The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer.

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection.

New Phenolic Glycosides from Coelogyne fuscescens Lindl. var. brunnea and Their Cytotoxicity against Human Breast Cancer Cells.

The phytochemical investigation of the whole plants of Coelogyne fuscescens Lindl. var. brunnea led to the discovery of three new phenolic glycosides, i.e., coelofusides A-C (1-3) and 12 known compounds (4-15). For the first time, we reported the nuclear magnetic resonance (NMR) data of 4-O-(6'-O-glucosyl-4″-hydroxybenzoyl)-4-hydroxybenzyl alcohol (4) in this study. The identification of the structures of newly discovered compounds was done through the analysis of their spectroscopic data [NMR, mass spectrometry, ultraviolet, Fourier transform infrared, optical rotation, and circular dichroism (CD)]. In comparison to anticancer drugs (i.e., etoposide and carboplatin), we evaluated anticancer potential of the isolated compounds on two different breast cancer cell lines, namely, T47D and MDA-MB-231. Human fibroblast HaCaT cells were used as the control cells. After a 48 h incubation, flavidin (8), coelonin (10), 3,4-dihydroxybenzaldehyde (11), and oxoflavidin (12) showed significant cytotoxic effects against breast cancer cells. Among them, oxoflavidin (12) exhibited the most potent cytotoxicity on MDA-MB-231 with an IC50 value of 26.26 ± 4.33 µM. In the nuclear staining assay, oxoflavidin induced apoptosis after 48 h in both T47D and MDA-MB-231 cells in a dose-dependent manner. Furthermore, oxoflavidin upregulated the expression of apoptotic genes, such as p53, Bax, poly(ADP-ribose) polymerase, caspase-3, and caspase-9 genes while significantly decreasing antiapoptotic protein (Bcl-2) expression levels.

CpG methylation changes in Alu repetitive sequences in normal aging due to diastolic hypertension in human dermal fibroblasts from the facial area.

Aging fibroblasts, an important factor contributing to skin aging, are affected by numerous mechanisms, including alterations in DNA methylation and age-related diseases. The current study aimed to investigate the role of Alu methylation in aging fibroblasts and hypertension. The Alu methylation levels in dermal fibroblasts obtained from patients of different ages and blood pressure status were analyzed using the combined bisulfite restriction analysis technique. An inverse correlation was observed between Alu methylation in dermal fibroblasts and patient age. Dermal fibroblasts from the high-normal diastolic blood pressure group had higher Alu methylation levels compared with those from the normal group. The findings of the present study suggest that Alu methylation alterations can be observed with chronological aging and hypertension, and are a potential aging marker or therapeutic target.

Immune-associated plasma proteins in oral and oropharyngeal cancer patients.

Background: Plasma protein patterns differ between cancer patients and healthy donors. This study aimed to examine the plasma levels of several cytokines and immunological checkpoint proteins between patients with oral and oropharyngeal cancer and healthy donors. Materials and methods: Plasma samples from healthy donors, oral cancer patients, and oropharyngeal cancer patients were analyzed using the Human Th Cytokine Panel 13-plex (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, and TNF-α) and Human Immune Checkpoint Panel1 12-plex [sCD25 (IL-2Ra), 4-1BB, sCD27, B7.2 (CD86), Free Active TGF-ß1, CTLA-4, PD-L1, PD-L2, PD-1, Tim-3, LAG-3, and Galectin-9]. The plasma 4-1BB levels were verified by Western blot method. In addition, the study of the receive operating curve (ROC) yielded the calculation of a number of diagnostically significant indicators. Results: Significantly increased levels of IL-6, 4-1BB, PDL-1, PD-1, and CTLA-4 and decreased levels of IL-13 and sCD27 were observed in cancer patients compared with healthy donors. These levels were highly significant, particularly for cancer patients in stage IV. Validation by Western blot revealed that cancer patients had higher plasma levels of 4-1BB than healthy donors (p < 0.05), and ROC curve analysis revealed that plasma 4-1BB had the highest cancer detection capability. Intriguingly, plasma levels of 4-1BB were significantly positively correlated with PDL-1 and PD-1 levels (p < 0.0001). Conclusion: This data provided descriptive knowledge of oral and oropharyngeal cancer immunity at a fundamental level. Additional research should concentrate on the significantly different factors, especially 4-1BB, PDL-1, and PD-1, which may contribute to the development of novel alternative diagnostic tools or therapies for patients with oral and oropharyngeal cancer.

