Loperamide, an FDA-approved antidiarrhea drug, effectively reverses the resistance of multidrug resistant MCF-7/MDR1 human breast cancer cells to doxorubicin-induced cytotoxicity.

Loperamide is an FDA-approved antidiarrhea drug which acts on the µ-opioid receptors in the mesenteric plexus of large intestine and exhibits limited side effects. We hypothesized that loperamide might reverse the multidrug resistance (MDR) of human cancer cells to chemotherapeutic agents. MCF-7/MDR1 cells express high level of MDR1 and are resistant to doxorubicin. We found that loperamide significantly enhanced the cytotoxicity of doxorubicin to MCF-7/MDR1 cells in a dose-dependent manner. In conclusion, loperamide reversed the resistance of MCF-7/MDR1 cells to doxorubicin, suggesting that chemotherapy in combination with loperamide may benefit patients with MDR tumors once applied in clinic.

Synthesis of amphiphilic triblock copolymers as multidentate ligands for biocompatible coating of quantum dots.

One barrier to apply current tri-octylphosphine oxide (TOPO) based quantum dots (QDs) to biomedical imaging is that the TOPO on TOPO-QDs can be replaced by the proteins in living system, which may cause the degradation of QDs and/or deactivation of protein. In order to develop biocompatible optical imaging agents, a novel triblock copolymer, designed as a multidentate ligand, was synthesized to coat quantum dot nanocrystals (QDs). The copolymer consists of a polycarboxylic acid block at one end and a polythiol block at the other end with an intervening cross-linked poly(styrene-co-divinylbenzene) block bridging the ends. The multiple mercapto groups from the polythiol block act as multidentate ligands to stabilize QDs, while the polycarboxylic acid block improves the water solubility of QDs and offers reaction sites for surface modification or conjugation with bimolecules. The cross-linked poly(styrene-co-divinylbenzene) block provides a densely compacted hydrophobic shell. This shell will act as a barrier to inhibit the degradation of QDs by preventing the diffusion of ions and small molecules into the core of QDs. This new multidentate polymer coating facilitates the transfer of QDs from organic solvent into aqueous phase. The QDs directly bound to multidentate mercapto groups instead of TOPO are less likely to be affected by the mercapto or disulfide groups within proteins or other biomolecules. Therefore, this research will provide an alternative coating material instead of TOPO to produce QDs which could be more suitable for in vivo use under complex physiological conditions.

A nanocomplex system as targeted contrast agent delivery vehicle for magnetic resonance imaging dynamic contrast enhancement study.

We have developed and tested a liposomal nanocomplex system, which contains Gd-DTPA as a payload and transferrin on the surface, as a tumor specific targeting MRI contrast agent for studying prostate cancer tumors in mice. In vivo, the probe significantly enhanced the MRI signal. The image contrast between the peripheral region of the tumor and the non-involved muscle was nearly 50% higher two hours after administration of the nanocomplex. The liposomal nanocomplex increased the amount of Gd accumulated in tumors by factor 2.8 compared to that accumulated by using Magnevist alone. Moreover, the heterogeneous MRI image features correlate well with the tumor pathology. The image enhancement patterns can be used for cancer prognosis and non-invasive monitoring of the response to therapy.

Salvianolic acid B inhibits growth of head and neck squamous cell carcinoma in vitro and in vivo via cyclooxygenase-2 and apoptotic pathways.

Overexpression of cyclooxygenase-2 (COX-2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti-inflammatory drug, which inhibits COX-2 but not COX-1. This selective COX-2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long-term chemoprevention and therapy. Salvianolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal-B inhibits HNSCC growth. Sal-B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal-B was assessed in HNSCC and other cell lines by estimating COX-2 expression, cell viability and caspase-dependent apoptosis. Sal-B inhibited growth of HNSCC JHU-022 and JHU-013 cells with IC(50) of 18 and 50 microM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal-B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX-2 inhibitors. Tumor volumes in Sal-B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal-B inhibited COX-2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal-B also caused dose-dependent inhibition of prostaglandin E(2) synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal-B shows promise as a COX-2 targeted anticancer agent for HNSCC prevention and treatment.

Vitamin E succinate induces ceramide-mediated apoptosis in head and neck squamous cell carcinoma in vitro and in vivo.

