Targeting ONECUT2 inhibits tumor angiogenesis via down-regulating ZKSCAN3/VEGFA.

OC-2 plays a vital role in tumor growth, metastasis and angiogenesis, but molecular mechanism how OC-2 regulates angiogenic factors is unclear. We found that OC-2 was highly expressed in HepG2, COLO, MCF-7, SKOV3 cells and rectum carcinoma tissues, and angiogenic factors levels were positively related to OC-2. Then OC-2 KD inhibited the tumor growth, metastasis and angiogenesis process in vitro and vivo. ChIP-Seq showed that 228 target genes of OC-2 were identified and they were associated with tumor growth, metastasis, angiogenesis and signal transduction; OC-2 bound to ZKSCAN3 at promoter region. Luciferase assays showed that ZKSCAN3 was identified as target gene of OC-2 and VEGFA was identified as target gene of ZKSCAN3; OC-2 promoted VEGFA expression via activating ZKSCAN3 transcriptional program. Importantly, OC-2 KD down-regulated VEGFA secretion to suppress tumor angiogenesis of HUVECs. Besides VEGFA, OC-2 was positively correlated with other angiogenic factors HIF-1α, FGF2, EGFL6 and HGF. Meanwhile, ERK1/2 and Smad1 signaling pathways might be related to function of OC-2 driving tumor aggressiveness. We revealed that OC-2 might regulate tumor growth, metastasis, angiogenesis via ERK1/2, Smad1 signaling pathways and regulate VEGFA expression for tumor angiogenesis via activating ZKSCAN3 transcriptional program, indicating that OC-2 was a convincing target to develop novel anti-tumor drugs based on angiogenesis.

A Morpholine Derivative N-(4-Morpholinomethylene)ethanesulfonamide Induces Ferroptosis in Tumor Cells by Targeting NRF2.

Small molecule drugs containing morpholine-based moieties have become crucial candidates in the tumor targeted therapy strategies, but the specific molecular mechanisms of these drugs causing tumor cell death require further investigation. The morpholine derivative N-(4-morpholinomethylene)ethanesulfonamide (MESA) was used to stimulate prostate and ovarian cancer cells and we focused on the ferroptosis effects, including the target molecule and signal pathways mediated by MESA. The results showed that MESA could induce ferroptosis to cause the proliferation inhibition and apoptosis effects of tumor cells according to the identification of ferroptosis inhibitor fer-1 and other cell death inhibitors. Further MESA could significantly increase the intracellular malondialdehyde (MDA), reactive oxygen species (ROS) and Fe2+ levels in tumor cells and mediate the dynamic changes of ferroptosis-relative molecules GPX4, nuclear factor erythroid2-related factor 2 (NRF2), ACSL4, SLC7A11 and P62-Kelch-like ECH-associated protein 1 (KEAP1)-NRF2-antioxidant response element (ARE) signal pathways. Further, NRF2 overexpression could reduce the tumor cell death and ROS levels exposure to MESA. Most importantly, it was confirmed that MESA could bind to NRF2 protein through molecular docking and thermal stability assays and NRF2 was a target molecule of MESA for inducing ferroptosis effects in tumor cells. Collectively, our findings indicated the ferroptosis effects of the morpholine derivative MESA in prostate and ovarian cancer cells and its function mechanism including targeted molecule and signal pathways, which would be helpful for developing MESA as a prospective small molecule drug for cancer therapy based on cell ferroptosis.

miR-129-2-3p mediates LPS-induced macrophage polarization and ferroptosis by targeting the SMAD3-GPX4 axis.

