Jeng-Jong Hwang

Gene Expression Profiles of Normal Human Fibroblasts after Exposure to Ionizing Radiation: A Comparative Study of Low and High Doses

Abstract Ding, L-H., Shingyoji, M., Chen, F., Hwang, J-J., Burma, S., Lee, C., Cheng, J-F. and Chen, D. J. Gene Expression Profiles of Normal Human Fibroblasts after Exposure to Ionizing Radiation: A Comparative Study of Low and High Doses. Radiat. Res. 164, 17–26 (2005). Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.

Diagnostic and therapeutic evaluation of 111In-vinorelbine-liposomes in a human colorectal carcinoma HT-29/luc-bearing animal model

Colorectal carcinoma is a highly prevalent and common cause of cancer in Taiwan. There is still no available cure for this malignant disease. To address this issue, we applied the multimodality of molecular imaging to explore the efficacy of diagnostic and therapeutic nanoradiopharmaceuticals in an animal model of human colorectal adenocarcinoma [colorectal cancer (CRC)] that stably expresses luciferase (luc) as a reporter. In this study, an in vivo therapeutic efficacy evaluation of dual-nanoliposome (100 nm in diameter) encaged vinorelbine (VNB) and (111)In-oxine on HT-29/luc mouse xenografts was carried out. HT-29/luc tumor cells were transplanted subcutaneously into male SCID mice. Multimodality of molecular imaging approaches including bioluminescence imaging (BLI), gamma scintigraphy, whole-body autoradiography (WBAR) and in vivo tumor growth tracing, histopathology and biochemistry/hematology analyses were applied on xenografted SCID mice to study the treatments with 6% polyethylene glycol (PEG) of (111)In-NanoX/VNB-liposomes. In vivo tumor growth tracing and BLI showed that tumor volume could be completely inhibited by the combination therapy with (111)In-VNB-liposomes and by chemotherapy with NanoX/VNB-liposomes (i.e., without Indium-111) (P<.01). The nuclear medicine images of gamma scintigraphy and WBAR also revealed the conspicuous inhibition of tumor growth by the combination therapy with (111)In-VNB-liposomes. Animal body weights, histopathology and biochemistry/hematology analyses were used to confirm the safety and feasibility of radiopharmaceuticals. A synergistic therapeutic effect on CRC xenografted SCID mice was proven by combining an Auger electron-emitting radioisotope (Indium-111) with an anticancer drug (VNB). This study further demonstrates the beneficial potential applications of multimodality molecular imaging as part of the diagnostic and therapeutic approaches available for the evaluation of new drugs and other strategic approaches to disease treatment.

Antitumor effect of orlistat, a fatty acid synthase inhibitor, is via activation of caspase-3 on human colorectal carcinoma-bearing animal.

We established a HT-29/tk-luc human colorectal carcinoma-bearing animal model for the study of the inhibition effect and mechanism of orlistat, a fatty acid synthase (FASN) inhibitor. The results showed that orlistat caused cell cycle arrest at G1 phase, and triggered apoptosis through caspase-3 activation. The tumor inhibition effect of orlistat may also due to the inhibition of fatty acid synthesis without altering FASN activity. The tumor size of orlistat-treated mice in vivo was significantly smaller than that of the controls with 55% inhibition. The therapeutic efficacy was further confirmed with the bioluminescent imaging and nuclear molecular imaging with ¹³¹I-FIAU/gamma scintigraphy and ¹¹C-acetate/microPET. We suggest that FASN is a potential target for the treatment of human colorectal carcinoma.

Therapeutic Efficacy Evaluation of 111In-Labeled PEGylated Liposomal Vinorelbine in Murine Colon Carcinoma with Multimodalities of Molecular Imaging

In our previous studies using combined radioisotopes with chemotherapeutic liposomal drugs (i.e., 111In-labeled polyethylene glycol (PEG)ylated liposomal vinorelbine) we have reported possible therapeutic efficiency in tumor growth suppression. Nevertheless, the challenge remains as to whether this chemotherapy has a therapeutic effect as good as that of combination therapy. The goal of this study was to investigate the real therapeutic effectiveness of 6 mol% PEG 111In-vinorelbine liposomes via the elevation of the radiation dosage and reduction in the concentration of chemotherapeutic agents. Methods: Murine colon carcinoma cells transfected with dual-reporter genes (CT-26/tk-luc) were xenografted into BALB/c mice. The biodistribution was estimated to determine the drug profile and targeting efficiency of 111In-vinorelbine liposomes. Bioluminescence imaging and 18F-FDG small-animal PET were applied to monitor the therapeutic response after drug administration. The survival in vivo was estimated and linked with the toxicologic and histopathologic analyses to determine the preclinical safety and feasibility of the nanomedicine. Results: Effective long-term circulation of radioactivity in the plasma was achieved by 6 mol% PEG 111In-vinorelbine liposomes, and this dose showed significantly lower uptake in the reticuloendothelial system than that of 0.9 mol% PEG 111In-vinorelbine liposomes. Selective tumor uptake was represented by cumulative deposition, and the maximum accumulation was at 48 h after injection. The combination therapy exhibited an additive effect for tumor growth suppression as tracked by caliper measurement, bioluminescence imaging, and small-animal PET. Furthermore, an improved survival rate and reduced tissue toxicity were closely correlated with the toxicologic and histopathologic results. Conclusion: The results demonstrated that the use of 6 mol% PEG 111In-vinorelbine liposomes for passively targeted tumor therapy displayed an additive effect with combined therapy, not only by prolonging the circulation rate because of a reduction in the phagocytic effect of the reticuloendothelial system but also by enhancing tumor uptake. Thus, this preclinical study suggests that 6 mol% PEG 111In-vinorelbine liposomes have the potential to increase the therapeutic index and reduce the toxicity of the passively nanotargeted chemoradiotherapies.

