Combination of automated sample preparation and micro-flow LC-MS for high-throughput plasma proteomics.

BACKGROUND: Non-invasive detection of blood-based markers is a critical clinical need. Plasma has become the main sample type for clinical proteomics research because it is easy to obtain and contains measurable protein biomarkers that can reveal disease-related physiological and pathological changes. Many efforts have been made to improve the depth of its identification, while there is an increasing need to improve the throughput and reproducibility of plasma proteomics analysis in order to adapt to the clinical large-scale sample analysis. METHODS: We have developed and optimized a robust plasma analysis workflow that combines an automated sample preparation platform with a micro-flow LC-MS-based detection method. The stability and reproducibility of the workflow were systematically evaluated and the workflow was applied to a proof-of-concept plasma proteome study of 30 colon cancer patients from three age groups. RESULTS: This workflow can analyze dozens of samples simultaneously with high reproducibility. Without protein depletion and prefractionation, more than 300 protein groups can be identified in a single analysis with micro-flow LC-MS system on a Orbitrap Exploris 240 mass spectrometer, including quantification of 35 FDA approved disease markers. The quantitative precision of the entire workflow was acceptable with median CV of 9%. The preliminary proteomic analysis of colon cancer plasma from different age groups could be well separated with identification of potential colon cancer-related biomarkers. CONCLUSIONS: This workflow is suitable for the analysis of large-scale clinical plasma samples with its simple and time-saving operation, and the results demonstrate the feasibility of discovering significantly changed plasma proteins and distinguishing different patient groups.

[Therapeutic effect of tadalafil on lower urinary tract symptoms with erectile dysfunction].

OBJECTIVE: To investigate the therapeutic effect of low-dose tadalafil on BPH-induced lower urinary tract symptoms (LUTS) complicated with ED. METHODS: We randomly assigned 126 patients with BPH-induced LUTS and ED to receive daily administration of tadalafil (5 mg) plus tamsulosin hydrochloride sustained-release capsules (0.2 mg) (treatment group Ⅰ ［T-Ⅰ］, n = 42), tadalafil (5 mg) only (treatment group Ⅱ ［T-Ⅱ］, n = 42) or placebo (control group, n = 42), all for 12 weeks. Before and after 6 and 12 weeks of medication, and at 4 and 8 weeks after drug withdrawal, we recorded and compared the IPSS sub-item scores in the voiding stage and urinary storage symptoms, total IPSS, IIEF-5 scores, and the frequency of sexual activities among the three groups of patients. RESULTS: As for the IPSS sub-item scores in the voiding stage symptoms, T-Ⅰ showed statistically significant differences between any two of the five time points (P < 0.05), but not T-Ⅱ between the baseline and 6-week medication or 8-week withdrawal (P > 0.05), or between 6-week medication and 8-week withdrawal (P > 0.05), nor did the control group among the five time points (P > 0.05). Statistically significant differences were observed between any two of the three groups at 12 weeks of medication and 4 weeks after drug withdrawal (P < 0.05), but not between the T-Ⅱ and control groups at 6 weeks of medication or 8 weeks after withdrawal (P > 0.05). As to the IPSS sub-item scores in the urinary storage symptoms, there were significant differences between any two time points in T-Ⅰ and T-Ⅱ (P < 0.05), but not in the control (P > 0.05), nor between T-Ⅰ and T-Ⅱ at 6 or 12 weeks of medication or at 4 or 8 weeks after drug withdrawal (P > 0.05). With regard to the total IPSS, statistically significant differences were found between any two of the three groups at 6 and 12 weeks of medication and 4 and 8 weeks after drug withdrawal (P < 0.05), but not between 6-week medication and 8-week withdrawal in T-Ⅰ or T-Ⅱ (P > 0.05), nor among the five time points in the control group (P > 0.05). Concerning the IIEF-5 scores, there was no significant difference in the frequency of sexual activities between the three groups （Ｐ>0.05）.There were statistically significant differences between any two of the three groups at 6 weeks of medication and 8 weeks after drug withdrawal (Ｐ<0.05), but not between 6-week medication and 4- or 8-week withdrawal (P > 0.05) or between 4- and 8-week withdrawal (P > 0.05) in T-Ⅰ, nor between 6-week medication and 8-week withdrawal in T-Ⅱ (P > 0.05) or among the five time points in the control (P > 0.05), nor between T-Ⅰ and T-Ⅱ at 12 weeks of medication (P > 0.05) or 4 weeks after drug withdrawal (P > 0.05). CONCLUSIONS: Daily administration of tadalafil at 5 mg can effectively improve the IPSS sub-item scores in the urinary storage symptoms and IIEF-5 scores, with a persistent effect after withdrawal similar to that of its combination with other drugs, and therefore can be recommended for the treatment of BPH-induced LUTS complicated with ED.

