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The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cell line to hyperthermia at 44 degrees C were investigated. The cell phase response as well as the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of 44 degrees C hyperthermia on A549 cells exhibited low thermosensitivity with a T(0) value of 12 min. The slope of the survival curve in the exponential phase, described semilogarithmically, in 44 degrees C hyperthermia combined treatment with AMROH (0.02 microg/ml) was approximately parallel to 44 degrees C hyperthermia alone. The initial shoulder shape portion of the survival curve from 44 degrees C hyperthermia alone, indicating the repair of sublethal thermal damage (SLTDR), was reduced with the sequential combined treatment of AMR or AMROH. Sequential treatments with AMR or AMROH prior to 44 degrees C hyperthermia resulted in additive thermo-enhancement effect by reducing not only survival but was shoulder wide. Furthermore, like AMR and AMROH, adriamycin (ADM) and etoposide (VP-16) are DNA topoisomerase II inhibitors, and the effects of these 4 agents on 44 degrees C hyperthermia were compared. All 4 agents exhibited comparable thermo-enhancement effects. Using synchronized A549 cells, AMR or AMROH did not elicit cell phase responses, irrespective of the concentration. The induction of apoptosis was investigated at 48 and 72 h after AMROH treatment, 44 degrees C hyperthermia or the combined treatment, in which apoptosis was not significantly induced after any treatment. Furthermore, the incidence of necrosis was examined as well as apoptosis. The incidence of necrosis at 48 and 72 h after AMROH was 2.4 and 4.3%, respectively; after 44 degrees C hyperthermia was 3.3 and 4.0%, respectively; and after the combined treatment it was 10.7 and 8.7%, respectively. The necrosis induced after the combined treatment was circa 3 times higher than that in either of the single treatments.

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

The third-order optical nonlinearities and responses of thin films containing the J-aggregates of a cyanine dye or a squarylium dye were measured using the degenerate four-wave mixing (DFWM) technique under resonant conditions. The sol-gel silica coating films containing the J-aggregates of the cyanine dye, NK-3261, are stable at room temperature and durable against laser beam irradiation. The temporal profiles of the DFWM signal were measured with a time resolution of 0.3 ps, and were found to consist of at least three components, i.e., the coherent instantaneous nonlinear response and the two slow responses with delay time constants of ca. 1.0 ps and ca. 5.6 ps. The contribution of the later was small. The electronic component of the effective third-order optical nonlinear susceptibility of the film had value of as high as ca. 3.0 x 10 -7 esu. We also studied the neat film of a squarylium dye J-aggregates. The temporal profile of the DFWM signal of the neat film of squarylium dye was also found to consist of at least three components, the coherent instantaneous nonlinear response and the delayed response with decay time constants of ca. 0.6 ps and ca. 6.5 ps. The contribution of the slow tail was also very small. The electronic component of effective third-order optical nonlinear susceptibility of the neat film of squarylium dye had value of as high as ca. 3.6 x 10 -8 esu.

We report the in vitro and in vivo characteristics of a new molecular conjugate vector for targeting and imaging of tumors. Its core is a cyclodecapeptide platform named RAFT, onto which two spatially independent functional domains can be covalently and stereospecifically linked: a cell-targeting domain for tumor targeting and a labeling domain able to carry two drugs and/or labeling agents. To prove the interest of this carrier, we used a well-known cRGD cyclopeptide, a ligand for the alphavbeta3 integrin. We demonstrate that this vector presenting four cRGD motifs very efficiently prevents alphavbeta3-mediated cell adhesion to vitronectin. Furthermore, it is actively endocytosed because of the multivalent cRGD presentation, a major advantage for drug delivery. In vivo experiments in nude mice reveal that repeated intratumoral injections of low doses of RAFT(cRGD)4 reduce tumor growth. Furthermore, RAFT(cRGD)4 significantly improves the targeting specificity of subcutaneous tumor masses as well as that of disseminated metastasis after intravenous injection. Thus, RAFT(cRGD)4 is specific, internalized, and perfectly controlled and can carry multiple biological functions on a single, spatially defined backbone, making it a powerful and versatile synthetic vector for drug delivery, molecular imaging, or both.

