Combinational therapy with Myc decoy oligodeoxynucleotides encapsulated in nanocarrier and X-irradiation on breast cancer cells

The Myc gene is the essential oncogene in triple-negative breast cancer(TNBC).This study investigates the synergistic effects of combining Myc decoy oligodeoxynucleotides-encapsulated niosomes-selenium hybrid nanocarriers with X-irradiation exposure on the MDA-MB-468 cell line.Decoy and scramble ODNs for Myc transcription factor were designed and synthesized based on promoter sequences of the Bcl2 gene.The nanocarriers were synthesized by loading Myc ODNs and selenium into chitosan(Chi-Se-DEC),which was then encapsulated in niosome-nanocarriers(NISM@Chi-Se-DEC).FT-IR,DLS,FESEM,and hemolysis tests were applied to confirm its characterization and physicochemical properties.Moreover,cellular uptake,cellular toxicity,apoptosis,cell cycle,and scratch repair assays were performed to evaluate its anticancer effects on cancer cells.All anticancer assessments were repeated under X-ray irradiation conditions(fractionated 2Gy).Physicochemical characteristics of niosomes containing SeNPs and ODNs showed that it is synthesized appropriately.It revealed that the anticancer effect of NISM@Chi-Se-DEC can be significantly improved in combination with X-ray irradiation treatment.It can be concluded that NISM@Chi-Se-DEC nanocarriers have the potential as a therapeutic agent for cancer treatment,particularly in combination with radiation therapy and in-vivo experiments are necessary to confirm the efficacy of this nano-drug.

Treatment of Hemangioma by Transfection of Antisense VEGF Gene

The effect of transfection of antisense vascular endothelial growth factor （VEGF） gene on the growth of hemangioma was studied. A total of 49 cases of capillary hemangiomas of the skin were collected. Immunohistochemical method was used to detect the expression of PCNA in hemangioma tissues. According to the finding, 49 cases of hemangiomas fell into proliferating phase （27 cases） and involuting phase （22 cases） respectively. Another 5 cases of normal skin tissues adjacent to the tumor tissues served as control. Immunohistochemical staining was performed to detect the expression of VEGF in the tumor tissues and the normal tissues. The average absorbance （A） values and the average positive area rate of VEGF were measured by image analysis system （HPIAS-2000）. Endothelial cells from the tumor tissues in proliferating phase were cultured. Eukaryotic expression vector was constructed by sub-cloning, and transfected into human hemangioma endothelial cells by using cation liposome as vector. The expression of VEGF mRNA and protein was detected by RT-PCR and indirect immunofluorescence assay （IFA）, respectively, and the biological characteristics of the transfected endothelial cells were examined by MTT assay and flow cytometry （FCM） after transfection. Immunohistochemical results showed that the expression of VEGF in proliferating endothelial cells was remarkably higher than those in involuting endothelial cells and normal endothelial cells （P〈0.01）, but there was no significant difference in the expression of VEGF between involuting endothelial cells and normal ones （P〉0.01）. Electrophoresis and sequencing indicated that the eukaryotic expression vector containing antisense VEGF gene, i.e. pcDNA3.1-VEGF, was success- fully constructed. After VEGF antisense RNA recombinant was transfected into hemangioma endothelial cells, RT-PCR revealed that the expression of VEGF mRNA in pcDNA-VEGF （V） group and blank group was obviously higher than that in pcDNA-VEGF （A） group, and that the expression of endogenous VEGF mRNA in pcDNA-VEGF （A） group was significantly inhibited. Immunohistochemical result demonstrated that, compared with blank group, there was statistically significant difference between pcDNA-VEGF （A） and pcDNA-VEGF （V） groups （P〈0.01）, but there was no significant difference between pcDNA-VEGF （V） group and blank group （P〉0.05）. The activity of endothelial cell proliferation was reduced significantly after transfection, and obvious apoptosis occurred in hemangioma endothelial cells after transfection of antisense VEGF. It was suggested that VEGF plays an important role in the pathological change of hemangiomas by promoting endothelial cell proliferation and angiogenesis. Antisense VEGF gene transfection could effectively inhibit the growth of hemanioma endothelial cells.

Local Delivery of C-myc Antisense Oligodeoxynucleotide by Gelatin Coated Platinium-Iridium Stent to Prevent Restenosis in a Normal Rabbit Carotid Artery

Objectives To investigate the feasibility and effect of local deliveryof c-myc antisense oligodeoxynucleotide (ASODN) by gelatin coated Platinium-Iridium stent to prevent restenosis in a normal rabbit carotid artery. Methods Gelatin coated Platinium-Iridium stent were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomized to the control group and the treated group receiving c-myc ASODN (n=16 respectively).7, 14, 30,90 days following the stenting procedure, morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical methods. Results 32 stents were successfully implanted into the right carotid arteries in 32 animals.Morphometric analysis showed that neointimal area and mean neointimal thickness siginificantly increased continuously up to 12 weeks after stent implantation,and at each time point , neointimal area and mean neointimal thickness were siginificantly smaller in the treated group than control group. (P<0.001,respectively).c-myc protein expression was weak positive or negative in treated group and positive in control group. Conclusions Gelatin coated Platinium-Iridium stent mediated local delivery of c- myc ASODN is feasibility , and it can inhibit neointimal hyperplasia to prevent restenosis in a normal rabbit carotid artery.

TELOMERASE: A NOVEL TARGET OF ANTITUMOR AGENTS

Telomerase activity was found to be high in various human cancers, but absent in most normal tissues. Its expression pattern made it a novel target for antitumor agents. Several strategies against telomerase were presented in this review. Targeting the telomerase RNA component by oligonucleotide/ribozyme was considered to be one of the most hopeful approaches. Some progresses were made in this area, such as the use of PANs and 2–5A antisense compounds. The relationships among telomerase activity and cell differentiation, signal transduction, oncogene, tumor suppressor gene as well as cell cycle modulation also provided a series of valuable ideas in designing anti-telomerase drugs for cancer therapy. In conclusion, although there is still a long way in understanding the mechanism and regulation of telomerase, the advance of studies on telomerase has allowed the development of numerous strategies for the treatment of cancer.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

Posttranscriptional Control of Gene Expression and Role of Small RNAs in Streptococcus mutans

Streptococcus mutansis a key contributor to the formation of biofilms associated with dental caries disease. Living in the oral environment and developing of disease require tight gene regulation. In bacteria, gene expression is most commonly regulated at the level of transcription initiation. This control involved interactions of specific DNA sequences with regulatory proteins. A second mechanism of control of gene expression is mediated at the RNA level by several mechanisms and is generally called posttranscriptional regulation. These mechanisms include cis- and trans-acting small, non-coding RNAs, RNA-binding proteins, riboswitches, thermosensors, RNases, and Type I toxin-antitoxin systems, and may result in changes in RNA stability, efficiency of ribosome binding, translation initiation, and transcript secondary structures. Here I review the posttranscriptional regulation in S. mutans.

Inhibitory effect of retroviral vector containing anti-sense Smad_4 gene on Ito cell line, LI90

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy