When type II diabetes mellitus meets COVID-19-Identification of the shared gene signatures and biological mechanism between the two diseases.

BACKGROUND: According to current studies, more than 20% of all patients diagnosed with COVID-19 globally have diabetes. Further, the mortality rate of these patients is 7.3%. Compared with non-diabetic COVID-19 patients, diabetic COVID-19 patients have higher rates of mortality and severe infection, suggesting that diabetes is associated with the severity of COVID-19 infection. This study aimed to analyse the relationship and susceptibility factors between COVID-19 and T2DM. METHODS: Using bioinformatics methods, potential targets for COVID-19 and T2DM were screened from GeneCards database. Potential targets of COVID-19 and T2DM were mapped to each other to identify overlapping targets, and a PPI network was constructed to extract the core target. The clusterProfiler package in R was used to analyse the function and pathway that core target involved. GO enrichment and KEGG pathway analysis were used to elucidate the correlation between COVID-19 and T2DM. RESULTS: A total of 277 potential pathogenic targets of COVID-19 were found, 282 potential targets were found for T2DM. Mapping of the potential COVID-19 and T2DM targets revealed 53 overlapping targets, with TNF as the core target. IL-17 signalling pathway was the most significant KEGG pathway involving TNF. CONCLUSIONS: The inflammatory cytokine, TNF, was identified as a core target between COVID-19 and T2DM, which induces inflammatory response mainly through the IL-17 signalling pathway, leading to aggravation of infection and increased difficulty in blood glucose control. This study provides a reference for further exploring the potential correlation and endogenous mechanisms between two seemingly independent and unrelated diseases-T2DM and COVID-19.

siAKR1C3@PPA complex nucleic acid nanoparticles inhibit castration-resistant prostate cancer in vitro.

Introduction: AKR1C3, as a crucial androgenic enzyme, implicates the androgen biosynthesis and promoting prostate cancer cell growth in vitro. This study provides a new gene therapy strategy for targeting AKR1C3 to treat castration-resistant prostate cancer. Methods: siAKR1C3@PPA is assembled from PEG3500, PAMAM, Aptamer-PSMA, and siRNA for AKR1C3. We analyzed the relationship between AKR1C3 expression and the survival rate of prostate cancer patients based on the GEPIA online database to perform disease-free survival, and found that AKR1C3 may be an important factor leading to poor prognosis in prostate cancer. Considering AKR1C3 as a therapeutic target for castration-resistant prostate cancer, we constructed a complex nucleic acid nanoparticle, siAKR1C3@PPA to investigate the inhibitory effect on castration-resistant prostate cancer. Results: Aptamer-PSMA acts as a target to guide siAKR1C3@PPA into PSMA-positive prostate cancer cells and specifically down regulate AKR1C3. Cyclin D1 was decreased as a result of siAKR1C3@PPA treatment. Changes in Cyclin D1 were consistent with decreased expression of AKR1C3 in LNCaP-AKR1C3 cells and 22RV1 cells. Furthermore, in the LNCaP-AKR1C3 group, 1070 proteins were upregulated and 1015 proteins were downregulated compared to the LNCaP group according to quantitative 4D label-free proteomics. We found 42 proteins involved in cell cycle regulation. In a validated experiment, we demonstrated that PCNP and CINP were up-regulated, and TERF2 and TP53 were down-regulated by western blotting. Conclusion: We concluded that siAKR1C3@PPA may arrest the cell cycle and affect cell proliferation.

Silencing of MALAT1 inhibits migration and invasion by sponging miR­1­3p in prostate cancer cells.

