Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker.

Exosomes are extracellular vesicles derived from the endosomal compartment that are potentially involved in intercellular communication. Here, we found that frequently used biomarkers of exosomes are heterogeneous, and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins, we used an unbiased and quantitative proteomic approach based on super-stable isotope labeling with amino acids in cell culture (super-SILAC), coupled to high-resolution mass spectrometry. In total, 1,212 proteins were quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. A cohort of 22 proteins was universally enriched. Fifteen proteins were consistently depleted in the proteome of exosomes compared to cells. Among the enriched proteins, we identified biogenesis-related proteins, GTPases and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified syntenin-1 as a consistently abundant protein in exosomes from different cellular origins. Syntenin-1 is also present in exosomes across different species and biofluids, highlighting its potential use as a putative universal biomarker of exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community.

Acute Kidney Injury Instigates Malignant Renal Cell Carcinoma via CXCR2 in Mice with Inactivated Trp53 and Pten in Proximal Tubular Kidney Epithelial Cells.

Renal cell carcinoma (RCC) is one of the most common urologic malignancies with the highest mortality rates worldwide. However, relevant mouse models that recapitulated the genetic alterations found in RCC have been lacking. In this study, we crossed Trp53 and Pten conditional knockout mice with Ggt1-Cre mice to generate a Ggt1-Cre; Trp53LoxP/LoxP ; PtenLoxP/LoxP ; YFPLoxP/LoxP (GPPY) mouse model, which resulted in the formation of dysplastic lesions involving kidney tubular epithelial cells (TEC), with only approximately 25% of mice developing RCC at an advanced age. Combining CRISPR/Cas9-mediated Vhl knockout in these mice increased the frequency of dysplasia, but failed to increase the incidence of RCC. Assessments of whether ischemic injury of TECs in the GPPY kidney without Vhl knockout influences the emergence of RCC revealed that advanced RCC predominantly emerged in the contralateral, noninjured kidney with 100% penetrance at a younger age, but rarely in the injured kidney due to severely damaged ischemic TEC. Injured TEC released CXCL1 into the microenvironment that traveled systemically to activate fibroblasts and recruit neutrophils to enable emergence of RCC in the contralateral kidney. Fibroblasts responded to CXCL1 via CXCR2 and recruited tumor-associated neutrophils, which in turn mediated tumor-promoting inflammation and angiogenesis. Treatment with anti-CXCR2 antibodies abolished the emergence of malignant RCC. Collectively, these results demonstrate a defining functional role of systemic inflammation and microenvironment in the emergence of malignant cancer from preestablished dysplastic precursor lesions. SIGNIFICANCE: These results identify a role for CXCL1/CXCR2 and the tumor microenvironment in the development of RCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2690/F1.large.jpg.See related commentary by Kusmartsev, p. 2584.

Carnitine palmitoyltransferase 1C contributes to progressive cellular senescence.

Stable transfection manipulation with antibiotic selection and passaging induces progressive cellular senescence phenotypes. However, the underlying mechanisms remain poorly understood. This study demonstrated that stable transfection of the empty vector induced PANC-1 cells into cellular senescence. Metabolomics revealed several acylcarnitines and their upstream regulatory gene, carnitine palmitoyltransferase 1C (CPT1C) involved in fatty acid ß-oxidation in mitochondria, were strikingly decreased in senescent PANC-1 cells. Low CPT1C expression triggered mitochondrial dysfunction, inhibited telomere elongation, impaired cell survival under metabolic stress, and hindered the malignance and tumorigenesis of senescent cells. On the contrary, mitochondrial activity was restored by CPT1C gain-of-function in senescent vector PANC-1 cells. PPARα and TP53/CDKN1A, crucial signaling components in cellular senescence, were downregulated in senescent PANC-1 cells. This study identifies CPT1C as a key regulator of stable transfection-induced progressive PANC-1 cell senescence that inhibits mitochondrial function-associated metabolic reprogramming. These findings confirm the need to identify cell culture alterations after stable transfection, particularly when cells are used for metabolomics and mitochondria-associated studies, and suggest inhibition of CPT1C could be a promising target to intervene pancreatic tumorigenesis.

Functional roles of eriocalyxin B in zebrafish revealed by transcriptome analysis.

