Search

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity; however the treatment approaches are still unsatisfactory. We used a luciferase-transfected animal model to evaluate the therapeutic effects of curcumin. Human oral squamous cell carcinoma SAS cell line was stably transfected with luc gene, named SAS/luc cells. For the in vivo study, they were inoculated subcutaneously to 6-week-old male NOD/SCID mice which were separated into four groups for intraperitoneal injection (i.p.) of curcumin: control, daily with 35 mg/kg, 70 mg/kg every 2 days, and 100 mg/kg every 3 days. We applied SAS/luc bearing animal model and bioluminescent imaging (BLI) to study the inhibition effect of curcumin on tumor growth. The cytotoxic effect of curcumin on SAS/luc cells was mainly at G2/M phase and a significant dose dependent increase of the apoptotic SAS/luc cells as represented by sub-G1 phase was shown. Therapeutic efficacy evaluated by both caliper assay and BLI showed a significant difference between curcumin-treated mice and the controls (p < 0.01). The significant inhibition effects of curcumin on the proliferation and the growth of human OSCC are observed both in vitro and in vivo. No significant body weight change (i.e. within 20%) was observed in all SAS/luc-bearing mice with or without curcumin treatment. This SAS/luc human OSCC bearing animal model combined with multimodalities of molecular imaging permits a sensitive and non-invasive approach to evaluate the therapeutic efficacy in vivo.

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin upregulated in G(1) phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G(1) cells. To investigate the role of cofilin in the control of G(1) phase progression, a tet-on gene expression system was introduced to overexpress different concentrations of cofilin in cells. The results showed that G(1) phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G(1) phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27(kip1) was the primary molecule induced by overexpressed cofilin in a time and dose dependent manner. Upregulated p27(kip1) repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27(kip1) expression in the cofilin overexpressing cells released the G(1) phase arrest. Furthermore, we found that overexpression of cofilin led to induction of p27(kip1) gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27(kip1) mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27(kip1) protein for ubiquitinyl-proteasomal mediated degradation was also involved in upregulation of p27(kip1). These data suggest that cofilin expression and its regulation of p27(kip1) expression is important for the control of G(1) phase progression.

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells.To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene.In these in vitro models, we showed that amelogenin can activate beta-catenin signaling.Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

Breast cancer is increasing with years in Taiwan because of dietary style, life behavior and several social-physiological factors. According to the record of Bureau of Health Promotion in Taiwan, the incidence of breast cancer is top one, and the mortality of that is top one cancer type in women. Compared with USA, most of breast cancer cases found in Taiwanese women have reached to stage 2 or 3. Current therapeutic strategies for breast cancer include surgery, radiation therapy, chemotherapy, hormone therapy and targeted therapy. However, these methods used for curing the late-stage breast cancer remains rare. Because the metastasis is the major problem of late-stage breast cancer, it is of interest to investigate whether a systemic therapy can reduce the symptoms of cancer. The immunotherapy, particularly an induction of autoimmune system, is probably important for the treatment of late-stage breast cancer. Glycated chitosan (GC) is derived from chitosan, a linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine through β–(1-4) linkage. Several lines of evidence have shown that GC is an immunoadjuvant that can target on primary and metastatic tumors formed in animal and human patients. In our previous data, GC was demonstrated to decrease the motility and invasion of mammalian breast cancer cells in vitro and in vivo. Radiofrequency ablation (RFA) is dependent on a small generator that delivers high frequency alternating electric current directly to burn a tumor lesion. Therefore, the temperature may reach up to above 60 °C. In this study, we used 4T1 mouse breast cancer cell that is the approximately equal to stage 4 of human breast cancer. And triple modality reporter gene (3R) was delivered into the cells using transfected piggyBac, a transposable element for observation of tumor growth and metastasis in vivo. Data showed that growth and metastasis of tumors smaller than 500mm3 were entirely suppressed by RFA-GC combination treatment. Plasma extraction from this group displayed an inhibitory effect on cultured 4T1 cells, suggesting some immunoreactions were triggered and therefore secreted some cytokines. Protein array data indicated that PF-4 may play a key role in GC-RFA caused tumor suppression but the further effects should be investigated. On the other hand, the survival rate of small tumor-bearing mice under RFA-GC treatment was higher than those of GC or RFA treatment only. In conclusion, this study confirmed that radiofrequency ablation combined with GC could trigger an autoimmune response to inhibit tumor metastasis and tumorigenesis. For metastatic cancers, this combination treatment may become the foundation for a feasible cancer treatment modality.

The Rho–LIM-kinase (LIMK) signaling pathway, believed to be involved in the regulation of tumor invasion, specifically regulates the activity of cofilin. However, it is unclear whether cofilin plays a pivotal role in tumor invasiveness. In this paper we show using a tet-on gene expression system that over-expression of cofilin inhibits the invasiveness of human lung cancer H1299 cells. Over-expressed cofilin disrupts the actin cytoskeleton at the leading edge of the cell and up-regulates p27kip1, which is known to be involved in regulating cell motility. Removal of cofilin over-expression normalizes the p27kip1 level and concomitantly restores the invasiveness of the cultured cells. These findings suggest that excessive cofilin production might prevent cancer cell invasion.

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1arrest without affecting mRNA expression of the other Cip or INK4 G1kinase inhibitors. To confirm the role of p21 in G1arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

Immunotherapy has been reported to effectively treat various cancers. In addition, scientists are dedicated in finding whether the combination of radiotherapy and immunotherapy can efficiently suppress cancer progression and recurrence. Although radiotherapy has been widely used for breast cancer, better strategies to overcome the latestage breast cancer remains explored. The glycated chitosan (GC), a novel immunological stimulant, was demonstrated to trigger local immune response facilitating the enhancement of radiosensitivity. Our previous study also revealed that the cell mortality and invasive ability were decreased under GC treatment, but the underlying mechanism remains unclear. In this study, we used 4T1-3R-L, a derived murine breast cancer cell line from the spontaneous metastasized liver lesion. We combined ionizing radiation with GC to treat 4T1-3R-L and found the expression of DNA damage-related genes such as gamma-H2AX was more than radiation alone In addition, the cell cycle distribution and colony forming assay showed an increased sub-G1 population and decreased cell survival rate after IR combined GC treatment. Taken together, we sought to elucidate the underlying mechanism by the investigation of DNA damage repair process when IR combined with GC, and to explore another advantage of GC to aid other cancer treatments. Based on our most updated results, the GC treatment is able to effectively increase the radiosensitivity through an immune-responsive signaling transduction, indicating that GC could be a valuable therapeutic strategy for treating against advanced breast cancers.