The roles of HMGB1-produced DNA gaps in DNA protection and aging biomarker reversal.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps "Youth-DNA-GAPs." The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high-mobility group box (HMGB) or the NAD-dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth-DNA-GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth-DNA-GAPs, Box A-produced DNA gaps avoided DNA double-strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth-DNA-GAPs are a biomarker determining the DNA aging stage (young/old). Box A-produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging-associated diseases.

Targeting multiple genes containing long mononucleotide A-T repeats in lung cancer stem cells.

BACKGROUND: Intratumour heterogeneous gene expression among cancer and cancer stem cells (CSCs) can cause failure of current targeted therapies because each drug aims to target the function of a single gene. Long mononucleotide A-T repeats are cis-regulatory transcriptional elements that control many genes, increasing the expression of numerous genes in various cancers, including lung cancer. Therefore, targeting A-T repeats may dysregulate many genes driving cancer development. Here, we tested a peptide nucleic acid (PNA) oligo containing a long A-repeat sequence [A(15)] to disrupt the transcriptional control of the A-T repeat in lung cancer and CSCs. METHODS: First, we separated CSCs from parental lung cancer cell lines. Then, we evaluated the role of A-T repeat gene regulation by counting the number of repeats in differentially regulated genes between CSCs and the parental cells of the CSCs. After testing the dosage and effect of PNA-A15 on normal and cancer cell toxicity and CSC phenotypes, we analysed genome-wide expression to identify dysregulated genes in CSCs. RESULTS: The number of A-T repeats in genes differentially regulated between CSCs and parental cells differed. PNA-A15 was toxic to lung cancer cells and CSCs but not to noncancer cells. Finally, PNA-A15 dysregulated a number of genes in lung CSCs. CONCLUSION: PNA-A15 is a promising novel targeted therapy agent that targets the transcriptional control activity of multiple genes in lung CSCs.

Lusianthridin targeting of lung cancer stem cells via Src-STAT3 suppression.

BACKGROUND: Cancer stem cells (CSCs) are well-recognized as a majority cause of treatment failure and can give rise to relapse. The discovery of compounds attenuating CSCs' properties is crucial for enabling advances in novel therapeutics to limit recurrence. CSCs' features in lung cancer are regulated through a reduction in Src-STAT3-c-Myc, which drives cancer progression, drug resistance, and metastasis. METHODS: The effect of lusianthridin suppresses CSC-like phenotypes was determined by 3D culture and anchorage independent growth. The expression of CSC markers and associated proteins were determined by Western blot analyses. Protein ubiquitination and degradation were assessed using immunoprecipitation. RESULTS: Herein, we report that lusianthridin, a pure compound from Dendrobium venustum, dramatically suppressed CSCs in lung cancer cells as verified by several CSC phenotype assessments and CSC markers. The CSC phenotypes in lusianthridin-treated cells were suppressed through downregulation of Src-STAT3-c-Myc pathways. Ectopic Src introduced by the transfection augmented CSC phenotypes in lung cancer cells through STAT3 (increased active p-STAT3Tyr705) and c-Myc signals, while the ShRNA-Src transfection or Src inhibitor dasatinib exhibited opposite results. Treatment of the Src-overexpressing cells with lusianthridin resulted in the reversal of active STAT3 (p-STAT3Tyr705) and c-Myc as well as the CSC marker CD133. Importantly, we confirmed the CSC-targeted activity of lusianthridin in CSC-rich primary lung cancer cells. The compound dramatically inhibited the formation of tumor spheres of primary lung cancer cells. Finally, we demonstrated that after CSC-attenuation by lusianthridin, the lung cancer cells exhibited significantly higher susceptibility to chemotherapeutic drugs. Such a sensitizing effect caused by pro-survival suppression and pro-apoptotic induction together with the abolishment of stemness indicated by the decrease in CSC markers CD133, ABCG2, and ALDH1A1. CONCLUSION: These findings revealed a novel pharmacological action and the underlying mechanism of lusianthridin in negatively regulating CSC-like phenotypes and sensitizing resistant cancer cells to cemetery.

Targeting high transcriptional control activity of long mononucleotide A-T repeats in cancer by Argonaute 1.