Seventeen strains with wideranging decolorization ability were screened from contaminated soil and were applied on the decoloring of dye wastewater. Three strains were selected due to their high decolorization capacity on azo dyes, anthraquinone dyes and triphenylmethane dyes. The three strains were named as Strain I, Strain II, Strain III. This three superior strains were identified as penicillium link (Strain Istrain II) and Cephalosporium corda (strain III). Using aqueous samples, the influences of several factors on decolorization were reported, such as pH, carbon source, temperature and so on. And then applying of these strains for treatment of actual wastewater have also been done. The results showed that the optimal pH, temperature and carbon source were 5-9, 18-37 degrees C and 0.5% respectively, under those conditions the decolorization removal rate was 70%. And these fungi have a good prospect for the treatment of dye wastewater.

To investigate the techniques and indications of treatment of degenerative scoliosis.The clinical data of 45 degenerative scoliosis patients, 18 males and 27 females, aged 41 - 77, all suffering from severe low back pain, were analyzed. Twenty of them underwent posterior lumbar interbody fusion with pedicle screws combined with correction of scoliosis. Sixteen patients underwent depression of vertebral canal and pure internal fixation and fusion of pedicle. Eight patients underwent pure depression, such as laminectomy or incision of intervertebral foramen. The corrective effects of scoliosis, condition of intervertebral fusion, and complications were observed.All operations were performed successfully and all the patients made a remarkable recovery. During the 13 months' follow-up, 88% of the patients showed remarkable relief from pain and claudication and improved life quality. No loss in correction angle and height of disc space was found among the patients who had undergone internal fixation and interbody fusion. The residual symptom of low back pain was more manifest in the patients who had not undergone internal fixation than in those who had.Posterior lumbar interbody fusion with pedicle screws is safe and effective in the treatment of severe degenerative scoliosis.

Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.

Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.

Both adriamycin (ADM) and hyperthermia show thermal chemo-enhancement. Tolerance induction against ADM in heated cells has been reported resulting in clinical difficulty of cancer therapy. We investigated thermo-enhancement induced with ADM (0.2 microg/ml) treatment alone or combined with ADM and 42 degrees C hyperthermia in Chinese hamster V79 cells in vitro. Intracellular accumulation of hsc70 and hsp72 proteins after hyperthermia or ADM was observed to examine the possible relationship between cell killing effect and their accumulations. Thermosensitivity of V79 cells at 42 degrees C after the simultaneous treatments with ADM showed marked thermo-enhancement within the short-term treatments for less than 1 h, while the combined treatments for longer than 1 h, the cells showed reduced thermosensitivity. Survival from the simultaneous treatments for less than 1 h was reduced markedly less than the single treatment both with ADM or 42 degrees C hyperthermia alone. Thermotolerance was markedly induced in a step-up hyperthermia (42 degrees C 2 h-44 degrees C). The combined treatments with ADM and 44 degrees C hyperthermia following the 42 degrees C preheating alone does not inhibit thermotolerance development. The combined treatments with ADM and 42 degrees C preheating showed markedly interactive cell killing, but no thermo-enhancement to the following 44 degrees C hyperthermia was shown. The leveling slope of the 44 degrees C heating period-survival curve was drawn. In the Western blot analyses, hsc70 existed constitutively in the V79 cells. Following the 42 or 44 degrees C hyperthermia alone, intracellular accumulation of hsp72 was determined. ADM treatment alone did not induce any accumulation of hsp72. In the simultaneous treatments with ADM and hyperthermia, the accumulation of hsp72 was markedly reduced. The accumulation of hsp72 after the combined treatment with ADM and hyperthermia was not observed as markedly as that after hyperthermia alone.