Background and purpose Cancer stem cells exhibit distinctive cellular metabolism compared with the more differentiated counterparts or normal cells. We aimed to investigate the impact of a novel radionuclide anti-cancer agent 188 Re-Liposome on stemness markers' expression and cellular metabolism in an ovarian cancer model. Material and methods A 2×2 factorial experiment was designed in which factor 1 represented the drug treatment comparing 188 Re-BMEDA, a free form of 188 Re, with 188 Re-Liposome, a nanoparticle-encapsulated form of 188 Re. Factor 2 represented the delivery route, comparing intravenous with intraperitoneal delivery. Results Intraperitoneal delivery of 188 Re-Liposome predominantly killed the CSCs-like cells in tumours and switched metabolism from glycolysis to oxidative phosphorylation. Further, intraperitoneal delivery of 188 Re-Liposome treatment was able to block epithelial-to-mesenchymal transition (EMT) and reactivate p53 function. Collectively, these molecular changes led to a striking tumour-killing effect. Conclusions Radionuclides encapsulated in liposomes may represent a novel treatment for ovarian cancer when delivered intraperitoneally (a type of loco-regional delivery). In the future, this concept may be further extended for the treatment of several relevant cancers that have been proved to be suitable for loco-regional delivery of therapeutic agents, such as colon cancer, gastric cancer, and pancreatic cancer.

Summary Background The treatment of gliomas poses significant clinical challenges due to resistance to chemo and radiation therapy, and treatment side effects. Metronomic photodynamic therapy (mPDT), which involves long treatment time with low fluence rate and multiple or continuous photosensitizer administrations, has potential in treating gliomas without threatening the quality of life and has been demonstrated in rats and rabbits. mPDT in small animals such as mouse is not yet shown due to lack of lightweight illumination device for long periods of time. Methods We presented low fluence rate (3 mW/cm2) and long duration (3.7 h) PDT treatment in a nude mouse model of human glioblastoma by using organic light emitting diode (OLED) with single dose of 5-aminolevulinic acid (ALA) administration as photosensitizer. Tumor volume was measured using bioluminescent imaging and the animal survival time was recorded. Additionally, we have performed limited PDT dosimetric measurements of PpIX fluorescence, tumor oxygenation and hemoglobin concentration in 3 PDT mice. Results For animals with similar pre- and immediate post-light tumor volume, the averaged total survival time of PDT mice is 40.5 ± 9.2 days that are significantly longer than the control mice (26.0 ± 2.0 days). The post-light survival time of PDT mice is 14.3 ± 5.9 days that are marginally longer than the control group (8.0 ± 0.0 days). In the dosimetric measurement, good maintenance of PpIX fluorescence in one PDT mouse has relatively improved survival time, compared with the other two PDT mice (i.e., 24 days versus 16 and 17 days). Conclusions This pilot study demonstrated the feasibility of low-fluence rate and long treatment time of ALA-PDT using OLED without anesthetization of animals. The response of PDT treated animals with similar pre- and post-light tumor volume is encouraging to show a longer survival time than the controls. The dosimetric indices such as photosensitizer fluorescence and tissue oxygenation would help understand the possible treatment barriers for further improvement of treatment plans.

The actin-depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia-induced renal disorders. We examine whether cofilin-1 is associated with acute kidney injury (AKI) using human urine samples. We exploited a 96-well based high-throughput biosensor that uses gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients (n=37 from 47 cases analyzed) was twofold higher than that of healthy adults (n=21 from 29 cases analyzed). The receiver operating characteristic (ROC) curve showed that cofilin-1 was acceptable for discriminating AKI patients from healthy adults. However, an increase of the sample size is required to conclude the importance of urine cofilin-1 on AKI diagnosis, and the high-throughput ultrasensitive biosensor used in this study would greatly accelerate the measurement of urine cofilin-1 in an increased sample size.

Securin overexpression correlates with poor prognosis in various tumours. We have previously shown that securin depletion promotes radiation-induced senescence and enhances radiosensitivity in human cancer cells. However, the underlying molecular mechanisms and the paracrine effects remain unknown. In this study, we showed that radiation induced senescence in securin-deficient human breast cancer cells involving the ATM/Chk2 and p38 pathways. Conditioned medium (CM) from senescent cells promoted the invasion and migration of non-irradiated cancer and endothelial cells. Cytokine assay analysis showed the up-regulation of various senescence-associated secretory phenotypes (SASPs). The IL-6/STAT3 signalling loop and platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor (PDGFR) pathway were important for CM-induced cell migration and invasion. Furthermore, CM promoted angiogenesis in the chicken chorioallantoic membrane though the induction of IL-6/STAT3- and PDGF-BB/PDGFR-dependent endothelial cell invasion. Taken together, our results provide the molecular mechanisms for radiation-induced senescence in securin-deficient human breast cancer cells and for the SASP responses.

A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

The actin depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia induced renal disorders. Here we examine if cofilin-1 is associated with acute kidney injury (AKI). We exploited a 96-well based fiber-optic biosensor that uses conjugated gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients was two-fold higher than that of healthy adults. The receiver operating characteristic (ROC) curve showed that cofilin-1 is a potential biomarker for discriminating AKI patients from healthy adults for intensive care patients.