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex.

Adverse life experiences increase the lifetime risk to several stress-related psychopathologies, such as anxiety or depressive-like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography-mass spectrometry (LC-MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu were upregulated, while Leu-enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, peptide PHI-27, somatostatin-28 (AA1-12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain-area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression-associated behaviors.

Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells.

BACKGROUND/AIMS: Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). METHODS: MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. RESULTS: MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. CONCLUSION: MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling.

LncRNA THOR promotes human renal cell carcinoma cell growth.

BACKGROUND: Recent studies have characterized a novel but extremely conserved long non-coding RNA (LncRNA) THOR. THOR directly associates with insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to promote mRNA stabilization of key pro-cancerous genes. RESULTS: Here, we show that THOR is expressed in human renal cell carcinoma (RCC) tissues and established/primary human RCC cells. It was not detected in normal renal tissues nor in HK-2 and primary human renal epithelial cells. THOR silencing (by targeted siRNAs) or CRISPR/Cas9 knockout inhibited RCC cell growth, viability and proliferation in vitro. Reversely, forced over-expression of THOR promoted RCC cell survival and proliferation. IGF2BP1-regulated genes, including IGF2, GLI1 and Myc, were downregulated by THOR silencing or knockout, but they were upregulated after THOR over-expression. In vivo, THOR-knockout 786-O tumors grew significantly slower than the control tumors in nude mice. CONCLUSION: THOR expression promotes RCC cell growth in vitro and in vivo. THOR could be a novel and important therapeutic target for human RCC.

Separation and identification of mouse brain tissue microproteins using top-down method with high resolution nanocapillary liquid chromatography mass spectrometry.

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top-down analytical method for simultaneous analysis of microproteins and endogenous peptides using high-resolution nanocapillary LC-MS/MS. This method is detergent-free and digestion-free, which allows for extracting and preserving intact microproteins and peptides for direct LC-MS analysis. Both higher energy collision dissociation and electron-transfer dissociation fragmentations were used in the LC-MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain-derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin-28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.

N-Butyl-4-hydroxy-1,8-naphthalimide: A new matrix for small molecule analysis by matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: The matrix plays an essential role in defining detection limits and ionization yields of analytes in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Small molecule MALDI-MS analyses commonly suffer from the high background interference generated from matrices. Moreover, the inhomogeneous crystallization of some matrices, such as 2,5-dihydroxybenzoic acid (DHB), is to the detriment of the quality or repeatability of detection. We have found that N-butyl-4-hydroxy-1,8-naphthalimide (BHN) can provide improved performance as a matrix for small molecule analysis. METHODS: BHN was evaluated in the low-mass region for its ionization efficiency, repeatability and background interference using O-acetyl-L-carnitine hydrochloride, Aß35-40, Aß35-42, and oxytocin as the model analytes. In addition, the modification effects of BHN on DHB were investigated for the in situ analysis of endogenous compounds in rat brain slices using Fourier transform ion cyclotron resonance (FTICR)-MS. RESULTS: BHN is capable of ionizing small molecules, including O-acetyl-L-carnitine hydrochloride and peptides, with high repeatability and low background interference signals. A low concentration of BHN (3 mM) modifies the crystallization state of DHB but still retains its ionization performance. The determination of small molecules desorbed from tissue slices was significantly improved by using a binary matrix of DHB and BHN, yielding superior signal-to-noise ratio and signal intensities. CONCLUSIONS: The new matrix BHN has exhibited suitability for the analysis of small molecules. Compared with the conventional matrices, CHCA and DHB, BHN provides a clean background in the low-mass region. In combination with DHB, the ability of BHN to form highly homogenous crystalline particles shows the clear beneficial effects of BHN for the reproducibility of MS detection.