PURPOSE: Vitamin E succinate (alpha-TOS) inhibits the growth of cancer cells without unacceptable side effects. Therefore, the mechanisms associated with the anticancer action of alpha-TOS, including ceramide-mediated apoptosis, were investigated using head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. EXPERIMENTAL DESIGN: Five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) were treated with alpha-TOS, and its effects on cell proliferation, cell cycle progression, ceramide-mediated apoptosis, and ceramide metabolism were evaluated. The anticancer effect of alpha-TOS was also examined on JHU-022 solid tumor xenograft growth in immunodeficient mice. RESULTS: Alpha-TOS inhibited the growth of all the HNSCC cell lines in vitro in a dose- and time-dependent manner. Thus, JHU-013 and JHU-022 cell lines were more sensitive to alpha-TOS than the other cell lines. Cellular levels of ceramide, sphingomyelinase activity, caspase-3, and p53 were elevated with increasing time of exposure to alpha-TOS. The degradation of poly(ADP-ribose) polymerase protein in JHU-022 cells treated with alpha-TOS provided evidence for apoptosis. The amounts of nuclear factor kappaB, Bcl-2, and Bcl-X(L) proteins were reduced in the cells treated with alpha-TOS for 6 hours. The levels of caspase-9, murine double minute-2, and IkappaB-alpha proteins were unchanged after alpha-TOS treatment. I.p. administration of alpha-TOS slowed tumor growth in immunodeficient mice. CONCLUSIONS: Alpha-TOS showed promising anticancer effects to inhibit HNSCC growth and viability in vivo and in vitro. The induction of enzymes involved in ceramide metabolism by alpha-TOS suggests that ceramide-mediated apoptosis may expand therapeutic strategies in the treatment of carcinoma.

Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation.

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the beta-casein gene promoter (p-344/-1betac-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction (R=0.99, P<0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous beta -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.

Bioluminescent animal models of human breast cancer for tumor biomass evaluation and metastasis detection.

INTRODUCTION: Convenient animal models are needed to study the progression and treatment of human tumors in vivo. Luciferase-based bioluminescent imaging (BLI) enables researchers to monitor tumors noninvasively and is sensitive to subtle changes in tumors. METHODS: Three human breast cancer models in nude mice were established by using luciferase-expressing MDA-MB-231-luc cells. They were subcutaneous xenografts (n = 8), mammary gland xenografts (n = 5), and lung metastases (n = 3). The tumors were imaged in live mice by using a highly sensitive BLI system. The relationship between the intensity of bioluminescence from the tumor was analyzed with respect to tumor volume. Bioluminescent signals from lung metastases were studied to determine the threshold of detectability. RESULTS: Tumors growing in the mice's backs and mammary gland fat pads were imaged dynamically after administration of D-luciferin. The bioluminescent intensity from the tumors gradually increased and then decreased in a one-hour span. The time to reach maximum signal intensity differed significantly among tumors and was independent of tumor volume and unrelated to maximum signal intensity. A significant correlation was observed between tumor volume and maximum signal intensity in tumors from both sites. Lung metastatic lesions of .3-.5 mm in diameter were clearly detectable through the entire animal imaging process. CONCLUSION: The animal models established with luciferase-expressing cancer cells in combination with BLI provide a system for rapid, noninvasive, and quantitative analysis of tumor biomass and metastasis. This biosystem simplifies in vivo monitoring of tumors and will be useful for noninvasive investigation of tumor growth and response to therapy.

Cytotoxicity of etidronic acid to human breast cancer cells.

INTRODUCTION: Bisphosphonates have been used to treat Paget's disease, osteoporosis, and cancer metastases to the bone. The cancer chemotherapeutic potential of a first-generation bisphosphonate, etidronic acid, was evaluated by using MCF-7 human breast cancer cells. METHODS: In vitro cytotoxicity of etidronic acid to MCF-7 cells was estimated on the basis of clonogenicity assays, while cell cycle effects were determined by using flow cytometry. Mutagenicity of etidronic acid was detected by using denaturing high-pressure liquid chromatography analysis of cellular DNA amplified by PCR with primers for exons 5 through 8 of the human p53 gene. RESULTS: A 24-hour treatment with etidronic acid (10 mM) with or without strontium chloride was cytototoxic to MCF-7 cells. Etidronic acid caused a decrease in the S-phase population and an increase in the G2/M population. Mutations in the p53 gene were detected in MCF-7 cells treated with etidronic acid. Strontium chloride was not cytotoxic to cells. CONCLUSIONS: Cytotoxicity of etidronic acid to breast cancer cells may complement its inhibitory effects on bone resorption at the site of bone metastasis. Within the cell cycle, late S-phase cells are the most radioresistant, while cells at the G2/M border are the most sensitive. Therefore the decrease in S-phase population with corresponding increase in G2/M would make the cells more radiosensitive. This may be useful if etidronic acid were combined with radioactive strontium (89Sr, metastron) or external-beam radiotherapy for treating bone metastases. Tumor cells that survive etidronic acid treatment may acquire drug resistance because of mutations in the p53 tumor-suppressor gene.