Tumor growth is dependent on a kinetic model that is based on the progression of cell proliferation and cell loss. The parameters for cell proliferation during tumor progression include the cell-cycle time (Tc), growth fraction (GF), and potential tumor-doubling time (Tpot). In contrast, the cell-loss factor is determined by Tpot and the actual time for doubling of the tumor volume (Td).1 The causes of cell loss include malnutrition and lack of oxygen caused by rapid proliferation, necrosis and apoptosis, immunological attack, escape from the primary site, and exfoliation.2 These conditions can be regarded as stresses for cells residing in a rapidly growing tumor. Whether cells escaping from these stresses inherit or obtain resistance abilities is unknown. The tracking and characterization of living cells in a tumor are important for cancer treatment. Reporter-gene imaging is an indirect approach to labeling cells for image-based in vivo tracking and targeting by different modalities.3 This method is especially important for tracking cell viability in vivo because gene transcription and translation occur only in living cells.4 In addition, the transmission of genes to progeny is in principle not diminished or diluted if the reporter genes can replicate within the genomes of host cells.5 Firefly luciferase and fluorescent proteins are canonical reporter genes used for bioluminescent imaging and optical imaging, respectively. For radionuclide-based reporter-gene imaging, herpes simplex virus type-1 thymidine kinase (HSV1-tk) is commonly used because it can uptake a broad range of radiolabeled nucleoside analogues by substrate phosphorylation for imaging the target cells in vivo.6 Expression of the HSV1-tk reporter gene can be used for living cell tracking by positron emission tomography (PET) or single-photon emission computed tomography (SPECT), depending on the types of radionuclide-labeled substrates. For instance, iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil (123I-FIAU) is the most reliable radiolabeled nucleoside analogues for SPECT imaging of HSV1-tk gene expression because it exhibits high tumor/background ratio in vivo.7, 8 As described above, the sensitivity and specificity of 123I-FIAU on cancer detection is based on the expression of HSV1-tk reporter gene in the transduced cancer cells. These cancer cells can be distinguished from the non-cancer cells without HSV1-tk genes using the radionuclide imaging modality. Technically, cultured cancer cells are stably transduced with HSV1-tk genes and implanted into animals for tumor formation. Using SPECT imaging, this cancer population can be distinguished from non-cancer portion in vivo by injecting 123I-FIAU as a radiotracer that are only accumulated in HSV1-tk gene expressing cancer cells.7 Multimodality reporter-gene imaging using coexpressed luciferase/fluorescent proteins and HSV1-tk has been reported to be a powerful tool for basic biological and preclinical research.9, 10 In addition, PET and SPECT can be merged with computed tomography (CT) to obtain functional/anatomic imaging with high sensitivity and spatial resolution. Although reporter-gene imaging is widely used for functional studies in vivo, concerns regarding the safety and transduction pathways of exogenous genes hamper the clinical application of this approach. Viral-mediated methods are commonly used for the transduction of reporter genes, but the uncontrolled infection and random genomic integration of genes of interest currently limit the clinical application of these methods.11 In contrast, nonviral gene delivery is more acceptable in the clinic because biocompatible materials can be used, and the safety of these approaches is relatively easily assessed by pharmacokinetic and pharmacodynamic studies.12 The piggyBac transposon system has recently attracted a great deal of attention because the system is a nonviral gene-delivery approach that uses DNA-transposition ability for the stable expression of exogenous genes via a ‘cut-and-paste' mechanism.13 The beauty of this system includes its known integration sites at ‘TTAA' sequences and intron-preferred positions, mammalian compatibility, large cargo capacity, and its ability to be removed from the integration site without changing the DNA sequence.14 In practice, this system requires a helper plasmid that encodes the piggyBac transposase gene to facilitate the transposition of exogenous gene.15 The piggyBac transposon system has been applied in induced pluripotent stem cells, cancer research, and immunotherapy.16, 17 However, the use of this system has been less frequently reported in multimodality reporter-gene imaging of tumor progression. Cancer stem cells (CSCs, or tumor-initiating cells) belong to the hierarchy model that a subset of rare cell population inherits stem cell-like characteristics, including self-renewal and generation of non-tumorigenic progeny.18 This theory has intrigued many researchers in recent years because CSCs are resistant to chemoradiotherapy and are likely to be the cause of tumor recurrence and metastasis.19 However, the identification of CSCs in vivo remains a challenge because of the lack of suitable markers for this purpose. If CSCs naturally resist environmental stresses, it would be speculated that this population may also escape from cell loss during tumor progression. More evidence is required to support this hypothesis. The percentage of cell loss during tumor progression is approximately 40–90%, depending on the cancer type.1 The remnant viable cells may be important for promoting tumor growth and metastasis. Reporter-gene imaging should be ideal to track these living cells for further investigation of their characteristics. In this study, we established a syngeneic tumor model derived from 4T1 murine breast carcinomas transduced with monomeric red fluorescent protein (mRFP)/HSV1-tk dual reporter genes using the piggyBac transposon system. A combination of optical imaging and SPECT/CT fusion imaging using 123I-FIAU as a probe was exploited to track the remnant living cells in late-stage primary tumors. Furthermore, ex vivo studies showed that the surviving cells exhibited CSC-like characteristics. These findings may contribute to therapeutic designs for cancer treatment.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with the characteristics of highly infiltrative growth and recurrent rate. In this study, we used animal imaging and molecular expressive profiles to investigate the characteristics of the primary tumor (GBM-3) cells and recurrent tumor (S1R1) cells from different GBM patients.Bioluminescent imaging and 3T magnetic resonance imaging (MRI) were used for assessing the orthotopical tumor development of GBM cells harboring a polycistronic reporter gene system. Western blot analysis and quantitative polymerase chain reaction were used to compare the molecular expressive profiles of two types of GBM cells.S1R1 cells exhibited apparent invasive ability compared to GBM-3 cells using in vitro invasion assay. In vivo bioluminescent imaging showed that intracranial tumors are formed by both types of GBM cells, but the bioluminescent signal was also detected in the lumbar region at late-stage tumor formed by S1R1 cells. The MRI showed that intracranial tumors formed by S1R1 cells were highly infiltrative compared to that formed by GBM-3 cells. Additionally, these two GBM types expressed different patterns of molecules associated with tumor development. Moreover, the suppressive effects of interleukine-23 (IL-23) on xenograft tumors formed by both GBM types were detected using bioluminescent imaging.The current data suggest that the in vivo growth behaviors and therapeutic responses of the primary and recurrent human GBMs were comparable using the reporter gene imaging, and different molecular expressive profiles exist between these two GBM types.

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.