Prostate cancer is a common malignancy with a high mortality rate. Long non­coding RNA metastasis associated with lung adenocarcinoma transcript 1 (MALAT1) has been reported to serve tumor­promoting roles. However, the underlying mechanism requires further examination. In the present study, it was demonstrated that MALAT1 was increased while microRNA (miR/miRNA)­1­3p was decreased in prostate cancer cell lines. The silencing of MALAT1 inhibited migration, invasion and epithelial­mesenchymal transition, when epithelial (E)­cadherin expression level was increased, and neural (N)­cadherin, vimentin, Slug and Snail expression levels were decreased. Dual­luciferase reporter assay results demonstrated that miR­1­3p bound to MALAT1 and coronin 1C (CORO1C) 3' untranslated region, and MALAT1 competed with CORO1C for the binding sites of miR­1­3p. MALAT1 inhibited the expression of miR­1­3p and vice versa. MALAT1 knockdown induced the decline of CORO1C, which was subsequently recovered by the miR­1­3p inhibitor. In addition, by inhibiting miR­1­3p or overexpressing CORO1C, the silencing of MALAT1­induced phenotypic alterations were restored. In conclusion, MALAT1 serving as a degradable miRNA sponge, may sequester miR­1­3p from CORO1C and by silencing MALAT1, migration, invasion and epithelial­mesenchymal transition may be inhibited in prostate cancer cells. MALAT1 and CORO1C may serve as novel clinical therapeutic targets for prostate cancer.

Silencing of lncRNA MALAT1 inhibits cell cycle progression via androgen receptor signaling in prostate cancer cells.

Prostate cancer is the second common cancer in men with high morbidity and mortality. Androgen receptor (AR) signaling plays a crucial role in occurrence and development of prostate cancer. In this study, we demonstrated that lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was increased in prostate cancer cells after androgen stimulation, as well as AR. The silencing of MALAT1 inhibited dihydrotestosterone (DHT) administration-induced acceleration of proliferation and cell cycle progression, and increase of AR expression in prostate cancer cells. MALAT1 bound to miR-320b and negatively regulated its expression, and vice versa. AR is a target of miR-320b. The phenotypic changes induced by silencing of MALAT1 were abolished by miR-320b inhibition or AR overexpression. Additionally, MALAT1 knockdown also suppressed the tumorigenesis of prostate cancer cells in nude mice. In summary, the silencing of MALAT1 inactivated AR signaling by sponging miR-320b, and inhibited proliferation and cell cycle progression in prostate cancer cells, suggesting that MALAT1 may be a new target in diagnosis and therapy of prostate cancer in clinic.

Ablation of Ggnbp2 impairs meiotic DNA double-strand break repair during spermatogenesis in mice.

Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30- and 60-day-old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double-strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co-immunoprecipitated with a testis-enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC-2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC-2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.

Identification of candidate genes that may contribute to the metastasis of prostate cancer by bioinformatics analysis.

To screen for marker genes associated with to the metastasis of prostate cancer (PCa), in silico analysis of the Gene Expression Omnibus dataset GSE27616, which included 4 metastatic and 5 localized PCa tissue samples, was performed. Differentially expressed genes (DEGs) were identified. Their potential functions were identified by Gene Ontology and Kyoto Encyclopedia of Gene Genomes pathway enrichment analyses. Furthermore, protein-protein interaction (PPI) networks for DEGs were constructed using Cytoscape. Module analysis of the PPI networks was performed with Cluster ONE. A total of 561 DEGs were screened, including 208 upregulated and 353 downregulated genes. Proliferating cell nuclear antigen (PCNA) and cluster of differentiation 4 (CD4) exhibited the highest degrees of connectivity in the PPI networks for up- and down-regulated DEGs, respectively. The DEGs in module A, including CD58, 2, 4 and major histocompatibility complex, class II DP-ß1 were enriched in 'cell adhesion molecules'. Anaphase promoting complex subunit 4, cell division cycle 20 and cell division cycle 16 in module B were primarily enriched in 'cell cycle'. The DEGs, including CD4, PCNA and baculoviral IAP repeat containing 5, may have critical roles in PCa metastasis and could thus be used as novel biomarker candidates for metastatic PCa. However, further studies are required to verify these results.

Comparison of miRNA and gene expression profiles between metastatic and primary prostate cancer.

