Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Targeted drug delivery using an aptamer against shared tumor-specific peptide antigen of MAGE-A3.

We developed a DNA aptamer, Ap52, against the shared tumor-specific MAGE-A3111-125 peptide antigen that was used to target multiple types of cancer cells. Here we report the in vivo study of mice implanted with pancreatic tumor cells AsPC-1, which demonstrates accumulation of phosphorothioate-modified Ap52 (ThioAp52) at the xenograft tumor following either intravenous or in situ injection. When complexed with antitumor drug doxorubicin (Dox), ThioAp52 achieves targeted delivery to four types of cancer cells, including breast, oral, pancreatic, and skin. Image analysis shows that ThioAp52-Dox complex selectively enters cancer cells, while free Dox is taken up by all cell lines. The cytotoxicity of ThioAp52-Dox for cancer cells is enhanced as compared to that for the corresponding normal/noncancerous cells. These results indicate that this aptamer against shared tumor-specific antigen can be a potential delivery vehicle for therapeutics to treat multiple cancers.

Musashi-1 promotes stress-induced tumor progression through recruitment of AGO2.

Carcinomatous progression and recurrence are the main therapeutic challenges frequently faced by patients with refractory tumors. However, the underlined molecular mechanism remains obscure. Methods: We found Musashi-1 (MSI1) transported into cytosol under stress condition by confocal microscopy and cell fractionation. Argonaute 2 (AGO2) was then identified as a cytosolic binding partner of MSI1 by Mass Spectrametry, immunoprecipitation, and recombinant protein pull-down assay. We used RNA-IP to determine the MSI1/AGO2 associated regions on downstream target mRNAs. Finally, we overexpressed C-terminus of MSI1 to disrupt endogenous MSI1/AGO2 interaction and confirm it effects on tmor progression. Results: Malignant tumors exhibit elevated level of cytosolic Musashi-1 (MSI1), which translocates into cytosol in response to stress and promote tumor progression. Cytosolic MSI1 forms a complex with AGO2 and stabilize or destabilize its target mRNAs by respectively binding to their 3´ untranslated region or coding domain sequence. Both MSI1 translocation and MSI1/AGO2 binding are essential for promoting tumor progression. Blocking MSI1 shuttling by either chemical inhibition or point mutation attenuates the growth of GBM-xenografts in mice. Importantly, overexpression of the C-terminus of MSI1 disrupts endogenous MSI1/AGO2 interaction and effectively reduces stress-induced tumor progression. Conclusion: Our findings highlight novel molecular functions of MSI1 during stress-induced carcinomatous recurrence, and suggest a new therapeutic strategy for refractory malignancies by targeting MSI1 translocation and its interaction with AGOs.

A quadrupole ion trap mass spectrometer for dry microparticle analysis.

In this work, we report a new design of a charge detection quadrupole ion trap mass spectrometer (QIT-MS) for the analysis of micro-sized dry inorganic and bioparticles including red blood cells (RBCs) and different sizes of MCF-7 breast cancer cells. The developed method is one of the fastest methods to measure the mass of micro-sized particles. This system allows the online analysis of various micro-sized particles up to 1 × 1017 Da. The calibration of the mass spectrometer has been done by using different sizes of polystyrene (PS) particles (2-15 µm). The measured masses of RBCs were around 1.8 × 1013 Da and MCF-7 cancer cells were between 1 × 1014 and 4 × 1014 Da. The calculated mass distribution profiles of the particles and cells were given as histogram profiles. The statistical data were summarized after Gaussian type fitting to the experimental histogram profiles. The new method gives very promising results for the analysis of particles and has very broad application.

High accuracy differentiating autoimmune pancreatitis from pancreatic ductal adenocarcinoma by immunoglobulin G glycosylation.