Combined nevi consisting of a Spitz nevus and an acquired nevus are unusual, and, to our knowledge, the combination of a spindle cell Spitz nevus and an overlying compound nevus has not been previously reported. We report a 17-year-old girl with a nodule on the left anterior lower extremity. The nodule was asymptomatic, firm, brown, symmetrical, dome-shaped, 8 mm in diameter, and not found with ulceration. Histological findings showed proliferation of spindle-shaped cells with an overlying compound nevus. The spindle-shaped cells were large, non-pigmented, uniform in size and shape, with rare mitoses and without nuclear atypia, and arranged in a storiform pattern in thick collagen bundles. They stained positively for S-100 and negatively for HMB-45. The lesion was considered to be a new type of combined nevus consisting of a spindle cell Spitz nevus and an overlying compound nevus.

In recent years, the effect of ultrasound (US) in combination with porphyrins such as a gallium-porphyrin complex (ATX-70) has been reported in the treatment of experimental cancers. Before clinical application of this method, further studies are needed. The transplanted squamous cell carcinomas (5CC) on C3H/He mice were irradiated by a tunable optical parametric oscillator (OPO) laser light or ultrasound, in the presence of several different photosensitizers. The tumor size and survival of mice after treatment were observed. Furthermore, we compared antitumor effects of combined irradiation of laser light and ultrasound with those of single laser light or ultrasound, in the presence of ATX-70 or a pheoforbide derivative (PH-1126). Besides PH-i 126 and ATX-70, the following sensitizers were tested: Photofrin II (Pf-II), hematoporphyrin oligomer (HpO). 5-aminolevulinic acid (5-ALA) and aluminum phthalocyanine tetrasuiphonate (AIPcS4). In the results, it was good effects of antitumor by the laser light treatment in the case of PH-1126 and AIPcS4, ATX-70, HpO nsd AIPcS4 produced betterantitumor effeets by irradiation with ultrasound. The antitumor effects of combined use of laser fight and ultrasound were stronger than those of single laser light or ultrasound in both cases of PH-i 126 and ATX-70. Key words: OPO laser light, ultrasound, PH-1126. ATX-70

In order to estimate the best concentrations of two photosensitizers and the dos of laser each lights for the anti-tumor effect of these photochemical combined treaments. We examined of anti-tumor effect of the photocheical treatment by optical parametric oscillator (OPO) laser lights with two photosensitizers administrations for the transplanted tumor of a cultivated murine leukemia (L5 178Y) cells to Balb/c mude mouse because there is an advantageousness of tunable wavelengths in the OPO laser. Different two photosensitizers of a 2.5ig/gbw pheophorbide-a derivative (PH-1126) and 1mM 5-aminolevulinic acid (5-ALA) as a protoporphyrin IX (Pp-IX) precursor were used when both sensitizers were administrated together. The tumor sizes, the survival fraction and the tumor histological changes of the nude mice were observed after the treatments. From those results, it was found that (1) it was observed the anti-tumor effect by PH-1126 also in the case of the single irrdiation at even 630nm of 5-ALA absorption peak, (2) the combined irradiations at 630nm and 6SOmn for the double administrations were obtained the best anti-tumor effect of the deepened necrosis and pyknosis or disappear-changes of nuclear of the tumor cells about 1 -1.5cm at the depth 1 weak after the laser treatments comparing that of the single treatment for the single photosensitizer. Key words:OPO-laser, two different wavelength irradiations, two different photosensitizers, anti-tumor effect of L5178Y, growth curve, survival fraction

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, 64 Cu-labeled cyclam-RAFT-c(-RGDfK-)4 peptide ( 64 Cu-RAFT-RGD), using this model. Varying degrees of avb3 integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing 64 Cu-RAFT-RGD and 18 F-fluoro-2-deoxy-D-glucose ( 18 F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly avb3 integrin specific. 64 Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of 64 Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, 64 Cu-RAFT-RGD accumulation provided better tumor contrast to background than 18 F-FDG. Our results suggest that 64 Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of avb3 integrin–expressing pancreatic tumors.

