Aminolevulinic Acid-Mediated Photodynamic Therapy Causes Cell Death in MG-63 Human Osteosarcoma Cells.

OBJECTIVE: The aim of this study was to test the efficacy of aminolevulinic acid-mediated photodynamic therapy (PDT) against the human osteosarcoma cell line MG-63. BACKGROUND DATA: Osteosarcoma is the most common type of primary malignant bone tumor diagnosed in the United States among adolescents and children. Treatments for osteosarcoma often result in diminished limb use or amputation. Because ALA-mediated PDT exhibits dual specificity in the context of tumor killing, this therapy could represent a less invasive, but effective, treatment for this disease. MATERIALS AND METHODS: To assess ALA dark toxicity in MG-63 cells, cells were incubated with varying concentrations of ALA, and cell viability was determined by crystal violet assay. Protoporphyrin IX (PpIX) accumulation was assessed subsequent to ALA incubation at various concentrations using spectrofluorometry. Cell death subsequent to ALA-PDT was determined by illuminating cells at a wavelength of 635 nm at various light intensities subsequent to ALA incubation. Cell viability was assessed using the MTT assay. RESULTS: ALA dark toxicity was observed only at the highest concentrations of 2, 5, and 10 mM. Maximal PpIX concentration was observed at 0.5 and 1 mM ALA, subsequent to a 24-h incubation. Maximal cell death with minimal light toxicity was observed at 0.5 and 1 mM ALA after illumination with 0.6 and 3 J/cm(2) light. CONCLUSIONS: Collectively, our data indicate that ALA-PDT can result in the death of MG-64 human osteosarcoma cells in vitro.

The carboxyl-terminal PDZ ligand motif of chemokine receptor CXCR2 modulates post-endocytic sorting and cellular chemotaxis.

Adaptor protein interaction with specific peptide motifs found within the intracellular, carboxyl terminus of chemokine receptor CXCR2 has been shown to modulate intracellular trafficking and receptor function. Efficient ligand-induced internalization of this receptor is dependent on the binding of adaptor protein 2 to the specific LLKIL motif found within the carboxyl terminus (1). In this study we show that the carboxyl-terminal type 1 PDZ ligand motif (-STTL) of CXCR2 plays an essential role in both proper intracellular receptor trafficking and efficient cellular chemotaxis. First, we show that CXCR2 is sorted to and degraded in the lysosome upon long-term ligand stimulation. We also show that receptor degradation is not dependent upon receptor ubiquitination, but is instead modulated by the carboxyl-terminal type I PDZ ligand of CXCR2. Deletion of this ligand results in increased degradation, earlier co-localization with the lysosome, and enhanced sorting to the Rab7-positive late endosome. We also show that deletion of this ligand effects neither receptor internalization nor receptor recycling. Furthermore, we demonstrate that deletion of the PDZ ligand motif results in impaired chemotactic response. The data presented here demonstrate that the type I PDZ ligand of CXCR2 acts to both delay lysosomal sorting and facilitate proper chemotactic response.

The lymphotoxin-beta receptor is an upstream activator of NF-kappaB-mediated transcription in melanoma cells.

The pleiotropic transcription factor nuclear factor-kappaB (NF-kappaB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-kappaB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-kappaB has not been elucidated in the tumors. We have previously shown that NF-kappaB-inducing kinase (NIK) protein and its association with Inhibitor of kappaB kinase alphabeta are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-kappaB. Moreover, expression of dominant negative NIK blocked this base-line NF-kappaB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-kappaB, only the lymphotoxin-beta receptor (LTbeta-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTbeta-R and constitutive NF-kappaB transcriptional activity. Moreover, we show that activation of the LTbeta-R can drive NF-kappaB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTbeta-R shRNA resulted in decreased NF-kappaB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTbeta-R constitutively induces NF-kappaB activation, and this event may be associated with autonomous growth of melanoma cells.

Role of chemokines in tumor growth.