BACKGROUND: Misdiagnosis of autoimmune pancreatitis (AIP) as pancreatic cancer (PDAC) or vice versa can cause dismal patents' outcomes. Changes in IgG glycosylation are associated with cancers and autoimmune diseases. This study investigated the IgG glycosylation profiles as diagnostic and prognostic biomarkers in PDAC and AIP. METHODS: Serum IgG-glycosylation profiles from 86 AIP patients, 115 PDAC patients, and 57 controls were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Classification and regression tree (CART) analysis was applied to build a decision tree for discriminating PDAC from AIP. The result was validated in an independent cohort. RESULTS: Compared with AIP patients and controls, PDAC patients had significantly higher agalactosylation, lower fucosylation, and sialylation of IgG1, a higher agalactosylation ratio of IgG1 and a higher agalactosylation ratio of IgG2. AIP patients had significantly higher fucosylation of IgG1 and a higher sialylation ratio of IgG subclasses 1, 2 and 4. Using the CART analysis of agalactosylation and sialylation ratios in the IgG to discriminate AIP from PDAC, the diagnostic accuracy of the glycan markers was 93.8% with 94.6% sensitivity and 92.9% specificity. There were no statistically significant difference of IgG-glycosylation profiles between diffuse type and focal type AIP. CONCLUSIONS: AIP and PDAC patients have distinct IgG-glycosylation profilings. IgG-glycosylation could different PDAC from AIP with high accuracy.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Nanobubble-embedded inorganic 808 nm excited upconversion nanocomposites for tumor multiple imaging and treatment.

Contrast reagents for ultrasound imaging are widely used in clinical medical diagnosis because ultrasound resolution is limited. Contrast agents must be utilized to enhance the image resolution. At present, microbubbles (MBs) and nanobubbles (NBs) are the main contrast reagent candidates for improving the signal resolution. Fluorescence upconversion nanoparticles provide high sensitivity and also function as nanocarriers. This can label tumor cells in a specific organ under irradiation of near-infrared (NIR) light. However, despite the use of NIR light, the penetration depth of NIR is only approximately 15 mm. Thus, we combine fluorescence with ultrasonic imaging to achieve the effect of multiple imaging and solve the low penetration depth of fluorescence imaging and the poor resolution of ultrasound imaging. The dual imaging modalities achieved higher resolution or signal to noise ratios. In this study, Nd3+-sensitized upconversion nanoparticles (UCNPs) are combined with graphitic carbon nitride quantum dots (CNs) and embedded in NBs (UCNP-CN@NBs). The UCNPs are excited by 808 nm light and emit visible and ultraviolet light. Then, the energy of the ultraviolet light is transferred to the CNs to produce reactive oxygen species (ROS) for photodynamic therapy. Ultrasonic waves are also used to promote NB bursting and the release of ROS molecules in photodynamic therapy, leading to cancer cell apoptosis.

Publisher Correction: EFHD2 promotes epithelial-to-mesenchymal transition and correlates with postsurgical recurrence of stage I lung adenocarcinoma.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

ESI MS for Microsized Bioparticles.

An ESI ion trap mass spectrometer was designed for high-throughput and rapid mass analysis of large bioparticles. Mass calibration of the instrument was performed using commercially available polystyrene (PS) microparticles with a size comparable to cancer cells. Different sizes of MCF-7 breast cancer cells (8 to 15 µm) were used in this study. The masses of different cancer cells were measured. This system allows for the analysis of all types of particles.

EFHD2 promotes epithelial-to-mesenchymal transition and correlates with postsurgical recurrence of stage I lung adenocarcinoma.

Surgery is the only curative treatment for early-stage non-small cell lung cancer (NSCLC) patients. However, approximately one-third of these patients develop recurrence, which remains the main cause of mortality in the postsurgical treatment of NSCLC. Many molecular markers have been proposed to predict recurrence of early-stage disease, but no marker has demonstrated sufficient reliability for clinical application. In the present study, the novel protein EF-hand domain-containing protein D2 (EFHD2) was identified as expressed in highly metastatic tumor cells. EFHD2 increased the formation of protrusive invadopodia structures and cell migration and invasion abilities and promoted the epithelial-to-mesenchymal transition (EMT) character of lung adenocarcinoma cells. We demonstrated that the mechanism of EFHD2 in enhancing EMT occurs partly through inhibition of caveolin-1 (CAV1) for cancer progression. The expression of EFHD2 was significantly correlated with postsurgical recurrence of patients with stage I lung adenocarcinoma in the Kaplan-Meier-plotter cancer database search and our retrospective cohort study (HR, 6.14; 95% CI, 2.40-15.74; P < 0.001). Multivariate Cox regression analysis revealed that EFHD2 expression was an independent clinical predictor for this disease. We conclude that EFHD2 expression is associated with increased metastasis and EMT and could serve as an independent marker to predict postsurgical recurrence of patients with stage I lung adenocarcinoma.

