The transcription factor Krüppel-like factor 5 promotes cell growth and metastasis via activating PI3K/AKT/Snail signaling in hepatocellular carcinoma.

The transcription factor Krüppel-like factor 5 (KLF5) is highly expressed in many cancers and serves as a prognostic factor. However, the function of KLF5 in hepatocellular carcinoma (HCC) is unclear. In this study, we found that KLF5 was significantly overexpressed in HCC cell lines and specimens, and high KLF5 expression predicted a poor prognosis for HCC patients. Then, we studied the effects of KLF5 on the proliferation, apoptosis, migration and invasion of HCC cells in vitro and vivo. The inhibition of KLF5 markedly inhibited HCC growth and metastasis, while KLF5 overexpression promoted these processes. In addition, we observed that KLF5 could promote the epithelial-mesenchymal transition (EMT) in HCC via the PI3K/AKT/Snail signaling pathway. The silencing of KLF5 in HCC cell lines downregulated the expression of N-cadherin, Vimentin and Snail and increased the expression of the epithelial marker E-cadherin. The expression of MMP2 and MMP9 was also decreased in KLF5-silenced HCC cells. However, opposite results were observed in the KLF5-overexpressing group. These results indicate that KLF5 plays a significant role in HCC progression and metastasis and induces EMT via activating PI3K/AKT/Snail signaling, and the inhibition of KLF5 may be a potential treatment modality for patients with HCC.

Genotype analysis of rotaviruses isolated from children during a phase III clinical trial with the hexavalent rotavirus vaccine in China.

The oral hexavalent live human-bovine reassortant rotavirus vaccine (RV6) developed by Wuhan Institute of Biological Products Co., Ltd (WIBP) has finished a randomized, placebo-controlled phase III clinical trial in four provinces of China in 2021. The trail demonstrated that RV6 has a high vaccine efficacy against the prevalent strains and is safe for use in infants. During the phase III clinical trial (2019-2021), 200 rotavirus-positive fecal samples from children with RV gastroenteritis (RVGE) were further studied. Using reverse transcription-polymerase chain reaction and high-throughput sequencing, VP7 and VP4 sequences were obtained and their genetic characteristics, as well as the differences in antigenic epitopes of VP7, were analyzed in detail. Seven rotavirus genotypes were identified. The predominant rotavirus genotype was G9P [8] (77.0%), followed by prevalent strains G8P [8] (8.0%), G3P [8] (3.5%), G3P [9] (1.5%), G1P [8] (1.0%), G2P [4] (1.0%), and G4P [6] (1.0%). The amino acid sequence identities of G1, G2, G3, G4, G8, and G9 genotypes of isolates compared to the vaccine strains were 98.8%, 98.2%-99.7%, 88.4%-99.4%, 98.2%, 94.2%-100%, and 93.9%-100%, respectively. Notably, the vaccine strains exhibited high similarity in amino acid sequence, with only minor differences in antigenic epitopes compared to the Chinese endemic strains. This supports the potential application of the vaccine in preventing diseases caused by rotaviruses.

Immunogenicity and safety of a new hexavalent rotavirus vaccine in Chinese infants: A randomized, double-blind, placebo-controlled phase 2 clinical trial.

Rotavirus remains a major cause of diarrhea among 5-y-old children, and vaccination is currently the most effective and economical measure. We conducted a randomized, double-blind, placebo-controlled phase II clinical trial designed to determine the dosage, immunogenicity, and safety profile of a novel hexavalent rotavirus vaccine. In total, 480 eligible healthy infants, who were 6-12 weeks of age at the time of randomization were randomly allocated (1:1:1) to receive 105.5 focus-forming unit (FFU) or 106.5FFU of vaccine or placebo on a 0, 28 and 56-d schedule. Blood samples were collected 28 d after the third dose to assess rotavirus immunoglobulin A (IgA) antibody levels. Adverse events (AEs) up to 28 d after each dose and serious adverse events (SAEs) up to 6 months after the third dose were recorded as safety measurements. The anti-rotavirus IgA seroconversion rate of the vaccine groups reached more than 70.00%, ranging from 74.63% to 76.87%. The postdose 3 (PD3) geometric mean concentrations (GMCs) of anti-rotavirus IgA among vaccine recipients ranged from 76.97 U/ml to 84.46 U/ml. At least one solicited AE was recorded in 114 infants (71.25%) in the high-dose vaccine group, 106 infants (66.25%) in the low-dose vaccine group and 104 infants (65.00%) in the placebo group. The most frequently solicited AE was fever. The novel oral hexavalent rotavirus vaccine was safe and immunogenic in infants support the conclusion to advance the candidate vaccine for phase 3 efficacy trials.