Nicotine decreases the cytotoxicity of doxorubicin towards MCF-7 and KB-3.1 human cancer cells in culture.

The harmful effects of tobacco use and nicotine are well known. There is strong epidemiological evidence for smoking as a risk factor for cancer of the lung and oral cavity. The evidence for second-hand smoke as a risk factor for breast cancer is rapidly accumulating. The anthracycline doxorubicin is used in the treatment of many types of malignancies, including breast cancer. The effect of nicotine on doxorubicin toxicity was evaluated in MCF-7 and KB-3.1 cancer cell cultures. Nicotine partially inhibited doxorubicin toxicity towards MCF-7 and KB-3.1 cells, as judged by clonogenicity and flow cytometry assays. Flow cytometric analysis showed that < 10% of cells treated with doxorubicin underwent apoptosis, while necrosis was the major mode of cell death. Inhibition of apoptosis and necrosis in cancer cells by nicotine can diminish the effectiveness of doxorubicin in cancer therapy.

Relationship Between Aerobic Fitness, the Serum IGF-1 Profiles of Healthy Young Adult African American Males, and Growth of Prostate Cancer Cells.

The growth of prostate tumors is mediated by the bioavailability of androgens and insulin-like growth factors. This study tested the hypothesis that healthy young adult African American men exhibiting low aerobic capacity (fitness) have serum insulin-like growth Factor-1 (IGF-1) and testosterone levels that promote growth of prostate cancer cells. A cross-sectional data research design was used to study groups of 18- to 26-year-old healthy men exhibiting low and moderate aerobic fitness, based on their peak oxygen consumption (VO2peak). The individual serum levels of IGF-1, IGF-1 binding protein-3 (IGFBP-3), and testosterone were measured. In vitro growth of androgen-dependent LNCaP prostate tumor cells was measured after incubation in culture medium fortified with each subject's serum. Aerobic capacity was significantly greater in the moderate-fitness group than in the low-fitness group without an intergroup difference in body mass index. The serum IGF-1 concentration was significantly higher in the low-fitness group in the absence of an intergroup difference in serum testosterone. The serum IGFBP-3 concentration was significantly lower in the low-fitness group. Prostate tumor cell growth was significantly greater in the cultures incubated in media containing the sera of the low-fitness group than in the sera of the moderate-fitness group. These findings suggest that moderate aerobic fitness in young adults may decrease the circulating levels of free IGF-1 and lower the potential to support growth of prostate cancer cells.

Cytotoxicity of liposomal alpha-tocopheryl succinate towards hamster cheek pouch carcinoma (HCPC-1) cells in culture.

There is compelling evidence for the cancer chemopreventive effects of vitamin E and related compounds. Of all the vitamin E derivatives that have been investigated to date, vitamin E acid succinate is the most effective anti-cancer agent. This report describes the preparation and testing of liposomal formulation of mono alpha-tocopheryl ester of succinic acid (alpha-TOS) for cytotoxicity against hamster cheek pouch carcinoma cell line (HCPC-1). Small unilamellar vesicles (SUV) of phosphatidylcholine incorporating 70 microM alpha-TOS were superior to alpha-TOS alone or SUV without incorporated alpha-TOS, as inducers of apoptosis in HCPC-1 cells. Liposomal alpha-TOS perturbed the lipid structure in cells, promoted apoptosis, and decreased cell viability. The mechanism of action of alpha-TOS appears to involve membrane damage and induction of ceramide mediated apoptosis.

Limits of stimulation of proliferation and differentiation of bone marrow cells of mice treated with swainsonine.