Cell cycle progression is dependent on a series of molecular regulation after cells are stimulated by growth factors. Growth factors bind to corresponding surface receptors and relay the signals through protein phosphorylation to trigger gene expression. Phosphorylation of retinoblastoma protein (Rb) is to release E2F family of transcription factors for DNA replication. In adherent cells, the actin filament plays an important role for anchorage, locomotion, morphological maintenance, and cell division (1). These mechanical characteristics influence cell cycle progression, and mediate cells responding to extracellular stimulations. The cyclin-dependent kinases (CDKs) are responsible for cell cycle transition through different phases. For G1 phase progression, the G1 cyclins associated CDKs can phosphorylate and inactivate Rb. Because the phosphorylation sites of Rb are multiple, they become a family of checkpoint to prevent release of E2F transcription factor under a stress condition, such as DNA damage. In addition, the CDKs activity and Rb phosphorylation are ablated by the family of CDK inhibitors (CKIs), including INK4 and CIP/KIP family proteins (2). The underlying mechanisms by which the intact actin filaments regulated cell cycle progression have been reviewed in literatures, although the pathways are diverse from different research results. However, it appears that Rb activity is commonly affected by destabilizing the actin cytoskeleton. Therefore, it is believed that growth factor stimulated actin cytoskeletal organization can regulate Rb activity for G1 phase progression and DNA replication.

N-Heterocyclic indolyl glyoxylamide compounds are derived from the antimicrotubule agent D-24851, which exhibits anticancer activity after oral administration. The actions of these compounds on lung cancer cells are still unknown. Here, we investigated the effects of two N-heterocyclic indolyl glyoxylamides, BPR0C259 and BPR0C123, on non-small human lung cancer cells.3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the half maximal inhibitory concentration (IC(50)), cell viability and radiation response of A549 cells and H1299 cells. Apoptosis was determined by sub-G(1) ratio, colony formation assay and caspase-3 activation. Cell cycle distribution was detected using flow cytometry.Both compounds were able to inhibit the viability of human lung cancer cells, although the IC(50) of BPR0C123 was lower than that of BPR0C259. Both compounds induced significant sub-G1 and caspase-3 activation as low as 0.1 μM in both cell lines. These effects were independent of p53 activation because the level of serine-15 phosphorylated p53 was not affected after drug treatment. Furthermore, both compounds induced similar levels of G(2)/M phase arrest and radiosensitivity in these lung cancer cells.Current data suggest that N-heterocyclic indolyl glyoxylamides can suppress the proliferation of and potentially increase radiosensitivity of human lung cancer cells.

Prostate-specific antigen (PSA) has been reported to be a potential biomarker of breast cancer. Serum PSA of normal women is around 1 pg/mL, which is usually undetectable by current assay methods; thus an ultrasensitive measurement of PSA expression in women's serum is necessary to distinguish normal from malignant breast diseases. To enhance the sensitivity of conventional immunoassay technology for the detection of PSA in sera, we adopted a localized surface plasmon coupled fluorescence fiber-optic biosensor, which combines a sandwich immunoassay with the localized surface plasmon technique. The concentration of total PSA (t-PSA) (from 0.1 to 1000 pg/mL) in phosphate-buffered saline solution and the normalized fluorescence signal exhibit a linear relationship where the correlation coefficient is 0.9574. In addition, the concentration of additional t-PSA in 10-fold-diluted healthly women's serum across a similar range was measured. The correlation coefficient for this measurement is 0.9142. In clinical serum samples, moreover, the experimental results of t-PSA detection show that both the mean value and median of normalized fluorescence signals in the breast cancer group (155.2 and 145.7, respectively) are higher than those in the noncancer group (46.6 and 37.1, respectively). We also examined the receiver operating characteristic curve for t-PSA, and the area under the curve (AUC) is estimated to be 0.9063, the AUC being used to measure the performance of a test to correctly identify diseased and nondiseased subjects.

Arsenic exerts its cytotoxicity via the generation of reactive oxygen species and inhibition of DNA repair. How arsenic disturbs oxidative DNA damage repair is, however, unclear. We found that arsenic trioxide (ATO), like ultraviolet (UV) irradiation, induced the expression of xeroderma pigmentosum group C (XPC) but not of xeroderma pigmentosum A in a human glioma cell line, U87. To explore the role of XPC in the toxic effects of ATO, small interfering RNA was used to silence XPC (siXPC) in U87 cells. siXPC cells were more susceptible to UV irradiation and ATO-induced cell death than control cells. Increased siXPC cell death induced by ATO was accompanied by increased senescence and autophagy. Because increased DNA strand breaks in siXPC cells were observed only when cells were concomitantly treated with ATO and DNA repair inhibitors, XPC silencing apparently did not interfere with repair of ATO-induced DNA damage. Although intracellular ROS levels were not significantly enhanced in siXPC cells, ATO treatment did result in increased 8-hydroxy-2'-deoxyguanosine and hyperoxidized peroxiredoxin. Enhanced superoxide production and autophagy by ATO in siXPC cells were suppressed by co-incubation with N-acetylcysteine (NAC). Furthermore, XPC silencing caused decreased glutathione levels and increased catalase and Mn-superoxide dismutase activities. Increased catalase activity in siXPC cells was suppressed by ATO treatment. XPC silencing also enhanced reporter activity of activator protein-1, whereas enhanced activity was suppressed by NAC. Taken together, our results indicate that XPC silencing causes increased ATO susceptibility by disturbing redox homeostasis rather than reducing DNA repair.

In this study, we demonstrated that an amplitude-sensitive paired surface plasma wave biosensor (PSPWB) is capable of real-time detection of prostate-specific antigen (PSA) in diluted human serum without labeling. Experimentally, the detection limit of PSPWB was 8.4 x 10(-9) refractive index unit (RIU) and the PSPWB could measure PSA in a phosphate buffered saline solution from 10 fg/mL ( approximately 300 aM) to 100 pg/mL ( approximately 3 pM) successfully, with demonstration of a linear relationship between PSA concentrations and surface plasmon resonance (SPR) signals. Therefore, results were obtained over a wide dynamic range 5 orders of magnitude for analyte concentration. In addition, the PSPWB successfully detected PSA in diluted human serum as well. These experimental results indicate that the PSPWB is capable of detection with high sensitivity over a wide range by using SPR-based biosensors and has a capability of detecting biological analytes in clinical sample without complicated operating procedures.