Neuroepithelial transforming protein 1 short interfering RNA-mediated gene silencing with microbubble and ultrasound exposure inhibits the proliferation of hepatic carcinoma cells in vitro.

OBJECTIVES: Short interfering RNA (siRNA) has been used to knock down the expression of targeted genes in a process known as RNA interference. However, the key to RNA interference is the efficient intracellular delivery of the siRNA. In this study, we sought to enhance the efficiency of transduction and find a novel therapy for hepatic carcinoma. METHODS: Three types of neuroepithelial transforming protein 1 (NET-1) siRNAs (labeled fluorescent) were designed and transduced into HepG2 cells. Then the most effective one in silencing NET-1 was determined. The HepG2 cells were divided into 5 groups: untreated control; delivery of siRNA; delivery of siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA; group L); delivery of siRNA using ultrasound exposure and microbubbles (group US); and delivery of siRNA using Lipofectamine, ultrasound exposure, and microbubbles (group LUS). The efficiency of siRNA transfer was determined by detection of luciferase activity on microscopy; NET-1 expression was assayed by reverse transcription-polymerase chain reaction and western blotting; and proliferation investigations of the HepG2 cells were performed. RESULTS- The transfection efficiency of microbubbles combined with ultrasound exposure was nearly equal to Lipofectamine-mediated transfection (P = .609). More importantly, the combination of Lipofectamine, microbubbles, and ultrasound exposure effectively reduced NET-1 expression compared with the other groups (P < .01). Furthermore, the proliferation of cells in groups L, US, and LUS was visibly inhibited between 24 and 72 hours. CONCLUSIONS: The use of a microbubble contrast agent combined with ultrasound exposure could be a potent physical method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer.

Efficacy, safety and immunogenicity of hexavalent rotavirus vaccine in Chinese infants.

A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy and safety of a hexavalent live human-bovine reassortant rotavirus vaccine (HRV) against rotavirus gastroenteritis (RVGE). A total of 6400 participants aged 6-12 weeks were enrolled and randomly assigned to either HRV (n â€‹= â€‹3200) or placebo (n â€‹= â€‹3200) group. All the subjects received three oral doses of vaccine four weeks apart. The vaccine efficacy (VE) against RVGE caused by rotavirus serotypes contained in HRV was evaluated from 14 days after three doses of administration up until the end of the second rotavirus season. VE against severe RVGE, VE against RVGE hospitalization caused by serotypes contained in HRV, and VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were also investigated. All adverse events (AEs) were collected for 30 days after each dose. Serious AEs (SAEs) and intussusception cases were collected during the entire study. Our data showed that VE against RVGE caused by serotypes contained in HRV was 69.21% (95%CI: 53.31-79.69). VE against severe RVGE and RVGE hospitalization caused by serotypes contained in HRV were 91.36% (95%CI: 78.45-96.53) and 89.21% (95%CI: 64.51-96.72) respectively. VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were 62.88% (95%CI: 49.11-72.92), 85.51% (95%CI: 72.74-92.30) and 83.68% (95%CI: 61.34-93.11). Incidences of AEs from the first dose to one month post the third dose in HRV and placebo groups were comparable. There was no significant difference in incidences of SAEs in HRV and placebo groups. This study shows that this hexavalent reassortant rotavirus vaccine is an effective, well-tolerated, and safe vaccine for Chinese infants.

Effect and Mechanism of miR-26a-5p on Proliferation and Apoptosis of Hepatocellular Carcinoma Cells.

