Design, synthesis and biological evaluations of niclosamide analogues against SARS-CoV-2.

Niclosamide, a widely-used anthelmintic drug, inhibits SARS-CoV-2 virus entry through TMEM16F inhibition and replication through autophagy induction, but the relatively high cytotoxicity and poor oral bioavailability limited its application. We synthesized 22 niclosamide analogues of which compound 5 was found to exhibit the best anti-SARS-CoV-2 efficacy (IC50 = 0.057 µ M) and compounds 6, 10, and 11 (IC50 = 0.39, 0.38, and 0.49 µ M, respectively) showed comparable efficacy to niclosamide. On the other hand, compounds 5, 6, 11 contained higher stability in human plasma and liver S9 enzymes assay than niclosamide, which could improve bioavailability and half-life when administered orally. Fluorescence microscopy revealed that compound 5 exhibited better activity in the reduction of phosphatidylserine externalization compared to niclosamide, which was related to TMEM16F inhibition. The AI-predicted protein structure of human TMEM16F protein was applied for molecular docking, revealing that 4'-NO2 of 5 formed hydrogen bonding with Arg809, which was blocked by 2'-Cl in the case of niclosamide.

In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide.

BACKGROUND: Current vaccination strategies and antiviral drugs only provide limited protection against influenza virus infection. In this study, we investigated the use of a novel antisense oligonucleotide (named IV-AS), which is specific for the 5'-terminal conserved sequence found in all eight viral RNA segments of influenza A virus. METHODS: The activity of IV-AS was monitored both in vitro, in Madin-Darby canine kidney (MDCK) cells, and in vivo using a mouse model. IV-AS was given intranasally to H5N1-infected mice once daily for 6 days starting 6 h after infection. A three-base mismatch of IV-AS was used as a control. RESULTS: IV-AS inhibited influenza virus A induced cytopathic effects in MDCK cells with the 50% effective concentration (EC50) ranging from 2.2 to 4.4 microM. IV-AS was effective against H5N1 virus in preventing death, lessening weight reduction, inhibiting lung consolidation and reducing lung virus titres. Dosages of 40 and 60 mg/kg/day provided 40% and 60% survival rates and prolonged mean survival days in comparison with the infected control group (P<0.05). The lung index in mice treated with IV-AS, at a dose of 20, 40 or 60 mg/kg/day, had been inhibited on day 4 or 6 (P<0.05 or P<0.01); virus titres in lung had declined to 2.42, 1.51 and 1.54 log10 TCID50/g of lung, respectively, whereas the yields in the infected control mice were 6.00 log10 TCID50/g of lung. CONCLUSIONS: Our results suggest that the 5'-terminal conserved region of influenza A virus RNA segments can be targeted using antisense technology; therefore, IV-AS is a potential drug for prophylaxis and control of influenza virus infections.

Inhibition of hepatocellular carcinoma growth by antisense oligonucleotides to type I insulin-like growth factor receptor in vitro and in an orthotopic model.

AIM: The type I insulin-like growth factor receptor (IGF-IR) is overexpressed in many tumors including human hepatocellular carcinoma (HCC). It is a critical signaling molecule for tumor cell proliferation and survival. In the present study, IGF-IR expression was down-regulated by phosphorothioate antisense oligonucleotides (AS[S]ODN) to evaluate their specific effects on growth of hepatoma cells in vitro and in vivo. METHODS: HepG2 cells were transfected with different doses of AS[S]ODN, sense [S]ODN, mismatch [S]ODN, or Lipofectin for 72 h, and cell proliferation was analyzed by MTS assay. In vivo, an orthotopic transplant model of HCC was established in nude mice, which were then injected with AS[S]ODN, sense [S]ODN, 5-fluorouracil or saline. At the endpoint of treatment, the tumors were excised and evaluated. RESULTS: Compared to sense and mismatched oligonucleotides, AS[S]ODN targeting to IGF-IR mRNA significantly inhibited hepatoma cell lines HepG2 proliferation and IGF-IR expression at both mRNA and protein levels. The in vivo results showed that systemic treatment also resulted in significant inhibition in tumor growth. Tumor growth in mice treated with AS[S]ODN (50 and 75 mg/kg per day) was significantly inhibited (71.81% and 61.74%, respectively) compared to the saline-treated group (P < 0.01) in a dose-dependent manner. The antitumor effect of IGF-IR AS[S]ODN was associated with down-regulation of IGF-IR in tumor xenografts. Furthermore, IGF-IR AS[S]ODN prevented liver recurrence tumor growth and metastasis in the lung, showing a dose-dependent response. The level of serum alpha-fetoprotein in AS[S]ODN-treated groups was also decreased in a dose-dependent manner, and a good correlation was observed between tumor volume and serum alpha-fetoprotein concentration. CONCLUSIONS: These data suggest that IGF-IR AS[S]ODN can effectively and specifically inhibit HCC growth in vitro and in vivo. Blockage of IGF-IR expression could be a promising therapeutic approach for the management of patients with HCC.