Extensive characterization of peptides from Panax ginseng C. A. Meyer using mass spectrometric approach.

Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS-based data mining and sequencing. The screen on the charge states of precursor ions indicated that Ginseng is a peptide-rich herb in comparison of a number of commonly used herbs. The Ginseng peptides were then extracted and submitted to nano-LC-MS/MS analysis using different fragmentation modes, including CID, high-energy collisional dissociation, and electron transfer dissociation. Further database search and de novo sequencing allowed the identification of total 308 peptides, some of which might have important biological activities. This study illustrates the abundance and sequences of endogenous Ginseng peptides, thus providing the information of more candidates for the screening of active compounds for future biological research and drug discovery studies.

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry.

The main goal of the present study is to develop a method to recognize and identify endogenous intrachain disulfide bonded peptide, which are rarely sequenced in current peptidomics studies. In order to achieve highly efficient detection of these peptides in a neuropeptidome analysis, we alkylated the peptides, mined the raw mass spectrometry data, and then recognized the candidates of untreated disulfide bonded peptides from unalkylated peptide extracts. After removing more than 90% features, targeted electron transfer dissociation fragmentation was performed for detecting and fragmenting disulfide bonded peptides, and even most of them were present in low abundance in the original sample. Diverse endogenous disulfide bonded peptides were then detected and sequenced, opening up new perspectives for comprehensively understanding the response of a neuropeptidome.

Identification and analysis of alkaloids in cortex Phellodendron amurense by high-performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection.

Alkaloids from Cortex Phellodendron amurense Rupr. were identified to determine the material basis for the bioactivity of this herb. HPLC-ESI-MS with photodiode array detection coupled to XCharge C18 column was applied to analyze the alkaloids qualitatively and quantitatively. A total of 37 alkaloids were identified and tentatively characterized from the ethanol extract by online ESI-MS(n) fragmentation and UV spectral analysis. A total of ten alkaloids, including four novel natural products, were tentatively identified for the first time in P. amurense. The fragmentation pathways for certain compounds were analyzed. The contents of a pair of isomers (columbamine and jatrorrhizine) and four main alkaloids (phellodendrine, magnoflorine, berberine, and palmatine) were simultaneously quantified using the aforementioned method. Results showed that the newly discovered and known components of P. amurense were helpful in determining the material basis for the bioactivity of the herb. The application of the XCharge C18 column is a suitable and practical method for the isolation of alkaloids in plants.

Deciphering intercellular signaling complexes by interaction-guided chemical proteomics.

Indirect cell-cell interactions mediated by secreted proteins and their plasma membrane receptors play essential roles for regulating intercellular signaling. However, systematic profiling of the interactions between living cell surface receptors and secretome from neighboring cells remains challenging. Here we develop a chemical proteomics approach, termed interaction-guided crosslinking (IGC), to identify ligand-receptor interactions in situ. By introducing glycan-based ligation and click chemistry, the IGC approach via glycan-to-glycan crosslinking successfully captures receptors from as few as 0.1 million living cells using only 10 ng of secreted ligand. The unparalleled sensitivity and selectivity allow systematic crosslinking and identification of ligand-receptor complexes formed between cell secretome and surfaceome in an unbiased and all-to-all manner, leading to the discovery of a ligand-receptor interaction between pancreatic cancer cell-secreted urokinase (PLAU) and neuropilin 1 (NRP1) on pancreatic cancer-associated fibroblasts. This approach is thus useful for systematic exploring new ligand-receptor pairs and discovering critical intercellular signaling events.