The limits of stimulation of the immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) were studied in inbred C57BL/6 mice for potential support of intense high dose cancer chemotherapy and/or radiation because of its attractive pharmacologic profile on the hematopoietic system. Specifically, the effects of swainsonine on bone marrow cellularity and on in vitro progenitor cell proliferation to total colony forming units (CFU) and differentiation to different lineages were studied as a function of number of days post drug administration. The lineages evaluated were colony forming units-granulocyte-macrophage (CFU-GM), erythroid-burst forming units (BFU-e) and CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM or CFU-Mix). Groups of mice were treated with swainsonine or plain vehicle, phosphate buffered saline for 10 consecutive days. The effects of these agents on the hematopoietic system were studied up to 60 days following their discontinuation. The magnitude of the effects of swainsonine on bone marrow system gradually declined with increasing duration of days following its discontinuation. Nevertheless, its residual stimulatory effects on bone marrow cellularity, total CFU, CFU-GM, BFU-e and CFU-Mix continued to be significant (P<0.0001) up to 45, 50, 50, 55 and 50 days, respectively, compared to those of diluent buffer or untreated controls. Since cancer chemotherapeutic agents or radiation are normally given in schedules and/or cycles, these results strongly suggest that swainsonine effects are sustained long enough to potentially support and facilitate hematopoietic recovery during anti-cancer cytotoxic treatment.

Enhanced proliferation of functionally competent bone marrow cells in different strains of mice treated with swainsonine.

The immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) has potential for overcoming the bone marrow suppressive effects of cancer chemotherapy and radiotherapy. An earlier study showed that multiple doses of swainsonine enhanced bone marrow cellularity in four different strains (C57BL/6; C3H-HEN; Balb/C and DBA-2 mice) of inbred mice which were not exposed to any chemotherapeutic agents or radiation. In vitro assessment of total colony formation capacity of bone marrow cells (BM CFUs) showed a 2- to 8-fold increase in swainsonine-treated mice compared to control mice that were given sham injections of physiological saline. In the current study, we have evaluated the functional competence of the bone marrow cells produced in response to swainsonine treatment of normal healthy mice. In particular, colony forming units-granulocyte-macrophage (CFU-GM), erythroid-burst forming units (BFUe) and CFU-Mix (or CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM)) levels, were determined using in vitro assays. The time course of the changes in CFU-GM, BFUe and CFU-Mix (CFU-GEMM) were also followed. Our results demonstrate that swainsonine bolsters the CFU capacity of BM cells without loss of function to levels which are several folds higher than in sham-treated control mice. Swainsonine treatment caused an increase in all lineages of marrow cells without loss of function. This effect was reproduced in all four strains of inbred mice in this investigation. Examination of the peripheral blood did not reveal increase in white blood cells or changes in the hematocrit levels. The long-term effects of swainsonine treatment are not known at present. Nonetheless, swainsonine-induced increase in CFU capacity of bone marrow cells and related cells along the different differentiation paths should find clinical application in cancer treatment with chemotherapeutic agents and/or radiation.

Strontium 89 in the treatment of pain due to diffuse osseous metastases: a university hospital experience.

More than two-thirds of the patients with osseous metastases experience debilitating bone pain, requiring some form of pain relief. Analgesics are limited in their efficacy. Palliative application of hemi-body external beam radiation therapy in the treatment of multiple osseous metastases also is limited due to toxicity associated with large treatment ports. Intravenous injections of bone seeking radioisotopes are effective in the palliation of pain with fewer side effects. Forty-one patients with multiple osseous metastases due to prostate and breast cancer were treated with strontium chloride 89 (89Sr) at the department of radiation oncology, in a university hospital. A retrospective analysis of these patients indicated that all subjects had severe pain that diminished their quality of life. Most of these patients had multiple co-morbid factors. Many were on opioids leading to adverse effects such as nausea, constipation, and drowsiness that required additional medication. Objective findings and evaluation of the responses were not always available for all patients. Following treatmentwith 89Sr, over two-thirds of the patients responded favorably and required lower doses of opioids.

Immediate breast reconstruction-impact on radiation management.

