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Organic molecules having emission in the NIR(II) region are emergent and receiving enormous attention. Unfortunately, attaining accountable organic emission intensity around the NIR(II) region is hampered by the dominant internal conversion operated by the energy gap law, where the emission energy gap and the associated internal reorganization energy λ

A series of NIR-II fluorescent TADF-incorporated polymer dots were successfully synthesized. The function of the TADF moiety was fully studied and the bio-applications of these polymer dots including bone imaging were also demonstrated.

Ionizing radiation is known to cause cell apoptosis at high dose range, but little is known about the cellular response to low dose radiation. In this study, we found that conditioned medium harvested from WI-38 lung fibroblasts and H1299 lung adenocarcinoma cells exposed to 0.1Gy to 1Gy could enhance the migration and invasion of unirradiated H1299 cells in both 2D and 3D culturing circumstances. Low dose radiation did not induce apoptosis, but induced senescence in irradiated cells. We next examined the expression of immediately early genes including c-Myc and K-Ras. Although both genes could be up-regulated by low dose radiation, induction of c-Myc was more specific to low dose range (0.5Gy) at transcriptional and translational levels. Knockdown of c-Myc by shRNA could repress the senescence induced by low dose radiation. The conditioned medium of irradiated cells induced migration of unirradiated cells was also repressed by knockdown of c-Myc. The c-Myc inhibitor 10058-F4 could suppress low dose radiation induced cell senescence, and the conditioned medium harvested from irradiated cells pretreated with 10058-F4 also lost the ability to enhance the migration of unirradiated cells. The cytokine array analysis revealed that immunosuppressive monocyte chemoattractant protein-1 increased by low dose radiation could be repressed by 10058-F4. We also showed that 10058-F4 could suppress low dose radiation induced tumor progression in a xenograft tumor model. Taken together, current data suggest that -Myc is involved in low dose radiation induced cell senescence and potent bystander effect to increase the motility of unirradiated cells.

Herbal medicines are attractive agents for human care. In this study, we found that the alcohol extract of Ajuga taiwanensis (ATE) screened from a chemical bank exhibited potent capacity for suppressing senescence associated biomarkers, including SA-β-gal and up-regulated p53 in old human dermal fibroblasts (HDFs) without induction of significant cytotoxicity up to 100 µg/ml. Concomitantly, cells re-entered the cell cycle by reducing G1 phase arrest and increasing cell growth rate. The ATE was further partitioned to obtain the sub-fractions of n-butanol (BuOH), ethyl acetate (EA) and water. The BuOH and water sub-fractions exhibited less effects on prohibition of cell growth than the EA sub-fraction. All of these sub-fractions exhibited the ability on suppressing SA-β-gal and p53 of old HDFs as low as 5–10 µg/ml. Under the activity guided fractionation and isolation, a major active constituent named AT-1 was isolated. The AT-1 was further identified as 8-O-acetylharpagide by structural analysis, and it could suppress SA-β-gal and p53 of old HDFs below 10 µM. In addition, the intracellular reactive oxygen species (ROS) levels of old HDFs were suppressed by ATE, the sub-fractions of BuOH and water, and AT-1. However, the EA sub-fraction showed little ability on suppression of ROS. Furthermore, we performed an in vivo study using aging mice to be fed with ATE and the sub-fractions followed by immunohistochemical (IHC) staining. The expression of p53 and SA-β-gal was significantly reduced in several tissue sections, including skin, liver, kidney, and spleen. Taken together, current data demonstrated that A. taiwanensis could suppress cellular senescence in HDFs, and might be used for health care.

Radiotherapy is routinely used for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the therapeutic efficacy is usually reduced by acquired radioresistance and locoregional recurrence. In this study, The Cancer Genome Atlas (TCGA) analysis showed that radiotherapy upregulated the miR-182/96/183 cluster and that miR-182 was the most significantly upregulated. Overexpression of miR-182-5p enhanced the radiosensitivity of HNSCC cells by increasing intracellular reactive oxygen species (ROS) levels, suggesting that expression of the miR-182 family is beneficial for radiotherapy. By intersecting the gene targeting results from three microRNA target prediction databases, we noticed that sestrin2 (SESN2), a molecule resistant to oxidative stress, was involved in 91 genes predicted in all three databases to be directly recognized by miR-182-5p. Knockdown of SESN2 enhanced radiation-induced ROS and cytotoxicity in HNSCC cells. In addition, the radiation-induced expression of SESN2 was repressed by overexpression of miR-182-5p. Reciprocal expression of the miR-182-5p and SESN2 genes was also analyzed in the TCGA database, and a high expression of miR-182-5p combined with a low expression of SESN2 was associated with a better survival rate in patients receiving radiotherapy. Taken together, the current data suggest that miR-182-5p may regulate radiation-induced antioxidant effects and mediate the efficacy of radiotherapy.