AIM: This study aimed to investigate the effect and mechanism of miR-26a-5p on proliferation and apoptosis of hepatocellular carcinoma (HCC) cells. METHODS: RT-PCR was used to analyze the expression of miR-26a-5p in HCC cells and its targeted gene HMGA2 mRNA determined by biological information prediction. The rate of proliferation, invasion, apoptosis, and expression levels of related proteins of HCC cells overexpressing miR-26a-5p or those after knocking down HMGA2 expression were detected by MTT, invasion and apoptosis rate tests. Moreover, the apoptosis-promoting protein bax was upregulated and the anti-apoptosis-related protein Bcl-2 was downregulated. RESULTS: RT-qPCR results showed that the level of miR-26a-5p was downregulated in HCC tissues and cells, and the expression of HMGA2 was upregulated; besides, the expression of miR-26a-5p and HMGA2 was negatively correlated; miR-26a-5p was correlated with tumor diameter, differentiation degree, TNM staging and lymph node metastasis. Cell tests confirmed that miR-26a-5p functioned in tumor suppression, including inhibiting cell proliferation and invasion in two hepatocellular carcinoma cell lines and promoting apoptosis. Bioinformatics prediction and subsequent experiments proved that HMGA2 was the direct target of miR-26a-5p; moreover, after knocking down HMGA2 expression in HCC cells, cell proliferation and invasion ability were significantly inhibited, and apoptosis rate increased significantly. CONCLUSION: miR-26a-5p can inhibit the proliferation and invasion of HCC cells and promote their apoptosis by directly targeting HMGA2. Abnormal decrease of miR-26a-5p and increase of its target HMGA2 are important factors that may participate in the occurrence and development of HCC. miR-26a-5p may be a new potential target for its treatment.

Cyclovirobuxine D Exerts Anticancer Effects by Suppressing the EGFR-FAK-AKT/ERK1/2-Slug Signaling Pathway in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the sixth most common malignancy worldwide, is characterized by a dismal prognosis due to high recurrence and metastasis rates. Thus, the need for the development of novel chemotherapeutic drugs is urgent. Cyclovirobuxine D (CVB-D), a steroidal alkaloid extracted from Buxus microphylla that has been extensively used to relieve the symptoms of cardiovascular diseases, has shown promising antineoplastic effects in recent studies. However, the therapeutic effects and underlying mechanisms of CVB-D on HCC remain largely unelucidated. This study experimentally indicated that CVB-D can repress HCC cell proliferation by arresting the cell cycle in G2 phase and can facilitate apoptosis. In addition, the migratory and invasive capabilities of HCC cells were noticeably attenuated by a nonlethal dose of CVB-D, and this attenuation was correlated with the inhibition of epithelial-mesenchymal transition (EMT). Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing nude mice. Regarding the molecular mechanisms of CVB-D activity, decreased Slug expression was determined to be associated with the aforementioned anti-HCC functions of this extract, which might be regulated by epidermal growth factor receptor (EGFR) through the focal adhesion kinase (FAK)-associated PI3K/AKT and MEK/ERK1/2 signaling pathways. Collectively, our results revealed the suppressive effects of CVB-D on progressive behaviors of HCC, including proliferation, migration, invasion, and EMT, in addition to its outstanding proapoptotic effects, which were correlated with the inhibition of the EGFR-FAK-AKT/ERK1/2-Slug signaling pathway. These discoveries provide an experimental and theoretical foundation for the use of CVB-D as a promising candidate for HCC therapy.

Polydatin executes anticancer effects against glioblastoma multiforme by inhibiting the EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway.

AIMS: Glioblastoma multiforme (GBM) is characterized by aggressive infiltration and terrible lethality. The overwhelming majority of chemotherapeutic drugs fail to exhibit the desired treatment effects. Polydatin (PD), which was initially extracted from Polygonum cuspidatum, is distinguished for its outstanding cardioprotective, hepatoprotective, and renal protective effects, as well as significant anticancer activities. However, the anti-GBM effect of PD is unclear. MATERIALS AND METHODS: Cell proliferation and apoptosis after PD intervention were estimated using MTT, colony formation and flow cytometry assays in vitro, while wound-healing and Transwell assays were applied to assess cell migration and invasion. In addition, the anti-GBM effects of PD in vivo were detected in the subcutaneous tumor model of nude mice. Moreover, Western blot, immunofluorescence and immunohistochemical staining assays were employed to elaborate the relevant molecular mechanisms. KEY FINDINGS: The present study demonstrated that PD repressed cell proliferation, migration, invasion and stemness and promoted apoptosis in GBM cells. Moreover, by correlating the molecular characteristics of cancer cells with different sensitivities to PD and employing diverse analytical methods, we ultimately verified that the cytotoxicity of PD was related to EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway inhibition, in which multiple components were vital therapeutic targets of GBM. SIGNIFICANCE: This work demonstrated that PD could inhibit proliferation, migration, invasion and stemness and induce apoptosis by restraining multiple components of the EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway in GBM cells.