Breast reconstruction is an option for women undergoing modified radical mastectomy due to a diagnosis of breast cancer. In certain patients, breast reconstruction is performed by insertion of a temporary tissue expander prior to the placement of permanent breast implants. Some of these patients, following mastectomy, may require chest wall irradiation to prevent loco regional relapse. The compatibility of radiation and tissue expanders placed in the chest wall is of major concern to the radiation oncologist. Clinically undetectable changes can occur in the tissue expander during the course of radiation therapy. This can lead to radiation treatment set-up changes, variation in tissue expansion resulting in unwanted cosmesis, and deviation from the prescribed radiation dose leading to over and/or under dosing of tumor burden. At Howard University hospital, a CT scan was utilized to evaluate the status of the temporary tissue expander during radiation treatment to enable us to prevent radiation treatment related complications resulting from dosimetric discrepancies. CT images of the tissue expander were obtained through the course of treatment. To avoid a 'geographic miss' the amount of fluid injected into the tissue expander was kept constant following patient's satisfaction with the size of the breast mound. The CT scans allowed better visualization of the prosthesis and its relation to the surrounding tumor bed. This technique ensured that anatomical changes occurring during radiation treatment, if any, were minimized. Repeated dosimetry evaluations showed no changes to the prescribed dose distribution. A CT of the reconstructed breast provides an important quality control. Further studies with greater number of patients are required for confirming this impact on radiation treatment.

Techniques for physicochemical characterization of nanomaterials.

Advances in nanotechnology have opened up a new era of diagnosis, prevention and treatment of diseases and traumatic injuries. Nanomaterials, including those with potential for clinical applications, possess novel physicochemical properties that have an impact on their physiological interactions, from the molecular level to the systemic level. There is a lack of standardized methodologies or regulatory protocols for detection or characterization of nanomaterials. This review summarizes the techniques that are commonly used to study the size, shape, surface properties, composition, purity and stability of nanomaterials, along with their advantages and disadvantages. At present there are no FDA guidelines that have been developed specifically for nanomaterial based formulations for diagnostic or therapeutic use. There is an urgent need for standardized protocols and procedures for the characterization of nanoparticles, especially those that are intended for use as theranostics.

Chemoprevention of skin cancer: effect of Lawsonia inermis L. (Henna) leaf powder and its pigment artifact, lawsone in the Epstein- Barr virus early antigen activation assay and in two-stage mouse skin carcinogenesis models.

In continuation of our studies with chemoprevention potential of plant-derived naphthoquinone derivatives, leaf powder of the medicinal plant Lawsonia inermis L, commonly known as 'henna', was evaluated by its inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Lawsone (2-hydroxy- 1,4-naphthoquinone), the reddish orange pigment artifact formed during the extraction or preparation of the dye from henna leaves and believed to be the active component, was also assessed in this in vitro assay. Both showed a profound inhibition (>88%) of EBV-EA activation. In the in vivo two-stage mouse skin carcinogenesis study using UV-B radiation for initiation and TPA for tumor promotion, oral feeding of henna (0.0025%) in drinking water ad libitum decreased tumor incidence by 66% and multiplicity by 40% when compared to the positive control at 10 weeks of treatment. Similarly, in the above mouse model, orally fed lawsone (0.0025%) decreased tumor incidence by 72% and multiplicity by 50%. The tumor inhibitory trend continued throughout the 20-week test period. Similar antitumor activities were observed when henna (0.5 mg/ml) was applied topically on the back skin in the UV-B initiated, TPA promoted and peroxynitrite initiated, TPA promoted mouse skin carcinogenesis models. Topically applied lawsone (0.015 mg/ml) also exhibited similar protection against tumor formation in the 7,12-dimtehylbenz(a)anthracene induced and TPA promoted skin cancer in mice. Also, there was a delay of 1 to 2 weeks in tumor appearance in both henna and lawsone treated groups compared to control in all three test models. This study ascertains the skin cancer chemopreventive activity of henna leaf powder and lawsone when administered by either oral (through drinking water) or topical (by application on the back skin) routes. Further, it emphasizes the need for the evaluation of these henna-derived green chemopreventive candidates in combination with currently used sunscreen agents for complementary anticancer potential against UV-induced skin carcinogenesis.

An anti-PSMA bivalent immunotoxin exhibits specificity and efficacy for prostate cancer imaging and therapy.

