A Phase II Clinical Trial on the Combination Therapy of PHY906 Plus Capecitabine in Hepatocellular Carcinoma.

LESSONS LEARNED: A PHY906 and capecitabine combination could be effective as a salvage therapy for patients with hepatocellular carcinoma (HCC) previously treated with multiple systemic therapies. This traditional Chinese medicine formulation can work with Western cancer chemotherapeutic agents to improve clinical outcomes or alleviate side effects for patients with advanced HCC. BACKGROUND: This study aimed to evaluate efficacy and safety of capecitabine combined with a PHY906 (a pharmaceutical-grade formulation of four traditional Chinese herbs) in the treatment of advanced hepatocellular carcinoma (HCC) in Asian patients who were positive for hepatitis B virus (HBV). METHODS: This study was an open-label, phase II safety and efficacy clinical trial of PHY906 and capecitabine in patients with advanced HCC. Patients received 750 mg/m2 capecitabine b.i.d. 14 days plus 800 mg of PHY906 b.i.d. on days 1-4 and days 8-11 every 21-day cycle. The primary endpoint was 6-month survival rate, and secondary endpoints were progression-free survival, overall survival, disease control rate, and safety. RESULTS: Thirty-nine subjects completed the study with a 46.2% stable disease rate. The median progression-free survival was 1.5 months, and median overall survival (mOS) was 6 months with a 51.3% 6-month survival rate. The most common adverse events included lower hemoglobin, diarrhea, pain, abdomen (not otherwise specified), fatigue, increased aspartate aminotransferase, and bilirubin. Patients who (a) had not received previous chemotherapies or targeted therapy or (b) had lower starting alpha-fetoprotein (AFP) levels or (c) had HBV infection showed better clinical outcome. CONCLUSION: Our data showed that PHY906 increases the therapeutic index of capecitabine by enhancing its antitumor activity and reduces its toxicity profile in advanced HCC.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Host immune response to anti-cancer camptothecin conjugated cyclodextrin-based polymers.

INTRODUCTION: Efficacy and safety are critical concerns when designing drug carriers. Nanoparticles are a particular type of carrier that has gained recent attention in cancer therapeutics. METHODS: In this study, we assess the safety profile of IT-101, a nanoparticle formed by self-assembly of camptothecin (CPT) conjugated cyclodextrin-based polymers. IT-101 delivers CPT to target cancer cells in animal models of numerous human cancers and in humans. Previous data from preclinical and clinical trials indicate that IT-101 has no notable immunological side effects. However, there have been no published studies focused on evaluating the effects of IT-101 on host immune systems. RESULTS: In this work, we demonstrate that IT-101 diminished initial host immune response following first injection of the nanopharmaceutical and induced NK cell activation and T cell proliferation upon further IT-101 exposure. Additionally, IT-101 could attenuate tumor growth more efficiently than CPT treatment only. CONCLUSIONS: Drugs administration in whole-body circulation may lead to poorly bioavailable in central nervous system and often has toxic effects on peripheral tissues. Conjugated with cyclodextrin-based polymers not only reduce adverse effects but also modulate the immune responses to elevate drug efficacy. These immune responses may potentially facilitate actions of immune blockage, such as PD1/PDL1 in cancer treatment.

Leptin OB3 peptide suppresses leptin-induced signaling and progression in ovarian cancer cells.

BACKGROUND: Obesity and its comorbidities constitute a serious health burden worldwide. Leptin plays an important role in diet control; however, it has a stimulatory potential on cancer cell proliferation. The OB3 peptide, a synthetic peptide, was shown to be more active than leptin in regulating metabolism but with no mitogenic effects in cancer cells. METHODS: In this study, we investigated the proliferative effects, gene expressions and signaling pathways modulated by leptin and OB3 in human ovarian cancer cells. In addition, an animal study was performed. RESULTS: Leptin, but not OB3, induced the proliferation of ovarian cancer cells. Interestingly, OB3 blocked the leptin-induced proliferative effect when it was co-applied with leptin. Both leptin and OB3 activated the phosphatidylinositol-3-kinase (PI3K) signal transduction pathway. In addition, leptin stimulated the phosphorylation of signal transducer and activator of transcription-3 (STAT3) Tyr-705 as well as estrogen receptor (ER)α, and the expression of ERα-responsive genes. Interestingly, all leptin-induced signal activation and gene expressions were blocked by the co-incubation with OB3 and the inhibition of extracellular signal-regulated kinase (ERK)1/2. Coincidently, leptin, but not OB3, increased circulating levels of follicle-stimulating hormone (FSH) which is known to play important roles in the initiation and proliferation of ovarian cancer cells. CONCLUSIONS: In summary, our findings suggest that the OB3 peptide may prevent leptin-induced ovarian cancer initiation and progression by disrupting leptin-induced proliferative signals via STAT3 phosphorylation and ERα activation. Therefore, the OB3 peptide is a potential anticancer agent that might be employed to prevent leptin-induced cancers in obese people.

Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy.

Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments.

Genomewide copy number analysis of Müllerian adenosarcoma identified chromosomal instability in the aggressive subgroup.

Müllerian adenosarcomas are malignant gynecologic neoplasms. Advanced staging and sarcomatous overgrowth predict poor prognosis. Because the genomic landscape remains poorly understood, we conducted this study to characterize the genomewide copy number variations in adenosarcomas. Sixteen tumors, including eight with and eight without sarcomatous overgrowth, were subjected to a molecular inversion probe array analysis. Copy number variations, particularly losses, were significantly higher in cases with sarcomatous overgrowth. Frequent gains of chromosomal 12q were noted, often involving cancer-associated genes CDK4 (six cases), MDM2, CPM, YEATS4, DDIT3, GLI1 (five each), HMGA2 and STAT6 (four), without association with sarcomatous overgrowth status. The most frequent losses involved chromosomes 13q (five cases), 9p, 16q and 17q (four cases each) and were almost limited to cases with sarcomatous overgrowth. MDM2 and CDK4 amplification, as well as losses of RB1 (observed in two cases) and CDKN2A/B (one case), was verified by FISH. By immunohistochemistry, all MDM2/CDK4-coamplified cases were confirmed to overexpress both encoded proteins, whereas all four cases with (plus an additional four without) gain of HMGA2 overexpressed the HMGA2 protein. Both cases with RB1 loss were negative for the immunostaining of the encoded protein. Chromothripsis-like copy number profiles involving chromosome 12 or 14 were observed in three fatal cases, all of which harbored sarcomatous overgrowth. With whole chromosome painting and deconvolution fluorescent microscopy, dividing tumor cells in all three cases were shown to have scattered extrachromosomal materials derived from chromosomes involved by chromothripsis, suggesting that this phenomenon may serve as visual evidence for chromothripsis in paraffin tissue. In conclusion, we identified frequent chromosome 12q amplifications, including loci containing potential pharmacological targets. Global chromosomal instability and chromothripsis were more frequent in cases with sarcomatous overgrowth. To our knowledge, this is the first time that evidence of chromothripsis has been demonstrated in paraffin-embedded clinical tissues and in adenosarcomas.

Characterization of FN1-FGFR1 and novel FN1-FGF1 fusion genes in a large series of phosphaturic mesenchymal tumors.

Phosphaturic mesenchymal tumors typically cause paraneoplastic osteomalacia, chiefly as a result of FGF23 secretion. In a prior study, we identified FN1-FGFR1 fusion in 9 of 15 phosphaturic mesenchymal tumors. In this study, a total of 66 phosphaturic mesenchymal tumors and 7 tumors resembling phosphaturic mesenchymal tumor but without known phosphaturia were studied. A novel FN1-FGF1 fusion gene was identified in two cases without FN1-FGFR1 fusion by RNA sequencing and cross-validated with direct sequencing and western blot. Fluorescence in situ hybridization analyses revealed FN1-FGFR1 fusion in 16 of 39 (41%) phosphaturic mesenchymal tumors and identified an additional case with FN1-FGF1 fusion. The two fusion genes were mutually exclusive. Combined with previous data, the overall prevalence of FN1-FGFR1 and FN1-FGF1 fusions was 42% (21/50) and 6% (3/50), respectively. FGFR1 immunohistochemistry was positive in 82% (45/55) of phosphaturic mesenchymal tumors regardless of fusion status. By contrast, 121 cases of potential morphologic mimics (belonging to 13 tumor types) rarely expressed FGFR1, the main exceptions being solitary fibrous tumors (positive in 40%), chondroblastomas (40%), and giant cell tumors of bone (38%), suggesting a possible role for FGFR1 immunohistochemistry in the diagnosis of phosphaturic mesenchymal tumor. With the exception of one case reported in our prior study, none of the remaining tumors resembling phosphaturic mesenchymal tumor had either fusion type or expressed significant FGFR1. Our findings provide insight into possible mechanisms underlying the pathogenesis of phosphaturic mesenchymal tumor and imply a central role of the FGF1-FGFR1 signaling pathway. The novel FN1-FGF1 protein is expected to be secreted and serves as a ligand that binds and activates FGFR1 to achieve an autocrine loop. Further study is required to determine the functions of these fusion proteins.

Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor.

BACKGROUND: Atypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT. METHODS: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT. RESULTS: RRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells. CONCLUSIONS: Collectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT.

Bridging Size and Charge Effects of Mesoporous Silica Nanoparticles for Crossing the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a highly selective cellular barrier that tightly controls the microenvironment of the central nervous system to restrict the passage of substances, which is a primary challenge in delivering therapeutic drugs to treat brain diseases. This study aimed to develop simple surface modifications of mesoporous silica nanoparticles (MSNs) without external stimuli or receptor protein conjugation, which exhibited a critical surface charge and size allowing them to cross the BBB. A series of MSNs with various charges and two different sizes of 50 and 200 nm were synthesized, which showed a uniform mesoporous structure with various surface zeta potentials ranging from +42.3 to -51.6 mV. Confocal microscopic results showed that 50 nm of strongly negatively charged N4-RMSN50@PEG/THPMP (∼-40 mV) could be significantly observed outside blood vessels of the brain in Tg(zfli1:EGFP) transgenic zebrafish embryos superior to the other negatively charged MSNs. However, very few positively charged MSNs were found in the brain, indicating that negatively charged MSNs could successfully penetrate the BBB. The data were confirmed by high-resolution images of 3D deconvoluted confocal microscopy and two-photon microscopy and zebrafish brain tissue sections. In addition, while increasing the size to 200 nm but maintaining the similar negative charge (∼40 mV), MSNs could not be detected in the brain of zebrafish, suggesting that transport across the BBB based on MSNs occurred in charge- and size-dependent manners. No obvious cytotoxicity was observed in the CTX-TNA2 astrocyte cell line and U87-MG glioma cell line treated with MSNs. After doxorubicin (Dox) loading, N4-RMSN50@PEG/THPMP/Dox enabled drug delivery and pH-responsive release. The toxicity assay showed that N4-RMSN50@PEG/THPMP could reduce Dox release, resulting in the increase of the survival rate in zebrafish. Flow cytometry demonstrated N4-RMSN50@PEG/THPMP had few cellular uptakes. Protein corona analysis revealed three transporter proteins, such as afamin, apolipoprotein E, and basigin, could contribute to BBB penetration, validating the possible mechanism of N4-RMSN50@PEG/THPMP crossing the BBB. With this simple approach, MSNs with critical negative charge and size could overcome the BBB-limiting characteristics of therapeutic drug molecules; furthermore, their use may also cause drug sustained-release in the brain, decreasing peripheral toxicity.

Thyroid Hormone Induces Oral Cancer Growth via the PD-L1-Dependent Signaling Pathway.

Oral cancer is a fatal disease, and its incidence in Taiwan is increasing. Thyroid hormone as L-thyroxine (T4) stimulates cancer cell proliferation via a receptor on integrin αvß3 of plasma membranes. It also induces the expression of programmed death-ligand 1 (PD-L1) and cell proliferation in cancer cells. Thyroid hormone also activates ß-catenin-dependent cell proliferation in cancer cells. However, the relationship between PD-L1 and cancer proliferation is not fully understood. In the current study, we investigated the role of inducible thyroid hormone-induced PD-L1-regulated gene expression and proliferation in oral cancer cells. Thyroxine bound to integrin αvß3 to induce PD-L1 expressions via activation of ERK1/2 and signal transducer and activator of transcription 3 (STAT3). Inactivated STAT3 inhibited PD-L1 expression and nuclear PD-L1 accumulation. Inhibition of PD-L1 expression reduced ß-catenin accumulation. Furthermore, nuclear PD-L1 formed a complex with nuclear proteins such as p300. Suppression PD-L1 expression by shRNA blocked not only expression of PD-L1 and ß-catenin but also signal transduction, proliferative gene expressions, and cancer cell growth. In summary, thyroxine via integrin αvß3 activated ERK1/2 and STAT3 to stimulate the PD-L1-dependent and ß-catenin-related growth in oral cancer cells.

Heteronemin and tetrac derivatives suppress non-small cell lung cancer growth via ERK1/2 inhibition.