To investigate the risk association and compare the onset age of hepatocellular carcinoma (HCC) patients in Taiwan with different genotypes of MDM2-SNP309.We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively. Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymorphism assay.The proportion of homozygous MDM2-SNP309 genotype (G/G) in cases and cancer-free healthy controls was similar (17.2% vs 16.7%). Multivariate analysis showed that the risk of G/G genotype of MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant (OR = 1.265, 95% CI = 0.074-21.77) after adjustment for sex, hepatitis B or C virus infection, age, and cardiovascular disease/diabetes. Nevertheless, there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus (OR = 2.568, 95% CI = 0.054-121.69). Besides, the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC.Current data suggest that the association between MDM2-SNP309 GG genotype and HCC is not significant, while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein–protein interactions in translational molecular–genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Methods: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. Results: Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome–dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP:dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. Conclusion: In translational molecular–genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.

Oxaliplatin, a chemotherapeutic drug, induces DNA double-strand breaks (DSBs) and apoptosis in colorectal cancer cells. It has been shown that gamma-H2AX acts as a marker of DSBs. However, the molecular events associated with oxaliplatin-mediated cell cycle arrest and cell death remain unclear. In this study, we investigated the roles of p53 and gamma-H2AX following oxaliplatin treatment, as they are important effector proteins for apoptosis and DSB repair, respectively. Both phosphorylated-p53 (Ser-15) and gamma-H2AX were up-regulated and accumulated in the nuclei of p53-wild type human colorectal cancer HCT116 cells after exposure to oxaliplatin. Concomitantly, oxaliplatin-induced G2/M arrest was associated with a reduction in both cyclin B1 expression and phosphorylated-CDC2 (Thr-161). Release of G2/M arrest by caffeine was accompanied by a decrease in the levels of p53/p21; however, gamma-H2AX levels were unchanged. Furthermore, inhibition of p53 phosphorylation by pifithrin-alpha was sufficient to reduce the oxaliplatin-induced up-regulation of gamma-H2AX and apoptosis. Oxaliplatin-induced gamma-H2AX via a p53-independent pathway but did not cause caspase-3 activation in p53-null HCT116 cells. Interestingly, no changes were observed in the H2AX gene knockdown with regards to oxaliplatin-induced G2/M arrest in p53-wild type and S phase arrest in p53-null HCT116 cells. Taken together, these data indicate that a molecular pathway involving p53, gamma-H2AX and cell cycle arrest plays a pivotal role in the cellular response to oxaliplatin.

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Background It has been suggested that the single nucleotide polymorphism 309 (SNP309, T -> G) in the promoter region of the MDM2 gene is important for tumor development; however, with regards to breast cancer, inconsistent associations have been reported worldwide. It is speculated that these conflicting results may have arisen due to different patient subgroups and ethnicities studied. For the first time, this study explores the effect of the MDM2 SNP309 genotype on Taiwanese breast cancer patients. Methods Genomic DNA was obtained from the whole blood of 124 breast cancer patients and 97 cancer-free healthy women living in Taiwan. MDM2 SNP309 genotyping was carried out by restriction fragment length polymorphism (RFLP) assay. The multivariate logistic regression and the Kaplan-Meier method were used for analyzing the risk association and significance of age at diagnosis among different MDM2 SNP309 genotypes, respectively. Results Compared to the TT genotype, an increased risk association with breast cancer was apparent for the GG genotype (OR = 3.05, 95% CI = 1.04 to 8.95), and for the TG genotype (OR = 2.12, 95% CI = 0.90 to 5.00) after adjusting for age, cardiovascular disease/diabetes, oral contraceptive usage, and body mass index, which exhibits significant difference between cases and controls. Furthermore, the average ages at diagnosis for breast cancer patients were 53.6, 52 and 47 years for those harboring TT, TG and GG genotypes, respectively. A significant difference in median age of onset for breast cancer between GG and TT+TG genotypes was obtained by the log-rank test (p = 0.0067). Conclusion Findings based on the current sample size suggest that the MDM2 SNP309 GG genotype may be associated with both the risk of breast cancer and an earlier age of onset in Taiwanese women.

In this study, we demonstrated that the fiber-optic biosensor based on localized surface plasmon coupled fluorescence (LSPCF) is capable of detecting alpha-fetoprotein (AFP) in human serum. The sensitivity of LSPCF fiber-optic biosensor is not only enhanced but also the specific selectivity is improved since the fluorophores are excited by the localized surface plasmon with high efficiency. Experimentally, this fiber-optic biosensor is able to detect AFP concentration in phosphate buffered saline (PBS) solution from 0.1ng/mL to 100ng/mL whereas the linear relationship between the AFP concentrations and the fluorescence signals is shown. Furthermore, a linear response between the fluorescence signals and the concentrations of AFP in human serum from 2.33ng/mL to 143.74ng/mL is also obtained. As a result, the detection limit of the LSPCF fiber-optic biosensor on AFP detection is comparable with the conventional enzyme-linked immunosorbent assay (ELISA). Additionally, the LSPCF fiber-optic biosensor benefits on inexpensive, disposable and simpler optical geometry that can become a high efficient immunoassay comparable with the conventional ELISA and radioimmunoassay (RIA) clinically.

Herpes simplex virus type 1 thymidine kinase (HSV1-TK) is a widely used reporter for in vivo noninvasive monitoring of therapeutic gene expression, immune cell trafficking, and protein‐ protein interactions in various animal systems. However, the stability of HSV1-TK limits its application in studies that require rapid turnover of the reporter. The purpose of this study was to create a destabilized HSV1-TK as a transcription reporter that allows for dynamic studies of short-time-scale gene expression events. Methods: A destabilized HSV1-TK was created by targeting inactivating mutations in the nuclear localization signal of HSV1-TK and fusing the degradation domain of mouse ornithine decarboxylase to the C-terminal end. The protein or enzyme stability was determined by Western blot analysis and HSV1-TK enzyme activity assay, respectively. The proteasome inhibition assay was used to test whether the rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome‐ dependent manner. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to a tetracyclineturnoff‐expressing system. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by destabilized HSV1-TK or by native HSV1-TK and were determined by an in vitro HSV1-TK enzyme activity assay and in vivo small-animal PET imaging. Results: The destabilized HSV1TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximideandhadashorthalf-lifeofproteinandenzymeactivity. The rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome‐dependent manner. Furthermore, thedestabilizedHSV1-TKhadlowcytotoxicitywhenitwashighly expressed in living cells. The results of dynamic gene expression studies invitro and invivo showed that thedestabilized HSV1-TK is an optimal reporter for monitoring short-time-scale dynamic transcriptional events mediating a series of doxycycline inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Conclusion: The use of destabilized HSV1-TK as a transcription reporter together with a molecular probe, which has a short physical and biologic half-life, allows more direct monitoring of transcription induction and easier monitoring of its coincidence with other biochemical changes.