Microarray analysis of differentially expressed genes in mouse bone marrow tissues after ionizing radiation.

PURPOSE: To identify differentially expressed genes in mouse bone marrow involved in radiation-induced injury. MATERIALS AND METHODS: Microarray analysis was used to identify the differentially expressed genes and other techniques, e.g., polymerase chain reaction (PCR), western-blotting and antisense were also used to validate the results. RESULTS: DNA microarray analysis demonstrated that the mRNA of 34 genes increased and 69 genes decreased in mouse bone marrow cells (BMC) from C57BL mice 6 h after a whole body dose of 6.5 Gy. These differentially expressed genes were involved in a number of processes including DNA replication/repair, proliferation/apoptosis, cell cycle control and RNA processing. In these experiments, a decline of the mammalian homolog Sir2a (an acronym for the silent mating type information regulation 2 homolog [SIRT1]) mRNA accompanied by an increase of P53 protein acetylation was observed in irradiated BMC. To determine whether the reduced SIRT1 is related to the higher acetylation status of P53 after irradiation, we designed and synthesized antisense oligonucleotides (AS) targeting human SIRT1 mRNA. Notably, AS transfection increased tumor protein 53 (P53) protein acetylation and bax-luciferase activity in human bone marrow stromal cell line (HS-5) after radiation. Furthermore, the AS transfer stimulated cell apoptosis in post-irradiation HS-5 cells. CONCLUSION: Ionizing radiation (IR) affects the expression of a series of genes including genes involved in G1/S transition and the P53 pathway. Among those, reduction of SIRT1 was seen to be involved in transactivation of P53.

[Screening and their anti-tumor activity of antisense oligodeoxynucleotides targeting KDR mRNA in breast cancer MCF-7 cells].

OBJECTIVE: To screen the antisense oligodeoxynucleotides (asONs) which could hybridize with KDR (kinase insert domain-containing receptor) mRNA in an effective and specific way and to explore their anti-tumor effects on breast cancer MCF-7 cell line in vitro. METHODS: The asONs were firstly selected using oligodeoxynucleotides library hybridization or computer prediction, then their hybridization ability with KDR mRNA was further tested with oligonucleotide microarray. The asONs with strong hybridization intensity were selected. Their inhibitory effects on MCF-7 cells proliferation and KDR expression were assayed by MTT, RT-PCR and Western blotting assay, respectively. RESULTS: In 13 asONs selected with oligodeoxynucleotides library hybridization, 8 (8/13, 61.5%) showed strong hybridization signals, while such was only 1 in 17 asONs designed by computer prediction. 9 asONs with strong hybridization intensity were selected and synthesized with phosphorothioated modification. All these asONs inhibited the MCF-7 cells proliferation in a dose-dependent manner, in which asON4 and asON7 screened by oligodeoxynucleotides library in combination with oligonucleotide microarray were the most effective, with inhibitory rates of 51.6% and 62.2% at 0.8 micromol/L, respectively. The KDR expression at mRNA and protein levels was reduced by both the two asONs, in a dose-dependent manner. CONCLUSION: asONs screened by oligodeoxynucleotides library hybridization are well consistent with that chosen with oligonucleotide microarray. The combination of oligodeoxynucleotides library with oligonucleotide microarray is an effective approach of asONs screening. The asONs targeting KDR mRNA showed prominent anti-tumor activity on breast cancer MCF-7 cells.

Identification of differentially expressed genes contributing to radioresistance in lung cancer cells using microarray analysis.