Chemokines play a paramount role in the tumor progression. Chronic inflammation promotes tumor formation. Both tumor cells and stromal cells elaborate chemokines and cytokines. These act either by autocrine or paracrine mechanisms to sustain tumor cell growth, induce angiogenesis and facilitate evasion of immune surveillance through immunoediting. The chemokine receptor CXCR2 and its ligands promote tumor angiogenesis and leukocyte infiltration into the tumor microenvironment. In harsh acidic and hypoxic microenvironmental conditions tumor cells up-regulate their expression of CXCR4, which equips them to migrate up a gradient of CXCL12 elaborated by carcinoma-associated fibroblasts (CAFs) to a normoxic microenvironment. The CXCL12-CXCR4 axis facilitates metastasis to distant organs and the CCL21-CCR7 chemokine ligand-receptor pair favors metastasis to lymph nodes. These two chemokine ligand-receptor systems are common key mediators of tumor cell metastasis for several malignancies and as such provide key targets for chemotherapy. In this paper, the role of specific chemokines/chemokine receptor interactions in tumor progression, growth and metastasis and the role of chemokine/chemokine receptor interactions in the stromal compartment as related to angiogenesis, metastasis, and immune response to the tumor are reviewed.

In situ analysis of breast cancer progression in murine models using a macroscopic fluorescence imaging system.

BACKGROUND AND OBJECTIVE: The goal of this study was to use an inexpensive macroscopic imaging system to monitor tumor progression in mouse models in real-time with minimal intervention. STUDY DESIGN/MATERIALS AND METHODS: Illumination is provided via a xenon arc lamp and a fiber optic probe which delivers white light or quasi-monochromatic excitation via specific bandpass filters. Fluorescence emission from SCID and nude mice following mammary fat pad injection of red fluorescence protein (RFP)-expressing human breast cancer cell lines was recorded and quantified using a single lens reflex (SLR) digital camera. RESULTS: This simple system enabled the verification of successful tumor take and temporal quantification of tumor progression in mouse models. CONCLUSION: The macroscopic fluorescence imaging system represents an inexpensive and portable tool to facilitate non-invasive in situ cancer detection with the potential to monitor fluorescent tumor formation and investigation of the efficacy of potential cancer therapeutics.

Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells.

INTRODUCTION: The metastatic progression of cancer is a direct result of the disregulation of numerous cellular signaling pathways, including those associated with adhesion, migration, and invasion. Members of the Rac family of small GTPases are known to act as regulators of actin cytoskeletal structures and strongly influence the cellular processes of integrin-mediated adhesion and migration. Even though hyperactivated Rac proteins have been shown to influence metastatic processes, these proteins have never been directly linked to metastatic progression. METHODS: To investigate a role for Rac and Cdc42 in metastatic breast cancer cell invasion and migration, relative endogenous Rac or Cdc42 activity was determined in a panel of metastatic variants of the MDA-MB-435 metastatic human breast cancer cell line using a p21-binding domain-PAK pull down assay. To investigate the migratory and invasive potential of the Rac isoforms in human breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed either dominant active Rac1 or dominant active Rac3 into the least metastatic cell variant. Dominant negative Rac1 or dominant negative Rac3 were stably expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1 or Rac3 were analyzed using in vitro adhesion, migration and invasion assays. RESULTS: We show that increased activation of Rac proteins directly correlates with increasing metastatic potential in a panel of cell variants derived from a single metastatic breast cancer cell line (MDA-MB-435). The same correlation could not be found with activated Cdc42. Expression of a dominant active Rac1 or a dominant active Rac3 resulted in a more invasive and motile phenotype. Moreover, expression of either dominant negative Rac1 or dominant negative Rac3 into the most metastatic cell variant resulted in decreased invasive and motile properties. CONCLUSION: This study correlates endogenous Rac activity with high metastatic potential and implicates Rac in the regulation of cell migration and invasion in metastatic breast cancer cells. Taken together, these results suggest a role for both the Rac1 and Rac3 GTPases in human breast cancer progression.