Macrophages has become a promising target of sepsis treatment because macrophages dysfunction contributes to the progress of sepsis. The targeted therapy of sepsis based on macrophages ferroptosis is drawing more and more attention, but the molecular mechanism involved is poorly understood. In this study, Mus musculus-derived macrophages were used for in-vitro experiments. We found that LPS could induce ferroptosis in macrophages via the detection of apoptosis, GSH, lipid peroxide and GPX4 levels. Meanwhile, miR-129-2-3p was up-regulated in macrophages exposure to LPS. Next, we confirmed that miR-129-2-3p promoted the LPS-induced polarization of M1 phenotype in macrophages via the detection of Arg-1 and iNOS levels; miR-129-2-3p promoted the LPS-induced ferroptosis in macrophages. Further, luciferase assay showed that SMAD3 was identified as a target gene of miR-129-2-3p and its expression was negatively regulated by miR-129-2-3p and LPS. SMAD3 could inhibit the LPS-induced polarization of M1 phenotype and ferroptosis in macrophages by targeting GPX4. Collectively, we demonstrated the target gene and molecular mechanism of miR-129-2-3p mediating LPS-induced polarization and ferroptosis in macrophages by targeting the SMAD3-GPX4 axis, which would provide a novel strategy for sepsis targeted therapy based on macrophages polarization and ferroptosis.

Feasibility and safety of bipolar-plasmakinetic transurethral enucleation and resection of the prostate in day surgery mode. / åŒæžç­‰ç¦»å­ç»å°¿é“å‰åˆ—è ºå‰œåˆ‡æ—¥é—´æ‰‹æœ¯ä¸´åºŠå®žè·µ.

OBJECTIVES: To evaluate the feasibility and safety of bipolar-plasmakinetic transurethral enucleation and resection of the prostate (B-TUERP) in day surgery. METHODS: From January 2021 to August 2022, 34 patients with benign prostatic hyperplasia (BPH) underwent B-TUERP in day surgery in the First Affiliated Hospital of Anhui Medical University. Patients completed the screening and anesthesia evaluation before admission and received the standard surgery which implements "anatomical enucleation of the prostate" and "absolute bleeding control" on the same day of admission, and by the same doctor. Bladder irrigation was stopped, catheter was removed and the discharge evaluation was performed on the first day after operation. The baseline data, perioperative conditions, time of recovery, treatment outcomes, hospitalization costs, and postoperative complications were analyzed. RESULTS: All operations were successfully conducted. The average age of the patients was (62.2±7.8) years, average prostate volume was (50.2±29.3) mL. The average operation time was (36.5±19.1) min, the average hemoglobin and blood sodium were decreased by (16.2±7.1) g/L and (2.2±2.0) mmol/L, respectively. The average postoperative length of hospital stay, and total length of hospital stay were (17.7±2.2) and (20.8±2.1) h, respectively, and the average hospitalization cost was (13 558±2320) CNY. All patients were discharged on the day after surgery except for one patient who was transferred to a general ward. Three patients received indwelling catheterization after catheter removal. The 3-month follow-up results showed a substantial improvement in the International Prostate Symptom Score, quality of life score and maximum urinary flow rate (all P<0.01). Three patients experienced temporary urinary incontinence, 1 patient experienced urinary tract infection, 4 patients were diagnosed with urethral stricture and 2 patients experienced bladder neck contracture. No complications above Clavien grade Ⅱ occurred. CONCLUSIONS: The preliminary results showed that B-TUERP ambulatory surgery is a safe, feasible, economical and effective treatment for appropriately selected patients with BPH.

Preparation of DSPE-PEG-cRGD Modified Cationic Liposomes for Delivery of OC-2 shRNA and The Antitumor Effects on Breast Cancer.

