Trilostane, a 3ß-hydroxysteroid dehydrogenase inhibitor, suppresses growth of hepatocellular carcinoma and enhances anti-cancer effects of sorafenib.

Background Human 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) is an enzyme associated with steroidogenesis, however its' role in hepatocellular carcinoma (HCC) biology is unknown. Trilostane is an inhibitor of HSD3B1 and has been tested as a treatment for patients with breast cancer but has not been studied in patients with HCC. Methods and Results The expression of HSD3B1 in HCC tumors in 57 patients were examined. A total of 44 out of 57 tumors (77.2%) showed increased HSD3B1 expression. The increased HSD3B1 in tumors was significantly associated with advanced HCC. In vitro, the knockdown of HSD3B1 expression in Mahlavu HCC cells by a short hairpin RNA (shRNA) led to significant decreases in colony formation and cell migration. The suppression of clonogenicity in the HSD3B1-knockdown HCC cells was reversed by testosterone and 17ß-estradiol. Trilostane-mediated inhibition of HSD3B1 in different HCC cells also caused significant inhibition of clonogenicity and cell migration. In subcutaneous HCC Mahlavu xenografts, trilostane (30 or 60 mg/kg, intraperitoneal injection) significantly inhibited tumor growth in a dose-dependent manner. Furthermore, the combination of trilostane and sorafenib significantly enhanced the inhibition of clonogenicity and xenograft growth, surpassing the effects of each drug used alone, with no documented additional toxicity to animals. HSD3B1 blockade was found to suppress the phosphorylation of extracellular signal-regulated kinase (ERK). The decreased ERK phosphorylation was reversed by testosterone or 17b-estradiol. Conclusions Trilostane significantly inhibited the growth of HCC by inhibiting HSD3B1 function and augmenting the efficacy of sorafenib.

A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma.

Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carcinoma (HCC). However, the therapeutic effect of sorafenib is transient, and patients invariably develop sorafenib resistance (SR). Recently, TYRO3, a member of the TYRO3-AXL-MER family of receptor tyrosine kinases, was identified as being aberrantly expressed in a significant proportion of HCC; however, its role in SR is unknown. In this study, we generated two functionally distinct sorafenib-resistant human Huh-7 HCC cell lines in order to identify new mechanisms to abrogate acquired SR as well as new potential therapeutic targets in HCC. Initially, we investigated the effects of a microRNA (miR), miR-7-5p (miR-7), in both in vitro and in vivo preclinical models of human HCC and identified miR-7 as a potent tumor suppressor of human HCC. We identified TYRO3 as a new functional target of miR-7, which regulates proliferation, migration, and invasion of Huh-7 cells through the phosphoinositide 3-kinase/protein kinase B pathway and is markedly elevated with acquisition of SR. Furthermore, miR-7 effectively silenced TYRO3 expression in both sorafenib-sensitive and sorafenib-resistant Huh-7 cells, inhibiting TYRO3/growth arrest specific 6-mediated cancer cell migration and invasion. CONCLUSION: We identified a mechanism for acquiring SR in HCC that is through the aberrant expression of the TYRO3/phosphoinositide 3-kinase/protein kinase B signal transduction pathway, and that can be overcome by miR-7 overexpression. Taken together, these data suggest a potential role for miR-7 as an RNA-based therapeutic to treat refractory and drug-resistant HCC. (Hepatology 2018;67:216-231).

Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer.

BACKGROUND: Protein tyrosine kinase 2 (PTK2) expression has been reported in various types of human epithelial cancers including lung cancer; however, the role of PTK2 in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) has not been elucidated. We previously reported that pemetrexed-resistant NSCLC cell line PC-9/PEM also acquired EGFR-TKI resistance with constitutive Akt activation, but we could not find a therapeutic target. METHODS: Cell viability in EGFR-mutant NSCLC cell lines was measured by the WST-8 assay. Phosphorylation antibody array assay for receptor tyrosine kinases was performed in PC-9 and PC-9/PEM cell lines. We evaluated the efficacy of EGFR and PTK2 co-inhibition in EGFR-TKI-resistant NSCLC in vitro. Oral defactinib and osimertinib were administered in mice bearing subcutaneous xenografts to evaluate the efficacy of the treatment combination in vivo. Both the PTK2 phosphorylation and the treatment combination efficacy were evaluated in erlotinib-resistant EGFR-mutant NSCLC cell lines. RESULTS: PTK2 was hyperphosphorylated in PC-9/PEM. Defactinib (PTK2 inhibitor) and PD173074 (FGFR inhibitor) inhibited PTK2 phosphorylation. Combination of PTK2 inhibitor and EGFR-TKI inhibited Akt and induced apoptosis in PC-9/PEM. The combination treatment showed improved in vivo therapeutic efficacy compared to the single-agent treatments. Furthermore, erlotinib-resistant NSCLC cell lines showed PTK2 hyperphosphorylation. PTK2 inhibition in the PTK2 hyperphosphorylated erlotinib-resistant cell lines also recovered EGFR-TKI sensitivity. CONCLUSION: PTK2 hyperphosphorylation occurs in various EGFR-TKI-resistant NSCLCs. Combination of PTK2 inhibitor and EGFR-TKI (defactinib and osimertinib) recovered EGFR-TKI sensitivity in the EGFR-TKI-resistant NSCLC. Our study result suggests that this combination therapy may be a viable option to overcome EGFR-TKI resistance in NSCLC.