Volumetric intensity-modulated Arc (RapidArc) therapy for primary hepatocellular carcinoma: comparison with intensity-modulated radiotherapy and 3-D conformal radiotherapy

BackgroundTo compare the RapidArc plan for primary hepatocellular carcinoma (HCC) with 3-D conformal radiotherapy (3DCRT) and intensity-modulated radiotherapy (IMRT) plans using dosimetric analysis.MethodsNine patients with unresectable HCC were enrolled in this study. Dosimetric values for RapidArc, IMRT, and 3DCRT were calculated for total doses of 45~50.4 Gy using 1.8 Gy/day. The parameters included the conformal index (CI), homogeneity index (HI), and hot spot (V107%) for the planned target volume (PTV) as well as the monitor units (MUs) for plan efficiency, the mean dose (Dmean) for the organs at risk (OAR) and the maximal dose at 1% volume (D1%) for the spinal cord. The percentage of the normal liver volume receiving ≥ 40, > 30, > 20, and > 10 Gy (V40 Gy, V30 Gy, V20 Gy, and V10 Gy) and the normal tissue complication probability (NTCP) were also evaluated to determine liver toxicity.ResultsAll three methods achieved comparable homogeneity for the PTV. RapidArc achieved significantly better CI and V107% values than IMRT or 3DCRT (p < 0.05). The MUs were significantly lower for RapidArc (323.8 ± 60.7) and 3DCRT (322.3 ± 28.6) than for IMRT (1165.4 ± 170.7) (p < 0.001). IMRT achieved a significantly lower Dmean of the normal liver than did 3DCRT or RapidArc (p = 0.001). 3DCRT had higher V40 Gy and V30 Gy values for the normal liver than did RapidArc or IMRT. Although the V10 Gy to the normal liver was higher with RapidArc (75.8 ± 13.1%) than with 3DCRT or IMRT (60.5 ± 10.2% and 57.2 ± 10.0%, respectively; p < 0.01), the NTCP did not differ significantly between RapidArc (4.38 ± 2.69) and IMRT (3.98 ± 3.00) and both were better than 3DCRT (7.57 ± 4.36) (p = 0.02).ConclusionsRapidArc provided favorable tumor coverage compared with IMRT or 3DCRT, but RapidArc is not superior to IMRT in terms of liver protection. Further studies are needed to establish treatment outcome differences between the three approaches.

Ki-67 expression as a prognostic marker in patients with hepatocellular carcinoma.

Ki‐67 expression in tumours has been shown to be associated with prognosis in patients with hepatocellular carcinoma (HCC). In this study, primary HCC samples were obtained from 67 patients undergoing surgical resection. None of these patients had been subjected previously to any other form of therapy, such as arterial embolization or chemotherapy. Histologically normal liver tissues from liver resection for metastatic colon cancer were taken as controls (n= 8). Monoclonal antibody against Ki‐67 was used for immunostaining and flow cytometry was used to measure tumour DNA ploidy. The mean Ki‐67 labelling index (percentage of Ki‐67‐positive cells) of the HCC (26 ± 22%; range 0.1–89%) was significantly higher than that of the normal controls (39 ± 0.8%, P < 0.05). The mean Ki‐67 labelling index (19 ± 15%; n= 28) of the tumours with diploid DNA pattern was significantly lower than those with aneuploid DNA pattern (32 ± 25%, n= 39; P= 0.01). Hepatocellular carcinoma patients (n= 47) with Ki‐67 index >10% had a significantly lower disease‐free and overall survival than those (n= 20) with Ki‐67 index ≤10% (P= 0.0009 and P= 0.02, respectively). Multivariate analysis showed that Ki‐67 expression and tumour node metastasis stage were two independent prognostic factors for disease‐free and overall survival rates. Our results suggest that the expression of Ki‐67 is an independent prognostic indicator for patients with HCC after resection and could be of assistance in the decision‐making of adjuvant therapy.