Cationic liposome delivery of interfering RNA (shRNA) plays an important role in tumor therapy. The cyclic Arg-Gly-Asp (cRGD) modified cationic liposomes (cRGD-CL) were designed for targeted delivery of ONECUT2 (OC-2) shRNA (pshOC-2) to breast cancer cells. The characterization analysis of cationic liposome showed that the prepared cRGD-CL/pshOC-2 lipoplexes had uniform particle size (150 ± 1.02 nm), moderate zeta potential (19.8 ± 0.249 mV) and high encapsulation efficiency (up to 96%). The results of flow cytometer showed that the introduction of cRGD could significantly promote the liposomes targeting tumor cells. In MCF-7 cells, the pshOC-2 could down-regulate expression of OC-2 and result in cell apoptosis, inhibition of the wound healing, migration and cell colony formation, in which the signal pathways of Bcl-xL, Bcl-2 were inhibited and the signal pathways of Bax and Cleaved Caspase-3 were promoted. In MCF-7 xenograft mice, intravenous administration of cRGD-CL/pshOC-2 lipoplexes could effectively reduce the expression of OC-2 in tumors and result in apparently antitumor effects, which suggested that the lipoplexes might be deeply penetrated into tumor through receptor-mediated transcytosis. The results revealed that the cationic liposome (cRGD-CL) was an effective delivery system for OC-2 shRNA, which might be an effective therapeutic candidate for breast cancer.

Association of Cytokine Gene Polymorphisms with Prostate Cancer Risk from a Study in Central China.

Our study intended to investigate five cytokine gene single nucleotide polymorphisms (SNPs) and their associations with prostate cancer risk. Genotypes of five cytokine gene SNPs were detected by MassARRAY for blood samples from a group of patients with prostate cancer (n = 90) and a control group (n = 140) in central China. The differences in tumor clinical stages, Gleason scores, and PSA values in patients with prostate cancer were also investigated. The frequencies of the five cytokine gene SNPs (L-1ß rs16944, IL-4 rs2070874, IL-4rs2227284, IL-16 rs7175701, and IL-16 rs11556218) genotypes were not found to be significantly mutated in prostate cancer patients compared with the control group. In addition, for five cytokine gene SNPs genotypic comparisons, patients with different Gleason scores, clinical stages, and PSA values were grouped into two subgroups. There was also no statistically significant association in all these subgroups. Our study suggests that cytokine gene polymorphisms may not be a risk factor for prostate cancer in a central Chinese population. Nevertheless, more large-scale studies on the Chinese population are necessary to examine our conclusions. The discovery of cytokine gene polymorphisms related to prostate cancer could update our understanding of the etiology and improve our knowledge of the early detection, diagnosis, and treatment of prostate cancer.

Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is an inflammatory immune disease characterized by intraprostatic leukocyte infiltration and pelvic or perineal pain. Macrophages play vital roles in the pathogenesis of CP/CPPS. However, the mechanisms controlling the activation and chemotaxis of macrophages in CP/CPPS remain unclear. This study aimed to investigate the roles of the CXCL10/CXCR3 pathway in the activation and chemotaxis of macrophages in CP/CPPS patients. The serums of CP/CPPS patients and healthy volunteers were collected and measured. Results showed that CXCL10 expression was significantly elevated and correlated with the severity of CP/CPPS patients. The experimental autoimmune prostatitis (EAP) model was generated, and adeno-associated virus and CXCR3 inhibitors were used to treat EAP mice. Immunofluorescence, flow cytometry, and Western blotting were used to analyze the functional phenotype and regulation mechanism of macrophages. Results showed that CXCL10 deficiency ameliorates EAP severity by inhibiting infiltration of macrophages to prostate. Moreover, CXCL10 could induce macrophage migrations and secretions of proinflammatory mediators via CXCR3, which consequently activated the downstream Erk1/2 and p38 MAPK signaling pathways. We also showed that prostatic stromal cell is a potential source of CXCL10. Our results indicated CXCL10 as an important mediator involved in inflammatory infiltration and pain symptoms of prostatitis by promoting the migration of macrophages and secretion of inflammatory mediators via CXCR3-mediated ERK and p38 MAPK activation.

hsa_circ_0062019 promotes the proliferation, migration, and invasion of prostate cancer cells via the miR-195-5p/HMGA2 axis.

