Effect of transformer noise on the neurophysiology of SD rats.

Transformer noise is a type of environmental sound that causes discomfort to individuals. The aim of the present study was to determine the effect of relatively long-term periods of transformer noise on the behavior and neurophysiology of SD rats. A total of 90 healthy SD rats with normal hearing were randomly divided into two experimental groups (65 and 60 dB group) and a control group. The experimental groups were exposed to recorded transformer noise for 8 weeks (sound level limits: 65 or 60 dB) and the control group was maintained under the same conditions without noise stimulation. Changes in physiological growth (weight tests), behavior (tail suspension and open field behavior tests) and neurophysiology (glutamate, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, the morphologies of hippocampi) following noise exposure were recorded and compared. The results revealed that rats exhibited normal physiological growth, with no significant difference between the experimental and control groups. Following noise exposure, no significant differences were observed in the results of behavioral experiments (tail suspension and open field behavior tests) between the experimental and control groups. In addition, there were no significant differences in glutamate, γ-aminobutyric acid, dopamine and 5-hydroxytryptamine levels or in the morphologies of hippocampi between groups. In conclusion, exposure to transformer noise with a sound level limit of 65 dB sound pressure level (SPL) or 60 dB SPL (spectral range, 100-800 Hz) for 8 weeks (10 h/day) had no significant impact on the behavior and neurophysiology of SD rats.

Estimation of the status of spiral ganglion neurons and Schwann cells in the auditory neural degeneration mouse using the auditory brainstem response.

CONCLUSION: The auditory brainstem response (ABR) wave I threshold, latency and amplitude are insensitive to spiral ganglion neurons (SGNs) degeneration, but are sensitive to the degeneration of Schwann cells and can estimate the status of Schwann cells in a neural degeneration mouse model. The thorough pre-operative ABR assessment would be helpful in predicting cochlear implant performance. OBJECTIVES: This study aimed in finding a non-invasive electrophysiological method to evaluate the status of the auditory nerve and the Schwann cells in sensorineural hearing loss (SNHL) and auditory neuropathy (AN) ears, and providing useful information for candidates screening and outcome prediction in cochlear implantation. METHODS: The frequency-specific acoustic ABR was recorded in mice. The immunohistochemical staining was performed to detect the SGNs and Schwann cells in mice cochlea. The correlations between ABR wave I metrics and SGNs, Schwann cells were investigated. RESULTS: In SNHL and AN mice cochlea, statistically significant correlations between ABR wave I thresholds, latencies and amplitudes at 8, 16, and 32 kHz and their corresponding SGNs densities were found only in wave I amplitude at 8 kHz. While the ABR wave I metrics at all three frequencies showed strong significant correlations with their corresponding Schwann cells densities.

Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea.

OBJECTIVE: This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL). METHODS: Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells. RESULTS: Ouabain at the dosages of 0.5 mM, 1 mM and 3 mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3 mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure. CONCLUSIONS: The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.

Effect of silencing key proteins in telomerase mechanism and alternative lengthening of telomeres mechanism in laryngeal cancer cells.

PURPOSE: To explore the influences of telomerase and alternative lengthening of telomeres mechanism on telomere length and laryngeal squamous cell carcinoma in vitro and in vivo. MATERIALS AND METHODS: Short hairpin RNA expression vectors targeting the messenger TERT, TRF2, RAD51 and NBS1 were constructed. The mRNA and protein expression of targeted genes in human laryngeal squamous carcinoma cell line HEp-2 was evaluated by reverse transcription polymerase chain reaction and Western blotting separately. The length of telomere was analyzed by fluorescent in-situ hybridization. Cell viability was examined by cell counting Kit-8. Effects on tumor growth were also investigated in vivo. RESULTS: The transfection of multiple short hairpin RNAs expression plasmid significantly inhibited the mRNA and protein expression of related genes. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes resulted in the shortening of telomere length in HEp-2 cell. However, silence of alternative lengthening of telomeres mechanism related genes could shorten the telomere length but had no significant difference. Both simultaneously and separately blocking telomerase mechanism and alternative lengthening of telomeres mechanism resulted in reduction of tumor cell viability. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes inhibited the tumor growth in vivo. CONCLUSIONS: The inhibition of telomere related gene may be a promising strategy for the treatment of laryngeal squamous cell carcinoma.

Phenethyl isothiocyanate induces apoptosis and inhibits cell proliferation and invasion in Hep-2 laryngeal cancer cells.

The dietary compound phenethyl isothiocyanate (PEITC), an important tumoricidal component found in cruciferous vegetables, exhibits strong anticancer and chemopreventive effects in a variety of tumors. However, its role in human laryngeal cancer is unclear. The aim of the present study was to investigate whether PEITC exhibits anticancer properties in human laryngeal carcinoma Hep-2 cells in vitro and to identify the potential molecular mechanisms. The results showed that treatment of Hep-2 cells with PEITC significantly inhibited cell proliferation in a dose- and time-dependent manner, promoted apoptosis with concurrent G2/M cell cycle arrest and inhibited cell invasion in a dose-dependent manner. These effects were accompanied by significant alterations in the expression levels of key proteins associated with pro-survival signaling pathways, including PI3K, Akt, ERK, NF-κB, Bcl, Bax, cyclin B, CDK4 and CDK6. Importantly, these effects were not reflected in 16HBE normal human bronchial epithelial cells, suggesting a safe range of treatment concentrations between 0 and 10 µM PEITC. In summary, PEITC exhibited significant anticancer effects against human laryngeal cancer cells in vitro with low toxicological impact on normal bronchial epithelial cells. This was achieved through dysregulation of key proteins involved in the occurrence and development of tumors, thereby offering a valuable contribution to future strategies for the treatment and screening of patients with laryngocarcinoma.