Prostate specific membrane antigen (PSMA) is overexpressed on prostate tumor cells and the neovascular endothelia various solid tumors. A bivalent immunotoxin generated by fusing a fold-back single-chain diabody derived from the Fv fragments of an anti-PSMA monoclonal antibody with a truncated diphtheria toxin (DT) containing the activity and translocation domains [A-dmDT390-scfbDb(PSMA)] might be suitable for targeted therapy of tumors that overexpress PSMA. In this study, a PSMA-positive and a PSMA-negative prostate cancer cell lines were treated with immunotoxin A-dmDT390-scfbDb(PSMA) in order to study the tumor targeting specificity and therapeutic potential of the immunotoxin. The cellular uptake and selective toxicity of the immunotoxin were evident in monolayer cultures of PSMA-positive LNCaP prostate cancer cells but not in cultures of PSMA-negative PC-3 prostate cancer cells. Cellular accumulation of A-dmDT390-scfbDb(PSMA) increased with increasing incubation times and concentrations in LNCaP cells. The proportion of apoptotic LNCaP cells increased upon incubation with increasing doses of the fold-back immunotoxin. Optical imaging and MRI with the Alexa Fluor 680-labeled A-dmDT390-scfbDb(PSMA) confirmed the specific targeting and therapeutic efficacy of this immunotoxin towards PSMA-positive LNCaP solid tumor xenografts in athymic nude mice.

Evaluation of Tumor Cell Response to Hyperthermia with Bioluminescent Imaging.

BACKGROUND: Hyperthermia is used in combination with radiotherapy and/or chemotherapy in the treatment of various types of cancer. Currently, the tumor cell response to hyperthermia is determined largely based on the size reduction of tumor mass, which is insensitive. METHODS: We tested the feasibility of bioluminescent imaging (BLI) in evaluation of the tumor cell response to hyperthermia by exposing luciferase-expressing MDA-MB-231-luc human breast cancer cells to high temperature (43 °C) for 10 minutes to 2 hours. The tumor cells were the imaged and the light signal generated by the tumor cells was quantified with BLI. To validate its usefulness, the light signal intensity was comparatively analyzed with the tumor cell clonogenicity and cell viability, which were measured with classic clonogenic and MTT assays. RESULTS: The light signal intensity determined by BLI was closely correlated with the absolute number of viable cells as well as the cell viability measured with the traditional MTT assay under normal culture condition. Relative to the clonogenicity of tumor cells after exposure to hyperthermia, however, BLI underestimated, while MTT assay overestimated the cell viability. Difference in the interpretation of tumor cell clonogenic ability following hyperthermia with BLI, MTT dye, and clonogenic assay may be due to the different mechanisms of the three measurements as well as the fact that hyperthermia can induce cell damage at levels of both transient and permanent. CONCLUSIONS: BLI is sensitive, convenient, and potentially valuable in the evaluation and monitoring of tumor cell response to treatments including hyperthermia.

Glucosamine-linked near-infrared fluorescent probes for imaging of solid tumor xenografts.

PURPOSE: Near-infrared fluorescence (NIRF) imaging is an attractive technique for studying diseases at the molecular level in vivo. Glucose transporters are often used as targets for in vivo imaging of tumors. The efficiency of a tumor-seeking fluorescent probe can be enhanced by attaching one or more glucosamine (GlcN) moieties. This study was designed to evaluate the use of previously developed GlcN-linked NIRF probes for in vitro and in vivo optical imaging of cancer. PROCEDURES: Cellular uptake of the probes (1 µM) was investigated in monolayer cultures of luciferase-expressing PC3 (PC3-luc) cells. The prostate tumors were established as subcutaneous xenografts using PC3-luc cells in nude mice. The biodistributions and tumor-targeting specificities of cypate (cyp), cypate-D: -(+)-glucosamine (cyp-GlcN), and D: -(+)-gluosamine-cypate-D: -(+)-gluosamine (cyp-2GlcN) were studied. The tumor, muscle, and major organs were collected for ex vivo optical imaging. RESULTS: The tumor cell uptake of the probe containing two glucosamine residues, cyp-2GlcN, was significantly higher than the uptake of both the probe with one glucosamine residue, cyp-GlcN, and the probe without glucosamine, cyp only. Similarly, in in vivo experiments, cyp-2GlcN demonstrated higher maximum fluorescence intensity and longer residence lifetime in tumors than cyp-GlcN or cyp. The ex vivo biodistribution analysis revealed that tumor uptake of cyp-2GlcN and cyp-GlcN was four- and twofold higher than that of cyp at 24 h post-injection, respectively. CONCLUSION: Both cyp-GlcN and cyp-2GlcN NIRF probes exhibited good tumor-targeting properties in prostate cancer cell cultures and live mice. The cyp-2GlcN probe showed the highest uptake with good retention characteristics in vivo. The uptake of cyp-2GlcN and cyp-GlcN is likely mediated by glucosamine-recognizing transporters. The uptake mechanism is being explored further for developing cypate-glucosamine-based probes for in vivo imaging.