Metastasis is the leading cause of death in non-small cell lung cancer patients (NSCLC). Actin cytoskeletal rearrangement is frequently accompanied by cancer cell invasion and metastasis induced by the epithelial-mesenchymal transition (EMT). The expression levels of the actin-associated protein cofilin-1 and the crucial EMT molecule Twist-1 in NSCLC tissues were investigated in this work. Immunohistochemical (IHC) staining was used to identify 67.4% of tissue locations that had reciprocal amounts of cofilin-1 and Twist-1 using lung cancer tissue arrays. This reciprocal expression pattern was also found in 10 out of 15 NSCLC cell lines and 21 out of 25 clinicopathological NSCLC tissue sections. Furthermore, in tissue arrays and clinicopathological tissue samples, high levels of cofilin-1 and low levels of Twist-1 accounted for 80% and 71.5% of the reciprocal expression pattern, respectively. This pattern was also found in normal lung tissues, lung cancer tissues in stages I and II, and NSCLC tissues with adenocarcinoma subtypes. Despite the fact that cofilin-1 and Twist-1 were expressed in opposite directions, there was a positive association between the two proteins in normal lung tissues and lung tumor tissues. In addition, induced expression of cofilin-1 in H1299 NSCLC cells may suppress Twist-1 expression. A public microarray dataset with a maximum of 1,926 NSCLC samples were made accessible through an online Kaplan-Meier survival analytic tool. The investigators discovered that high levels of expression of both the cofilin-1 and Twist-1 genes were linked to lower NSCLC patient survival, particularly in the adenocarcinoma subtype. The multivariate Cox regression model was used in the analysis. Even though more data is required to confirm the reciprocal relationship between cofilin-1 and Twist-1 expression levels and NSCLC survival rates, it could be a useful indication of NSCLC progression.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27Kip1 , but not on the p53 and p16INK4 expressions. The knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27Kip1 gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.

Hypopharyngeal cancer (HPC) accounts for the lowest survival rate among all types of head and neck cancers (HNSCC). However, the therapeutic approach for HPC still needs to be investigated. In this study, a theranostic 188Re-liposome was prepared to treat orthotopic HPC tumors and analyze the deregulated microRNA expressive profiles. The therapeutic efficacy of 188Re-liposome on HPC tumors was evaluated using bioluminescent imaging followed by next generation sequencing (NGS) analysis, in order to address the deregulated microRNAs and associated signaling pathways. The differentially expressed microRNAs were also confirmed using clinical HNSCC samples and clinical information from The Cancer Genome Atlas (TCGA) database. Repeated doses of 188Re-liposome were administrated to tumor-bearing mice, and the tumor growth was apparently suppressed after treatment. For NGS analysis, 13 and 9 microRNAs were respectively up-regulated and down-regulated when the cutoffs of fold change were set to 5. Additionally, miR-206-3p and miR-142-5p represented the highest fold of up-regulation and down-regulation by 188Re-liposome, respectively. According to Differentially Expressed MiRNAs in human Cancers (dbDEMC) analysis, most of 188Re-liposome up-regulated microRNAs were categorized as tumor suppressors, while down-regulated microRNAs were oncogenic. The KEGG pathway analysis showed that cancer-related pathways and olfactory and taste transduction accounted for the top pathways affected by 188Re-liposome. 188Re-liposome down-regulated microRNAs, including miR-143, miR-6723, miR-944, and miR-136 were associated with lower survival rates at a high expressive level. 188Re-liposome could suppress the HPC tumors in vivo, and the therapeutic efficacy was associated with the deregulation of microRNAs that could be considered as a prognostic factor.

