Force degradation study on aligner plates immersed in various solutions.

Background/purpose: The strength of aligners themselves has a high decay rate and is susceptible to accelerated degradation in the environment. The purpose of this study was to compare three types of invisible aligner films after being immersed in coffee, tea, cola, and red wine for seven days and to evaluate the changes in their strengths. Materials and methods: Three types of invisible aligner plates with a thickness of 0.75 mm, i.e., Duran T (polyethylene terephthalate glycol, PETG), Biolon (polyethylene terephthalate, PET), and Zendura FLX (polyurethane, PU), were soaked in artificial saliva and four drinks (coffee, tea, cola, red wine) for 1, 4, and 7 days. The strength test was performed by using the three-point bending test method. The residual strength ratio for the same type of invisible correction film at the same time was separately recorded. The independent t-test was used to indicate significant differences at P < 0.05. Results: The Biolon invisible correction film soaked in cola, red wine and artificial saliva showed significant differences on the 1st and 4th days (P < 0.05). The Duran T invisible correction film soaked in coffee and artificial saliva showed significant differences on the first day (P < 0.05). The Zendura FLX invisible correction film had a waterproof layer on the surface, and there was no significant difference between soaking in any drink and soaking in saliva (P > 0.05). Conclusion: Invisible correction films with different ingredients soaked in solutions show a strength decay phenomenon, except for those with TPU ingredients.

An in vitro evaluation of aligner force decay in artificial saliva.

Background/purpose: The present study aimed to compare the force decay of invisible aligners for maxillary anterior teeth with 0.1 mm (D1), 0.2 mm (D2), and 0.3 mm (D3) labial movement within a simulated oral environment over 7 days. Materials and methods: The prepared invisible aligners were immersed in saliva (S) and subjected to applied force (F) for 7 days. The aligners were set and placed on the maxillary right central incisor with 0.1 mm (D1), 0.2 mm (D2), and 0.3 mm (D3) labial movement. Thin-film pressure sensors were used to measure the aligner force changes. The data were collected and analyzed by statistical methods. Results: Significant differences were observed in the initial and first-day force between the D2 and D3 groups under simulated oral environment force (SF) (P < 0.05). There was a significant difference in force decay between Day 1 and Day 7 for all groups (P < 0.05). The SFD1 group showed a significant decrease in force on Day 5 (P < 0.05), while the SFD2 and SFD3 groups showed significant force decay on Day 4 (P < 0.05). The force decay ratio on Day 7 was higher in the SFD3 group than in the SFD1 and SFD2 groups, but no significant difference was observed. Conclusion: Larger labial movement of the aligners resulted in higher force decay under artificial saliva environments, and the force decay of invisible aligners was increased by immersion time in artificial saliva.

Collagen-binding peptides for the enhanced imaging, lubrication and regeneration of osteoarthritic articular cartilage.

Treatments for osteoarthritis would benefit from the enhanced visualization of injured articular cartilage and from the targeted delivery of disease-modifying drugs to it. Here, by using ex vivo human osteoarthritic cartilage and live rats and minipigs with induced osteoarthritis, we report the application of collagen-binding peptides, identified via phage display, that are home to osteoarthritic cartilage and that can be detected via magnetic resonance imaging when conjugated with a superparamagnetic iron oxide. Compared with the use of peptides with a scrambled sequence, hyaluronic acid conjugated with the collagen-binding peptides displayed enhanced retention in osteoarthritic cartilage and better lubricated human osteoarthritic tissue ex vivo. Mesenchymal stromal cells encapsulated in the modified hyaluronic acid and injected intra-articularly in rats showed enhanced homing to osteoarthritic tissue and improved its regeneration. Molecular docking revealed WXPXW as the consensus motif that binds to the α1 chain of collagen type XII. Peptides that specifically bind to osteoarthritic tissue may aid the diagnosis and treatment of osteoarthritic joints.