Oxaliplatin, a chemotherapeutic drug, induces DNA strand breaks leading to apoptosis in colorectal cancer cells. gamma-H2AX is a phosphorylated histone H2AX that can act as a marker of DNA double-strand breaks (DSBs). It has been shown that securin proteins were over-expressed in a variety of cancer cells. However, the roles of gamma-H2AX and securin on the oxaliplatin-induced apoptosis in human colorectal cancer cells remain unclear. Treatment of oxaliplatin (1-10 microM for 6-24h) persistently induced gamma-H2AX formation and inhibited securin protein expression via a time- and concentration-dependent manner in HCT116 securin-wild type colorectal cancer cells. Compared with HCT116 securin-wild type cells, the induction of apoptosis and persistent gamma-H2AX formation by oxaliplatin was reduced in the HCT116 securin-null colorectal cancer cells. Furthermore, the blockage of caspases by specific caspase inhibitors reduced the levels of gamma-H2AX proteins and cytotoxicity but increased securin protein expression in the oxaliplatin-exposed cells. The gene knockdown of H2AX by transfection with a short interfering RNA of H2AX enhanced the oxaliplatin-induced cell death. Interestingly, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was markedly increased by oxaliplatin. Pre-treatment of a specific p38 MAPK inhibitor SB202190 reduced gamma-H2AX proteins and increased securin protein expression in the oxaliplatin-treated cells. Our findings suggest that p38 MAPK may oppositely regulate securin protein expression and gamma-H2AX formation in the oxaliplatin-induced apoptosis of human colorectal cancer cells.

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.

The significance of actin cytoskeleton on cell growth was historically studied using toxic drugs, such as cytochalasin. However, it is possible that unpredictable effects of these agents may have influenced the reported observations. In our study, we have established a drug-free system using cofilin overexpression to investigate the relationship between actin filaments and cell cycle progression. Cofilin is a member of the actin depolymerization factor (ADF)/cofilin family, cofilin cDNA was cloned to a tetracycline-inducible gene expression vector and stably transfected to human lung cancer H1299 epithelial cells. Destabilization of actin filaments and morphological change was detected in cofilin overexpressing cells by actin analysis and microscopy, respectively. Measurements of growth rates showed that cell proliferation was retarded in cells with overexpressed cofilin. Also, cell cycle analysis showed that approx 90% of cofilin overexpressing cells were arrested in G1 phase, which is consistent with previous reports that drug-mediated disruption of actin filaments can cause G1 phase arrest. Taken together, cofilin overexpression cell model provides evidence that the effects of actin cytoskeletal destabilization on cell cycle progression can be studied using molecular approach instead of drug.

Cellular sensitivity to ionizing radiation (IR) treatment is a complex biologic phenomenon that is affected by several processes, namely the ability of the cell to detect and repair DNA damage, regulate cell cycle division, and execute apoptosis. Because the p53 tumor suppressor protein is implicated in the regulation of each of these processes, radiation sensitivity of H1299 p53-null human lung carcinoma cells was evaluated after restoration of wild-type p53. Expression of wild-type p53 in radiation-resistant H1299 cells reinstated a radiation-sensitive phenotype that was not fully explained by cell death resulting from p53-mediated apoptosis. In addition, we show that p53 alters radiation sensitivity only in the G1 phase of the cell cycle, whereas S- and G2/M-phase cells were unaffected by p53 status. To determine the mechanism of p53-induced G1-phase radiation sensitivity, we investigated the G1/S checkpoint response to IR in H1299/p53 cells. We show that H1299/p53 cells arrest in the G1 phase in a p53-dependent manner as a result of transcriptional activation of p21WAF1/Cip1. To determine if p53-induced radiation sensitivity was the result of a reproductive death from accumulated p21 protein expression, p21 was independently induced in H1299 parental cells. However, induction of p21 was not sufficient to account for the enhanced radiation sensitivity in H1299/p53 cells. Together, these data indicate that p53 modulates radiation sensitivity in the G1 phase of the cell cycle through mechanisms independent of p53-mediated transcriptional activation of p21 and cell cycle arrest.

A Phase I/II clinical study using pulsed low-dose paclitaxel and radiation for thoracic malignancy was conducted. The study was based on preclinical research of the effects of paclitaxel on apoptosis and the cell cycle in human cancer cell lines.Three human epithelial cancer cell lines were investigated for preclinical study. Cells were analyzed for apoptosis and cell cycle characteristics after paclitaxel treatment. The Phase I/II clinical trial for non-small cell lung cancer used pulsed low-dose paclitaxel three times/week with the starting dose of 15 mg/m(2). Daily thoracic radiotherapy was delivered in 1.8 Gy/fraction to 60-65 Gy for gross disease and to 45-58 Gy for microscopic disease. Timing of radiotherapy was delayed to allow for a minimum of 4 h for cell cycle progression.Forty-one patients have enrolled and 33 completed treatments. Seventeen patients completed the Phase I study, with an average primary tumor shrinkage of 83 +/- 8% (95% confidence interval). Tumor response rate was 100% for the Phase I study. Overall local control was 98%, and the survival rate was 46% at 1 year, 33% at 2 years, and 18% at 3 years. Toxicity was low with 3 of 18 patients having grade 3 pneumonitis and 3 of 18 patients having grade 3 esophagitis. There was no grade 4 pneumonitis, esophagitis, or hematological toxicity.Pulsed low-dose paclitaxel radiosensitization for non-small cell lung cancer resulted in a superior local control rate and comparable survival rate when compared with chemoradiation regimens using systemic dose chemotherapy. The regimen is associated with low toxicity and deserves additional investigation, particularly in patients with poor performance or older age, who cannot tolerate standard chemoradiation regimens.