[Effects of sildenafil on bladder compliance and endothelin-1 in rabbit model of partial bladder outlet obstruction].

OBJECTIVE: To explore the effects of sildenafil on bladder compliance and endothelin-1 in the rabbit model of partial bladder outlet obstruction. METHODS: A total of 24 adult male New Zealand white rabbits were randomly divided into group A, group B, group C and group D (n = 6 each). The rabbit model of partial bladder outlet obstruction was established in groups C and D while groups A and B underwent a sham operation. Daily sildenafil (10 mg/kg) was dosed to groups B and C by lavage. Daily normal saline was dosed similarly to groups A and D. Bladder urodynamic examinations were conducted in each group at Week 16. Then bladder was isolated and weighed from each group. And ET-1 in bladder tissue was measured by ELISA. RESULTS: Pressure thresholds for voiding (PT) in A-D groups were (10.6 ± 2.0), (11.6 ± 2.7), (14.0 ± 4.2) and (20.4 ± 6.1) cm H2O respectively. Compared with groups A, B and C, PT in group D was significantly higher (all P < 0.01). Bladder compliance in 4 groups were (2.75 ± 0.51), (2.78 ± 0.46), (4.98 ± 2.15) and (1.22 ± 0.25) ml/cm H2O respectively. Compared with groups A, B and C, bladder compliance was significantly lower in group D (all P < 0.01). Bladder compliance in group C was higher than that in groups A and B (both P < 0.01). The weights of bladder specimens in 4 groups were (5.0 ± 0.4), (4.6 ± 0.4), (8.2 ± 1.3) and (17.9 ± 2.3) g respectively. Compared with groups A and B, the weights of groups C and D were significantly heavier (all P < 0.01). And the weight of group D was much greater than that of group C (P < 0.01). The contents of ET-1 in bladder tissue of 4 groups were (72 ± 19), (69 ± 18), (76 ± 21) and (106 ± 29) pg/g respectively. Compared with groups A, B and C, ET-1 in bladder tissue was significantly higher in group D (all P < 0.05). CONCLUSIONS: Daily sildenafil can effectively alleviate the damage of rabbit bladder compliance from partial bladder outlet obstruction and protect bladder functions. Its mechanism may be related with the down-regulation of ET-1 in bladder tissue of partial bladder outlet obstruction.

Targeting drug delivery and efficient lysosomal escape for chemo-photodynamic cancer therapy by a peptide/DNA nanocomplex.

A peptide/DNA nanocomplex was developed for the targeted delivery of chemotherapeutics and photosensitizers to cancer cells for efficient combination therapy. The chemotherapeutic drug doxorubicin (DOX) and the photosensitizer 5,10,15,20-tetra-(1-methylpyridine-4-yl)-porphyrin (TMPyP4) were physically incorporated by an aptamer (AS1411)-modified tetrahedral DNA nanostructure, where the tetrahedral DNA and aptamer-induced G-quadruplex provide binding sites of DOX and TMPyP4. The co-loaded 3A-TDN/DT displayed a targeted uptake by HeLa cancer cells through the high affinity and specificity between AS1411 and nucleolin, a protein overexpressed on many types of cancer cells. A polycationic polymer, mPEG-PAsp(TECH), was synthesized to complex with the DNA nanostructure to efficiently escape from lysosomes via the proton sponge effect upon the enhanced internalization by tumor cells. Under the irradiation of 660 nm laser light, TMPyP4 induced an upregulation of intracellular reactive oxygen species, which combined with DOX to fulfill the efficient inhibition of HeLa cells. Our study demonstrated a biocompatible peptide/DNA composite nanoplatform for combinational cancer therapy via the targeted delivery of therapeutic agents and efficient lysosomal escape.