Erratum: Polydatin inhibits hepatocellular carcinoma via the AKT/STAT3-FOXO1 signaling pathway.

[This corrects the article DOI: 10.3892/ol.2019.10123.].

Polydatin inhibits hepatocellular carcinoma via the AKT/STAT3-FOXO1 signaling pathway.

Polydatin, extracted from Polygonum cuspidatum, is known for its anti-platelet aggregation and anti-inflammatory effects. However, studies on the association of polydatin with cancer are limited, particularly with regards to epithelial-mesenchymal transition (EMT)-associated migration and invasion of cancer cells. The purpose of the present study was to reveal the potential anticancer effects of polydatin on hepatocellular carcinoma (HCC) cells, particularly its effects on EMT. MTT assay was used to determine cell viability. Migration and invasion were evaluated through wound healing and transwell assays. Colony formation efficiency assay was conducted to detect proliferation. Flow cytometric analyses of apoptosis and cell cycle progression were performed following cells staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and PI alone, respectively. Western blotting was used to investigate relevant molecular mechanisms. The results indicated that polydatin inhibited proliferation via G2/M arrest, suppressed migration and invasion of HCC cells, and promoted their apoptosis. In addition, phosphorylated (p)-protein kinase B (AKT), p-Janus kinase 1 and p-signal transducer and activator of transcription 3 (STAT3) levels were decreased as polydatin concentrations increased, and forkhead box protein O1 (FOXO1) expression was upregulated. Furthermore, the expression levels of various markers of EMT were reversed following treatment with polydatin. In conclusion, the present study validated that polydatin may inhibit proliferation via G2/M arrest, and suppressed EMT-associated migration and invasion of HCC cells. The results also suggested that polydatin may promote HCC cell apoptosis by blocking the AKT/STAT3-FOXO1 signaling pathway.

Oncogenic MSH6-CXCR4-TGFB1 Feedback Loop: A Novel Therapeutic Target of Photothermal Therapy in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) has been considered the most aggressive glioma type. Temozolomide (TMZ) is the main first-line chemotherapeutic agent for GBM. Decreased mutS homolog 6 (MSH6) expression is clinically recognized as one of the principal reasons for GBM resistance to TMZ. However, the specific functions of MSH6 in GBM, in addition to its role in mismatch repair, remain unknown. Methods: Bioinformatics were employed to analyze MSH6 mRNA and protein levels in GBM clinical samples and to predict the potential cancer-promoting functions and mechanisms of MSH6. MSH6 levels were silenced or overexpressed in GBM cells to assess its functional effects in vitro and in vivo. Western blot, qRT-PCR, and immunofluorescence assays were used to explore the relevant molecular mechanisms. Cu2(OH)PO4@PAA nanoparticles were fabricated through a hydrothermal method. Their MRI and photothermal effects as well as their effect on restraining the MSH6-CXCR4-TGFB1 feedback loop were investigated in vitro and in vivo. Results: We demonstrated that MSH6 is an overexpressed oncogene in human GBM tissues. MSH6, CXCR4 and TGFB1 formed a triangular MSH6-CXCR4-TGFB1 feedback loop that accelerated gliomagenesis, proliferation (G1 phase), migration and invasion (epithelial-to-mesenchymal transition; EMT), stemness, angiogenesis and antiapoptotic effects by regulating the p-STAT3/Slug and p-Smad2/3/ZEB2 signaling pathways in GBM. In addition, the MSH6-CXCR4-TGFB1 feedback loop was a vital marker of GBM, making it a promising therapeutic target. Notably, photothermal therapy (PTT) mediated by Cu2(OH)PO4@PAA + near infrared (NIR) irradiation showed outstanding therapeutic effects, which might be associated with a repressed MSH6-CXCR4-TGFB1 feedback loop and its downstream factors in GBM. Simultaneously, the prominent MR imaging (T1WI) ability of Cu2(OH)PO4@PAA could provide visual guidance for PTT. Conclusions: Our findings indicate that the oncogenic MSH6-CXCR4-TGFB1 feedback loop is a novel therapeutic target for GBM and that PTT is associated with the inhibition of the MSH6-CXCR4-TGFB1 loop.