Human head and neck squamous cell carcinoma (HNSCC) is usually treated with chemoradiotherapy, but the therapeutic efficacy could be hampered by intrinsic radioresistance and early relapse. Repeated administrations of rhenium-188 (188Re)-conjugated radiopharmaceutical has been reported to escalate the radiation doses for better control of advanced human cancers. Here we found that high dosage of 188Re-liposome, the liposome-encapsulated 188Re nanoparticles exhibited significant killing effects on HNSCC FaDu cells and SAS cells but not on OECM-1 cells. To investigate the biological and pharmaceutical responses of high 188Re-liposomal dosage in vivo, repeated doses of 188Re-liposome was injected into the orthotopic tumor model. FaDu cells harboring luciferase reporter genes were implanted in the buccal positions of nude mice followed by intravenous injection of 188Re-liposome. The Cerenkov luminescence imaging (CLI) was performed to demonstrate an increased accumulation of 188Re-liposome in the tumor lesion of nude mice with repeated doses compared to a single dose. Repeated doses also enhanced tumor growth delay and elongated the survival of tumor-bearing mice. These observations were associated with significant loss of Ki-67 proliferative marker and epithelial–mesenchymal transition (EMT) markers in excised tumor cells. The body weights of mice were not significantly changed using different doses of 188Re-liposome, yet repeated doses led to lower blood counts than a single dose. Furthermore, the pharmacokinetic analysis showed that the internal circulation of repeated 188Re-liposomal therapy was elongated. The biodistribution analysis also demonstrated that accumulations of 188Re-liposome in tumor lesions and bone marrow were increased using repeated doses. The absorbed dose of repeated doses over a single dose was about twofold estimated for a 1 g tumor. Together, these data suggest that the radiopharmacotherapy of 188Re-liposome can enhance tumor suppression, survival extension, and internal circulation without acute toxicity using repeated administrations.

// Liang-Ting Lin 1, 10, * , Shin-Yi Liu 2, * , Jyh-Der Leu 3, 4, * , Chun-Yuan Chang 1 , Shih-Hwa Chiou 5, 6, 7 , Te-Chang Lee 7, 8 and Yi-Jang Lee 1, 9 1 Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 2 Department of Radiation Oncology, MacKay Memorial Hospital, Taipei, Taiwan 3 Division of Radiation Oncology, Taipei City Hospital Ren Ai Branch, Taipei, Taiwan 4 Institute of Neuroscience, National Chengchi University, Taipei, Taiwan 5 Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan 6 Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan 7 Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan 8 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 9 Biophotonics and Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taipei, Taiwan 10 Current address: Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong * These authors have contributed equally to this work Correspondence to: Te-Chang Lee, email: bmtcl@ibms.sinica.edu.tw Yi-Jang Lee, email: yjlee2@ym.edu.tw Keywords: arsenic trioxide; sestrin 2; miR-182; oxidative stress; anti-oxidant effect Received: April 12, 2017 Accepted: February 24, 2018 Published: March 23, 2018 ABSTRACT Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 ( SESN2 ) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3’- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

Fluorescence probes emitting in the second near-infrared (NIR-II, 1000-1700 nm) window with the ability for deep-tissue imaging in mammals herald a new era in surgical methodology. However, the brightness of these NIR-II probes is still far from satisfactory due to their low fluorescence quantum yields (QYs), preventing the observation of high-resolution images such as whole-organ vascular networks in real time. Described here is the molecular engineering of a series of semiconducting polymer dots (Pdots) incorporated with aggregation-induced emission moieties to exhibit the QYs as high as 14% in the NIR-II window. Benefiting from the ultrahigh brightness, a 1400 nm long-pass filter is utilized to realize in vivo 3D tumor mapping in mice. To further understand how the geometrical and electron structures of the semiconducting polymers affect their optical properties, the in-depth and thorough density-functional theory calculations are performed to interpret the experimental results. This study lays the groundwork for further molecular design of highly bright NIR-II Pdots.