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Deubiquitination and Stabilization of PD-L1 by CSN5.

Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. In the current study, we identified tumor necrosis factor alpha (TNF-α) as a major factor triggering cancer cell immunosuppression against T cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1). We demonstrated that COP9 signalosome 5 (CSN5), induced by NF-κB p65, is required for TNF-α-mediated PD-L1 stabilization in cancer cells. CSN5 inhibits the ubiquitination and degradation of PD-L1. Inhibition of CSN5 by curcumin diminished cancer cell PD-L1 expression and sensitized cancer cells to anti-CTLA4 therapy.

Interleukin-18 deteriorates Fabry cardiomyopathy and contributes to the development of left ventricular hypertrophy in Fabry patients with GLA IVS4+919 G>A mutation.

RATIONALE: A high incidence of GLA IVS4+919 G>A mutation in patients with Fabry disease of the later-onset cardiac phenotype, has been reported in Taiwan. However, suitable biomarkers or potential therapeutic surrogates for Fabry cardiomyopathy (FC) in such patients under enzyme replacement treatment (ERT) remain unknown. OBJECTIVE: Using FC patients carrying IVS4+919 G>A mutation, we constructed an induced pluripotent stem cell (iPSC)-based disease model to investigate the pathogenetic biomarkers and potential therapeutic targets in ERT-treated FC. RESULTS AND METHODS: The iPSC-differentiated cardiomyocytes derived from FC-patients (FC-iPSC-CMs) carried IVS4+919 G>A mutation recapitulating FC characteristics, including low α-galactosidase A enzyme activity, cellular hypertrophy, and massive globotriaosylceramide accumulation. Microarray analysis revealed that interleukin-18 (IL-18), a pleiotropic cytokine involved in various myocardial diseases, was the most highly upregulated marker in FC-iPSC-CMs. Meanwhile, IL-18 levels were found to be significantly elevated in the culture media of FC-iPSC-CMs and patients' sera. Notably, the serum IL-18 levels were highly paralleled with the progression of left ventricular hypertrophy in Fabry patients receiving ERT. Finally, using FC-iPSC-CMs as in vitro FC model, neutralization of IL-18 with specific antibodies combined with ERT synergistically reduced the secretion of IL-18 and the progression of cardiomyocyte hypertrophy in FC-iPSC-CMs. CONCLUSION: Our data demonstrated that cardiac IL-18 and circulating IL-18 are involved in the pathogenesis of FC and LVH. IL-18 may be a novel marker for evaluating ERT efficacy, and targeting IL-18 might be a potential adjunctive therapy combined with ERT for the treatment of advanced cardiomyopathy in FC patients with IVS4+919 G>A mutation.

Near-Infrared Light-Mediated Photodynamic Therapy Nanoplatform by the Electrostatic Assembly of Upconversion Nanoparticles with Graphitic Carbon Nitride Quantum Dots.

Photodynamic therapy (PDT) is a promising antitumor treatment that is based on photosensitizers. This therapy kills cancer cells by generating reactive oxygen species (ROS) after irradiation with specific laser wavelengths. Being a potential photosensitizer, graphitic carbon nitride (g-C3N4) quantum dots (QDs) are noncytotoxic. Although the use of g-C3N4 QDs is challenged by the limited tissue penetration of UV light, g-C3N4 QDs display excellent ultraviolet (UV) light-triggered cytotoxicity. The g-C3N4 QDs were synthesized using a solid-phase hydrothermal method. The well-distributed hydrophilic g-C3N4 can be combined with NaYF4:Yb3+/Tm3+ upconversion nanoparticles via the positive ligand poly(l-lysine) to produce the final nanocomposite, NaYF4:Yb/Tm-PLL@g-C3N4. Upconversion nanoparticles can transfer IR light into UV light and promote g-C3N4 to release blue-to-green visible light to generate different images. Moreover, g-C3N4 is a promising photosensitizer in PDT because g-C3N4 can transfer oxygen into toxic ROS. The singlet oxygen formed by g-C3N4 displays great potential for use in the treatment of cancer.