Epigenetic regulatory changes alter the gene regulation function of DNA repeat elements in cancer and consequently promote malignant phenotypes. Some short tandem repeat sequences, distributed throughout the human genome, can play a role as cis-regulatory elements of the genes. Distributions of tandem long (≥10) and short (<10) A-T repeats in the genome are different depending on gene functions. Long repeats are more commonly found in housekeeping genes and may regulate genes in harmonious fashion. Mononucleotide A-repeats around transcription start sites interact with Argonaute proteins (AGOs) to regulate gene expression. miRNA-bound AGO alterations in cancer have been reported; consequently, these changes would affect genes containing mononucleotide A- and T-repeats. Here, we showed an unprecedented hallmark of gene regulation in cancer. We evaluated the gene expression profiles reported in the Gene Expression Omnibus and found a high density of 13-27 A-T repeats in the up-regulated genes in malignancies derived from the bladder, cervix, head and neck, ovary, vulva, breast, colon, liver, lung, prostate, kidney, thyroid, adrenal gland, bone, blood cells, muscle and brain. Transfection of cell-penetrating protein tag AGO1 containing poly uracils (CPP-AGO1-polyUs) to the lung cancer cell lines altered gene regulation depending on the presence of long A-T repeats. CPP-AGO1-polyUs limited cell proliferation and the ability of a cancer cell to grow into a colony in lung cancer cell lines. In conclusion, long A-T repeats up-regulated many genes in cancer that can be targeted by AGO1 to change the expression of many genes and limited cancer growth.

Chrysotobibenzyl inhibition of lung cancer cell migration through Caveolin-1-dependent mediation of the integrin switch and the sensitization of lung cancer cells to cisplatin-mediated apoptosis.

BACKGROUND: A Lung cancer death account for approximately 1 in 5 of all cancer-related deaths and is particularly virulent due to its enhanced metastasis and resistance to chemotherapy. Chrysotobibenzyl has been reported to decrease cell metastasis, according to the results of an anchorage-independent growth assay; however, its underlying mechanism has not been investigated yet. PURPOSE: The aim of this study was to investigate the effect of chrysotobibenzyl on lung cancer cell migration and drug sensitization and its mechanism. METHODS: Cell viability, cell proliferation and drug sensitization were determined by MTT assay. Cell migration was analyzed using a wound-healing assay. Transwell migration and invasion were analyzed using Boyden chamber assay. Mechanisms of chrysotobibenzyl against metastasis including cell migration, invasion, and epithelial to mesenchymal transition (EMT) were evaluated by Western blot analysis and immunofluorescence. RESULTS: Treatment with chrysotobibenzyl was applied at concentrations of 0-50 µM and the results showed non-cytotoxicity in human lung cancer cells (H460, H292, A549, and H23) and other non-cancerous human cells (HCT116, primary DP1 and primary DP2). However, 50 µM of chrysotobibenzyl significantly altered cell proliferation in H292 cells at 48 h. In addition, 1-50 µM of chrysotobibenzyl significantly inhibited H460 and H292 cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 48 h, which were correlated with reduced protein levels of integrins ß1, ß3, and αν, p-FAK, p-AKT, Cdc42, and Cav-1. We also established shRNA-Cav-1-transfected (shCav-1) H460 and H292 cells. shCav-1 transfected cells can decrease cell migration and downregulate the expression of integrins ß1, ß3, and αν when compared with the control. Moreover, chrysotobibenzyl was shown to suppress EMT indicated by the reduction of EMT markers (Vimentin, Snail, and Slug), and sensitize lung cancer cells to cisplatin-mediated apoptosis. CONCLUSION: Treatment with chrysotobibenzyl inhibited lung cancer cell migration via Cav-1, integrins ß1, ß3, and αν, and EMT suppressions. The downregulation of integrins in response to the compound not only inhibited cell metastasis, but also sensitized lung cancer cells to cisplatin-mediated apoptosis.

Ti0.8O2 Nanosheets Inhibit Lung Cancer Stem Cells by Inducing Production of Superoxide Anion.