The current study aims to investigate whether MDM2-SNP309 and p53R72P polymorphisms were associated with the risk of bladder cancer in Mongolian populations. These polymorphisms were evaluated in 79 controls and 63 bladder cancer cases using a PCR-restriction fragment length polymorphism assay, followed by analysis using multivariate logistic regression model and the Kaplan-Meier model to determine the odds ratio (OR) and age at onset of bladder cancer, respectively. The results revealed that the homozygous (G/G) genotype of MDM2-SNP309 increased the risk of bladder cancer compared to the wild-type (T/T) genotype [OR=1.629; 95% confidence interval (CI)=0.622–4.266] among Mongolians. On the other hand, the homozygous (P/P) genotype of p53R72P tended to protect the population from bladder cancer compared with the wild-type (R/R) genotype (OR=0.445; 95% CI=0.1727–2.147). It also showed that G/G genotype of MDM2-SNP309 increased the risk of bladder cancer when combined with the R/R genotype of p53R72P (OR=3.355; 95% CI=0.3914–28.766). Stratification by smoking and history of chronic urinary tract diseases tended towards increasing the risk association of the G/G (OR=2.3704; 95% CI=0.4308–3.044) and T/G genotypes (OR=5; 95% CI=0.8442–30.4088) of MDM2-SNP309 with bladder cancer, respectively. The protective role of P/P of p53R72P remained following stratification. MDM2-SNP309 and p53R72P were not involved in early age onset of bladder cancer in Mongolian patients. Taken together, MDM2-SNP309 and p53R72P had no significant association with bladder cancer in Mongolian patients. The two SNPs were also not able to predict early age at onset of bladder cancer.

Radiation is a widely used therapeutic method for treating breast cancer. N-dihydrogalactochitosan (GC), a biocompatible immunostimulant, is known to enhance the effects of various treatment modalities in different tumor types. However, whether GC can enhance the radiosensitivity of cancer cells remains to be explored. In this study, triple-negative murine 4T1 breast cancer cells transduced with multi-reporter genes were implanted in immunocompetent Balb/C mice to track, dissect, and identify liver-metastatic 4T1 cells. These cells expressed cancer stem cell (CSC) -related characteristics, including the ability to form spheroids, the expression of the CD44 marker, and the increase of protein stability. We then ex vivo investigated the potential effect of GC on the radiosensitivity of the liver-metastatic 4T1 breast cancer cells and compared the results to those of parental 4T1 cells subjected to the same treatment. The cells were irradiated with increased doses of X-rays with or without GC treatment. Colony formation assays were then performed to determine the survival fractions and radiosensitivity of these cells. We found that GC preferably increased the radiosensitivity of liver-metastatic 4T1 breast cancer cells rather than that of the parental cells. Additionally, the single-cell DNA electrophoresis assay (SCDEA) and &gamma, H2AX foci assay were performed to assess the level of double-stranded DNA breaks (DSBs). Compared to the parental cells, DNA damage was significantly increased in liver-metastatic 4T1 cells after they were treated with GC plus radiation. Further studies on apoptosis showed that this combination treatment increased the sub-G1 population of cells, but not caspase-3 cleavage, in liver-metastatic breast cancer cells. Taken together, the current data suggest that the synergistic effects of GC and irradiation might be used to enhance the efficacy of radiotherapy in treating metastatic tumors.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

Background: The accurate determination of cell confluence is a critical step for generating reasonable results of designed experiments in cell biological studies. However, the cell confluence of the same culture may be diversely predicted by individual researchers. Herein, we designed a systematic quantification scheme implemented on the Matlab platform, the so-called &ldquo, Confluence-Viewer&rdquo, program, to assist cell biologists to better determine the cell confluence. Methods: Human normal oral fibroblasts (hOFs) seeded in 10 cm culture dishes were visualized under an inverted microscope for the acquisition of cell images. The images were subjected to the cell segmentation algorithm with top-hat transformation and the Otsu thresholding technique. A regression model was built using a quadratic model and shape-preserving piecewise cubic model. Results: The cell segmentation algorithm generated a regression curve that was highly correlated with the cell confluence determined by experienced researchers. However, the correlation was low when compared to the cell confluence determined by novice students. Interestingly, the cell confluence determined by experienced researchers became more diverse when they checked the same images without a time limitation (up to 1 min). Conclusion: This tool could prevent unnecessary human-made mistakes and meaningless repeats for novice researchers working on cell-based studies in health care or cancer research.