Glioblastoma multiforme (GBM) is classified the fourth grade of human glioma that indicates the most malignant brain tumor with poor five-year survival (5%). Accumulated evidence showed that GBM cells were resistant to various therapeutic strategies. Recently, use of arsenic trioxide (ATO) to induce apoptosis and to increase the radiosensitivity was reported for the benefit in treating GBMs. However, the responsible signaling cascades are still remain investigation. MicroRNA, a category of RNA interference, regulates its downstream genes post transcriptionally by binding to the 3′untraslated region (UTR) of its targets. In this study, we used S1 and S1R1 GBM cells which were derived from the primary and the recurrent lesion respectively. Firstly, we identified that an oncogenic microRNA, miR-182, was down-regulated after the treatment of ATO in both GBM cells. In addition, one of the miR-182 targeted coding genes, Sestrin 2 (SESN2), plays an important role in relieving intracellular oxidative stress and participates in TP53 tumor suppressive pathway. Use of luciferase-based reporter assay confirmed that miR-182 directly bound to SESN2 3′UTR and interrupted the further translation. Intriguingly, the basal level of miR-182 in the primary S1 GBM was significantly lower than the recurrent S1R1 GBM, supporting the oncogenic evidence in the previous studies. The counteracted SESN2 expression was also determined by real-time qPCR and western blot. We then hypothesized that ATO may retarded the growth of GBM through a miR-182-SESN2 related regulatory axis. Overexpression of miR-182 in S1 GBM resulted in a decrease of SESN2 and insensitive to ATO treatment. On the opposite, inhibition of miR-182 in S1R1 GBM dramatically retarded the growth rate and sensitized the cells to ATO. Moreover, pretreatment of an antioxidant, N-acetyl-cysteine (NAC), rescued the miR-182 suppression, suggesting that ATO-induced oxidative stress may be dominant in this responsive axis. Concordant expression of miR-182/96/182 cassette was revealed, indicating the transcriptional regulation was unveiled. Further investigation on the detailed machinery still remain elucidating. Based on our findings, we displayed that ATO-induced oxidative stress triggered a tumor suppressive response through a miR-182-SESN2 dependent machinery. Citation Format: Liang-Ting Lin, Shih-Hwa Chiou, Yi-Jang Lee. Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2014-3548

Cellular senescence is a state of irreversible growth arrest; however, the metabolic processes of senescent cells remain active. Our previous studies have shown that radiation induces senescence of human breast cancer cells that display low expression of securin, a protein involved in control of the metaphase–anaphase transition and anaphase onset. In this study, the protein expression profile of senescent cells was resolved by two-dimensional gel electrophoresis to investigate associated metabolic alterations. We found that radiation induced the expression and activation of glyceraldehyde-3-phosphate dehydrogenase that has an important role in glycolysis. The activity of lactate dehydrogenase A, which is involved in the conversion of pyruvate to lactate, the release of lactate and the acidification of the extracellular environment, was also induced. Inhibition of glycolysis by dichloroacetate attenuated radiation-induced senescence. In addition, radiation also induced activation of the 5′-adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor kappa B (NF-κB) pathways to promote senescence. We also found that radiation increased the expression of monocarboxylate transporter 1 (MCT1) that facilitates the export of lactate into the extracellular environment. Inhibition of glycolysis or the AMPK/NF-κB signalling pathways reduced MCT1 expression and rescued the acidification of the extracellular environment. Interestingly, these metabolic-altering signalling pathways were also involved in radiation-induced invasion of the surrounding, non-irradiated breast cancer and normal endothelial cells. Taken together, radiation can induce the senescence of human breast cancer cells through metabolic alterations.

Stimulation of the host immune system is crucial in cancer treatment. In particular, nonspecific immunotherapies, when combined with other traditional therapies such as radiation and chemotherapy, may induce immunity against primary and metastatic tumors. In this study, we demonstrate that a novel, non-toxic immunoadjuvant, glycated chitosan (GC), decreases the motility and invasion of mammalian breast cancer cells in vitro and in vivo. Lung metastatic ratios were reduced in 4T1 tumor-bearing mice when intratumoral GC injection was combined with local high-intensity focused ultrasound (HIFU) treatment. We postulate that this treatment modality stimulates the host immune system to combat cancer cells, as macrophage accumulation in tumor lesions was detected after GC-HIFU treatment. In addition, plasma collected from GC-HIFU-treated tumor-bearing mice exhibited tumor-specific cytotoxicity. We also investigated the effect of GC on epithelial-mesenchymal transition-related markers. Our results showed that GC decreased the expression of Twist-1 and Slug, proto-oncogenes commonly implicated in metastasis. Epithelial-cadherin, which is regulated by these genes, was also upregulated. Taken together, our current data suggest that GC alone can reduce cancer cell motility and invasion, whereas GC-HIFU treatment can induce immune responses to suppress tumor metastasis in vivo.

D-Amino-acid oxidase (DAO2; EC 1.4.3.3) catalyses the oxidative deamination of D-amino acids to alpha-keto acids and ammonia. The purified DAO protein from Rhodosporidium toruloides was used to determine its amino acid sequence. Three internal peptide sequences, YCQYLARELQ, IAGIDDQAAEPIR and RCTMDSSDP, were obtained and used to synthesize four fully degenerated oligonucleotides for cloning of the DAO gene. Both cDNA and genomic DNA encoding R. toruloides DAO were cloned and sequenced. Comparison of these two DNA sequences revealed that the DAO gene contains six exons and five introns. The gene encodes a polypeptide of 368 amino acids with a calculated molecular mass of 40,079 Da. Using an Escherichia coli protein expression system, the DAO protein of R. toruloides can easily be produced in an active form and purified in a large quantity.

Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Förster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovative-fluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo.

The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells. Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 440. doi:10.1158/1538-7445.AM2013-440 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Metallothionein ( MT ) cDNAs were cloned and sequenced from two genera of ducks, Muscovy ( Cairina muschata ) and Tsai ya ( Anas platyrhynchos ). The two cDNAs show an extremely high sequence homology and contain an open reading frame encoding 63 amino acids. MT mRNA expressions were studied after metal induction using the cloned cDNA as a probe. Cadmium and copper induced MT gene efficiently, whereas zinc showed a markedly less effect. In addition, the MT mRNA accumulations in various developmental stages were also investigated. The result reveals a different pattern of expression from that of mammals. The discrepancy in MT gene between Tsai ya and Muscovy was further explored by examining genomic DNA structures. The duck MT showed three exons and two introns. The most significant variation of the genes occurs at intron II in which Tsai ya MT has 24 bases more than Muscovy MT . Moreover, MT expressions in the hybrids of Muscovy and Tsai ya were investigated using a reverse transcriptase-polymerase chain reaction. Those results demonstrated that both parental MT genes are expressed in the hybrids after metal induction.