Circular RNA (circRNA) is a new class of non-coding RNA. It was reported that circRNA involves in the metastasis of cancer. The aim of this study is to explore the role and mechanism of circRNA hsa_circ_0062019 in the development of prostate cancer (PCa). Our results showed that hsa_circ_0062019 was highly expressed in PCa cell lines. Cell Counting Kit-8 assay revealed that upregulation of hsa_circ_0062019 boosted PCa cell proliferation, and silencing of hsa_circ_0062019 inhibited cell proliferation. Meanwhile, transwell assay proved that upregulation of hsa_circ_0062019 facilitated PCa cell invasion and migration, while downregulation of hsa_circ_0062019 inhibited these malignant phenotypes. Furthermore, luciferase reporter assay proved the binding of hsa_circ_0062019 with miR-195-5p and the binding between miR-195-5p and high mobility group AT-hook 2 (HMGA2), suggesting that hsa_circ_0062019 promoted the expression of HMGA2 by sponging miR-195-5p. In addition, our results revealed that the hsa_circ_0062019-induced PCa cell malignant phenotypes were notably reversed by the downregulation of HMGA2. Overall, our study demonstrated that hsa_circ_0062019 promoted PCa cell proliferation, migration, and invasion via upregulation of HMGA2 expression by sponging miR-195-5p. Our study proved a novel molecular mechanism of PCa development and provided a potential target for the treatment of PCa.

Exosomal LINC00355 derived from cancer-associated fibroblasts promotes bladder cancer cell proliferation and invasion by regulating miR-15a-5p/HMGA2 axis.

We have previously demonstrated that exosomes derived from cancer-associated fibroblasts (CAFs) promote bladder cancer (BC) cell proliferation and invasion by transferring LINC00355. In this study, the molecular mechanisms underlying the pro-bladder cancer action of exosomal LINC00355 were explored. CAFs were obtained from BC tumor tissues, and normal fibroblasts (NFs) were obtained from adjacent normal tissues. Human BC cell lines (T24 and 5367) were incubated with NF-Exo (exosomes from NFs), CAF-Exo (exosomes from CAFs), CAFsi-Ctrl-Exo (exosomes from si-Ctrl-transfected CAFs), and CAFsi-LINC00355-Exo (exosomes from si-LINC00355-transfected CAFs). BC cell proliferation and invasion were evaluated by MTT and Transwell assays, respectively. The interaction between miR-15a-5p and LINC00355 or HMGA2 was examined by online bioinformatics analysis and luciferase activity assay. Results showed that HMGA2 is a direct target of miR-15a-5p, and LINC00355 functions as a sponge of miR-15a-5p to upregulate HMGA2 expression. The promoting effects of CAF-Exo on HMGA2 expression, cell proliferation, and cell invasion were hindered when LINC00355 expression was inhibited in BC cells. These promoting effects were also hindered when miR-15a-5p was overexpressed or HMGA2 was silenced in BC cells. In conclusion, exosomal LINC00355 derived from CAFs promotes BC cell proliferation and invasion by regulating miR-15a-5p/HMGA2 axis.

Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2.

Angiogenesis is considered one of the hallmarks of cancer and plays a critical role in the development of tumor. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family and participates in excessive cancer cell proliferation and tumor angiogenesis. Thus, targeting FGF-2 was considered to be a promising anti-tumor strategy. A disulfide-stabilized diabody (ds-Diabody) against FGF-2 was produced in Pichia pastoris (GS115) by fermentation and the anti-tumor activity was analyzed. The novel 10-L fed batch fermentation with newly designed media was established, and the maximum production of the ds-Diabody against FGF-2 reached 210.4 mg/L. The ds-Diabody against FGF-2 was purified by Ni2+ affinity chromatography and DEAE anion exchange chromatography. The recombinant ds-Diabody against FGF-2 could effectively inhibit proliferation, migration, and invasion of melanoma and glioma tumor cells stimulated by FGF-2. Furthermore, xenograft tumor model assays showed that the ds-Diabody against FGF-2 had potent antitumor activity in nude mice by inhibiting tumor growth and angiogenesis. The tumor growth inhibition rate of melanoma and glioma was about 70 and 45%, respectively. The tumor angiogenesis inhibition rate of melanoma and glioma was about 64 and 51%, respectively. The results revealed that the recombinant ds-Diabody against FGF-2 may be a promising anti-tumor drug for cancer therapy.