As cancer stem cells (CSCs) are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.

Introduction We report here the in vitro and in vivo characteristics of a new molecular conjugate vector for targeting and imaging of tumors. Its core is a cyclo-decapeptide plateform named RAFT, onto which two spatially independent functional domains can be cova-lently and stereo-specifically linked : a cell targeting domain for tumor targeting and a labeling domain able to carry two drugs and/or labeling agent. In order to prove the interest of this carrier, we used a well known cRGD cyclopeptide, a ligand for the α v β 3 integrin. Methods Using competition assays and confocal microscopy in vitro , as well as in vivo tumor formation and near-infrared optical imaging in mice, we analyzed the characteristics of the RAFT(cRGD)4 vector. Results We demonstrate that this vector presenting 4 cRGD motifs very efficiently prevents (α v β 3 -mediated cell adhesion to vit-ronectin. Furthermore, it is actively endocytosed because of the multivalent cRGD presentation, a major advantage for drug delivery. In vivo experiments in nude mice reveal that repeated intratu-moral injections of low doses of RAFT(cRGD)4 reduce tumor growth by reducing the number of tumor blood vessels. Furthermore, RAFT(cRGD)4 significantly improves the targeting specifi-city of subcutaneous tumor masses as well as disseminated metastasis after intravenous injection. Conclusion Thus, RAFT (cRGD)4 is specific, internalized, perfectly controlled and can carry multiple biological functions on a single, spatially defined backbone, making of it a powerful and versatile synthetic vector for drug delivery, molecular imaging or both. This versatility is a major advantage since we can load it with multiple functions. This was demonstrated by the addition of a cytotoxic KL peptide on the cargo domain of the RAFT. RAFT-RGD-KL shows a very promissing specific antitumoral activity when injected intravenously in tumors bearing mice. Finally, recent developments in the optical System allowed us to follow these optical probes using a new 3D optical tomographic system in lung metastasis. This allowed us to detect lung metastasis in 3D for the first time to our knowledge using optical NIR probes.

64Cu-cyclam-RAFT-c(-RGDfK-)4 is a novel multimeric positron emission tomography (PET) probe for αVβ3 integrin imaging. Its uptake and αVβ3 expression in tumors showed a linear correlation. Since αVβ3 integrin is strongly expressed on activated endothelial cells during angiogenesis, we aimed to determine whether 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used to image tumor angiogenesis and monitor the antiangiogenic effect of a novel multi-targeted tyrosine kinase inhibitor, TSU-68. Athymic nude mice bearing human hepatocellular carcinoma HuH-7 xenografts, which expressed negligible αVβ3 levels on the tumor cells, received intraperitoneal injections of TSU-68 or the vehicle for 14 days. Antiangiogenic effects were determined at the end of therapy in terms of 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake evaluated using PET, biodistribution assay, and autoradiography, and they were compared with microvessel density (MVD) determined by CD31 immunostaining. 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET enabled clear tumor visualization by targeting the vasculature, and the biodistribution assay indicated high tumor-to-blood and tumor-to-muscle ratios of 31.6 ± 6.3 and 6.7 ± 1.1, respectively, 3 h after probe injection. TSU-68 significantly slowed tumor growth and reduced MVD; these findings were consistent with a significant reduction in the tumor 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake. Moreover, a linear correlation was observed between tumor MVD and the corresponding standardized uptake value (SUV) (r = 0.829, P = 0.011 for SUVmean; r = 0.776, P = 0.024 for SUVmax) determined by quantitative PET. Autoradiography and immunostaining showed that the distribution of intratumoral radioactivity and tumor vasculature corresponded. We concluded that 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used for in vivo angiogenesis imaging and monitoring of tumor response to antiangiogenic therapy. Electronic supplementary material The online version of this article (doi:10.1007/s10456-012-9281-1) contains supplementary material, which is available to authorized users.