The present study aimed to identify the regulatory mechanisms associated with the metastasis of prostate cancer (PC). The microRNA (miRNA/miR) microarray dataset GSE21036 and gene transcript dataset GSE21034 were downloaded from the Gene Expression Omnibus database. Following pre-processing, differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between samples from patients with primary prostate cancer (PPC) and metastatic prostate cancer (MPC) with |log2 fold change (FC)| >1 and a false discovery rate <0.05 were selected using the Linear Models for Microarray and RNA-seq Data 4 package of R. Next, a DEM-DEG regulatory network was constructed by downloading miRNA-DEG pairs from the miRNA.org database. Finally, functional annotation of each DEM-DEG module was performed using the Database for Annotation, Visualization and Integrated Discovery based on the Gene Ontology database. The upregulated miRNAs, including miR-144, miR-494 and miR-181a, exhibited a higher degree of connections compared with other nodes, including in the DEM-DEG regulatory network, and regulated a number of downregulated DEGs. According to the functional annotation of the DEM-DEG modules, miR-144 and its targeted DEGs enriched the highest number of biological process terms (36 terms), followed by miR-494 (24 terms), miR-30d (18 terms), miR-181a (15 terms), hsa-miR-196a (8 terms), miR-708 (7 terms) and miR-486-5p (2 terms). Therefore, these miRNAs may serve roles in the metastasis of PC cells via downregulation of their corresponding target DEGs.

Relationship between the expression of CD133, HIF-1α, VEGF and the proliferation and apoptosis in hypoxic human prostate cancer cells.

This study measured the levels of expression of CD133, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in human prostate cancer cells grown under hypoxic and non-hypoxic conditions to compare the values to resulting amounts of proliferation and apoptosis in the cells. Human prostate cancer cell line LNCaP cells were routinely thawed, cultured and passaged. Actively growing cells were divided into batches. Cells in the control group were grown under 5% CO2 + 20% O2, and those in the hypoxia group were grown under 5% CO2 + 1% O2. The experiments were performed after 12, 24 and 72 h under each growth condition. The percentages of CD13+ cells were detected by flow cytometry, the expression of HIF-1α and VEGF was detected by western blot analysis, the cell proliferation rate was detected by the MTT assay, and the apoptotic rate was detected by flow cytometry. The results showed that the percentage of CD133+ cells, and the expressions of HIF-1α and VEGF for the cells in the hypoxia group increased gradually from 12 to 24, to 72 h, while there were no equivalent changes in the control group. Cell proliferation in the two groups increased gradually from 12 to 24, to 72 h, but was significantly higher at all time-points in the hypoxia group (p<0.05). There was no significant difference in terms of the amount of apoptotic cells at any of the three different time-points in either group, but the apoptotic cells in the hypoxia group were significantly less than those in the control group at each time-point, and the difference was statistically significant (p<0.05). We conclude that the expression of CD133+, HIF-1α and VEGF in human prostate cancer cells is related to conditions of hypoxia, which ultimately promotes the proliferation and reduces apoptosis in these cells.

Inhibition of prostate cancer RM1 cell growth in vitro by hydroxyapatite nanoparticle­delivered short hairpin RNAs against Stat3.

The present study investigated the effect of signal transducer and activator of transcription 3 (Stat3) interference on RM1 prostate cancer cell viability in vitro, using plasmid­based Stat3 specific short hairpin RNA (sh­Stat3) delivered by hydroxyapatite nanoparticles (HAP). HAP carrying sh­Stat3 plasmids were transfected into tumor cells. MTT assays were used to measure RM1 cell viability 24 and 48 h following transfection, and the apoptosis rate and cell cycle phase distribution were determined by flow cytometry. Stat3 mRNA expression levels were measured by reverse transcription­quantitative polymerase chain reaction and Stat3, Cyclin D1, B cell lymphoma 2 apoptosis regulator (Bcl­2), vascular endothelial growth factor (VEGF), Bcl­2 associated X apoptosis regulator (Bax) and cleaved­caspase­3 protein expression levels were detected using western blot analysis. The results demonstrated that HAP­delivered sh­Stat3 significantly decreased RM1 cell viability through the promotion of cell cycle arrest and apoptosis. Stat3 mRNA and protein expression levels were significantly downregulated in RM1 cells. Bcl­2, VEGF and Cyclin D1 were also significantly downregulated, but cleaved­caspase­3 and Bax mRNA and protein expression levels were significantly upregulated. HAP­delivered sh­Stat3 decreased RM1 cell viability in vitro, and HAP assisted plasmid­based delivery of shRNA into tumor cells. The present results suggest that HAP may be a useful method for successful shRNA delivery into tumors.

Immune analysis of expression of IL-17 relative ligands and their receptors in bladder cancer: comparison with polyp and cystitis.