Exosomal LINC00355 derived from cancer-associated fibroblasts promotes bladder cancer cell resistance to cisplatin by regulating miR-34b-5p/ABCB1 axis.

Cisplatin resistance is a major challenge for bladder cancer (BC). Evidence indicates that exosome derived from cancer-associated fibroblasts (CAF-Exo) can promote chemotherapy resistance in various human tumors by delivering bioactive molecules. We have previously demonstrated that CAF-derived exosomal LINC00355 promotes BC cell proliferation and invasion. However, the underlying mechanisms are still unclear. In this study, we aimed to investigate the role and mechanisms of CAF-derived exosomal LINC00355 in BC cell resistance to cisplatin. Exosomes were isolated from normal fibroblasts (NFs) and BC tumor-derived CAFs, namely, NF-Exo and CAF-Exo. CAFs were transfected with si-Ctrl or si-LINC00355 and then different exosomes were isolated, namely, CAFsi-Ctrl-Exo and CAFsi-LINC00355-Exo. The human BC cell lines (T24 and 5367) were incubated with NF-Exo, CAF-Exo, CAFsi-Ctrl-Exo, and CAFsi-LINC00355-Exo in the presence of cisplatin. MTT proliferation assay and flow cytometric analysis showed that CAF-Exo promoted BC cell resistance to cisplatin and upregulated ABCB1 expression in BC cells by transferring LINC00355 to BC cells. Luciferase activity assay confirmed the interaction between miR-34b-5p and LINC00355 or ABCB1. qRT-PCR and western blot analysis further showed that LINC00355 sponged miR-34b-5p to upregulate ABCB1 expression. However, the promoting effects of CAF-Exo on BC cell resistance to cisplatin were abolished by miR-34b-5p overexpression and ABCB1 silencing. In conclusion, exosomal LINC00355 derived from CAFs promotes BC cell resistance to cisplatin by regulating the miR-34b-5p/ABCB1 axis.

Characterization of DPP-IV Inhibitory Peptides Using an In Vitro Cell Culture Model of the Intestine.

Here, we characterize the activities of two depeptidyl peptidase-IV (DPP-IV) inhibitory peptides, VLATSGPG and LDKVFER, using the Caco-2 monolayer model for the intestine. VLATSGPG and LDKVFR inhibited the DPP-IV in the cells via a mixed-type inhibition mode, with in situ IC50 values of 207.3 and 148.5 µM, respectively. Furthermore, VLATSGPG and LDKVFR were transported intact across the cells, with Papp values of 2.41 ± 0.16 and 4.23 ± 0.29 × 10-7 cm/s, respectively. Fragmented peptides were identified in the basolateral side of the membrane. Two of these, GPG and VLA, exhibited high inhibitory activities of 83.6 ± 3.3 and 58.5 ± 2.5%, respectively, at 100 µM concentration. Although 3 mM VLATSGPG and LDKVFR were transported across the epithelium in a concentration-dependent manner, their transport did not damage the tight junction proteins, ZO-1 and occludin. This study demonstrates that the two peptides potentially regulate DPP-IV activity in the intestine.

Nucleolar and spindle-associated protein 1 accelerates cellular proliferation and invasion in nasopharyngeal carcinoma by potentiating Wnt/ß-catenin signaling via modulation of GSK-3ß.