Recent research into the cancer stem cell (CSC) concept has driven progress in the understanding of cancer biology and has revealed promising CSC-specific targets for drug discovery efforts. As malignancies of lung cancer have been shown to be strongly associated with activities of CSCs, we examined the effects of Ti0.8O2 nanosheets on these cells. Here we show that the nanosheets target lung CSCs but not normal primary dermal papilla (DP) stem cells. Whereas Ti0.8O2 caused a dramatic apoptosis along with a decrease in CSC phenotypes, in primary human DP cells such effects of nanosheets have been minimal. Nanosheets reduced the ability of lung cancer cells to generate three-dimensional tumor spheroids, lung CSC markers (CD133 and ALDH1A1), and CSC transcription factors (Nanog and Oct-4). Ti0.8O2 nanosheets reduced CSC signaling through mechanisms involving suppression of protein kinase B (AKT) and Notch-1 pathways. In addition, the nanosheets inhibited the migration and invasive activities of lung cancer cells and reduced epithelial-to-mesenchymal transition (EMT) markers as N-cadherin, vimentin, and Slug, as well as metastasis-related integrins (integrin-αv and integrin-ß1). Importantly, we found that the selectivity of the Ti0.8O2 nanosheets in targeting cancer cells was mediated by induction of cellular superoxide anion in cancerous but not normal cells. Inhibition of nanosheet-induced superoxide anion restored the suppression of CSC and EMT in cancer cells. These findings demonstrate a promising distinctive effect of Ti0.8O2 nanosheets on lung CSC that may lead to opportunities to use such a nanomaterial in cancer therapy.

Nitric oxide promotes cancer cell dedifferentiation by disrupting an Oct4:caveolin-1 complex: A new regulatory mechanism for cancer stem cell formation.

Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.

Cancer Stem Cell-Suppressing Activity of Chrysotoxine, a Bibenzyl from Dendrobium pulchellum.

Cancer stem cells (CSCs) have been recognized as rare populations driving cancer progression, metastasis, and drug resistance in leading cancers. Attempts have been made toward identifying compounds that specifically target these CSCs. Therefore, investigations of novel therapeutic strategies for CSC targeting are required. The cytotoxic effects of chrysotoxine on human non-small cell lung cancer-derived H460 and H23 cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effects of chrysotoxine suppression of CSC-like phenotypes were determined in CSC-rich populations and primary CSCs in three-dimensional (3D) culture and in an extreme limiting dilution assay. Expression of CSC markers and associated proteins was determined by Western blot analyse and flow cytometry. We have reported herein the CSC-suppressing activity of chrysotoxine, a bibenzyl compound isolated from Dendrobium pulchellum We have shown, to our knowledge for the first time, that chrysotoxine dramatically suppresses CSC-like phenotypes of H460 and H23 cells. Treatment with chrysotoxine significantly reduced the viability of 3D CSC-rich populations and concomitantly decreased known CSC markers. Chrysotoxine suppressed CSC phenotypes through downregulation of Src/protein kinase B (Akt) signaling. Active (phosphorylated Y416) Src was shown to regulate cancer stemness, since ectopic overexpression of Src strongly activated Akt and subsequently enhanced pluripotency transcription factor SRY (sex-determining region Y)-box 2 (Sox2)- mediating CSC phenotypes, whereas the short hairpin RNA of Src and an Src inhibitor (dasatinib) suppressed Akt, Sox2, and CSC properties. Importantly, chrysotoxine was shown to suppress active Src/Akt signaling and in turn depleted Sox2-mediated CSCs. Our findings indicate a novel CSC-targeted role of chrysotoxine and its regulation by Src/Akt and Sox2, which may be exploited for cancer treatment.

Gigantol Suppresses Cancer Stem Cell-Like Phenotypes in Lung Cancer Cells.

As cancer stem cells (CSCs) contribute to malignancy, metastasis, and relapse of cancers, potential of compound in inhibition of CSCs has garnered most attention in the cancer research as well as drug development fields recently. Herein, we have demonstrated for the first time that gigantol, a pure compound isolated from Dendrobium draconis, dramatically suppressed stem-like phenotypes of human lung cancer cells. Gigantol at nontoxic concentrations significantly reduced anchorage-independent growth and survival of the cancer cells. Importantly, gigantol significantly reduced the ability of the cancer cells to form tumor spheroids, a critical hallmark of CSCs. Concomitantly, the treatment of the compound was shown to reduce well-known lung CSCs markers, including CD133 and ALDH1A1. Moreover, we revealed that gigantol decreased stemness in the cancer cells by suppressing the activation of protein kinase B (Akt) signal which in turn decreased the cellular levels of pluripotency and self-renewal factors Oct4 and Nanog. In conclusion, gigantol possesses CSCs suppressing activity which may facilitate the development of this compound for therapeutic approaches by targeting CSCs.

A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3ß protein-dependent activation of the Wnt/ß-catenin signaling pathway.

Estrogen promotes growth in many tissues by activating Wnt/ß-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/ß-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced ß-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of ß-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of ß-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3ß. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/ß-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3ß-dependent activation of ß-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.