[Effects and mechanisms of phosphodiesterase type 5 inhibitors on rats with overactive bladder].

OBJECTIVE: To investigate the effects and the possible mechanistic pathway of phosphodiesterase type 5 (PDE5) inhibitors on rats with overactive bladder. METHODS: A total of 24 adult male spontaneously hypertensive rats (SHRs) were randomly divided into 3 groups: daily lavage group, discontinuous lavage group and blank group (n = 8 each). Daily vardenafil (10 mg×kg(-1)×d(-1)), discontinuous vardenafil (10 mg×kg(-1)×d(-1)) and daily normal saline were administered respectively to 3 groups by lavage. And 8 adult male SD rats were included into the control group. Bladder urodynamic examinations were conducted in each group 2 weeks later. Then bladder detrusor muscle strips isolated from each group were further divided into two parts. One part was first pre-contracted and then the relaxant effects of sodium nitroprusside and Y-27632 were observed. For another part, enzyme-linked immunosorbent assay was used to measure cyclic guanosine monophosphate (cGMP). RESULTS: As compared with the control group, the values of bladder inter contraction interval (ICI) and bladder capacity (BC) were significantly lower [(409 ± 36) s vs (568 ± 60) s, (284 ± 25) µl vs (395 ± 42) µl, P < 0.01] while the bladder non voiding contraction (NVC) was significantly higher in the blank group [(2.03 ± 0.49) number/min vs(1.07 ± 0.30) number/min, P < 0.01]. Compared with the blank group, the values of ICI and BC were elevated. NVC decreased obviously in the discontinues and daily lavage groups [(486 ± 53) s and (564 ± 44) s; (337 ± 37) µl and (392 ± 30) µl; (1.82 ± 0.32) number/min and (0.52 ± 0.23) number/min, P < 0.05]. The effects were more significant in the daily lavage group (P < 0.01). The maximal relaxant effect of sodium nitroprusside was obviously enhanced in the discontinues and daily lavage groups [(50.6 ± 2.1)% and (67.9 ± 4.1)% vs(25.3 ± 5.0)%, P < 0.01]. However the sensitivity of Y-27632 decreased significantly [(35.8 ± 2.5)% and (20.2 ± 2.3)% vs (71.6 ± 2.8)%, P < 0.01], while the level of cGMP was significantly higher in the bladder detrusor muscle [(20.6 ± 4.1) fmol/mg and (29.4 ± 4.3) fmol/mg vs (12.9 ± 2.1) fmol/mg, P < 0.01]. The effects of the daily lavage group were more pronounced (P < 0.01). CONCLUSION: The phenomenon of bladder overactivity is observed in the SHRs. The PDE5 inhibitors are effective in treating overactive bladder. And the effect of daily supplement is much better. In addition, the mechanism may operate through the cGMP-dependent protein kinase G-RhoA/Rho kinase signaling pathway.

The influence of lncRNAs on the prognosis of prostate cancer based on TCGA database.

