Yi-Jang Lee

Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum.

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.

Autophagy inhibition enhances apoptosis triggered by BO-1051, an N-mustard derivative, and involves the ATM signaling pathway.

In a previous study, BO-1051, an N-mustard linked with a DNA-affinic molecule, was shown to target various types of cancer cell lines. In the present study, we aimed to investigate the cytotoxicity, as well as the underlying mechanism, of BO-1051. We found that BO-1051 simultaneously induced apoptosis and autophagy in hepatocellular carcinoma cell lines. DNA double strand breaks induced by BO-1051 activated the ATM signaling pathway and subsequently resulted in caspase-dependent apoptosis. When autophagy was inhibited in its early or late stages, apoptosis was significantly enhanced. This result indicated autophagy as a cytoprotective effect against BO-1051-induced cell death. We further inhibited ATM activation using an ATM kinase inhibitor or ATM-specific siRNA and found that while apoptosis was blocked, autophagy also diminished in response to BO-1051. We not only determined a signaling pathway induced by BO-1051 but also clarified the linkage between DNA damage-induced apoptosis and autophagy. We also showed that BO-1051-induced autophagy acts as a cytoprotective reaction and downstream target of the ATM-signaling pathway. This research revealed autophagy as a universal cytoprotective response against DNA damage-inducing chemotherapeutic agents, including BO-1051, cisplatin, and doxorubicin, in hepatocellular carcinoma cell lines. Autophagy contributes to the remarkable drug resistance ability of liver cancer.

Discrimination of Breast Cancer by Measuring Prostate-Specific Antigen Levels in Women's Serum

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 womens 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 womens 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.

Radiation induces senescence and a bystander effect through metabolic alterations

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.

Radiation-induced senescence in securin-deficient cancer cells promotes cell invasion involving the IL-6/STAT3 and PDGF-BB/PDGFR pathways

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.

Targeting Protective Autophagy Exacerbates UV-Triggered Apoptotic Cell Death

Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV) irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II) expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment). The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.

Detection of prostate-specific antigen with a paired surface plasma wave biosensor.

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.

Therapeutic efficacy evaluation of curcumin on human oral squamous cell carcinoma xenograft using multimodalities of molecular imaging

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.

Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice

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-induced gamma-H2AX activation via both p53-dependent and -independent pathways but is not associated with cell cycle arrest in human colorectal cancer cells.

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.