Radiotherapy has played a key role in the control of tumor growth in many cancer patients. It is usually difficult to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. The response of tumor cells to radiation is believed to be accompanied by complex changes in the gene expression pattern. It may be possible to use these to sensitize radioresistant tumor cells and improve radiocurability. Based on the biological effects of ionizing radiation, in the present study, we developed one oligonucleotide microarray to analyze the expression of 143 genes in cells of two lung cancer cell lines with different radiosensitivities. Compared to NCI-H446 cells, expression of 18 genes significantly increased the basal levels in the radioresistant A549 cells, in which eight genes were up-regulated and 10 genes were down-regulated. In A549 cells irradiated with 5 Gy, 22 (19 up-regulated and three down-regulated) and 26 (eight up-regulated and 18 down-regulated) differentially expressed genes were found 6 and 24 h after irradiation, respectively. In NCI-H446 cells, the expression of 17 (nine up-regulated and eight down-regulated) and 18 (six up-regulated and 12 down-regulated) genes was altered 6 and 24 h after irradiation, respectively. RT-PCR was performed, and we found that MDM2, BCL2, PKCZ and PIM2 expression levels were increased in A549 cells and decreased in NCI-H446 cells after irradiation. Genes involved in DNA repair, such as XRCC5, ERCC5, ERCC1, RAD9A, ERCC4 and the gene encoding DNA-PK, were found to be increased to a higher level in A549 cells than in NCI-H446 cells. Antisense suppression of MDM2 resulted in increased radiosensitivity of A549 cells. Taken together, these results demonstrate the possibility that a group of genes involved in DNA repair, regulation of the cell cycle, cell proliferation and apoptosis is responsible for the different radioresistance of these two lung cancer cells. This list of genes may be useful in attempts to sensitize the radioresistant lung cancer cells.

Combined effects of Cantide and chemotherapeutic drugs on inhibition of tumor cells' growth in vitro and in vivo.

AIM: To investigate the combination effect of hTERT antisense oligonucleotide "Cantide" and three chemotherapeutic drugs (cisplatin, 5-fluorouracil (5-FU) and adriamycin (ADM)) on inhibiting the proliferation of HepG2, BGC and A549 cell lines in vitro, and to investigate the efficacy of Cantide used in combination with cisplatin (DDP) in vivo. METHODS: Cantide was transfected into these tumor cells by Lipofectin, and cell growth activity was calculated by microcytotoxicity assay. In vivo study, cells of HepG2 were implanted in Balb/c nude mice for 4 d. Then Cantide, DDP and Cantide+DDP were given intraperitoneally for 24 d respectively. The body weights of the tumor-bearing animals and their tumor mass were measured later to assess the effect of combination therapy in the nude mice. To evaluate the interaction of Cantide and these chemotherapeutic drugs, SAS software and Jin Zhengjun method were used. RESULTS: Combination treatments with 0.1 micromol/L Cantide reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29 microg/mL to 0.25, 1.52 and 0.12 microg/mL respectively. The inhibition ability of DDP, 5-FU and ADM respectively in combination with Cantide in these tumor cells was higher than that of these drugs alone (P<0.0001). And synergism (Q > or = 1.15) was observed at the lower concentration of DDP (< or = 1 microg/mL), 5-FU (< or = 10 microg/mL) and ADM (< or = 0.1 microg/mL) with combination of Cantide. In vivo, combination treatment with Cantide and DDP produced the greater growth inhibition of human liver carcinoma cells HepG2 in nude mice (0.65+/-0.19 g tumor) compared with that when only one of these drugs was used (Cantide group: 1.05+/-0.16 g tumor, P = 0.0009<0.001; DDP group: 1.13+/-0.09 g tumor, P = 0.0001<0.001). CONCLUSION: These findings indicate that Cantide may enhance therapeutic effectiveness of chemotherapeutic drugs over a wide range of tumor cells in vitro, and the combination use of Cantide and DDP can produce much higher inhibition rates, as compared with when either of these drugs was used only in vivo.

Effective siRNA targets screening for human telomerase reverse transcriptase.

AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence. METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4 sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000. At 48-72 h after siRNAs transfection, MTT assay, RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively. RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2 cells. CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method, synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

Proteomic analysis of ionizing radiation-induced proteins at the subcellular level.