Fisetin (3,7,3′,4′-tetrahydroxyflavone), which belongs to the flavonoid group of polyphenols and is found in a wide range of plants, has been reported to exhibit a number of biological activities in human cancer cells, including antioxidant, anti-inflammatory, antiangiogenic, anti-invasive and antiproliferative effects. Although previous in vitro studies have shown that fisetin treatment increases the apoptotic rate and enhances the radiosensitivity of human colorectal cancer cells, the in vivo effects of fisetin on tumor growth remain unclear. In the present study a murine xenograft tumor model was employed to investigate the therapeutic effects of fisetin in combination with radiation on CT-26 colon cancer cells and human HCT116 colorectal cancer cells. This revealed that intratumoral injection of fisetin significantly suppressed the growth of CT-26 tumors compared with the untreated control group, but had little effect on the growth of HCT116 tumors. However, fisetin in combination with 2-Gy radiation enhanced tumor suppressor activity in murine colon and human colorectal xenograft tumors, as compared with 2-Gy fractionated radiation administered alone for 5 days and fisetin alone. Interestingly, fisetin downregulated the expression of the oncoprotein securin in a p53-independent manner. However, securin-null HCT116 tumors showed only moderate sensitivity to fisetin treatment, and the combination of fisetin and radiation did not significantly suppress securin-null HCT116 tumor growth compared with normal HCT116 tumors. Therefore, the role of securin in mediating the effect of fisetin on colorectal cancer growth warrants further investigation. In conclusion, the results of the current study provide important preclinical data for evaluating the efficacy of fisetin and radiation combination treatment as an adjuvant chemoradiotherapy for human colorectal cancers.

// Liang-Ting Lin 1, 2 , Chun-Yuan Chang 1 , Chih-Hsien Chang 3 , Hsin-Ell Wang 1 , Shih-Hwa Chiou 2, 4, 5 , Ren-Shyan Liu 1 , Te-Wei Lee 3 , Yi-Jang Lee 1, 6 1 Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 2 Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan 3 Isotope Application Division, Institute of Nuclear Energy Research, Taoyuan, Taiwan 4 Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan 5 Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan 6 Biophotonics and Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taipei, Taiwan Correspondence to: Te-Wei Lee, email: tewei123456@gmail.com Yi-Jang Lee, email: yjlee2@ym.edu.tw Keywords: 188 Re-liposome, HNSCC, orthotopic tumor model, microarray analysis, let-7 microRNA Received: May 20, 2016 Accepted: August 13, 2016 Published: August 29, 2016 ABSTRACT Human head and neck squamous cell carcinoma (HNSCC) is usually treated by surgical resection with adjuvant radio-chemotherapy. In this study, we examined whether the radiopharmaceutical 188 Re-liposome could suppress the growth of HNSCC followed by an investigation of molecular mechanisms. The orthotopic HNSCC tumor model was established by human hypopharyngeal FaDu carcinoma cells harboring multiple reporter genes. The drug targeting and therapeutic efficacy of 188 Re-liposome were examined using in vivo imaging, bio-distribution, pharmacokinetics, and dosimetry. The results showed that 188 Re-liposome significantly accumulated in the tumor lesion compared to free 188 Re. The circulation time and tumor targeting of 188 Re-liposome were also longer than that of free 188 Re in tumor-bearing mice. The tumor growth was suppressed by 188 Re-liposome up to three weeks using a single dose treatment. Subsequently, microarray analysis followed by Ingenuity Pathway Analysis (IPA) showed that tumor suppressor let-7 microRNA could be an upstream regulator induced by 188 Re-liposome to regulate downstream genes. Additionally, inhibition of let-7i could reduce the effects of 188 Re-liposome on suppression of tumor growth, suggesting that let-7 family was involved in 188 Re-liposome mediated suppression of tumor growth in vivo . Our data suggest that 188 Re-liposome could be a novel strategy for targeting HNSCC partially via induction of let-7 microRNA.