Paracrine Senescence of Mesenchymal Stromal Cells Involves Inflammatory Cytokines and the NF-κB Pathway.

It has been known that senescence-associated secretory phenotype (SASP) triggers senescence of the surrounding normal cells. However, SASP signaling regarding mesenchymal stromal cell aging remains to be fully elucidated. Therefore, the present study aimed to clarify the molecular mechanism of late (passage) MSC-induced paracrine SASP-mediated senescence of early (passage) MSCs during ex vivo expansion. Here, we conducted an extensive characterization of senescence features in bone-marrow (BM)-derived MSCs from healthy human donors. Late MSCs displayed an enlarged senescent-like morphology, induced SASP-related proinflammatory cytokines (IL-1α and IL-8), and reduced clonogenic capacity and osteogenic differentiation when compared to early MSCs. Of note, paracrine effects of SASP-related IL-1α and IL-8 from late MSCs induced cellular senescence of early MSCs via an NF-κB-dependent manner. Moreover, cellular senescence of early MSCs was promoted by the synergistic action of IL-1α and IL-8. However, inhibition of NF-κB by shRNA transfection or using inhibitors in early MSCs blocked early MSCs cellular senescence caused by paracrine SASP of late MSCs. In conclusion, these findings reveal that late MSCs display features of senescence and that, during ex vivo expansion, SASP-related proinflammatory cytokines contribute to activate a cellular senescence program in early MSCs that may ultimately impair their functionality.

Snail induces dormancy in disseminated luminal type A breast cancer through Src inhibition.

Breast cancer includes biologically distinct subtypes, and the time between rise in distant metastases and overall survival for the subtypes are different. The mechanisms involved in these differences in tumor metastasis remain to be elucidated. Here, we demonstrated that, luminal type A breast cancer cells, such as MCF7 and T47D, when overexpressed with active mutant form of Snail (6SA-Snail) increased in the expression of EMT markers such as Vimentin, N-cadherin and Fibronectin but decreased in the expression of E-cadherin, compared to control vectors or wild type Snail. Moreover, this mutant increased in migration and invasion ability, while decreased in the capacity to survive and form spheres in tumor spheroid medium. Luciferase reporter assay and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) analysis revealed that Snail downregulated Src by binding to the E-box of Src promoter. Human luminal type A breast cancer specimens showed an inverse correlation between Vimentin and Src expression. Most importantly, downregulation of Src by Snail was not found in breast cancer cell types other than luminal type A. Therefore, elucidation of the differences in signaling pathways involved in controlling migration, invasion and colonization may have a therapeutically beneficial effect on breast cancer treatment.

Mesenchymal Stem Cells From a Hypoxic Culture Can Improve Rotator Cuff Tear Repair.

A rotator cuff tear is an age-related common cause of pain and disability. Studies including our previously published ones have demonstrated that mesenchymal stem cells cultured under hypoxic conditions [hypoxic multipotent stromal cells (MSCs)] facilitate the retention of transplanted cells and promote wound healing. However, there are very few, if any, reports targeting the punctured supraspinatus tendons to create more or equally serous wounds as age-related tears of rotator cuff. It remains to be determined whether transplantation of bone-marrow-derived hypoxic MSCs into the punctured supraspinatus tendon improves tendon repair and, when combined with ultrasound-guided delivery, could be used for future clinical applications. In this study, we used a total of 33 Sprague-Dawley rats in different groups for normal no-punched control, hypoxic MSC treatment, nontreated vehicle control, and MSC preparation, and then evaluated treatment outcomes by biomechanical testing and histological analysis. We found that the ultimate failure load of the hypoxic MSC-treated group was close to that of the normal tendon and significantly greater than that of the nontreated vehicle control group. In vivo tracking of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles revealed an enhanced retention of transplanted cells at the tear site. Our study demonstrates that hypoxic MSCs improve rotator cuff tear repair in a rat model.