Irradiation induces a series of liver diseases. However the molecular mechanisms involving in the process of liver diseases induced by irradiation are still unclear. Subcellular proteomics provides a method to understand regional differences in protein expression levels. With accumulating evidence in the literature that new proteins are implicated in radiation response, in the present study, C57BL/6 mice were treated with irradiation, liver cell homogenates were subfractionated by differential ultracentrifugation into nuclei, mitochondria and cytosol, which were subjected to 2-DE to generate the proteomic maps of these fractions. The differentially expressed proteins in the nuclei, mitochondria and cytosol compartment of liver at 24 and 48 h after exposure to 20 Gy irradiation compared to control were identified by MALDI-TOF MS respectively. Total 37 proteins at 24 h and 29 proteins at 48 h were matched with known proteins after database searching in nuclei, mitochondria and cytosol, respectively, among which nine proteins exhibited changes at both time points. Most of these proteins are involved in antioxidant response, energy metabolism, molecular chaperones and inflammatory response. More antioxidant-associated proteins were induced at 48 h than 24 h. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting further validated 2-DE results of two of these proteins. It is feasible that the differential proteins identified in this study have a biological significance and may provided clues for understanding the mechanism of injury in liver induced by irradiation.

SIRT1 interacts with p73 and suppresses p73-dependent transcriptional activity.

The tumor suppressor p53-related p73 shares significant amino-acid sequence identity with p53. Like p53, p73 recognizes canonical p53 DNA-binding sites and activates p53-responsive target genes and induces apoptosis. Moreover, SIRT1 binds to p53 while repressing the expression of their target genes. Here, we report that SIRT1 also binds to p73 and suppresses p73-dependent transcriptional activity. SIRT1 in human cells reduces the transcriptional activity of p73, and partly inhibits apoptosis induced by p73. Furthermore, SIRT1 can deacetylate p73 protein acetylation both in vivo and in vitro. Collectively, these data suggest that SIRT1 can modulate p73 activity via deacetylation.

Inhibition of tumor growth and metastasis with antisense oligonucleotides (Cantide) targeting hTERT in an in situ human hepatocellular carcinoma model.

AIM: To evaluate the in vivo antitumor effects of Cantide and the combined effect with 5-fluorouracil. METHODS: An in situ human hepatocellular carcinoma model was established in mice livers orthotopically. Drugs were administered intravenously and tumor sizes were monitored with calipers. Plasma alpha-fetoprotein(AFP) were detected by radiation immunoassay. Morphology of tumors was evaluated by hematoxylin-eosin (H and E) staining of histological sections. Human telomerase reverse transcriptase (hTERT) protein levels were detected by Western blotting. RESULTS: Cantide significantly inhibit in situ human hepatocellular carcinoma growth in mice with a 75 and 50 mg.kg(-1).d(-1) administration of Cantide compared to the saline group in a dose-dependent manner, which included injecting Cantide 25 mg.kg(-1).d(-1) by iv for 20 d after surgically removing the tumor in liver. Cantide was also found to prevent tumor recurrence in the liver and metastasis in the lung, showing a dose-dependent response. When Cantide was administered by iv combined with 5-fluorouracil, it resulted in a significant reduction in tumor growth compared to either agent alone treatment group. After the treatment with Cantide alone or combined with 5-fluorouracil, plasma AFP concentration decreased in a dose-dependent manner. CONCLUSION: These results demonstrated that Cantide was an effective antitumor antisense oligonucleotide in vivo and has the potential to be developed into a clinical anti-cancer drug.

Antisense oligodeoxynucleotides targeting the serine/threonine kinase Pim-2 inhibited proliferation of DU-145 cells.

AIM: To investigate the effect of antisense oligodeoxynucleotides (ASODN) targeting Pim-2 on cell proliferation of DU-145 cells. METHODS: Three ASODN targeting Pim-2 were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, Pim-2 mRNA, protein levels, and cell cycles were examined. RESULTS: The ASODN designed and synthesized by our laboratory significantly reduced Pim-2 mRNA level and protein content in DU-145 cells. After transfection with ASODN for 48 h, a marked reduction in cell viability was observed in DU-145 cells in a dose-dependent manner. No remarkable apoptosis occurred in cells treated with ASODN compared with control cells. However, it should be noted that G1 phase arrest was clearly observed in ASODN-treated cells. CONCLUSION: ASODN targeting Pim-2 resulted in a marked reduction in DU-145 cell proliferation, and induction of G1 phase cell cycle arrest is one of the important mechanisms for ASODN to reduce cell growth. Moreover, antisense inhibition of Pim-2 expression provides a new promising therapy target for prostate cancer.