It has been demonstrated that by using doping and co-doping strategies the lifetimes as well as luminescence intensities of the doping species in the lanthanide oxysulfide nanoparticles (NPs) Ln2O2S:Ln’3+ (Ln=Gd, Tb, Eu; Ln׳=Tb, Eu, 1% and 5%) could be tuned. The lanthanide oxysulfide NPs, i.e., Gd2O2S, Tb2O2S and Gd2O2S:Tb3+(5%) were synthesized as matrices and doped with Eu3+(1% or 5%), by the thermal decomposition method and characterized by TEM, XRD, photoluminescence, cellular and in vivo animal imaging studies for potential luminescence bioimaging applications. Of these materials, Gd2O2S:Eu3+(5%) NPs possess the highest photo-stability and strongest luminescent intensity at 625 nm with a lifetime 853 μs in hexane. Surface modification of the Gd2O2S:Eu3+(5%) NPs with mPEG-APTES to increase its aqueous solubility resulted in almost complete luminescence quenching. Calcination of the host Gd2O2S:Eu3+(5%) NPs at 400 °C to increase their crystallinity and maintained some of their luminescence properties in aqueous solution. Further surface modification of the Gd2O2S:Eu3+(5%)-APTES NPs with mPEG and Alexa Fluor 660 allowed their effective cellular and in vivo animal luminescence imaging with low bio-toxicity. These novel imaging materials with tunable lifetimes would be potentially useful for luminescence bioimaging applications, particularly in the time-resolved, up-conversion, and/or multiplex modes.

Metastasis is the primary cause of mortality in patients with non‑small cell lung cancer (NSCLC). Actin cytoskeletal reorganization is usually accompanied by the epithelial‑mesenchymal transition (EMT)‑induced invasion and metastasis of cancer cells. In the present study, expression levels of the actin‑associated protein cofilin‑1 and of the pivotal EMT molecule Twist‑1 were determined in NSCLC tissues. Using lung cancer tissue arrays, the identification of 67.4% of tissue spots that exhibited reciprocal levels of cofilin‑1 and Twist‑1 was achieved by immunohistochemical (IHC) staining. This reciprocal expression pattern was also detected in 21 out of 25 clinicopathological NSCLC tissue sections, and in 10 out of 15 NSCLC cell lines. In addition, high levels of cofilin‑1 and low levels of Twist‑1 accounted for 80 and 71.5% of the reciprocal expression pattern in tissue arrays and clinicopathological tissue samples, respectively. This pattern was also detected in normal lung tissues, stage I and II lung cancer tissues, and adenocarcinoma subtypes of NSCLC tissues. Although cofilin‑1 and Twist‑1 were expressed inversely, a positive correlation of these two proteins was present in normal lung tissues and lung tumor tissues. Furthermore, enforced expression of cofilin‑1 suppressed the expression level of Twist‑1 in NSCLC H1299 cells. An on‑line Kaplan‑Meier survival analytic tool allowed access to a public microarray dataset with a maximum of 1,926 NSCLC samples. The analysis revealed that high expression levels of both cofilin‑1 (CFL1) and Twist‑1 (TWIST1) genes were associated with decreased survival of NSCLC patients, notably with regard to the adenocarcinoma subtype. The analysis was conducted using the multivariate Cox regression model. Although the reciprocal association of the expression levels of cofilin‑1 and Twist‑1 with the survival rate of NSCLC patients requires additional information, it may be a significant indicator of the progression of NSCLC.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin‐binding proteins, that the post‐translational up‐regulation of cofilin‐1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up‐regulated cofilin‐1 mainly existed in the serine‐3 phosphorylated form, according to the 2D gel immunoblotting assay. The up‐regulation of cofilin‐1 was also detected in aged mammalian tissues. The over‐expression of wild‐type cofilin‐1 and constitutively phosphorylated cofilin‐1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin‐1, which led to a decrease in phosphorylated cofilin‐1. The senescence induced by the over‐expression of cofilin‐1 was dependent on p27Kip1, but not on the p53 and p16INK4 expressions. The knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over‐expression of cofilin‐1 induced the expression of p27Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27Kip1 gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down‐regulation of TEAD1 was accompanied by the up‐regulation of cofilin‐1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin‐1/TEAD1/p27Kip1 signaling is involved in senescence‐related morphological change and growth arrest., Triggers of senescence, such as replicative stress or oxidative stress, can induce the expression of cofilin‐1 to influence of cell morphology. Up‐regulated cofilin‐1 would enhance the expression of p27kip1 gene by suppressing the TEAD‐1 that transrepresses p27kip1 and lead to senescence‐associated growth delay.

Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 (SESN2) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3’- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

Despite standard treatment, about 70% of ovarian cancer will recur. Cancer stem cells (CSCs) have been implicated in the drug-resistance mechanism. Several drug resistance mechanisms have been proposed, and among these, autophagy plays a crucial role for the maintenance and tumorigenicity of CSCs. Compared to their differentiated counterparts, CSCs have been demonstrated to display a significantly higher level of autophagy flux. Moreover, mitophagy, a specific type of autophagy that selectively degrades excessive or damaged mitochondria, is shown to contribute to cancer progression and recurrence in several types of tumors. Nanomedicine has been shown to tackle the CSCs problem by overcoming drug resistance. In this work, we developed a nanomedicine, 188Re-liposome, which was demonstrated to target autophagy and mitophagy in the tumor microenvironment. Of note, the inhibition of autophagy and mitophagy could lead to significant tumor inhibition in two xenograft animal models. Lastly, we presented two cases of recurrent ovarian cancer, both in drug resistance status that received a level I dose from a phase I clinical trial. Both cases developing drug resistance showed drug sensitivity to 188Re-liposome. These results suggest that inhibition of autophagy and mitophagy by a nanomedicine may be a novel strategy to overcome drug resistance in ovarian cancer.

Aim Effects of radiofrequency ablation (RFA) combining immunoadjuvant glycated chitosan (GC) on tumor control and potent cytokine responses were investigated in a syngeneic breast tumor model. Materials and methods Murine 4T1 breast carcinoma cells harboring the luciferase reporter gene were used to evaluate the tumor growth rate and metastasis in vivo using bioluminescent imaging. Plasma of RFA/GC-treated tumor-bearing mice was collected for ex vivo cytotoxicity analysis and mouse chemokine array assays. Results Tumor growth and systemic metastasis were suppressed by combined RFA and GC when tumor size reached 300 mm3, not detected, however, when tumor size reached 800 mm3 The survival rate of mice bearing small tumors was also higher than that of large ones after RFA-GC treatment. Plasma extracted from RFA-GC-treated small tumor-bearing mice exhibited cytotoxicity on cultured 4T1 cells. Moreover, reduced tumor growth-related cytokines and increased antitumor-related cytokines were detected in the plasma collected. Conclusion RFA combining GC could control tumor progression with induced potent antitumor cytokine responses.

Non–small cell lung cancer (NSCLC) is a highly morbid and mortal cancer type that is difficult to eradicate using conventional chemotherapy and radiotherapy. Little is known about whether radionuclide-based pharmaceuticals can be used for treating NSCLC. Here we embedded the therapeutic radionuclide 188Re in PEGylated (PEG is polyethylene glycol) liposomes and investigated the biodistribution, pharmacokinetics, and therapeutic efficacy of this nanoradiopharmaceutical on NSCLC using a xenograft lung tumor model and the reporter gene imaging techniques. Methods: Human NSCLC NCI-H292 cells expressing multiple reporter genes were used in this study. 188Re was conjugated to N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA) and loaded into the PEGylated liposome to form a 188Re-liposome. The tumor growth rates and localizations were confirmed using bioluminescent imaging and SPECT/CT after the 188Re-BMEDA or 188Re-liposome was intravenously injected. The accumulation of the nanodrug in various organs was determined by the biodistribution analysis and the nano-SPECT/CT system. The pharmacokinetic and dosimetric analyses were further determined using WinNonlin and OLINDA/EXM, respectively. Results: The biodistribution and nano-SPECT/CT imaging showed that PEGylated 188Re-liposome could efficiently accumulate in xenograft tumors formed by NCI-H292 cells that were subcutaneously implanted in nude mice. Pharmacokinetic analysis also showed that the retention of 188Re-liposome was longer than that of 188Re-BMEDA. In an orthotopic tumor model, ex vivo γ counting revealed that the uptake of 188Re-liposome was detected in tumor lesions but not in surrounding normal lung tissues. Moreover, we evaluated the therapeutic efficacy using bioluminescent imaging and showed that the lung tumor growth was suppressed but not eradicated by 188Re-liposome. The life span of 188Re-liposome–treated mice was 2-fold longer than that of untreated control mice. Conclusion: The results of biodistribution, pharmacokinetics, estimated dosimetry, nano-SPECT/CT, and bioluminescent imaging suggest that the PEGylated liposome–embedded 188Re could be used for the treatment of human lung cancers.