BACKGROUND: Eriocalyxin B (EriB) is a natural ent-kaurane diterpenoid obtained from Isodon eriocalyx var. laxiflora (family Lamiaceae), which has multiple biological activities (e.g. anti-tumor and anti-inflammatory) via the alteration of gene expression and signaling transduction. Recently, RNA sequencing (RNA-seq) has been developed as a dynamic transcriptome approach to analyze the transcriptional profile and in addition use such gene expression profiles to identify novel candidate genes in a zebrafish model. In the present study, a transcriptome analysis was performed to identify differentially expressed genes (DEGs) in an EriB-exposed zebrafish model. RESULTS: RNA sequencing was conducted on zebrafish embryos after EriB (10 µM and 15 µM) treatment for 72 h. A total of 1570 (405 up-regulated and 1165 down-regulated) and 2511 genes (543 up-regulated and 1968 down-regulated) were identified in the 10 µM and 15 µM groups, respectively. Gene ontology analysis was then performed to elucidate the mechanism of action and effects of EriB. We found that 4 pathways were significantly enriched, which include glutathione metabolism, the metabolism of xenobiotics by cytochrome P450, tight junctions, and phototransduction. The critical transcriptional regulators for the DEGs were also identified by Ingenuity Pathway Analysis after the construction of a protein-protein network, which involves p53, c-myc, binding transcription factor 2, sterol regulatory element binding transcription factor 2, nuclear factor erythroid 2 like 2, and interferon regulatory factor 3. CONCLUSION: In summary, this is the first study to comprehensively explore the effects of EriB in a zebrafish model using a transcriptome analysis approach. Several important genes with substantial changes in expression levels were discovered. The results of this study will provide insights for the future investigation on the biological activities or toxic effects of EriB.

Elaborating the Role of Natural Products on the Regulation of Autophagy and their Potentials in Breast Cancer Therapy.

Autophagy is an intracellular lysosomal/vacuolar degradation system, in which the inner cytoplasmic cell membrane is degraded by the lysosomal hydrolases, followed by the resulting products released back into the cytosol. It is involved in many physiological processes which are crucial for cell growth and survival. However, disturbance in the autophagic process is often associated with a variety of human diseases, such as cancer. Breast cancer is one of the most malignant tumors characterized by the imbalanced cell proliferation, apoptosis as well as disordered autophagy regulation. The alterations of autophagy related genes or protein levels in breast cancer cells also suggested a potential implication of autophagy in breast cancer development and progression. Many natural products had been reported as potential anti-cancer agents or being considered as direct or indirect sources of new chemotherapy adjuvants to enhance the efficacy or to ameliorate the side effects through the modulation of autophagy. Investigation of the underlying mechanism of these compounds could be crucial for the development of new therapeutic or chemopreventive options for breast cancer treatment. In this review, a summary of those natural products that can regulate autophagy in breast cancer is presented and the potential value of such autophagy modulators on the development of anti-cancer drugs is also discussed.

Ginsenoside-Rb1-Mediated Anti-angiogenesis via Regulating PEDF and miR-33a through the Activation of PPAR-γ Pathway.

Angiogenesis is the formation of new blood vessels from the existing vasculature, which is involved in multiple biological processes, including atherosclerosis, ischemic heart disease, and cancer. Ginsenoside-Rb1 (Rb1), the most abundant ginsenoside isolated form Panax ginseng, has been identified as a promising anti-angiogenic agent via the up-regulation of PEDF. However, the underlying molecular mechanisms still unknown. In the present study, human umbilical vein endothelial cells (HUVECs) were selected to perform in vitro assays. Rb1 (0-20 nM) treatment induced pigment epithelial-derived factor (PEDF) protein expression in concentration and time-dependent manners. Interestingly, it was also demonstrated that the exposure of Rb1 (10 nM) could increase PEDF protein expression without any alteration on mRNA level, suggesting the involvement of posttranscriptional regulation. Furthermore, bioinformatics predictions indicated the regulation of miR-33a on PEDF mRNA 3'-UTR, which was further confirmed by luciferase reporter gene assay and real-time PCR. Over-expression of pre-miR-33a was found to regress partly Rb1-mediated PEDF increment and anti-angiogenic effect in HUVECs. Additionally, Rb1-reduced miR-33a and increased PEDF expression was prevented by pre-incubation with peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist (GW9662) or transfection with PPAR-γ siRNA in HUVECs. Taken together, our findings demonstrated that Rb1 exerted anti-angiogenic effects through PPAR-γ signaling pathway via modulating miR-33a and PEDF expressions. Thus, Rb1 may have the potential of being developed as an anti-angiogenic agent, however, further appropriate studies are warranted to evaluate the effect in vivo.