The most common cancer, lung cancer, causes deaths worldwide. Most lung cancer patients have non-small cell lung carcinomas (NSCLCs) with a poor prognosis. The chemotherapies frequently cause resistance therefore search for new effective drugs for NSCLC patients is an urgent and essential issue. Deaminated thyroxine, tetraiodothyroacetic acid (tetrac), and its nano-analogue (NDAT) exhibit antiproliferative properties in several types of cancers. On the other hand, the most abundant secondary metabolite in the sponge Hippospongia sp., heteronemin, shows effective cytotoxic activity against different types of cancer cells. In the current study, we investigated the anticancer effects of heteronemin against two NSCLC cell lines, A549 and H1299 cells in vitro. Combined treatment with heteronemin and tetrac derivatives synergistically inhibited cancer cell growth and significantly modulated the ERK1/2 and STAT3 pathways in A549 cells but only ERK1/2 in H1299 cells. The combination treatments induce apoptosis via the caspases pathway in A549 cells but promote cell cycle arrest via CCND1 and PCNA inhibition in H1299 cells. In summary, these results suggest that combined treatment with heteronemin and tetrac derivatives could suppress signal transduction pathways essential for NSCLC cell growth. The synergetic effects can be used potentially as a therapeutic procedure for NSCLC patients.

Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism.

Basal macroautophagy/autophagy has recently been found in anucleate platelets. Platelet autophagy is involved in platelet activation and thrombus formation. However, the mechanism underlying autophagy in anucleate platelets require further clarification. Our data revealed that LC3-II formation and SQSTM1/p62 degradation were noted in H2O2-activated human platelets, which could be blocked by 3-methyladenine and bafilomycin A1, indicating that platelet activation may cause platelet autophagy. AMPK phosphorylation and MTOR dephosphorylation were also detected, and block of AMPK activity by the AMPK inhibitor dorsomorphin reversed SQSTM1 degradation and LC3-II formation. Moreover, autophagosome formation was observed through transmission electron microscopy and deconvolution microscopy. These findings suggest that platelet autophagy was induced partly through the AMPK-MTOR pathway. In addition, increased LC3-II expression occurred only in H2O2-treated Atg5f/f platelets, but not in H2O2-treated atg5-/- platelets, suggesting that platelet autophagy occurs during platelet activation. atg5-/- platelets also exhibited a lower aggregation in response to agonists, and platelet-specific atg5-/- mice exhibited delayed thrombus formation in mesenteric microvessles and decreased mortality rate due to pulmonary thrombosis. Notably, metabolic analysis revealed that sphingolipid metabolism is involved in platelet activation, as evidenced by observed several altered metabolites, which could be reversed by dorsomorphin. Therefore, platelet autophagy and platelet activation are positively correlated, partly through the interconnected network of sphingolipid metabolism. In conclusion, this study for the first time demonstrated that AMPK-MTOR signaling could regulate platelet autophagy. A novel linkage between AMPK-MTOR and sphingolipid metabolism in anucleate platelet autophagy was also identified: platelet autophagy and platelet activation are positively correlated.Abbreviations: 3-MA: 3-methyladenine; A.C.D.: citric acid/sod. citrate/glucose; ADP: adenosine diphosphate; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy-related; B4GALT/LacCS: beta-1,4-galactosyltransferase; Baf-A1: bafilomycin A1; BECN1: beclin 1; BHT: butylate hydrooxytoluene; BSA: bovine serum albumin; DAG: diacylglycerol; ECL: enhanced chemiluminescence; EDTA: ethylenediamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GALC/GCDase: galactosylceramidase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/GluSDase: glucosylceramidase beta; GPI: glycosylphosphatidylinositol; H2O2: hydrogen peroxide; HMDB: human metabolome database; HRP: horseradish peroxidase; IF: immunofluorescence; IgG: immunoglobulin G; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPV: mean platelet volume; MTOR: mechanistic target of rapamycin kinase; ox-LDL: oxidized low-density lipoprotein; pAb: polyclonal antibody; PC: phosphatidylcholine; PCR: polymerase chain reaction; PI3K: phosphoinositide 3-kinase; PLS-DA: partial least-squares discriminant analysis; PRP: platelet-rich plasma; Q-TOF: quadrupole-time of flight; RBC: red blood cell; ROS: reactive oxygen species; RPS6KB/p70S6K: ribosomal protein S6 kinase B; SDS: sodium dodecyl sulfate; S.E.M.: standard error of the mean; SEM: scanning electron microscopy; SGMS: sphingomyelin synthase; SM: sphingomyelin; SMPD/SMase: sphingomyelin phosphodiesterase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; UGT8/CGT: UDP glycosyltransferase 8; UGCG/GCS: UDP-glucose ceramide glucosyltransferase; ULK1: unc-51 like autophagy activating kinase 1; UPLC: ultra-performance liquid chromatography; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; WBC: white blood cell; WT: wild type.

Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Synthesis and characterization of Gd-DTPA/fucoidan/peptide complex nanoparticle and in vitro magnetic resonance imaging of inflamed endothelial cells.

P-selectin overexpressed on activated endothelial cells and platelets is a new target for treatment of cancers and cardiovascular diseases such as atherosclerosis and thrombosis. In this study, depolymerized low molecular weight fucoidan (LMWF8775) and a thermolysin-hydrolyzed protamine peptide (TPP1880) were prepared. TPP1880 and LMWF8775 were able to form self-assembled complex nanoparticles (CNPs). The formation of TPP1880/LMWF8775 CNPs was characterized by Fourier-transform infrared spectra, circular dichroism spectra and isothermal titration calorimetry. The CNPs selectively targeted PMA-stimulated, inflamed endothelial cells (HUVECs) with high expression of P-selectin. Gd-DTPA MRI contrast agent was successfully loaded in the CNPs with better T1 relaxivity and selectively accumulated in the activated HUVECs with increased MRI intensity and reduced cytotoxicity as compared to free Gd-DTPA. Our results suggest that the TPP1880/LMWF8775 CNPs may have potential in future for early diagnosis of cardiovascular diseases and cancers in which the endothelium is inflamed or activated.

Author Correction: The role of sentrin-specific protease 2 substrate recognition in TGF-ß-induced tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy.

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

Alteration of mesenchymal stem cells polarity by laminar shear stimulation promoting ß-catenin nuclear localization.

Mesenchymal stem cell (MSC) is mechanosensitive and the respond to mechanical force is pattern specific. We previously reported that oscillatory shear stress at 0.5 ±â€¯4 dyne/cm2 guided MSCs polarity vertical to net flow direction before apolaric stage at 30 min resulting in phosphorylation of ß-catenin and inhibition of Wnt signaling. This time, we explored laminar shear stress (LS) at 0.5 dyne/cm2 polarized MSCs by guiding F-actin orientation parallel to the flow direction before apolarity at 30 min accompanied with activation of Wnt signaling. Time-dependent microarray analysis supported cell-cell junctional complex of MSCs was the major mechanosensor on MSCs to respond 0.5 dyne/cm2 LS. Three-dimensional immunofluorescence image confirmed LS promoting ß-catenin nuclear localization during 15 min to 1 h with a peak at 30 min. Functional analysis of proteomic study on MSC with 30 min LS stimulation indicated that upregulation of ß-catenin downstream proteins related to cardiovascular development, endothelial cell protection and angiogenesis. Conditioned medium from MSCs with 30 min LS stimulation improved the viability of human endothelial cells from oxidative damage. In conclusion, 0.5 dyne/cm2 LS on MSCs for 30 min guides MSCs lack of polarity and promotes ß-catenin nuclear translocation favoring Wnt activation and paracrine cardiovascular support.

Thyroid hormone-induced expression of inflammatory cytokines interfere with resveratrol-induced anti-proliferation of oral cancer cells.

Thyroid hormone, L-thyroxine (T4), induces inflammatory genes expressions and promotes cancer growth. It also induces expression of the checkpoint programmed death-ligand 1 (PD-L1), which plays a vital role in cancer progression. On the other hand, resveratrol inhibits inflammatory genes expressions. Moreover, resveratrol increases nuclear inducible cyclooxygenase (COX)-2 accumulation, complexes with p53, and induces p53-dependent anti-proliferation. In this study, we investigated the effect of T4 on resveratrol-induced anti-proliferation in oral cancer. T4 increased the expression and cytoplasmic accumulation of PD-L1. Increased expressions of pro-inflammatory genes, interleukin (IL)-1ß and transforming growth factor (TGF)-ß1, were shown to stimulate PD-L1 expression. T4 stimulated pro-inflammatory and proliferative genes expressions, and oral cancer cells proliferation. In contrast, resveratrol inhibited those genes and activated anti-proliferative genes. T4 retained resveratrol-induced COX-2 in cytoplasm and prevented COX-2 nuclear accumulation when resveratrol treated cancer cells. A specific signal transducer and activator of transcription 3 (STAT3) inhibitor, S31-201, blocked T4-induced inhibition and restored resveratrol-induced nuclear COX-2 accumulation. By inhibiting the T4-activated STAT3 signal transduction axis with S31-201, resveratrol was able to sequentially reestablish COX-2/p53-dependent gene expressions and anti-proliferation. These findings provide a novel understanding of the inhibitory effects of T4 on resveratrol-induced anticancer properties via the sequential expression of PD-L1 and inflammatory genes.