BACKGROUND: Bladder cancer, cystitis and bladder polyp are the most common urinary system diseases all over the world. Our former research results show that IL-17A and IL-17 F contribute to the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer (Pca) while IL-17E interacting with IL-17RB might have an anti-tumor effect. RESULTS: Using imunohistochemistry, we systemically compared immunoreactivity of ligands (IL-17A, E and F) and receptors (IL-17RA, IL-17RB and IL-17RC) of IL-17 family, infiltration of inflammatory cells and changes of structural cells (fibroblast cells, smooth muscle and vascular endothelial cells) in sections of bladder tissues from subjects with bladder cancer, cystitis and bladder polyp. Compared with subjects with cystitis, immunoreactivity for IL-17A, IL-17 F and IL-17RC was significantly elevated in the group of bladder cancer (p < 0.01), while immunoreactivity of IL-17E, IL-17RA and IL-17RB, and the infiltrating neutrophils were decreased (p < 0.05). The numbers of infiltrating lymphocytes and phagocytes and CD31+ blood vessels and immunoreactivity of CD90+ fibroblasts were also elevated in patients with bladder cancer compared with those of cystitis. The patterns of IL-17 ligands and receptors, and inflammatory cells and structural cells varied in cystitis, bladder polyp and bladder cancer. In bladder cancer, immunoreactivity of IL-17E and IL-17 F was positively correlated with smooth muscles and lymphocytes, respectively. In addition, immunoreactivity of IL-17A and IL-17E was positively correlated with their receptors IL-17RA and IL-17RB respectively. CONCLUSIONS: The data suggest that changed patterns of expression of the IL-17 cytokine family ligands and receptors might be associated with infiltration of inflammatory cells and structural cells (CD90+ fibroblasts and CD31+ blood vessels), which might also contribute to occurrence and development in bladder cancer.

Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling.

Atrazine, a widely used pesticide, is frequently detected in soil and surface water, which alarms epidemiologists and medical professionals because of its potential deleterious effects on health. Indeed, atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. Both animal and human studies have suggested that atrazine is possibly carcinogenic, although discrepant results have been reported. In this study, RM1 cells were used to explore the atrazine effects on prostate cancer. Proliferation, migration and invasion of RM1 cells were assessed by colony formation, wound-healing and invasion assays, respectively, after in vitro exposure to atrazine. In addition, an RM1 cell xenograft model was generated to evaluate the effects of atrazine in vivo. To explore the molecular mechanisms, qRT­PCR, immunohistochemistry, and western blot analyses were employed to detect mRNA and protein levels of STAT3 signaling and cell cycle related proteins, including p53, p21, cyclin B1 and cyclin D1. Interestingly, RM1 cell proliferation was increased after treatment with atrazine, concomitantly with STAT3 signaling activation. These results suggest that atrazine promotes RM1 cell growth in vitro and in vivo by activating STAT3 signaling.

Overexpression of Aquaporin 1 on cysts of patients with polycystic liver disease.

BACKGROUND AND OBJECTIVE: Polycystic liver disease (PCLD) represents a group of genetic disorders that include autosomal dominant polycystic kidney disease (ADPKD) and isolated polycystic liver disease (iPCLD). There is currently no definitive treatment except for liver transplantation. The aim of this study was to assess the expression level of aquaporin 1 (AQP1) on the PCLD cysts with different sizes and provide the potential therapeutic target. METHODS: We collected 3 normal bile ducts, and recruited 8 patients with simple liver cyst disease, 24 patients with ADPKD, and 17 patients with iPCLD. AQP1 expression in different types of cyst walls and in normal bile ducts was detected using real time quantitative PCR, western blot and immunofluorescence staining. We also compared AQP1 expression levels in cysts of different sizes. Besides, ionic concentrations, pH and osmolality of cyst fluid were analyzed. RESULTS: The results showed that AQP1 expression in PCLD cysts was significantly higher than that in simple liver cysts and the normal bile ducts. In addition, a comparable increasing trend was found in cysts of smaller sizes to cysts of larger sizes. pH values, the sodium and chloride concentrations were higher in cyst fluid than that in the serum. CONCLUSIONS: AQP1 was overexpressed in cystic cholangiocytes. A tendency of increased AQP1 protein expression in correlation with the cyst size was also found. These observations offered a direction into the molecular mechanisms of cyst expansion and maybe provide new treatment strategies to reduce fluid secretion into liver cysts.