BACKGROUND: Prostate adenocarcinoma (PRAD) is a common male urinary system cancer globally with a poor prognosis. Our research aims to explore the role of LncRNA in the occurrence and prognosis of prostate cancer and its underlying mechanism. METHODS: The biomaRt package screened for the differentially expressed lncRNA. The survival package was used to identify lncRNAs related to prognosis. The survminer package completed the Kaplan-Meier survival curves. WGCNA (Weighted Gene Co-Expression Network Analysis) screened for the co-expressed genes. The ClusterProfiler package implemented the analysis results of GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes). RESULTS: We performed differential expression analysis on the TCGA (The Cancer Genome Atlas) database to determine the association between LncRNA and Prostate cancer. The data of 500 Prostate cancer patients were tested. 6 LncRNAs (AC245884.1, LINC01524, AL807752.4, AP000844.2, AC016590.1, LINC00641) were selected as independent prognostic factors using statistical analysis methods, and their value was tested through multivariate COX analysis and Kaplan-Meier survival analysis. Through the study of co-expressed genes, the biological processes of these lncRNA enrichment are the perception and conduction of smell and taste. The specific carcinogenic and cancer-promoting mechanisms need further study. CONCLUSIONS: This study shows that lncRNA has a certain predictive effect on prostate cancer occurrence and prognosis and can be a new biomarker for prostate cancer survival and potential treatment targets.

Androgen receptor regulates eIF5A2 expression and promotes prostate cancer metastasis via EMT.

Androgen receptor (AR) is an androgen-activated transcription factor of the nuclear receptor superfamily. AR plays a role in the development and progression of prostate cancer (PCa). However, the exact role of AR in PCa metastasis remains unclear. In the present study, we aimed to elucidate the function of AR in PCa. We found that eukaryotic translation initiation factor (EIF) 5A2, an elongation factor that induces epithelial-to-mesenchymal transition (EMT) in PCa cells, was significantly upregulated after 5α-dihydrotestosterone (DHT) stimulation and downregulated after anti-androgen bicalutamide treatment in PCa cells with high AR expression, but not in cells with low AR expression. Moreover, eIF5A2 knockdown could eliminate DHT-induced invasion and migration of AR-positive PCa cells. DHT treatment decreased epithelial expression of E-cadherin and ß-catenin but increased the expression of the mesenchymal marker proteins Vimentin and N-cadherin. DHT therefore induced EMT, and knockdown of eIF5A2 inhibited DHT-induced EMT. Moreover, in vivo study, Luciferase signals from the lungs of the eIF5A2 plasmid group indicated higher metastasis ability, and the eIF5A2 siRNA group had lower metastasis ability. Our results suggest that AR positively regulates eIF5A2 expression in androgen-dependent cells, and stimulation of AR expression and signaling in prostate tumors promotes PCa metastasis by EMT induction and upregulation of eIF5A2.

Zinc finger C3H1 domain-containing protein (ZFC3H1) evaluates the prognosis and treatment of prostate adenocarcinoma (PRAD): A study based on TCGA data.

The present study was aimed to evaluate the expression profile of Zinc finger C3H1 domain-containing protein (ZFC3H1) using bioinformatic analysis of public datasets from The Cancer Genome Atlas database (TCGA). The results showed that the expression levels of ZFC3H1 were notably lower than the corresponding non-cancerous tissues in prostate adenocarcinoma (PRAD), and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. We predicted the regulatory target and protein interaction partner network of ZFC3H1, and identified a PPI network composed of 26 node genes in PRAD. Furthermore, we found that the expression levels of MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein S31) were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we knocked-down the ZFC3H1 expression and found that the inhibition of ZFC3H1 significantly inhibited PRAD cell migration and invasion. Furthermore, ZFC3H1 siRNA treatment could reduce cell viability and increase the number of apoptotic cells in PRAD cells. Taken together, ZFC3H1 could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.

Isoalantolactone Increases the Sensitivity of Prostate Cancer Cells to Cisplatin Treatment by Inducing Oxidative Stress.

