 24 hours and after 3 and 7 days for dye uptake. The peak concentration of hypericin in murine organs was reached at 4 hours with uptake per gram of tissue as follows: lung>spleen>liver>blood>kidney>heart>gut>tumor>stomach>skin>muscle>brain. Elimination of hypericin was rapid in most murine organs with residual dye under 10% of maximum by 7 days compared to 25% to 30% retention for the squamous cell tumors and several normal tissues. These results suggest that hypericin may be a useful photosensitizer for KTP/532 laser interstitial therapy of human cancer.

Intratumoral hypericin and KTP laser therapy for transplanted squamous cell carcinoma.

Objectives/Hypothesis To test intratumoral photodynamic therapy (IPDT) as a new treatment for squamous cell carcinoma in a preclinical tumor model.