Nucleolar and spindle-associated protein 1 (NUSAP1) is a pivotal tumor-related protein that has been implicated in the progression of broad spectrum of tumors. However, no detailed study of the role of NUSAP1 in nasopharyngeal carcinoma (NPC) has been reported. The aim of this work is to enhance our understanding of NUSAP1 in the progression of NPC. By analyzing data available within the Oncomine database, we found that NUSAP1 expression was elevated in NPC relative to normal tissues. Further, we showed that NUSAP1 expression in clinical specimens of NPC and several NPC cell lines was elevated. Down-regulation of NUSAP1 by gene silencing markedly depleted the capacity of NPC cells to proliferate and invade. Contrastingly, overexpression of NUSAP1 potentiated the proliferative and invasive abilities of NPC cells. Further mechanistic research revealed that NUSAP1 knockdown decreased levels of Wnt/ß-catenin signaling in NPC cells via a mechanism associated with downregulation of glycogen synthase kinase-3ß (GSK-3ß) phosphorylation. However, suppression of GSK-3ß markedly abolished the inhibitory effect of NUSAP1 knockdown on Wnt/ß-catenin signaling. Further, inhibition of Wnt/ß-catenin signaling partially reversed NUSAP1-mediated tumor growth in NPC cells. In addition, NUSAP1 knockdown restrained tumorigenesis of NPC in vivo, and was associated with down-regulation of Wnt/ß-catenin signaling. In conclusion, these findings demonstrate that NUSAP1 is capable of accelerating proliferation and invasion in NPC cells by potentiating Wnt/ß-catenin signaling. Our study unveils a potential role of NUSAP1 in promoting NPC tumors and suggests that the protein is an attractive antitumor target for NPC treatment.

Actin-like 6A enhances the proliferative and invasive capacities of laryngeal squamous cell carcinoma by potentiating the activation of YAP signaling.

Overexpression of Actin-like 6A (ACTL6A) has been observed in a wide spectrum of tumors and exerts an outstanding oncogenic function throughout cancer progression. However, the detailed relevance of ACTL6A for laryngeal squamous cell carcinoma (LSCC) is not fully understood. In this work, we aimed to elucidate the precise role of ACTL6A in LSCC. Here, we show that ACTL6A expression was significantly upregulated in LSCC versus normal tissues. In vitro functional assays demonstrated that silencing of ACTL6A significantly diminished the capacity of LSCC cells to proliferate and invade, whilst up-regulation of ACTL6A drove proliferation and invasion in LSCC cells. Further investigation revealed that knockdown of ACTL6A repressed the activation of Yes-associated protein (YAP)-mediated signaling in LSCC cells, while reactivation of YAP markedly reversed ACTL6A-silencing-mediated inhibition of proliferation and invasion in LSCC cells. Moreover, suppression of YAP markedly diminished ACTL6A-overexpression-induced promotion of tumor formation in LSCC. In addition, ACTL6A silencing diminished the tumorigenicity of LSCC cells in vivo. To summarize, our work indicates that ACTL6A may enhance LSCC progression via potentiating the activation of YAP signaling. Thus, ACTL6A may be an attractive anticancer target for the treatment of LSCC.

CRISPR/Cas9-Mediated OC-2 Editing Inhibits the Tumor Growth and Angiogenesis of Ovarian Cancer.