Eriocalyxin B, a novel autophagy inducer, exerts anti-tumor activity through the suppression of Akt/mTOR/p70S6K signaling pathway in breast cancer.

Eriocalyxin B (EriB), a natural ent-kaurane diterpenoid presented in the plant Isodon eriocalyx var. laxiflora, has been reported to diminish angiogenesis-dependent breast tumor growth. In the present study, the effects of EriB on human breast cancer and its underlying mechanisms were further investigated. The in vitro anti-breast cancer activity of EriB was determined using MCF-7 and MDA-MB-231 cell lines. MDA-MB-231 xenograft model of human breast cancer was also established to explore the anti-tumor effect in vivo. We found that EriB was able to induce apoptosis accompanied by the activation of autophagy, which was evidenced by the increased accumulation of autophagosomes, acidic vesicular organelles formation, the microtubule-associated protein 1A/1B-light chain 3B-II (LC3B-II) conversion from LC3B-I and p62 degradation. Meanwhile, EriB treatment time-dependently decreased the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K), leading to the inhibition of Akt/mTOR/p70S6K signaling pathway. Moreover, the blockage of autophagy obviously sensitized EriB-induced cell death, which suggested the cytoprotective function of autophagy in both MCF-7 and MDA-MB-231 cells. Interestingly, the autophagic features and apoptosis induction were prevented by reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine, indicating that ROS played an essential role in the mediation of EriB-induced cell death. Furthermore, in MDA-MB-231 xenograft model, EriB displayed a significant anti-tumor effect via the activation of autophagy and apoptosis in breast tumor cells. Taken together, our findings firstly demonstrated that EriB suppressed breast cancer cells growth both in vitro and in vivo, and thus could be developed as a promising anti-breast tumor agent.

Bigelovin triggered apoptosis in colorectal cancer in vitro and in vivo via upregulating death receptor 5 and reactive oxidative species.

Colorectal cancer (CRC) is the third most prevalent cancer and the third highest cancer-related mortality in the United States. Bigelovin, a sesquiterpene lactone isolated from Inula helianthus aquatica, has been proven to induce apoptosis and exhibit anti-inflammatory and anti-angiogenic activities. However, the effects of bigelovin on CRC and underlying mechanisms have not been explored. The present study demonstrated that bigelovin exhibited potent anti-tumor activities against CRC in vitro and in vivo. Bigelovin suppressed cell proliferation and colony formation and induced apoptosis in human colorectal cancer HT-29 and HCT 116 cells in vitro. Results also revealed that bigelovin activated caspases, caused the G2/M cell cycle arrest and induced DNA damage through up-regulation of death receptor (DR) 5 and increase of ROS. In HCT 116 xenograft model, bigelovin treatment resulted in suppression of tumor growth. Bigelovin at 20 mg/kg showed more significant tumor suppression and less side effects than conventional FOLFOX (containing folinic acid, 5-fluorouracil and oxaliplatin) treatment. In addition, in vivo data confirmed that anti-tumor activity of bigelovin in CRC was through induction of apoptosis by up-regulating DR5 and increasing ROS. In conclusion, these results strongly suggested that bigelovin has potential to be developed as therapeutic agent for CRC patients.

Eriocalyxin B, a natural diterpenoid, inhibited VEGF-induced angiogenesis and diminished angiogenesis-dependent breast tumor growth by suppressing VEGFR-2 signaling.

