De-Ming Yang

Targeting Signal Transducer and Activator of Transcription 3 Pathway by Cucurbitacin I Diminishes Self-Renewing and Radiochemoresistant Abilities in Thyroid Cancer-Derived CD133+ Cells

Anaplastic thyroid cancer (ATC) is a lethal solid tumor with poor prognosis because of its invasiveness and its resistance to current therapies. Recently, ATC-CD133+ cells were found to have cancer stem cell (CSC) properties and were suggested to be important contributors to tumorigenicity and cancer metastasis. However, the molecular pathways and therapeutic targets in thyroid cancer-related CSCs remain undetermined. In this study, ATC-CD133+ cells were isolated and found to have increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared with ATC-CD133− cells. Microarray bioinformatics analysis suggested that the signal transducer and activator of transcription 3 (STAT3) pathway could be important in regulating the stemness signature in ATC-CD133+ cells; therefore, the effect of the potent STAT3 inhibitor cucurbitacin I in ATC-CD133+ cells was evaluated in this study. Treatment of ATC-CD133+ cells with cucurbitacin I diminished their CSC-like abilities, inhibited their stemness gene signature, and facilitated their differentiation into ATC-CD133− cells. Of note, treatment of ATC-CD133+ cells with cucurbitacin I up-regulated the expression of thyroid-specific genes and significantly enhanced radioiodine uptake. Furthermore, cucurbitacin I treatment increased the sensitivity of ATC-CD133+ cells to radiation and chemotherapeutic drugs through apoptosis. Finally, xenotransplantation experiments revealed that cucurbitacin I plus radiochemotherapy significantly suppressed tumorigenesis and improved survival in immunocompromised mice into which ATC-CD133+ cells were transplanted. In summary, these results show that the STAT3 pathway plays a key role in mediating CSC properties in ATC-CD133+ cells. Targeting STAT3 with cucurbitacin I in ATC may provide a new approach for therapeutic treatment in the future.

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.

Involvement of Rab3A in Vesicle Priming During Exocytosis: Interaction with Munc13‐1 and Munc18‐1

Rab3A is a small G‐protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13‐1 and Munc18‐1 during vesicle priming. Phorbol 12‐myristate 13‐acetate (PMA) is known to enhance the percentage of fusion‐competent vesicles and this is mediated by protein kinase C (PKC)‐independent Munc13‐1 activation and PKC‐dependent dissociation of Munc18‐1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13‐1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab‐interacting molecule (RIM)‐binding deficient Munc13‐1 mutant, 128‐Munc13‐1, the effects of Rab3A on PMA‐induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP‐Rab3A (Q81L), could be reversed by co‐expressing Munc18‐1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18‐1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18‐1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13‐1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18‐1 acts downstream of Munc13‐1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18‐1 promotes Rab3A dissociation from vesicles, which then results in fusion.

Calcification of senile cataractous lens determined by Fourier transform infrared (FTIR) and Raman microspectroscopies

A calcified plaque on the surface of a senile cataractous lens (CL) isolated from a 79‐year‐old male patient was identified and its chemical composition quantified using Fourier transform infrared (FTIR) and confocal Raman microspectroscopies. The noncalcified area of the same CL and hydroxyapatite (HA) were selected as a control. Several unique absorption bands, at 960, 1034 and 1090 cm−1 assigned to the ν1 and ν3 stretching modes of phosphate and at 875 cm−1 attributed to carbonate, were clearly displayed in the infrared (IR) spectra of calcified plaque and HA. A peak at 961 cm−1 due to the ν1 stretching mode of phosphate was also evidenced in the Raman spectra of calcified plaque and HA. The calcified plaque formed within the lens protein was found to mainly consist of a mature HA, in which type‐A carbonate apatites (11.4%), type‐B carbonate apatites (55.6%) and liable surface carbonate ions (33.0%) were presented. A higher content of the liable carbonate implies that the calcification or mineralization in this calcified lens was incomplete and still in progress. Moreover, calcification seems not to influence the secondary structure of lens protein because both IR and Raman spectra for the lens protein in the noncalcified area and calcified plaque were similar. The result suggests that both microscopic FTIR and Raman spectroscopies were easy to perform and capable of determination of the chemical composition of a calcified CL.