Ovarian cancer is the leading cancer-related cause of death in women worldwide. It is of great relevance to understand the mechanism responsible for tumor progression and identify unique oncogenesis markers for a higher chance of preventing this malignant disease. The high-expression OC-2 gene has been shown to be a potential candidate for regulating oncogenesis and angiogenesis in ovarian cancer. Hence, we wished to investigate the impact of OC-2 gene on ovarian cancer aggressiveness. CRISPR/Cas9, a gene editing tool, allows for direct ablation of OC-2 at the genomic level, and we successfully generated OC-2 KO cell lines from SKOV3 and CAOV3 cells. In an apoptosis assay, OC-2 KO induced the apoptosis activation of tumor cells, with the up-regulation of Bax/Caspase-8 and the down-regulation of Bcl-2. Consequently, the proliferation, migration, and invasion of OC-2 KO cell lines were significantly inhibited. Assays of qRT-PCR and Western blotting showed that the expression levels of pro-angiogenic growth factors VEGFA, FGF2, HGF, and HIF-1α and the activation of Akt/ERK pathways were significantly down-regulated at the loss of OC-2. In the xenograft model, OC-2 KO potently suppressed the subcutaneous tumor growth, with the inhibition exceeding 56%. The down-regulation of CD31 and relevant pro-angiogenic growth factors were observed in OC-2 KO tumor tissues. Taken together, OC-2 depletion negatively regulated the ovarian cancer progression possibly by apoptosis activation and angiogenesis inhibition. This work revealed a pivotal regulator of apoptosis and angiogenesis networks in ovarian cancer, and we applied the CRISPR/Cas9 system to the transcription factor pathway for developing a broad-acting anti-tumor gene therapy.

Large-scale production, purification, and function of a tumor multi-epitope vaccine: Peptibody with bFGF/VEGFA.

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10-L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation-exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high-titer anti-bFGF and anti-VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large-scale process we developed could support its industrial production and pre-clinical study in the future.

Barriers and Strategies of Cationic Liposomes for Cancer Gene Therapy.

Cationic liposomes (CLs) have been regarded as the most promising gene delivery vectors for decades with the advantages of excellent biodegradability, biocompatibility, and high nucleic acid encapsulation efficiency. However, the clinical use of CLs in cancer gene therapy is limited because of many uncertain factors in vivo. Extracellular barriers such as opsonization, rapid clearance by the reticuloendothelial system and poor tumor penetration, and intracellular barriers, including endosomal/lysosomal entrapped network and restricted diffusion to the nucleus, make CLs not the ideal vector for transferring extrinsic genes in the body. However, the obstacles in achieving productive therapeutic effects of nucleic acids can be addressed by tailoring the properties of CLs, which are influenced by lipid compositions and surface modification. This review focuses on the physiological barriers of CLs against cancer gene therapy and the effects of lipid compositions on governing transfection efficiency, and it briefly discusses the impacts of particle size, membrane charge density, and surface modification on the fate of CLs in vivo, which may provide guidance for their preclinical studies.

Effect of Eriocalyxin B on prostatic inflammation and pelvic pain in a mouse model of experimental autoimmune prostatitis.

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common disease in males. Eriocalyxin B (EriB), a natural diterpenoid purified from Isodon eriocalyx var. laxiflora, was previously reported to have antitumor effects via multiple immune-related pathways. In this study, we investigated the effect of EriB on CP/CPPS using a mouse model of experimental autoimmune prostatitis (EAP) and explored its potential mechanisms. METHODS: The EAP model was established in nonobese diabetic mice by intradermal injecting a mixture of prostate antigens and Complete Freund's Adjuvant on days 0 and 28. Then, EAP mice received daily intraperitoneal injections of EriB (5 or 10 mg/kg/d) for 14 days, from days 28 to 42 (EAP+EriB5 or EAP+EriB10 groups). The histopathological appearance of the prostate tissues was evaluated. Chronic pelvic pain development was assessed by cutaneous allodynia. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay tests. We then explored anti-inflammatory potential mechanisms of EriB by studying the effects of PI3K inhibitor wortmannin (EAP+EriB10+Wort group) and NF-κB inhibitor SC75741 (EAP+EriB10+SC group) on prostate inflammation and pelvic pain using this model. RESULTS: Histological analyses revealed significant prostate inflammation in EAP mice compared with control mice. Significantly increased pelvic pain was detected in EAP mice (P < .05). Compared with the EAP+Veh group, chronic pain development, histological appearance, and cytokine levels demonstrated that EriB could alleviate the severity of EAP in a dose-dependent manner though upregulation of the PI3K/Akt/mTOR pathway and downregulation of the NF-κB pathway. Further mechanism research demonstrated that the PI3K/AKT/mTOR pathway could be blocked by wortmannin, but was not affected by SC75741. In addition, the NF-κB pathway could be further inhibited by SC75741 compared with the EAP+EriB10+Veh group. However, wortmannin could reactivate the NF-κB pathway, indicating that the PI3K/AKT/mTOR pathway negatively regulates the NF-κB pathway during EriB treatment. CONCLUSIONS: The results of the present study suggested that EriB could alleviate the severity of prostatic inflammation and pelvic pain in an EAP mouse model. These findings may broaden the value of EriB as a promising candidate for the treatment of CP/CPPS.