Mono- and Polyassociation Processes of Pentavalent Biotinylated PEI Glycopolymers for the Fabrication of Biohybrid Structures with Targeting Properties.

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.

Effects of microRNA-1271 on ovarian cancer via inhibition of epithelial-mesenchymal transition and cisplatin resistance.

AIM: Efficacy of platinum based-chemotherapy is limited by cisplatin (DDP) resistance, however, the underlying mechanism of cisplatin resistance remains unclear. We aimed to investigate the role of miR-1271 in cisplatin-resistant ovarian cancer cells. METHODS: Transfection of miR-1271 mimic and inhibitor was performed to study the role of miR-1271 in ovarian cancer. Cell viability was assessed by Cell Counting Kit (CCK)-8 assay. Flow cytometry was used to determine the apoptosis rates. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect mRNA and protein expressions. Target predicted by Targetscan7.2 was confirmed by dual-luciferase activity assay. Mammalian target of rapamycin (mTOR) siRNA (simTOR) co-transfection was performed to verify the role of mTOR in the suppression effect of miR-1271 on ovarian cancer. RESULTS: In SKOV3 cells, miR-1271 overexpression significantly decreased cell viability and up-regulated apoptosis rate (from 5.54% of control to 24.03%). MiR-1271 adversely affected SKOV3 cell migration and invasion, and induced the upregulation of E-cadherin and downregulation of N-cadherin and alpha-smooth muscle actin (α-SMA). Moreover, SKOV3/DDP cells had a lower miR-1271 level, and enhancing miR-1271 contributed strongly to cisplatin-induced apoptosis through altering the expressions of B-cell lymphoma-2 associated X protein (BAX), cleaved caspase-3 and B-cell lymphoma 2 (Bcl-2). In contrast, the opposite result was observed in miR-1271 inhibitor. mTOR was identified to be a target of miR-1271. SimTOR partially reversed the increased cell viability under the effect of miR-1271 inhibitor. CONCLUSION: Our data indicate that miR-1271 can inhibit the ovarian cancer epithelial-mesenchymal transition (EMT) and sensitize resistant cells to cisplatin-induced apoptosis through blocking mTOR expression.

Anti-angiogenic effects of CD73-specific siRNA-loaded nanoparticles in breast cancer-bearing mice.

CD73 facilitates tumor growth by upregulation of the adenosine (immunosuppressive factor) in the tumor microenvironment, however, its precise molecular mechanisms is not precisely understood. Regarding the importance of angiogenesis in tumor development and spreading, we decided to assign the anti-angiogenic effects of CD73 suppression. We used chitosan lactate (ChLa) nanoparticles (NPs) to deliver CD73-specific small interfering RNA (siRNA) into cancer cells. Our results showed that treatment of the 4T1 cells with CD73-specific siRNA-loaded NPs led to potent inhibition of cancer cell proliferation and cell cycle arrest, in vitro. This growth arrest was correlated with downregulation of angiogenesis-related molecules including vascular endothelial growth factor (VEGF)-A, VEGF-R2, interleukin (IL)-6, and transforming growth factor (TGF)-ß. Moreover, administration of NPs loaded with CD73-siRNA into 4T1 breast cancer-bearing mice led to tumor regression and increased mice survival time accompanied with downregulation of angiogenesis (VEGF-A, VEGF-R2, VE-Cadherin, and CD31) and lymphangiogenesis (VEGF-C and LYVE-1)-related genes in the tumor site. Furthermore, the expression of angiogenesis promoting factors including IL-6, TGF-ß, signal transducer, and activator of transcription (STAT)3, hypoxia inducible factor (HIF)-1α, and cyclooxygenase (COX)2 was decreased after the CD73 suppression in mice. Moreover, analysis of leukocytes derived from the tumor samples, spleen, and regional lymph nodes showed that they had lower capability for secretion of angiogenesis promoting factors after CD73-silencing. These results indicate that suppression of tumor development by downregulation of CD73 is in part related to angiogenesis arrest. These findings imply a promising strategy for inhibiting tumor growth accompanied with suppressing the angiogenesis process.

Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.

Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs.

One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma-Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin™), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a ∼100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs.

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2).

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200µM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3h of exposure, returning to normality at 24h. Additionally, BZL increased glutathione peroxidase activity at 12h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.

Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis.

[Effect of CD40 siRNA on inflammatory response of MRL/Lpr mice].

OBJECTIVE: To observe the effect of CD40 siRNA on expression of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE) animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice. METHODS: In the study, 16 female MRL/Lpr mice were randomly divided into control group (n=4), empty vector group (n=4), CD40-siRNA1 group (n=4) and CD40-siRNA2 group (n=4). The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice, while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively. The injection was given six times and every one day. The mice were sacrificed 14 d after injection, and the spleen tissue was weighed. The pGFP-V-RS was labeled by green fluorescent protein (GFP) and the tissue sections were observed whether siRNA expressed in the spleen. The expression levels of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd, 5th, 8th, 11th, and 14th days after last injection, and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method. RESULTS: The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr. The spleens in CD40-siRNA1 group [(78.85±5.61) mg] and CD40-siRNA2 group [(80.25±4.07) mg] were lower than those in control [(141.88±7.81) mg] and empty vector group [(153.10±7.60) mg]. The levels of IL-17, IFN-γ and anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd, 5th and 8th days after last injection than on the 1st day before the first time (P<0.05). The levels of IFN-γ in CD40-siRNA1 group were (118.74±10.32) ng/L, (115.24±8.26) ng/L and (113.71±5.02) ng/L in turn, the levels of IFN-γ in CD40-siRNA2 group were (117.83±6.83) ng/L, (114.07±0.97) ng/L and (112.67±9.66) ng/L in turn. The levels of IL-17 in CD40-siRNA1 group were (7.05±0.41) ng/L, (6.34±0.76) ng/L and (5.83±0.43) ng/L in turn, the levels of IL-17 in CD40-siRNA2 group were (7.07±0.22) ng/L, (6.35±0.49) ng/L and (6.12±0.80) ng/L in turn. The levels of anti-dsDNA antibody in CD40-siRNA1 group were (7.51±0.29) ng/L, (6.74±0.45) ng/L and (6.32±0.39) ng/L in turn, the levels of anti-dsDNA antibody in CD40-siRNA2 group were (8.19±0.38) ng/L, (7.14±0.50) ng/L and (6.48±0.29) ng/L in turn. The levels of IL-4 in CD40-siRNA1 group were (26.51±1.81)ng/L (27.80±1.72) ng/L and (28.08±2.21) ng/L in turn, the level of IL-4 in CD40-siRNA2 group were (26.28±2.03) ng/L, (28.15±2.95) ng/L and (28.37±1.71) ng/L in turn. The expression levels of IL-17 and IFN-γ antibody increased gradually and the levels of IL-4 decreased gradually in CD40-siRNA1 group and CD40-siRNA2 group on the 11th and 14th days after last injection, then reached to the levels of control group and empty vector group (P>0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day, there was more significance than those in control group and empty vector group (P<0.05). There was no significance between the 4 groups on the 14th day. The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P<0.05). CONCLUSION: CD40-siRNA can reduce the concentration of IL-17, IFN-γ and of anti-dsDNA antibody in serum, and at the same time, it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr. Meanwhile after suppressing CD40 mRNA and protein, it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr, suggesting that CD40-siRNA has therapy effect on SLE.

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

UNLABELLED: Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/"duct-like" structures, which was blocked by treatment with JTE-013. CONCLUSION: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.

Sevoflurane promotes the expansion of glioma stem cells through activation of hypoxia-inducible factors in vitro.