Protein phosphatase 2A inactivation induces microsatellite instability, neoantigen production and immune response.

Microsatellite-instable (MSI), a predictive biomarker for immune checkpoint blockade (ICB) response, is caused by mismatch repair deficiency (MMRd) that occurs through genetic or epigenetic silencing of MMR genes. Here, we report a mechanism of MMRd and demonstrate that protein phosphatase 2A (PP2A) deletion or inactivation converts cold microsatellite-stable (MSS) into MSI tumours through two orthogonal pathways: (i) by increasing retinoblastoma protein phosphorylation that leads to E2F and DNMT3A/3B expression with subsequent DNA methylation, and (ii) by increasing histone deacetylase (HDAC)2 phosphorylation that subsequently decreases H3K9ac levels and histone acetylation, which induces epigenetic silencing of MLH1. In mouse models of MSS and MSI colorectal cancers, triple-negative breast cancer and pancreatic cancer, PP2A inhibition triggers neoantigen production, cytotoxic T cell infiltration and ICB sensitization. Human cancer cell lines and tissue array effectively confirm these signaling pathways. These data indicate the dual involvement of PP2A inactivation in silencing MLH1 and inducing MSI.

Disruption of Cytosolic Folate Integrity Aggravates Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and Modulates Metastatic Properties in Non-Small-Cell Lung Cancer Cells.

Patients with advanced-stage non-small-cell lung cancer (NSCLC) are susceptible to malnutrition and develop folate deficiency (FD). We previously found that folate deprivation induces drug resistance in hepatocellular carcinoma; here, we assessed whether disrupted cytoplasmic folate metabolism could mimic FD-induced metastasis and affect the sensitivity of NSCLC cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We examined whether cytosolic folate metabolism in NSCLC cells was disrupted by FD or the folate metabolism blocker pemetrexed for 1-4 weeks. Our results revealed an increase in NF-κB overexpression-mediated epithelial-mesenchymal transition biomarkers: N-cadherin, vimentin, matrix metalloproteinases (MMPs), SOX9, and SLUG. This finding suggests that the disruption of folate metabolism can drastically enhance the metastatic properties of NSCLC cells. Cytosolic FD also affected EGFR-TKI cytotoxicity toward NSCLC cells. Because SLUG and N-cadherin are resistance effectors against gefitinib, the effects of SLUG knockdown in folate antagonist-treated CL1-0 cells were evaluated. SLUG knockdown prevented SLUG/NF-κB/SOX9-mediated invasiveness and erlotinib resistance acquisition and significantly reduced pemetrexed-induced gelatinase activity and MMP gene expression. To summarize, our data reveal two unprecedented adverse effects of folate metabolism disruption in NSCLC cells. Thus, the folic acid status of patients with NSCLC under treatment can considerably influence their prognosis.

Combination of Mesenchymal Stem Cell-Delivered Oncolytic Virus with Prodrug Activation Increases Efficacy and Safety of Colorectal Cancer Therapy.

Although oncolytic viruses are currently being evaluated for cancer treatment in clinical trials, systemic administration is hindered by many factors that prevent them from reaching the tumor cells. When administered systemically, mesenchymal stem cells (MSCs) target tumors, and therefore constitute good cell carriers for oncolytic viruses. MSCs were primed with trichostatin A under hypoxia, which upregulated the expression of CXCR4, a chemokine receptor involved in tumor tropism, and coxsackievirus and adenovirus receptor that plays an important role in adenoviral infection. After priming, MSCs were loaded with conditionally replicative adenovirus that exhibits limited proliferation in cells with a functional p53 pathway and encodes Escherichia coli nitroreductase (NTR) enzymes (CRAdNTR) for targeting tumor cells. Primed MSCs increased tumor tropism and susceptibility to adenoviral infection, and successfully protected CRAdNTR from neutralization by anti-adenovirus antibodies both in vitro and in vivo, and specifically targeted p53-deficient colorectal tumors when infused intravenously. Analyses of deproteinized tissues by UPLC-MS/QTOF revealed that these MSCs converted the co-administered prodrug CB1954 into cytotoxic metabolites, such as 4-hydroxylamine and 2-amine, inducing oncolysis and tumor growth inhibition without being toxic for the host vital organs. This study shows that the combination of oncolytic viruses delivered by MSCs with the activation of prodrugs is a new cancer treatment strategy that provides a new approach for the development of oncolytic viral therapy for various cancers.

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

AIMS: This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ­glutamylcysteine synthetase heavy chain (γ­GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. MAIN METHODS: Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ­GCSh activity were measured by HPLC methods. γ­GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. KEY FINDINGS: The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ­GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ­GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. SIGNIFICANCE: Our data unequivocally demonstrate that γ­GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells.

Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

Novel roles of folic acid as redox regulator: Modulation of reactive oxygen species sinker protein expression and maintenance of mitochondrial redox homeostasis on hepatocellular carcinoma.

We provide herein several lines of evidence to substantiate that folic acid (or folate) is a micronutrient capable of functioning as a novel redox regulator on hepatocellular carcinoma. First, we uncovered that folate deficiency could profoundly downregulate two prominent anti-apoptotic effectors including survivin and glucose-regulated protein-78. Silencing of either survivin or glucose-regulated protein-78 via small interfering RNA interfering technique established that both effectors could serve as reactive oxygen species sinker proteins. Second, folate deficiency-triggered oxidative-nitrosative stress could strongly induce endoplasmic reticulum stress that in turn could provoke cellular glutathione depletion through the modulation of the following two crucial events: (1) folate deficiency could strongly inhibit Bcl-2 expression leading to severe suppression of the mitochondrial glutathione pool and (2) folate deficiency could also profoundly inhibit two key enzymes that governing cellular glutathione redox regulation including γ-glutamylcysteinyl synthetase heavy chain, a catalytic enzyme for glutathione biosynthesis, and mitochondrial isocitrate dehydrogenase 2, an enzyme responsible for providing nicotinamide adenine dinucleotide phosphate necessary for regenerating oxidized glutathione disulfide back to glutathione via mitochondrial glutathione reductase. Collectively, we add to the literature new data to strengthen the notion that folate is an essential micronutrient that confers a novel role to combat reactive oxygen species insults and thus serves as a redox regulator via upregulating reactive oxygen species sinker proteins and averting mitochondrial glutathione depletion through proper maintenance of redox homeostasis via positively regulating glutathione biosynthesis, glutathione transporting system, and mitochondrial glutathione recycling process.

Cisplatin-induced regulation of signal transduction pathways and transcription factors in p53-mutated subclone variants of hepatoma cells: Potential application for therapeutic targeting.

Cisplatin is commonly recognized as a DNA-damaging drug; however, its versatile antitumor effects have been demonstrated to extend beyond this narrow functional attribute. The present study determined how cisplatin regulates alternative pathways and transcription factors to exert its additional antitumor actions. Cisplatin was observed to be able to trigger an endoplasmic reticulum stress response through aggravated nitrosative stress coupled to perturbed mitochondrial calcium (Ca2+) homeostasis, which substantially downregulated glucose-regulated protein (GRP) 78 expression by suppressing the cleavage of activating transcription factor (ATF) 6α (90 kDa) to its active 50 kDa subunit. Concomitantly, the ATF4-ATF3-C/emopamil binding protein homologous protein axis was activated by cisplatin, which triggered cellular glutathione (GSH) depletion by strongly inhibiting γ-glutamylcysteine synthetase heavy chain (γ-GCSh), a key enzyme in GSH biosynthesis. The present study also demonstrated that cisplatin substantially inhibited ß-catenin, causing a marked downregulation of survivin and B-cell lymphoma (Bcl)-2. Taken together, the present results uncovered a novel mechanism of cisplatin that could simultaneously trigger the inhibition of three prominent antiapoptotic effector molecules (Bcl-2, survivin and GRP78) and effectively promote GSH depletion by inhibiting γ-GCSh. These newly discovered functional attributes of cisplatin can provide an avenue for novel combined therapeutic strategies to kill hepatocellular carcinoma cells effectively.