The Design, Characterizations, and Tumor Angiogenesis Inhibition of a Multi-Epitope Peptibody With bFGF/VEGFA.

Tumor angiogenesis is dependent on growth factors, and inhibition of their pathways is one of the promising strategies in cancer therapy. However, resistance to single pathway has been a great concern in clinical trials so that it necessitates multiple targetable factors for developing tumor angiogenesis inhibitors. Moreover, the strategy of Fc fusion protein is an attractive platform for novel peptide agents, which gains increasing importance with FDA approval because of better immunogenicity and stability. Here, we applied the Fc fusion protein concept to bFGF/VEGFA pathways and designed a multi-epitope Peptibody with immunogenic peptides derived from human bFGF and VEGFA sequences. Immunization with Peptibody could elicit high-titer anti-bFGF and anti-VEGFA antibodies, activate T cells, and induce Th1/Th2-type cytokines. In in vitro experiments, the isolated anti-Peptibody antibody inhibited the proliferation and migration of A549 cells and human umbilical vein endothelial cells (HUVECs) by decreasing the MAPK/Akt/mTOR signal pathways. In the murine tumor model, pre-immunization with Peptibody suppressed the tumor growth and neovascularization of lung cancer by decreasing the production of bFGF/VEGFA/PDGF, the MAPK/Akt/mTOR signal pathways, and the activation of suppressive cells in tumor sites. Further, the biological characterizations of the recombinant Peptibody were investigated systematically, including protein primary structure, secondary structure, stability, and toxicity. Collectively, the results highlighted the strategy of bFGF/VEGFA pathways and Fc fusion protein in suppressing tumor progression and angiogenesis, which emphasized the potential of multiple targetable factors for producing enduring clinical responses in tumor patients.

CaMK4-dependent phosphorylation of Akt/mTOR underlies Th17 excessive activation in experimental autoimmune prostatitis.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is a complicated syndrome characterized by genitourinary pain in the absence of bacterial infection. Th17 cell-driven autoimmunity has been proposed as a cause of CP/CPPS. However, the factors that promote Th17-driven autoimmunity in experimental autoimmune prostatitis (EAP) and the molecular mechanisms are still largely unknown. Here, we showed that Th17 cells were excessively activated, and blockade of IL-17A could effectively ameliorate various symptoms in EAP. Furthermore, we revealed that calcium/calmodulin-dependent kinase Ⅳ (CaMK4), especially Thr196 p-CaMK4 was increased in the Th17 cells of the EAP group, which were activated by intracellular cytosolic Ca2+ . Pharmacologic and genetic inhibition of CaMK4 decreased the proportion of Th17 cells, and the protein and mRNA level of IL-17A, IL-22, and RORγt. The phosphorylation of CaMK4 was dependent on the increase in intracellular cytosolic Ca2+ concentration in Th17 cells. A mechanistic study demonstrated that inhibition of CaMK4 reduced IL-17A production by decreasing the phosphorylation of Akt-mTOR, which was well accepted to positively regulate Th17 differentiation. Collectively, our results demonstrated that Ca2+ -CaMK4-Akt/mTOR-IL-17A axis inhibition may serve as a promising therapeutic strategy for CP/CPPS.