Overexpression of Aquaporin 1 on cysts of patients with polycystic liver disease

Background and objective: Polycystic liver disease (PCLD) represents a group of genetic disorders that include autosomal dominant polycystic kidney disease (ADPKD) and isolated polycystic liver disease (iPCLD). There is currently no definitive treatment except for liver transplantation. The aim of this study was to assess the expression level of aquaporin 1 (AQP1) on the PCLD cysts with different sizes and provide the potential therapeutic target. Methods: We collected 3 normal bile ducts, and recruited 8 patients with simple liver cyst disease, 24 patients with ADPKD, and 17 patients with iPCLD. AQP1 expression in different types of cyst walls and in normal bile ducts was detected using real time quantitative PCR, western blot and immunofluorescence staining. We also compared AQP1 expression levels in cysts of different sizes. Besides, ionic concentrations, pH and osmolality of cyst fluid were analyzed. Results: The results showed that AQP1 expression in PCLD cysts was significantly higher than that in simple liver cysts and the normal bile ducts. In addition, a comparable increasing trend was found in cysts of smaller sizes to cysts of larger sizes. pH values, the sodium and chloride concentrations were higher in cyst fluid than that in the serum. Conclusions: AQP1 was overexpressed in cystic cholangiocytes. A tendency of increased AQP1 protein expression in correlation with the cyst size was also found. These observations offered a direction into the molecular mechanisms of cyst expansion and maybe provide new treatment strategies to reduce fluid secretion into liver cysts (AU)

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47,XY,+der(X)t(X;18)(p11.4;p11.22): A Unique Aneuploidy Associated with Klinefelter Syndrome due to an Extra Derivative X Chromosome Inherited Maternally.

A derivative X chromosome formed by translocation involving an X chromosome and a chromosome 18 in a Klinefelter syndrome (KS) patient with a 47,XXY karyotype has not been reported before. In this study, we present the clinical and molecular cytogenetic characteristics. The patient presented with small testes and azoospermia. G-banding analysis identified the karyotype as 47,XY,del(X)(p?11.4). Array CGH detected a 10.36-Mb duplication of chromosome region 18p11.22p11.32 (14,316-10,377,516) and a 111.18-Mb duplication of chromosome region Xp11.4q28 (61,931, 689-155,111,583), in addition to the normal chromosome 18 and an X chromosome. FISH results further revealed the extra 18p located at the end of the short arm of a deleted X chromosome, forming a derivative X chromosome. Finally, we identified the karyotype of the patient as 47,XY,+der(X)t(X;18)(p11.4;p11.22). The derivative X chromosome was maternally inherited. To our knowledge, this rare karyotype has not yet been reported in the literature. The present study may suggest a novel karyotype associated with KS.

[Protective effect of lycopene on human spermatozoa during cryopreservation and its mechanism].

OBJECTIVE: To investigate the protective effect of lycopene against cryopreservation injury of post-thawing human sperm and its mechanism. METHODS: Semen samples were collected from 25 volunteers, each sample equally divided into four parts to be cryopreserved with cryoprotectant only (Ly0 control) or cryoprotectant + lycopene at the concentrations of 2 (Ly2), 5 (Ly5), and 10 µmol/L (Ly10), respectively. Before and after thawing, the semen samples were subjected to computer-assisted semen analysis ( CASA) for sperm kinematics, flow cytometry for sperm apoptosis, thiobarbituric acid assay for malondialdehyde (MDA) concentration, and JC-1 fluorescent staining for the sperm mitochondrial membrane potential (MMP). RESULTS: After cryopreservation, sperm motility was markedly decreased in all the groups (P < 0.01). The rate of sperm apoptosis was significantly lower in the Ly5 group than in the Ly0 control ([25.68 ± 4.36]% vs [33.26 ± 4.78]%, P < 0.05), while sperm MMP remarkably higher in the former than in the latter ([66.18 ± 14.23]% vs [55.24 ± 12.31]%, P < 0.05). The Ly2, Ly5 and Ly10 groups showed no statistically significance differences in the MDA level from the Ly0 control (P > 0.05). CONCLUSION: Addition of lycopene at a proper concentration to cryoprotectant may reduce oxidative damage to sperm mitochondria in the freezing-thawing process, attenuate oxidative stress injury induced by reactive oxygen species to sperm plasma membrane, and improve the anti-apoptosis ability of sperm.