Biodistribution study of [123I] ADAM in mice: correlation with whole body autoradiography

Iodine-123 labeled 2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine ([(123)I] ADAM) has been suggested as a promising serotonin transporter (SERT) imaging agent. Much research has been accomplished, mainly focusing on the SERT binding sites in the central nervous system (CNS). However, the biodistribution of [(123)I] ADAM using whole body autoradiography (WBAR) has never been previously described, to the best of our knowledge. In this study, we assayed the biodistribution of [(123)I] ADAM in tissues/organs removed from mice, and measured their radioactivity with a scintillation counter (SC). The results showed that the liver has the highest uptake. On the other hand, the WBAR clearly demonstrated that [(123)I] ADAM was bound to SERT-rich sites including those in the brain stem, lung, adrenal glands and intestinal mucosa. This radiotracer also accumulated in the liver, kidney, and thyroid. The results from both methods were compared; each has its own complementary role in the biodistribution studies. The SC method revealed the total amount of radiotracer accumulation in each organ, and the WBAR demonstrated more anatomical details of the radiotracers distribution. The whole body distribution results of the radioligand using both methods explore the usage of this novel radioligand for most possible SERT binding sites, not only in the CNS but also in the peripheral nervous system and neuroendocrine tissues. These findings suggest that [(123)I] ADAM is a potentially useful imaging agent for SERT.

Evaluation of Pharmacokinetics of 111In-Labeled VNB-PEGylated Liposomes After Intraperitoneal and Intravenous Administration in a Tumor/Ascites Mouse Model

Nanoliposomes are important drug carriers that can passively target tumor sites by the enhanced permeability and retention (EPR) effect in neoplasm lesions. This study evaluated the biodistribution and pharmacokinetics of 111In-labeled vinorelbine (VNB)-encapsulated PEGylated liposomes (IVNBPL) after intraperitoneal (i.p.) and intravenous (i.v.) administration in a C26/tk-luc colon carcinoma ascites mouse model. IVNBPL was prepared by labeling VNB-encapsulated PEGylated liposomes with 111In-oxine. BALB/c mice were i.p. inoculated with 2 x 10(5) C26/tk-luc cells in 500 muL of phosphate-buffered saline. Peritoneal tumor lesions were confirmed by 124I-FIAU/micro-PET (positron emission tomography) and bioluminescence imaging. Ascites production was examined by ultrasound imaging on day 10 after tumor cell inoculation. The pharmacokinetics and biodistribution studies of IVNBPL in a tumor/ascites mouse model were conducted. The labeling efficiency was more than 90%. The in vitro stability in human plasma at 37 degrees C for 72 hours was 83% +/- 3.5%. For i.p. administration, the areas under curves (AUCs) of ascites and tumor were 6.78- and 1.70-fold higher, whereas the AUCs of normal tissues were lower than those via the i.v. route. This study demonstrates that i.p. administration is a better approach than i.v. injection for IVNBPL, when applied to the treatment of i.p. malignant disease in a tumor/ascites mouse model.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β -glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (βG) on CT26 tumors. The fluorescent intensity in βG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

Sorafenib sensitizes human colorectal carcinoma to radiation via suppression of NF-κB expression in vitro and in vivo.

Over-expression of transcription factor nuclear factor-κB (NF-κB) in the residual tumor after the treatment is often observed at the later period of cancer radiotherapy, results in tumor radioresistance and poor treatment outcome. In this study, we combined sorafenib, a multikinase inhibitor, with ionizing radiation to evaluate the therapeutic effect, and to elucidate the possible mechanism responsible for the radiosensitization of sorafenib on a human HT29/tk-luc colorectal carcinoma in vitro and in vivo. Clonogenic survival and cell cycle analysis were used to evaluate the cytotoxicity of sorafenib in vitro. The combination effect and the role of NF-κB in therapeutic efficacy with respects in apoptosis and tumor cell invasion were studied with HT29/tk-luc tumor-bearing animal model. The expression of NF-κB and its downstream-related proteins were assayed with electrophoretic mobility shift assay (EMSA) and Western blot. Sorafenib combined with radiation shows the synergistic cytotoxicity on HT29/tk-luc cells and increased tumor cell apoptosis. Both EMSA and Western blot show that the NF-κB activity induced by radiation is significantly suppressed by sorafenib. Combination of sorafenib and radiation shows the maximum tumor growth inhibition as compared to that of sorafenib alone or radiation alone in vivo (P<0.001). In conclusion, the effect of sorafenib combined with radiation for the treatment of human colorectal carcinoma is synergistic. The mechanism of synergism is through the inhibition of radiation-induced NF-κB expression and its regulated downstream gene products.