Cellular senescence is an irreversible growth arrest mechanism that prevents indefinite proliferation of mammalian cells. Change of morphology is a common characteristic of senescent cells, while the underlying mechanism has not been completely addressed. In this study, we found that the flattened and enlarged cell shape in aged WI-38 fibroblast was accompanied by reduced actin cytoskeletal organization and up-regulated cofilin-1, a member of actin depolymerization factor family. Up-regulation of cofilin-1 was also detected in various tissues (lung, kidney, heart and liver) of one-year old BALB/c mice compared to that of 6-week old mice. Forced expression of cofilin-1 in early passaged cells accelerated senescence, while knockdown of cofilin-1 in late passaged cells inhibited senescence. Hydrogen peroxide mediated stress-induced premature senescence (SIPS) also induced cofilin-1 in human lung cancer cell lines, and this phenomenon was suppressed by knockdown of cofilin-1. Up-regulation of cofilin-1 in SIPS was independent of p53, while p27kip1 was essential for cofilin-1 induced senescence. Knockdown of p27kip1 in cells treated with hydrogen peroxide or in that with over-expressed cofilin alleviated senescence. Moreover, forced expression of cofilin-1 was sufficient to induce senescence in human lung cancer cells and repress the growth of xenograft tumor in NOD-SCID mice. Taken together, we found that cofilin-1 is able to mediate the cell senescence that is important for repressing the cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2011-1243

CD133 (Prominin-1) have been reported as one of CNS cancer stem cell markers, and is used for isolating cancer stem cells via fluorescence-activating cell sorting. However, more and more new evidence showed that CD133 cannot totally indicate to cancer stem cells, and the role of CD133 plays in glioblastoma still remains controversial. Here, we tend to use multiple imaging modalities to investigate CD133-positive and CD133-negative cells on tumorigenesis and recurrence in vivo. We sorted patient tumor-derived GBM cells by FACS with CD133-PE staining, and delivered multicistronic reporter system into each group of cells by lentivirus. The established cells stably expressed green fluorescent protein, firefly luciferase and thymidine kinase, and each of them was assayed in vitro. GFP expression was used for collecting cells with better reporter efficiency. Our in vitro results showed that CD133-positive cells revealed shorter doubling time and better transgene expression than CD133-negative cells. Furthermore, both groups of CD133 expression cells were xenografted orthotropically into left brain of NOD/SCID mice, and the tumorigenesis phenomenon were evaluated by magnetic resonance imaging and bioluminescence. Tumor formation was observed in both groups, suggested that CD133 may not be the key role in tumorigenesis of glioblastoma. Moreover, to demonstrate the relationship between cancer recurrence and cancer stem cells, we also treated the xenografted mice with ganciclovir to generate cytotoxicity by tk gene for 2 weeks, and in vivo imaging was recorded for upcoming 2 weeks. For future work, we plan to figure out whether the CD133 takes the most responsibility in tumor recurrence, and look forward to the other stemness-related markers such as Sox2, that may also provide a novel approach in cancer prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3354.

Cofilin is a member of actin depolymerizing factor (ADF)/cofilin family that can regulates actin dynamics for cancer growth and invasion/metastasis. Several lines of evidence have demonstrated that cofilin is associated with the cell cycle regulation, while the underlying mechanisms remain to be addressed. To better understand the involvement of cofilin in cell cycle progression, we dissected the cofilin expression and activity in different phases of the cell cycle. We synchronized human non-small lung A549 cancer cells in G1, S and G2/M phase by serum starvation, double thymidine blockage and nocodazole treatment, respectively. It was found that the expression of endogenous cofilin was weakest in G1 phase, while it was strongest in S phase and was modest in G2/M phase. This observation was further confirmed by detecting the down-regulation of highly expressed cofilin in S phase-synchronized cells entering mid-late S, G2/M and G1 phase of the cell cycle. Interestingly, it also showed that inactive form of cofilin (ser-3 phosphorylated) weakly expressed in S phase and gradually increased when cells progress through G2/M and G1 phases. These results suggest that cofilin expression and activity are coordinately regulated during the cell cycle progression. It is also consistent with our previous findings that enforced expression of cofilin results in G1 phase arrest but not S and G2/M phases. Moreover, the level of cofilin transcripts was not significantly altered in different phases of cell cycle. On the other hand, pretreatment of the proteasome inhibitor MG132 resulted in increase of cofilin in G1 phase, suggesting that the fluctuation of cofilin during cell cycle progression is associated with the protein stability. In addition, transduction of undegradable Y68F form of mutant cofilin potentially led to increase of the G1 percentage. Taken together, precisely regulation of cofilin expression and activity is important for cell cycle progression. Targeting on cofilin may provide alternative therapeutic strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1087.

We adopted the FeCl 3 and Grignard metathesis (McCullough) methods to synthesize three poly(3-hexylthiophene)s (P3HTs) exhibiting different degrees of regioregularity and then blended them with [6,6]-phenyl-C 61 -butyric acid methyl ester (PCBM) to obtain bulk heterojunction phases on the top of an acid-doped polyaniline nanotube (a-PANINT) interfacial layer. From integration of 1 H NMR spectra, we determined that the three P3HTs had head-to-tail coupling contents of 67, 81, and 96%, respectively. The photovoltaic (PV) performance of P3HT:PCBM-based devices fabricated without the a-PANINT interfacial layer increased as the regioregularity of the P3HT increased. The presence of the a-PANINT interfacial layer resulted in improved PV performances of the P3HT:PCBM-based devices. This improvement in the PV performance resulted from the highly conductive, controlled one-dimensional tubular nanoscale morphology of the annealed a-PANINT interfacial layer, which mediated the efficient migration of photogenerated holes to the buffer layer and suppressed exciton recombination.