FOXO1 inhibits the invasion and metastasis of hepatocellular carcinoma by reversing ZEB2-induced epithelial-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) program is critical for epithelial cell cancer progression and fibrotic diseases. FOXO1 influences a broad range of physiological and pathological processes. However, the mechanism by which FOXO1 inhibits EMT is not fully understood. In this study, we demonstrated that FOXO1 overexpression inhibited cell motility and invasiveness in vitro and inhibited lung metastasis in vivo. In addition, we found that FOXO1 couldreverse the EMT program. FOXO1 silencing by siRNA in hepatocellular carcinoma (HCC) cell lines enhanced the expression of mesenchymal markers and decreased the expression of the epithelial markers. Consistent with these findings, FOXO1 overexpression exerted opposite effects. Furthermore, we found that FOXO1 levels were inversely correlated with the levels of EMT inducers, including Snail, Slug, ZEB1, ZEB2 and Twist1 in HCC cells. Co-immunoprecipitation and immunohistochemistry assays revealed that an interaction between FOXO1 and ZEB2. A dual-luciferase reporter assay and a ChIP assay further demonstrated that FOXO1 binds to the ZEB2 promoter. Together, these findings suggest that FOXO1 overexpression or ZEB2 inhibition might be potential therapeutic strategies for treating HCC.

Antinociceptive activity of a polysaccharide from the roots of Sophora flavescens.

A polysaccharide (SFWP), with a molecular weight of 51kDa, was successfully purified from the roots of Sophora flavescens and the antinociceptive actions of SFWP were evaluated using acetic acid induced writhing, tail flick, and formalin tests in mice. GC-MS results showed that SFWP had a backbone composed of (1→2)-linked Glc, (1→2,6)-inkedGal and (1→3,6)-inked Man residues, which were terminated with (1→)-inked Xyl and (1→)-inked Ara at O-6 of (1→2,6)-inkedGal and (1→3,6)-inked Man along the main chain, in the ratio of 2.0: 1.02: 1.09: 1.10: 0.98. Data showed that SFWP (100, 200 and 400mg/kg) significantly reduced the number of writhings induced by acetic acid in a dose-dependent manner. However, SFWP did not produce analgesia in tail-flick test. Moreover SFWP strongly attenuated the formalin-induced flinching behaviour in the second phases but not in the first phase in a dose-dependent manner. These results revealed that SFWP could be used safely to attenuate both inflammatory and peripheral neuropathic pain.

Galactosylated poly-L-lysine targeted microbubbles for ultrasound mediated antisense c-myc gene transfection in hepatocellular carcinoma cells.

INTRODUCTION: The aim of the study was to investigate the efficiency of delivery and targeted binding of c-myc antisense oligodeoxynucleotide (ASODN) and find a novel therapy for hepatic carcinoma. MATERIAL AND METHODS: A targeted ultrasound microbubble compound was synthesized to deliver the c-myc ASODN by ultrasound-targeted microbubble destruction (UTMD) and applied in hepatocellular carcinoma cells (HCC) and cancer bearing mice. Lipid microbubbles were conjugated with biotinylated galactosylated poly-L-lysine (G-PLL) and SonoVue to target the hepatocellular carcinoma SMMC7721 cells with asialoglycoprotein receptors. There were four groups in both in vitro and in vivo studies: control group (group A); c-myc ASODN + G-PLL (CG group, group B); c-myc ASODN + SonoVue (CUS group, group C); c-myc ASODN + G-PLL + SonoVue (CGUS group, group D). The expression of c-myc mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and proliferation investigations of the SMMC7721 cells were also performed. In addition, the tumor volume was calculated and compared among different groups. RESULTS: The level of c-myc mRNA in the three experimental groups was significantly lower than that in the control group in vitro (p < 0.05). Furthermore, c-myc gene expression was suppressed more strongly in the CGUS group compared with other groups in both in vitro and in vivo studies (p < 0.05). In addition, ultrasound mediation of targeted microbubbles yielded the highest inhibition of tumor growth and cell proliferation among the four groups. CONCLUSIONS: The use of a G-PLL targeted microbubble contrast agent combined with ultrasound exposure could be a potential method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer.