Ze-Zhang Tao

Regulation of telomerase activity by apparently opposing elements

Telomeres, the ends of chromosomes, undergo frequent remodeling events that are important in cell development, proliferation and differentiation, and neoplastic immortalization. It is not known how the cellular environment influences telomere remodeling, stability, and lengthening or shortening. Telomerase is a ribonucleoprotein complex that maintains and lengthens telomeres in the majority of cancers. Recent studies indicate that a number of factors, including hormones, cytokines, ligands of nuclear receptor, vitamins and herbal extracts have significantly influence telomerase activity and, in some instances, the remodeling of telomeres. This review summarizes the advances in understanding of the positive and negative regulation by extracellular factors of telomerase activity in cancer, stem cells and other systems in mammals.

Effect of blocking VEGF, hTERT and Bcl-xl by multiple shRNA expression vectors on the human laryngeal squamous carcinoma xenograft in nude mice

In our previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human telomerase reverse transcriptase (hTERT) messenger RNA, and we demonstrated that these vectors could inhibit the growth of cancer cells by 76.5% in vivo. In order to maximize the efficiency and versatility of the vector-based siRNA approach, here we have constructed multiple shRNA expression vectors that simultaneously targeted 3 different genes in cancer cells, and then investigated their effect on human laryngeal squamous carcinoma (Hep-2) in vivo. Materials and methods Short hairpin RNA expression vectors targeting the mRNA of VEGF, hTERT and Bcl-xl were constructed and subsequently transfected by direct injections into the tumors formed by Hep-2 cells implanted in nude mice. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. Results We found that expression of multiple shRNAs led to a significant reduction in VEGF, hTERT and Bcl-xl RNA and protein expression. Tumor growth curves showed that those tumors treated with the shRNAs were obviously smaller than control, non-transfected tumors. Analysis at 14 days following the final injection showed a tumor inhibition ratio of 91.2%. However, the control shRNA vectors showed none of these effects. Conclusions Our results demonstrate that the application of vector-based RNAi technology that involves blocking multiple targets will be a promising therapeutic modality in the gene therapy of human cancers. The data strongly suggest that simultaneously blocking multiple genes in human cancers using an RNAi approach should be considered in cancer therapy.

Application of combination of short hairpin RNA segments for silencing VEGF, TERT, and Bcl-xl expression in laryngeal squamous carcinoma

Objective To develop an RNA-interference (RNAi) approach involving the hitting of multiple targets by a recombinant plasmid and evaluate its antitumor effect on laryngeal squamous carcinoma in vitro and in vivo. Materials and methods A plasmid containing 3 different short hairpin RNA (shRNA) segments termed pEGFP-shVEGF-shTERT-shBcl-xl was constructed. Plasmids containing single shRNA against each target (VEFG, TERT, BCL-xl alone) individually were also constructed as control. Cells were treated with these plasmids. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. The mRNA and protein expression were determined by RT-PCR and western blotting. Cell viability was examined using the MTT assay. Apoptotic morphological alterations were observed by Hoechst staining and electron microscopy. The in vivo antitumor effect was characterized in a nude mice model of laryngeal squamous carcinoma. Results. we demonstrated that a recombinant plasmid containing multiple shRNAs could effectively and simultaneously inhibit VEGF, TERT and Bcl-xl mRNA and protein expression in the HEp-2 cells; the plasmid containing the 3 different shRNAs exhibited a potent antitumor effect on LSCC both in vitro and in vivo, and could much more effectively induced cell apoptosis than each single shRNA.. We also demonstrated that the simultaneous blockage of these 3 genes have a better inhibitory effect on human HEp-2 cells than the blockage of each single shRNA. Conclusions Our study demonstrates that the application of vector-based RNAi technology that involves hitting multiple targets will be a promising therapeutic modality in the gene therapy of human laryngeal cancers; further, it provides experimental evidence for the clinical application of this technology in the future.

Suppression of the notch signaling pathway by γ-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation

Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch-3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC.

Intranasal Administration of Lentiviral miR-135a Regulates Mast Cell and Allergen-Induced Inflammation by Targeting GATA-3

Mast cell (MC) degranulation is the foundation of the acute phase of allergic rhinitis (AR). Previously, downregulation of GATA binding protein 3 (GATA-3) was shown to suppress MC activation in an AR mouse model. Binding of microRNA-135a (miR-135a) to GATA-3 was also observed, and overexpression of this miRNA decreased GATA-3 mRNA and protein expression. However, the effects of miR-135a on MCs during AR are currently unknown. In the present study, we utilized a lentiviral (LV) vector to intranasally administer miR-135a to ovalbumin (OVA)-sensitized AR mice. Following miR-135a treatment, the total serum IgE concentration observed during AR was significantly reduced. In the nasal mucosa, the expression of T-box expressed in T cells (T-bet) was higher, whereas that of GATA-3 was lower in the AR mice following miRNA treatment. Notably, during AR, the ratio of type 1 T-helper cells (Th1) to type 2 (Th2) cells in the spleen is unbalanced, favoring Th2. However, administering miR-135a to the AR mice appeared to balance this ratio by increasing and decreasing the percentage of Th1 and Th2 cells, respectively. MiR-135a also appeared to strongly suppress the infiltration of eosinophils and MCs into the nasal mucosa, and it was specifically localized in the MCs, suggesting that its influence is modulated through regulation of GATA-3 in these cells. Additional work identifying the full therapeutic potential of miR-135a in the treatment of AR and diseases involving allergen-induced inflammation is warranted.

Protective effect of melatonin against gentamicin ototoxicity.

OBJECTIVE To research the protective effect of melatonin against gentamicin ototoxicity. METHODS Guinea pigs were randomly divided into four groups. The first group received intramuscular gentamicin (120 mg/kg body weight/day) for 17 days. Over the same time period, a second group simultaneously received intramuscular gentamicin (120 mg/kg body weight/day) plus (on the other side) intramuscular melatonin (0.3 ml kg body weight/day). Two groups of controls were treated for 17 days with either intramuscular melatonin or intramuscular saline. After the 17 days, each animal underwent distortion product otoacoustic emission testing (both ears). The guinea pigs were sacrificed by decapitation just after the final injection. Their cochleae were used to produce a tissue section, surface preparation and scanning electron microscope preparation. RESULTS Distortion product otoacoustic emission testing indicated gentamicin-induced hearing loss at 3, 4, 6 and 8 kHz in gentamicin-treated animals. Animals receiving melatonin co-therapy had significantly attenuated hearing loss and their cochleae showed lower rates of outer hair cell loss (comparing the same cochlear turns), compared with gentamicin-treated animals (p < 0.01). CONCLUSION These findings confirm the occurrence of outer hair cell loss after gentamicin treatment, and the attenuation of such loss following simultaneous melatonin injection, using the method of morphological evaluation. These results suggest that melatonin protects against gentamicin ototoxicity by interfering with cytotoxic mechanisms.

miR-512-5p suppresses tumor growth by targeting hTERT in telomerase positive head and neck squamous cell carcinoma in vitro and in vivo.

Telomerase activation has very important implications for head and neck squamous cell carcinoma (HNSCC), but the regulatory mechanisms of telomerase in HNSCC remain unclear. In our present study, we found that miR-512-5P was markedly downregulated in telomerase-positive HNSCC cell lines. Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation. Furthermore, the dual-luciferase reporter gene assay results demonstrated that hTERT was a direct target of miR-512-5P. We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC. Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

Alternative lengthening of telomeres in hTERT‐inhibited laryngeal cancer cells

In most human malignancies, telomere homeostasis is maintained by the reactivation of telomerase. While inhibiting telomerase provides a novel approach to the treatment of many cancers, telomere maintenance can occur in the absence of telomerase activity by the alternative lengthening of telomeres (ALT) mechanism. Therefore, it must be determined if inhibiting telomerase selects for cancer cells that activate ALT. Here, we report that Hep‐2 cells that survived anti‐telomerase treatments showed sustained proliferation in culture with down‐regulated human telomerase reverse transcriptase (hTERT) expression and significantly enhanced levels of ALT‐specific promyelocytic leukemia (PML) bodies. Analysis of the telomere lengthening kinetics also demonstrated elevated telomeric sister‐chromatid exchange (T‐SCE) in surviving Hep‐2 cells, consistent with their long and heterogeneous telomeres. Similar to ALT cells, the surviving cells showed evidence of ALT telomere homeostasis. Furthermore, proteomic analysis identified several proteins differentially expressed between the untreated Hep‐2 cells and surviving cells that may provide new insight for understanding these two telomere maintenance mechanisms. Thus, the findings in this study may help to improve telomerase‐based therapy for cancer. (Cancer Sci 2010)

Association between single nucleotide polymorphisms of surfactant protein D and allergic rhinitis in Chinese patients

The development of allergic rhinitis is considered to be determined by the interaction between genetic and environmental factors. Surfactant protein D (SP-D) has been proposed to offer protection against allergenic challenge at various levels in allergic responses. The present study aimed to investigate whether polymorphisms within the SFTPD gene (Met11Thr, Ala160Thr, and Ser270Thr) are associated with allergic rhinitis. Genotyping of SFTPD polymorphisms was performed using the pyrosequencing method. The study population comprised 216 patients with allergic rhinitis and 84 normal controls. The frequency of 11Thr/Thr genotype and Thr allele in the patient group was significantly higher than that in the control group after applying Bonferroni corrections (P = 0.007 and P = 0.006, respectively). Our subjects with the 11Thr/Thr genotype are more susceptible to allergic rhinitis. There were no significant differences between the patient group and the control group for frequencies of genotypes and alleles in either Ala160Thr or Ser270Thr single nucleotide polymorphisms (P > 0.05). No significant associations could be detected between any of these three SFTPD gene polymorphisms and the skin prick test response (P > 0.05). Meanwhile, there was a lack of association between the three loci and the levels of serum total immunoglobulin E (P > 0.05). In summary, our results suggest that the Met11Thr polymorphism in SP-D plays a major role in the genetic predisposition to allergic rhinitis in Chinese adult population, whereas the other two SP-D polymorphisms displayed no significant association with allergic rhinitis.

Indole-3-Carbinol Inhibits Nasopharyngeal Carcinoma Growth through Cell Cycle Arrest In Vivo and In Vitro

Nasopharyngeal carcinoma is a common malignant tumor in the head and neck. Because of frequent recurrence and distant metastasis which are the main causes of death, better treatment is needed. Indole-3-carbinol (I3C), a natural phytochemical found in the vegetables of the cruciferous family, shows anticancer effect through various signal pathways. I3C induces G1 arrest in NPC cell line with downregulation of cell cycle-related proteins, such as CDK4, CDK6, cyclin D1 and pRb. In vivo, nude mice receiving I3C protectively or therapeutically exhibited smaller tumors than control group after they were inoculated with nasopharyngeal carcinoma cells. The expression of CDK4, CDK6, cyclin D1 and pRb in preventive treatment group and drug treatment group both decreased compared with the control group. We conclude that I3C can inhibit the growth of NPC in vitro and in vivo by suppressing the expression of CDK and cyclin families. The drug was safe and had no toxic effects on normal tissues and organs.