Selective killing of cancer cells by nanoparticle-assisted ultrasound.

BACKGROUND: Intense ultrasound, such as that used for tumor ablation, does not differentiate between cancerous and normal cells. A method combining ultrasound and biocompatible gold or magnetic nanoparticles (NPs) was developed under in vitro conditions using human breast and lung epithelial cells, which causes ultrasound to preferentially destroy cancerous cells. RESULTS: Co-cultures of BEAS-2B normal lung cells and A549 cancerous lung cells labeled with green and red fluorescent proteins, respectively, were treated with focused ultrasound beams with the addition of gold and magnetic nanoparticles. There were significantly more necrotic A549 cells than BEAS-2 cells when gold nanoparticles were added to the culture medium [(50.6 ± 15.1) vs. (7.4 ± 2.9) %, respectively, P < 0.01]. This selective damage to cancer cells was also observed for MDA-MB231 breast cancer cells relative to MCF-10A normal breast cells after treatment with magnetic nanoparticles. CONCLUSIONS: The data obtained for different cell lines indicate that nanoparticle-assisted ultrasound therapy (NAUT) could be an effective new tool for cancer-specific treatment and could potentially be combined with conventional methods of cancer diagnosis and therapy to further increase the overall cancer cure rate.

Analysis of Urinary Prostate-Specific Antigen Glycoforms in Samples of Prostate Cancer and Benign Prostate Hyperplasia.

Glycans of prostate-specific antigen (PSA) in prostate cancer were found to be different from that in benign disease. It is difficult to analyze heterogeneous PSA glycoforms in each individual specimen because of low protein abundance and the limitation of detection sensitivity. We developed a method for prostate cancer diagnosis based on PSA glycoforms. Specific glycoforms were screened in each clinical sample based on liquid chromatography-tandem mass spectrometry with ion accumulation. To look for potential biomarkers, normalized abundance of each glycoform in benign prostate hyperplasia (BPH) and in prostate cancer was evaluated. The PSA glycoform, Hex5HexNAc4NeuAc1dHex1, and monosialylated, sialylated, and unfucosylated glycoforms differed significantly between the prostate cancer and BPH samples. The detection sensitivity (87.5%) and specificity (60%) for prostate cancer identification are higher than those of the serum PSA marker. As low as 100 amol PSA could be detected with the ion accumulation method which has not been reported before. The improved detection specificity can help reduce unnecessary examinations.

Sciellin mediates mesenchymal-to-epithelial transition in colorectal cancer hepatic metastasis.

Hepatic metastasis is the major cause of mortality in colorectal cancer (CRC) patients. Using proteomic analysis, we found sciellin (SCEL) to be specifically expressed in hepatic metastatic CRC cell lines. SCEL knockdown increased CRC cell migration and invasion, while overexpression had the opposite effect. SCEL knockdown also caused cancer cells to form more invasive structures within 3D cultures, increased the mesenchymal marker vimentin, and attenuated the epithelial marker E-cadherin. SCEL increased WNT signaling by activating ß-catenin and its downstream target c-myc, and activated mesenchymal-to-epithelial transition (MET) through a SCEL-ß-catenin-E-cadherin axis. SCEL showed higher expression in late stage primary CRC than in its hepatic metastatic counterpart. SCEL expression is dynamically modulated by TGF-ß1 and hypoxia, revealing a plastic MET mechanism for tumor colonization. Intrahepatic injection in immunodeficient mice revealed that SCEL is necessary for metastatic CRC tumor growth in the liver. These results demonstrate that SCEL is a MET inducer dynamically regulated through the metastasis process. They suggest SCEL may be a useful therapeutic target for preventing or eliminating CRC hepatic metastasis.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors.

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. One PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with mutations in BRCA genes. BRCA1 and BRCA2 have essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. Here we show that the receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (PARP1 pTyr907 or pY907). PARP1 pY907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. The combination of c-Met and PARP1 inhibitors synergized to suppress the growth of breast cancer cells in vitro and xenograft tumor models, and we observed similar synergistic effects in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients whose tumors show high c-Met expression and who do not respond to PARP inhibition alone.