Eriocalyxin B (EriB), a natural ent-kaurane diterpenoid isolated from the plant Isodon eriocalyx var. laxiflora, has emerged as a promising anticancer agent. The effects of EriB on angiogenesis were explored in the present study. Here we demonstrated that the subintestinal vein formation was significantly inhibited by EriB treatment (10, 15 µM) in zebrafish embryos, which was resulted from the alteration of various angiogenic genes as shown in transcriptome profiling. In human umbilical vein endothelial cells, EriB treatment (50, 100 nM) could significantly block vascular endothelial growth factors (VEGF)-induced cell proliferation, tube formation, cell migration and cell invasion. Furthermore, EriB also caused G1 phase cell cycle arrest which was correlated with the down-regulation of the cyclin D1 and CDK4 leading to the inhibition of phosphorylated retinoblastoma protein expression. Investigation of the signal transduction revealed that EriB inhibited VEGF-induced phosphorylation of VEGF receptor-2 via the interaction with the ATP-binding sites according to the molecular docking simulations. The suppression of VEGFR-2 downstream signal transduction cascades was also observed. EriB was showed to inhibit new blood vessel formation in Matrigel plug model and mouse 4T1 breast tumor model. EriB (5 mg/kg/day) treatment was able to decrease tumor vascularization and suppress tumor growth and angiogenesis. Taken together, our findings suggested that EriB is a novel inhibitor of angiogenesis through modulating VEGFR-2 signaling pathway, which could be developed as a promising anti-angiogenic agent for treatment of angiogenesis-related human diseases, such as cancer.

Resveratrol suppresses the inducible expression of CYP3A4 through the pregnane X receptor.

The pregnane X receptor (PXR, NR1I2), a member of the nuclear receptor superfamily, is activated by a number of clinically prescribed drugs and herbal extracts. The inducible expression of several important cytochrome P450 (CYP450) enzymes has been shown to be regulated by the activation of PXR in the liver. In the current study, reporter gene-transfected cells were used to identify potential antagonists of PXR. Here, we showed that resveratrol (RES), a natural polyphenolic compound could significantly suppress the rifampicin-induced PXR transactivation of the CYP3A4 promoter. Treatment of hPXR-over-expressed cells with RES reduced the rifampicin-inducible expression of CYP3A4 in a concentration-dependent manner. Moreover, the induction of mRNA and protein expression of CYP3A11 by pregnenolone 16α-carbonitrile was also significantly reduced when RES was applied in primary cultures of mouse hepatocytes. Taking together, these findings suggest that RES can attenuate the PXR-mediated induction of CYP3A enzyme. Therefore, it would be possible for RES to antagonize the elevation in CYP3A-mediated drug metabolism by identified PXR activators.

Low dose of oleanolic acid protects against lithocholic acid-induced cholestasis in mice: potential involvement of nuclear factor-E2-related factor 2-mediated upregulation of multidrug resistance-associated proteins.

Oleanolic acid (OA) is a natural triterpenoid and has been demonstrated to protect against varieties of hepatotoxicants. Recently, however, OA at high doses was reported to produce apparent cholestasis in mice. In this study, we characterized the protective effect of OA at low doses against lithocholic acid (LCA)-induced cholestasis in mice and explored further mechanisms. OA cotreatment (5, 10, and 20 mg/kg, i.p.) significantly improved mouse survival rate, attenuated liver necrosis, and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; more importantly, serum total bile acids and bilirubin, as well as hepatic total bile acids were also remarkably reduced. Gene and protein expression analysis showed that hepatic expression of multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4 was significantly increased by OA cotreatment, whereas other bile acid metabolism- and transport-related genes, including Na+/taurocholate cotransporter, organic anion transporter 1b2, bile salt export pump, multidrug resistance protein 3, Cyp3a11, Cyp2b10, Sulfotransferase 2a1 (Sult2a1), and UDP-glucuronosyltransferase 1a1 (Ugt1a1), were only slightly changed. OA also caused increased nuclear factor-E2-related factor (Nrf2) mRNA expression and nuclear protein accumulation, whereas nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor were not significantly influenced by OA. Luciferase (Luc) assays performed in HepG2 cells illustrated that OA was a strong Nrf2 agonist with moderate PXR and weak FXR agonism. Finally, in mouse primary cultured hepatocytes, OA dose- and time-dependently induced expression of Mrp2, Mrp3, and Mrp4; however, this upregulation was abrogated when Nrf2 was silenced. In conclusion, OA produces a protective effect against LCA-induced hepatotoxicity and cholestasis, possibly due to Nrf2-mediated upregulation of Mrp2, Mrp3, and Mrp4.

Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII-MDR1 and Caco-2 cell monolayer models.

AIM: To investigate the effects of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on P-glycoprotein-mediated efflux of digoxin in two cell transport models. METHODS: Caco-2 cells, wild MDCKII cells (MDCKII-WT) and MDCKII cells transfected stably with human MDR1-gene encoding P-gp (MDCKII-MDR1) were examined. Cell viability was evaluated with MTT assay. Bidirectional transport of digoxin was evaluated in these cells. Intracellular ATP level was measured using ATP assay. P-gp ATPase activity was analyzed using a Pgp-Glo(TM) assay. RESULTS: PMA (10 µmol/L) did not reduce the viability of the 3 types of cells. In Caco-2 and MDCKII-MDR1 cell monolayers, PMA (1, 10 and 100 nmol/L) dose-dependently inhibited the basolateral to apical transport of digoxin, but did not change the apical to basolateral transport. In addition, PMA did not affect both the basolateral to apical and apical to basolateral transport of digoxin in MDCKII-WT cell monolayer. In agreement with the above results, PMA dose-dependently reduced intracellular ATP level and stimulated P-gp ATPase activity in both Caco-2 and MDCKII-MDR1 cells. Verapamil (a positive control, 100 µmol/L) caused similar inhibition on digoxin efflux as PMA did, whereas 4α-PMA (a negative control, 100 nmol/L) had no effect. CONCLUSION: PMA significantly inhibited P-gp-mediated efflux of digoxin in both Caco-2 and MDCKII-MDR1 cell monolayers via PKC activation.

Effects of praeruptorin A and praeruptorin C, a racemate isolated from Peucedanum praeruptorum, on MRP2 through the CAR pathway.

Praeruptorin A and praeruptorin C, racemic to each other, are main bioactive constituents of the species Peucedanum praeruptorum, traditionally used as a Chinese herbal medicine (also known as Bai-Hua Qian Hu). In the present study, the ability of praeruptorins A and C to activate the constitutive androstane receptor and induce human multidrug resistance-associated protein 2 expressions in HepG2 cells was investigated. The changes in mRNA level, protein expression, and transport activity of multidrug resistance-associated protein 2 were determined by quantitative real-time PCR, Western blot, and the CDF uptake assay, respectively. The effects of constitutive androstane receptor knockdown on multidrug resistance-associated protein 2 mRNA and protein expression were also measured by transient transfection of a specific constitutive androstane receptor siRNA. The results showed that praeruptorin A and praeruptorin C significantly induced the multidrug resistance-associated protein 2 mRNA and protein expression, and enhanced the transport activity of multidrug resistance-associated protein 2. A further study showed that mRNA and protein upregulation were attenuated by transient transfection of a specific constitutive androstane receptor siRNA, suggesting that the upregulation of multidrug resistance-associated protein 2 was mediated by the constitutive androstane receptor. Taken together, our findings indicate that praeruptorin A and praeruptorin C can significantly upregulate multidrug resistance-associated protein 2 expression via the constitutive androstane receptor-mediated pathway in vitro, and this should be taken as an herb-drug interaction.

[Induction of UGT1A1 expression by praeruptorin A and praeruptorin C through hCAR pathway].

This study is purposed to investigate the effects of praeruptorin A (PA) and praeruptorin C (PC) on UGT1A1 in HepG2 cells through hCAR pathway. PA and PC were incubated with HepG2 cells for 24 h and 48 h, mRNA and protein expressions of UGT1A1 were determined by real-time PCR and Western blotting assays. Additionally, effects of PA and PC on UGT1A1 mRNA and protein expressions were also measured after transient transfection of a specific CAR siRNA for 72 h in HepG2 cells. UGT1A1 mRNA and protein expression levels were significantly increased by PA and PC after incubation for 48 h. Moreover, the mRNA and protein up-regulations of UGT1A1 were attenuated by transient transfection of a specific CAR siRNA, suggesting the induction was mediated by CAR. The results suggest that PA and PC can significantly up-regulate UGT1A1 expression partially via the CAR-mediated pathway.