Methylselenocysteine preventing castration-resistant progression of prostate cancer.

BACKGROUND: Castration-resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration-resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration-resistant progression of prostate cancer. METHODS: The regrowth of LNCaP prostate cancer xenografts after castration was monitored. The levels of prostate-specific antigen in mouse serum were measured by ELISA. Tumor cell proliferation and apoptosis were analyzed via Ki-67 immunohistochemistry and TUNEL assay, respectively. Intratumoral angiogenesis was assessed by immunohistochemistry staining of vascular endothelial growth factor and CD31. RESULTS: We showed that methylselenocysteine delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation. This was accompanied by decreased serum levels of prostate-specific antigen, inhibition of prostate cancer cell proliferation and tumor angiogenesis, as well as downregulation of androgen receptor and induction of apoptosis in the relapsed tumors. CONCLUSIONS: The present study represents the first to show the preclinical efficacy of methylselenocysteine in delaying castration-resistant progression of prostate cancer. The findings provide a rationale for evaluating the clinical application of combining methylselenocysteine with androgen deprivation therapy for the treatment of advanced prostate cancer.

mTOR inhibitor AZD8055 inhibits proliferation and induces apoptosis in laryngeal carcinoma.

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, against all class I phosphatidylinositol3-kinase (PI3K) and other members of the PI3K-like kinase family. The study was to determine the effect of AZD8055 on proliferation and apoptosis on Hep-2, a human laryngeal cancer cell line and to investigate the underlying mechanism(s) of action. Hep-2 cells were treated with AZD8055 for 24, 48 or 72 h. MTT was used to determine cell proliferation. Rhodamine 123 and TUNEL staining were used to determine mitochondrial membrane potential and cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Protein expressions were examined by western blotting. Treatment with AZD8055 inhibited proliferation and induced apoptosis in Hep-2 cells in a dose- and time-dependent manner. During the prolonged treatment with AZD8055, AZD8055 inhibits the mammalian target of rapamycin mTOR. Further experiments showed which signaling cascade p-4EBP1 and substrate EIF4E as well as downstream proteins were down regulated. Furthermore, our study showed that the expression profiles of various BH3-only proteins including Bid, Bad, and Bim, apoptosis regulatory protein cleaved caspase3 was up regulated in a time-dependent manner in Hep-2 cells treated with AZD8055. Thus, in vitro, AZD8055 potently inhibits proliferation and induces apoptosis in head and neck squamous cell carcinoma.

Delivery of the co-expression plasmid pEndo-Si-Stat3 by attenuated Salmonella serovar typhimurium for prostate cancer treatment.

OBJECTIVES: To investigate the therapeutic utility of an attenuated bacterium carrying a plasmid that co-expresses Endostatin, an inhibitor of tumor neovasculogenesis, and a shRNA that targets Stat3 to suppress prostate cancer growth. METHODS: Plasmid pEndo-Si-Stat3 was constructed and introduced into an attenuated strain of Salmonella enterica serovar typhimurium. The resultant recombinant bacterium was used as a vector to deliver the plasmid to tumor cells growing in vivo. Tumor-associated gene and protein expression changes were measured by using RT-PCR and Western blot analyses. Expression of Endostatin in tumor tissue was detected by ELISA. The presence of vector bacteria in tissues was monitored and tumor destruction was assessed by using TUNEL and H&E staining assays. RESULTS: Bacterially delivered pEndo-Si-Stat3 decreased Stat3 levels and increased Endostatin expression in mouse tumors, resulting in a significant suppression of tumor growth (P < 0.01). Expression of Bcl-2 and PCNA was down-regulated and Caspase3 expression was up-regulated to promote apoptosis of tumor cells. CONCLUSIONS: Successful delivery by attenuated Salmonella of the combination therapeutic plasmid simultaneously knocked down the expression of Stat3 and resulted in over-expression of Endostatin, which synergistically inhibited prostate cancer growth.