Dominant Role of Orai1 with STIM1 on the Cytosolic Entry and Cytotoxicity of Lead Ions

Pb(2+) ions cause severe damages to living cells. In particular, our previous study showed that the Orai-STIM1 (stromal interacting protein 1)-formed store-operated Ca(2+) channels (SOCs) allow Pb(2+) entry. In relation to this, the present study investigates the molecular gating mechanism of Pb(2+) entry by Orai1 with STIM1, as well as the resulting cytotoxicity on human embryonic kidney HEK293 cells. The store-operated Ca(2+) entry (SOCE, activity of SOCs) and Pb(2+) entry were measured using the fura-2 imaging method and indo-1 quenching strategy, as well as through an atomic absorption spectrophotometer. The results of RT-PCR, Western blot, fast confocal, and fluorescent lifetime imaging microscopy indicated the endogenous expression of Orai1 and STIM1 in HEK cells and the functional interaction between these two proteins during SOCE. Both SOCE and Pb(2+) entry largely increased when Orai1 and STIM1 were overexpressed (3- and 1.64-folds, respectively) compared with nonfluorescent cells, and they were significantly attenuated when the E106Q mutation of Orail with STIM1 was cotransfected (6- and 2.25-folds decrease, respectively) compared with Orai1-STIM1 coexpressed cells. The ion gating for Pb(2+) could be governed by the E106 region of Orai1. After sorting and subsequent cultures, the Orai1-STIM1 positive expressed cells behaved more sensitively to Pb(2+) than the Orai1-STIM1 negative cells. In summary, the data suggest that Orai1, together with STIM1, plays a critical role in Pb(2+) entry and the toxicity of Pb(2+).

Orai1–STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells

Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This studys main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.

Intracellular Pb2+ Content Monitoring Using a Protein-Based Pb2+ Indicator

Lead ion (Pb(2+)) is one of the most hazardous heavy metals to almost all life forms. The components of store-operated Ca(2+) entry as a molecular gateway have been previously found to participate in the cytotoxic entry of Pb(2+). However, the safe levels of intracellular Pb(2+) hiding in blood Pb(2+) levels are still not determined with full certainty. The present study aimed to construct protein-based Pb(2+) indicators to help establish a reliable setting for the content monitoring of intracellular Pb(2+). A series of Pb(2+) indicators based on fluorescence resonance energy transfer, Met-leads, were developed. The Pb(2+)-binding protein PbrR (from Cupriavidus metallidurans CH34) was applied between the fluorescent protein pair ECFP(ΔC11) and cp173Venus. The spectral patterns and sensing ranges of all Met-leads were characterized in vitro. Among these constructs, Met-lead 1.59 had relatively high ion selectivity and broad dynamic range (3.3-5.7). Consequently, this Met-lead was adopted in the cellular Pb(2+) biosensing. The intracellular Pb(2+) content in human embryonic kidney cells was successfully monitored using Met-lead 1.59 under both short- and long-term treatments. The existence of intracellular Pb(2+) can be significantly sensed using Met-lead 1.59 after 3 h 0.5μM (10 μg/dl) exposure, which is 200 times more improved than previous live-cell indicators. In summary, a new Pb(2+) indicator, Met-lead 1.59, was successfully developed for advanced research on Pb(2+) toxicology.

Clodronate-Induced Cell Apoptosis in Human Thyroid Carcinoma Is Mediated via the P2 Receptor Signaling Pathway

Clodronate, a halogenated bisphosphonate, can inhibit the growth of human thyroid carcinoma (TC) cells. Previously, we found that a clodronate-induced Ca2+ transient was correlated with clodronate-induced growth inhibition in TC cells. However, the details of the signaling process underlying the antiproliferative effect of clodronate on TC cells are not clear. In this study, we investigated the antiproliferative mechanism of clodronate on papillary TC (PTC) cells and xenotransplanted animals using a combination of pharmacological drugs. Reverse transcription-polymerase chain reaction analysis confirmed the endogenous expression of P2Y receptor isoforms in PTC cells. The P2 antagonist suramin not only inhibited the antiproliferative effect of clodronate and ATP on TC cells but also blocked all the Ca2+ transients induced by clodronate and ATP. The release of Ca2+ from the endoplasmic reticulum and membrane depolarization of mitochondria was observed during the clodronate-induced Ca2+ transients. The results of terminal deoxynucleotidyltransferase dUTP nick-end labeling assays and flow cytometry with annexin V and caspase-3 staining suggest that both ATP and clodronate induce apoptosis. Significant inhibition of tumor invasion and colony formation was also observed in clodronate-treated PTC cells. We further demonstrated that only the cAMP inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), and not inhibitors of phospholipase C [1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] or store-operated Ca2+ entry (2-aminoethyl diphenylborinate), can significantly reverse the effect of clodronate. Finally, in vivo animal and green fluorescent protein imaging studies further proved that the tumor inhibitory effect of clodronate on xenotransplanted CG3 cells can be reversed by treatment with suramin. In conclusion, we demonstrated that clodronate-induced PTC cell apoptosis and tumor inhibition are partially mediated by the P2Y receptor-cAMP cascade.

In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions

The high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)-labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)-labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (taupeak) and the left side of the half-peak width (tau1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

Visualization of the Orai1 Homodimer and the Functional Coupling of Orai1-STIM1 by Live-Cell Fluorescence Lifetime Imaging

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or delta1-90+delta267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.