BACKGROUND: Growing evidences indicate that inhalational anaesthetics can enhance the growth and malignant potential of tumour cells and may affect tumour recurrence after surgery. Tumour stem cells play a vital role in tumour recurrence. This study investigates the effect of sevoflurane on glioma stem cells (GSCs) in vitro and the underlying molecular mechanisms in this process. METHODS: Cultured GSCs were exposed to clinically relevant concentrations and durations of sevoflurane exposure. Cell proliferation and self-renewal capacity were determined. Expression of the stem cell marker CD133, vascular endothelial growth factor (VEGF), hypoxia-inducible factors (HIFs), and phosphorylated Akt, which is a protein kinase invoved in multiple cellular processes, were measured using western blotting. Small interfering RNAs and an Akt inhibitor were used to investigate specific pathways. RESULTS: Compared with controls, cells exposed to 2% sevoflurane for 6 h induced a larger number of proliferated cells (31.2±7.6% vs 19.0±5.8%; P<0.01). Levels of CD133, VEGF, HIF-1α, HIF-2α, and p-Akt were up-regulated by sevoflurane in a time- and concentration-dependent manner. Small interfering RNA against HIFs decreased the percentage of proliferating GSCs after sevoflurane exposure and pre-treatment of cells with an Akt inhibitor abrogated the expression of HIFs induced by sevoflurane. CONCLUSIONS: Sevoflurane can promote the expansion of human GSCs through HIFs in vitro. Inhaled anaesthetics may enhance tumour growth through tumour stem cells.

Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1.

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl2-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8 %). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.

Using adenovirus armed short hairpin RNA targeting transforming growth factor ß1 inhibits melanoma growth and metastasis in an ex vivo animal model.

The transforming growth factor ß (TGF-ß) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-ß can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-ß provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-ß1-RNA interference (RNAi) which mediated the RNAi for TGF-ß1 gene silencing. We examined the effects of TGF-ß1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-ß1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-ß1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-ß1 expression was reduced in B16F0/TGF-ß1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-ß1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFß1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-ß1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-ß1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-ß1-RNAi tumors. The blood vessel density of the tumors markedly reduced in B16F0/TGF-ß1-RNAi tumors. Our results showed that Ad/TGF-ß1-RNAi could induce silencing of the TGF-ß1 gene effectively. Silencing of TGF-ß1 expression in B16F0 cells by RNAi technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. This kind of adenoviral vector based on RNAi might be a promising vector for cancer therapy.

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove.

Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration.

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

Clusterin contributes to hepatitis C virus-related hepatocellular carcinoma by regulating autophagy.

AIMS: To explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). MAIN METHODS: Patients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. KEY FINDINGS: DGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. SIGNIFICANCE: CLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.

Anticancer effect of celecoxib via COX-2 dependent and independent mechanisms in human gastric cancers cells.

Cyclooxygenase-2 (COX-2) inhibitors cause growth inhibition of human gastric carcinoma cells, but it remains unclear whether this is both COX-2 dependent and independent. The related mechanisms remain to be determined. Both low COX-2 expressing gastric carcinoma and high COX-2 expressing gastric carcinoma cells were used to study the effect and mechanisms of celecoxib on gastric carcinoma cell growth. Celecoxib resulted in comparable growth inhibition in AGS cells with stable transfections of small interfering RNA (siRNA) against COX-2 (SAC) and negative control vector (NC) cells. Simultaneously, celecoxib resulted in significant reduction of Bcl-2 and significant increase of p21(WAF1) and p27(KIP1) in SAC and NC cells. The present study shows that celecoxib causes growth inhibition of gastric carcinoma cells by decreasing Bcl-2 of cyclooxygenase-2-dependent pathway, and by increasing p21(WAF1) and p27(KIP1) of cyclooxygenase-2-independent pathway. These data extend our knowledge on the effect and mechanisms of celecoxib-induced inhibition of gastric carcinoma cell growth.

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells.

Exposure to environmental tobacco smoke (ETS) is a known risk factor for the development of chronic lung diseases, cancer, and the exacerbation of viral infections. Extracellular vesicles (EVs) have been identified as novel mediators of cell-cell communication through the release of biological content. Few studies have investigated the composition/function of EVs derived from human airway epithelial cells (AECs) exposed to cigarette smoke condensate (CSC), as surrogates for ETS. Using novel high-throughput technologies, we identified a diverse range of small noncoding RNAs (sncRNAs), including microRNA (miRNAs), Piwi-interacting RNA (piRNAs), and transfer RNA (tRNAs) in EVs from control and CSC-treated SAE cells. CSC treatment resulted in significant changes in the EV content of miRNAs. A total of 289 miRNAs were identified, with five being significantly upregulated and three downregulated in CSC EVs. A total of 62 piRNAs were also detected in our EV preparations, with five significantly downregulated and two upregulated in CSC EVs. We used TargetScan and Gene Ontology (GO) analysis to predict the biological targets of hsa-miR-3913-5p, the most represented miRNA in CSC EVs. Understanding fingerprint molecules in EVs will increase our knowledge of the relationship between ETS exposure and lung disease, and might identify potential molecular targets for future treatments.