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

A Novel Microtubule-Disrupting Agent Induces Endoplasmic Reticular Stress-Mediated Cell Death in Human Hepatocellular Carcinoma Cells.

Here, we present evidence of a novel microtubule-disrupting agent, N-deacetyl-N-(chromone-2-carbonyl)-thiocolchicine (TCD), exhibiting potent antitumor activity (with IC50 values in the nanomolar range) against hepatocellular carcinoma cell lines. Cell cycle analysis revealed that TCD induced G2/M cell-cycle arrest in a dose- and time-dependent manner in both Hep-J5 and Mahlavu HCC cell lines. TCD also induced a decrease in mitochondrial membrane potential (ΔΨm) and caused DNA damage. Mechanistically, TCD activated protein kinase RNA-like endoplasmic reticular kinase and several transcription factors, including activating transcription factor (ATF) 6, ATF4, ATF3, and the CCAAT-enhancer binding protein homologous protein. These data clearly demonstrate that the antitumor activity of TCD is mechanistically linked to its capacity to trigger both intrinsic and extrinsic apoptotic cell death via endoplasmic reticular stress pathway. The potent antitumor activity of TCD was similarly demonstrated in a hepatocellular carcinoma xenograft model, where 5 and 10 mg/kg doses of TCD significantly arrested Hep-J5 and Mahlavu tumor growth. Our finding suggests that TCD is a promising therapeutic agent against hepatocellular carcinoma; further translational assessment of its clinical usage is warranted.

Folate deficiency-triggered redox pathways confer drug resistance in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) are prone to folate deficiency (FD). Here we showed that, in cell line-specific manner, FD caused resistance to FD-induced oxidative stress and multi-drug resistance (MDR). This resistance was due to upregulation of glucose-regulated protein 78 (GRP78) and Survivin. Using siRNA and Epigallocatechin gallate (EGCG), we found that GRP78 and Survivin cooperatively conferred MDR by decreasing FD-induced ROS generation. Our data showed that FD increases GRP78 and Survivin, which serve as ROS inhibitors, causing MDR in HCC. We suggest that folate supplementation may enhance the efficacy of chemotherapy.

Paclitaxel pretreatment overcomes hypoxia inducible factor-1α-induced radioresistance acquisition of human hepatoma and lung adenocarcinoma cells.

AIMS: This study delineated the mechanisms of paclitaxel (PTX) assistance in overcoming radioresistance in hepatoma and human lung adenocarcinoma (HLAC) cells. MAIN METHODS: The TUNEL assay was used as an index of radiosensitivity, and the MTT assay assessed the efficacy of various combined PTX/RT treatments. The efficacy of PTX disruptions of hypoxia-inducible factor-1 alpha (HIF-1α) was assessed using Western blotting. KEY FINDINGS: Normoxically overexpressed HIF-1α in hepatoma J5 cells was mechanistically linked to activation of the bFGF/PI3K/Akt pathway because the viability of these cells was strongly inhibited by either Akt inhibitors or an HIF-1α inhibitor. All of the cell lines used were extremely sensitive to PTX, and these effects also correlated excellently with HIF-1α suppression. We designed five combined radiation-PTX protocols of varying dose duration and treatment sequences against CL1-1 cells based on the gathered data. Pretreatment of CL1-1 cells with PTX (100nM) for 24h before irradiation (2.5Gy) was the best combined protocol to achieve maximum radiosensitizing effects. SIGNIFICANCE: Our data clearly indicate that PTX pretreatment is an effective radiosensitizing procedure against HIF-1α-expressing hepatoma and HLAC cells, which are constitutively endowed with radioresistance.