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remain to be addressed. Here we demonstrated, after a survey of various actin-binding proteins, that up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization in senescent cells, as well as their morphological enlargement . Additionally, the up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form according to the 2D gel immunoblotting assay. Accumulation of cofilin-1 in senescent cells depended on the post-translational mechanism, as the ubiquitin-proteasomal degradation of cofilin-1 was reduced without any change in mRNA level. Up-regulation of cofilin-1 was not only found in replicative senescence but also in oxidative stress- and K-Ras oncogene-induced senescence. Over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with increased cell size. Additionally, senescent phenotypes were reduced by knockdown of total cofilin-1. The senescence induced by over-expression of cofilin-1 was dependent on p27Kip1 but not p53 and p16INK4 expression. Knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that over-expression of cofilin-1 induced expression of p27Kip1 through transcriptional suppression of the TEAD1 transcription factor. The TEAD1 transcription factor played a transrepressive role on the p27Kip1 gene promoter as determined by the promoter deletion reporter gene assay. Interestingly, TEAD1 down-regulation was accompanied by up-regulation of cofilin-1 in senescence. Knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and cell senescence, respectively. Furthermore, senescence induced by the over-expression of cofilin-1 could be alleviated by ectopic expression of TEAD1. Taken together, current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest. Keywords: Cofilin-1, TEAD1, p27Kip1, morphology, growth arrest, senescence Citation Format: Yi-Jang Lee, Cheng-Han Tsai, Chun-Yuan Chang. The cofilin-TEAD1-p27Kip1 pathway ablates cell senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 893.

Purpose188Re-liposome has been used for evaluating the theranostic efficacy in various human cancers. However, whether the therapeutic efficacy of 188Re-liposome directly related to microRNAs regulation and specific signaling pathways are largely unknown. In this project, we investigated the 188Re-liposome induced systemic microRNA expressive responses in human head and neck cancer (HNSCC) cells. Methods The FaDu-3R cells were implanted into the nude mice at the position of buccal cavity. After two weeks of implantation, the tumors were palpable and the bioluminescent imaging indicated the location of this orthotopic tumor model. The PEGylated 188Re-liposome was intravenously injected single and dual dose respectively. We harvested all of the tumors after 188Re-liposome injection one month ago, and used Taqman Openarray MicroRNA (Thermo, USA) to detect the expression of miRNAs in different groups. The dataset we observed was further analyzed by using Ingenuity Pathway Analysis (IPA) software to establish the molecular signaling pathway of 188Re-liposome radiopharmaceutical. Results Quantification of the photon signals showed that the tumor growth of the dual 188Re-liposome treated mice was slower than that of control ones. The survival rate of 188Re-liposome treated animals were also better than control ones. The microRNA Openarray showed that, compared to untreated control, 17 miRNA were significantly changed in expressive levels over 5 fold after 188Re-liposome treatment. Subsequently, we used IPA to determine the role of these miRNA, and found these miRNA with oncogenic and tumor suppressive properties were down-regulated and up-regulated, respectively. Among them, we focused on miR-152-5p that is closed related to cancer occurrence and development, and showed that knockdown of miR-152-5p could decreased the effects of 188Re-liposome on suppressing the growth of HNSCC xenograft tumors. Conclusion HNSCC tumor model treated with dual dose of 188Re-liposome exhibited significant response to this radiopharmaceutical. We also found that 17 miRNA could be gsignificantly re-regulated, and miR-152-5p is one example to be involved in mediating the efficacy of 188Re-liposome. Targeting on specific miRNA may enhance the application value of 188Re-liposome.in cancer treatment. Citation Format: Bing-Ze Lin, Chun-Yuan Chang, Chih-Hsien Chang, Yi-Jang Lee. Openarray profiling of the systemic change of microRNAs in rhenium-188 liposome treated human head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1803.

Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against Pseudomonas aeruginosa within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by P. aeruginosa. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.

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.