Prostate cancer is the most common malignancy among men worldwide. Platinum (II)-based chemotherapy has been used to treat a number of malignancies including prostate cancer. However, the potential of cisplatin for treating prostate cancer is restricted owing to its limited efficacy and toxic side effects. Combination therapies have been proposed to increase the efficacy and reduce the toxic side effects. In the present study, we investigated how isoalantolactone (IATL), a sesquiterpene lactone extracted from the medicinal plant Inula helenium L., acts synergistically with cisplatin on human prostate cancer cells. We show that IATL significantly increased cisplatin-induced growth suppression and apoptosis in human prostate cancer cells. Mechanistically, the combined treatment resulted in an excessive accumulation of intracellular reactive oxygen species (ROS), which leads to the activation of endoplasmic reticulum (ER) stress and the JNK signaling pathway in human prostate cancer cells. Pretreatment of cells with the ROS scavenger N-acetylcysteine (NAC) significantly abrogated the combined treatment-induced ROS accumulation and cell apoptosis. In addition, the activation of ER stress and the JNK signaling pathway prompted by IATL and cisplatin was also reversed by NAC pretreatment. In vivo, we found that IATL combined with cisplatin showed the strongest antitumor effects compared with single agents. These results support the notion that IATL and cisplatin combinational treatment may be more effective for treating prostate cancer than cisplatin alone.

Significance of TP53 and immune-related genes to prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of most common male neoplasms. TP53 is the tumor suppressor gene with the highest correlation with human tumorigenesis discovered so far. Besides the TP53, immune-related genes attracted much attention since the clinical application of PD-1/PD-L1 (programmed death 1/programmed cell death-ligand 1) related drugs. There is currently a lack of studies that combine TP53 with immune-related genes to analyze the prognosis of prostate cancer patients. METHODS: Differentially expressed genes were filtered out by R package (edgeR) based on the TCGA-PRAD (The Cancer Genome Atlas-Prostate adenocarcinoma) data set. Using the R package (coxph), we distinguished which ones were related to survival prognosis. Constructing high and low risk groups, we used GEO (Gene Expression Omnibus) data set to verify the prediction performance. Subsequently, we explored the functional differences in gene expression between high and low risk groups. RESULTS: A total of six immune-related genes can be seen as prognostic factors in individuals with TP53 mutations. In the high-risk group, genes related to macrophage activation, epithelial cell apoptosis, and inflammation of the skin should be highly expressed. In the low-risk group, highly expressed genes are mainly involved in nucleotide phosphorylation, tRNA metabolism, and mitochondrial metabolism. CONCLUSIONS: Mutations in the TP53 gene can adversely affect the prognosis of prostate cancer and prostate cancer patients with mutations in some immune-related genes together have a worse prognosis. Compared with any other single clinical index, the prognostic score we proposed gave a more accurate forecast. In order to assist clinicians in making predictive assessments, we have also drawn a nomogram of the prognosis of prostate cancer patients.

A tumor extracellular pH-sensitive PD-L1 binding peptide nanoparticle for chemo-immunotherapy of cancer.

Chemo-immunotherapy is a promising model for the combination treatment of cancer. Many solid tumors overexpress programmed cell death ligand (PD-L1) for immune suppression. In this study, a PD-L1 binding peptide conjugate (DCS) nanoparticle with tumor extracellular pH-responsiveness was developed for efficient chemo-immunotherapy of colon cancer. A hydrophilic D-type polypeptide (D-PPA) and two hydrophobic stearyl chains were linked with a pH-sensitive linker to obtain amphiphilic DCS, which could self-assemble into nanoparticles (NPs). Anticancer agent doxorubicin (DOX) was loaded to obtain DOX@DCS NPs, which could accumulate at the tumor site by enhanced permeability and retention effect and release D-PPA at tumor extracellular pH. The release of D-PPA could not only lead to instability and aggregation of NPs for enhanced tumor retention but also block PD-1/PD-L1 to avoid immune escape and elicit enhanced immune response. In addition, DOX could induce immunogenic cell death (ICD) of cancer cells and promote anti-tumor immune response with the combination of PD-1/PD-L1 blocking. DOX@DCS showed efficient inhibition of CT26 tumors and induced immune response both in vitro and in vivo. Overall, our study reported a facile yet robust nanosystem based on an immune blocking peptide and a chemotherapeutic ICD inducer for efficient chemo-immunotherapy of cancer.