ERK signaling mediates long-term low concentration 3,3'-diindolylmethane inhibited nasopharyngeal carcinoma growth and metastasis: An in vitro and in vivo study.

It is well known that crucifers have antitumor effects and 3,3'-diindolylmethane (DIM) is one of the major bioactive components, and the associated molecular mechanisms in a short-term high-dose manner are widely discussed. However, the antitumor effects of DIM in a long-term low-dose manner in nasopharyngeal carcinoma (NPC) has not been reported yet, as to the potential mechanisms in the human body. In the present study, NPC cells were induced by 20 µmol/l DIM for over a month, and the proliferation, apoptosis, migration and in vivo metastasis were investigated. The results showed that DIM significantly reduced the proliferation and migration; however, changes in apoptosis were not observed. In vivo study showed the metastasis was significantly reduced. Compared to the short-term high-dose manner, incomplete similar qualities were observed; next we explored the possible signal pathway revolved, the ERK signaling showed similar changes, while the PI3K/Akt, NF-κB, P38, JNK pathways were significantly altered in the short-term high-dose manner (our previous study) showed no obvious change, indicating the ERK signaling may be the main effector of DIM.

Pro-apoptotic and anti-proliferative effects of 3,3'-diindolylmethane in nasopharyngeal carcinoma cells via downregulation of telomerase activity.

The pro-apoptotic and anti-proliferative effects of 3,3'-diindolylmethane (DIM) in various tumor cell types have been widely investigated. The underlying mechanisms were suggested to include cell cycle arrest, cell signaling inhibition and downregulation of the androgen receptor. The present study demonstrated that DIM induced apoptosis and inhibited proliferation in nasopharyngeal carcinoma cells by downregulating the activity of telomerase. The nasopharyngeal carcinoma cell line 5­8F was selected for this purpose. A cell counting kit­8 assay and flow cytometry were performed to detect apoptosis and proliferation of 5­8F cells, respectively, which revealed the pro­apoptotic and anti­proliferative effects of DIM. Telomerase activity was detected using a telomeric repeat amplification protocol assay, which revealed that the telomerase activity was inhibited by DIM in a dose­dependent manner. Reverse transcription polymerase chain reaction was used to detect the mRNA expression levels of human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR), and western blot analysis was used to detect the protein expression of hTERT. The results showed that the mRNA and protein expression of hTERT were downregulated in 5­8F cells following treatment with DIM; however, the mRNA expression of hTR remained unchanged, suggesting that hTERT was the target of DIM. To further identify the target, the length of telomeres was continually measured using a telomere length detection kit, revealing that the telomeres were shortened by DIM in an concentration­dependent manner. The present study confirmed that DIM had pro­apoptotic and anti­proliferative effects in nasopharyngeal carcinoma cells by regulating telomerase.

Nuclear translocation of telomerase reverse transcriptase is a critical process in lymphatic metastasis of nasopharyngeal carcinoma.

Telomerase reverse transcriptase (TERT) is the predominant functional unit of telomerase and maintains the telomere length and the stability of chromosomes. Recently, TERT has been shown to be a critical factor in a number of other biological processes, including cell proliferation and cancer metastasis. In addition, although numerous studies have been conducted, the subcellular localization of the TERT protein and the association of such with cancer metastasis remains unclear. To investigate the involvement of TERT in in vivo metastasis, quantum dots-based immunofluorescence and western blot analysis were conducted to detect changes in the subcellular localization of TERT in human nasopharyngeal carcinoma (NPC) tissues and metastatic lymph nodes. To further investigate, metastatic and non-metastatic models of NPC were generated using 5-8F (high metastasis capability) and 6-10B (low metastasis capability) cell lines, respectively. It was found that TERT protein was overexpressed in NPC tissue samples and metastatic lymph nodes and TERT was predominantly located in the cytoplasm of primary NPC tissues, while TERT was predominantly located in the nucleus of the metastatic lymph nodes. The ratio of cytoplasmic TERT/nuclear TERT for the primary tumor of the 6-10B cell line was almost six-fold higher than that of the metastatic lymph nodes of the 5-8F cell line. TERT translocation from the cytoplasm to nucleus may present a critical step in the lymphatic metastasis of NPC. Thus, TERT translocation may be more useful than TERT expression level and telomerase activity for predicting the metastasis of NPC.

Subtelomeric demethylation deregulated hTERT expression, telomerase activity, and telomere length in four nasopharyngeal carcinoma cell lines.

Global DNA hypomethylation, in particular that of the gene promoter sequence in gene hypermethylation, is a well-known characteristic of human cancer. Subtelomeres are enriched CpG islands; methylation is believed to be a potential epigenetic regulator. However, regulation on the telomere length remains largely unknown. To demonstrate this correlation, four nasopharyngeal carcinoma cell lines (CNE, CNE1, CNE2, 5-8F) were treated for 72 hours with 0, 1, or 2.5 µM of the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Subtelomeric (D4Z4) level methylation was evaluated with a bisulfite assay, the human telomerase catalytic subunit (hTERT) expression was assayed by reverse transcription-polymerase chain reaction, the telomerase activity was detected using a telomeric repeat amplification protocol assay, and the telomere length was measured by Southern blot terminal restriction fragment analysis. There was significant demethylation following 5-aza-dC treatment, and a strongly repressed hTERT expression decreased the telomerase activity and remarkably shortened telomeres. Thus, partial subtelomeric methylation does not repress hTERT expression; conversely, demethylation may downregulate hTERT expression and shorten telomeres.

The efficacy of traditional Chinese Medicine as an adjunctive therapy in nasopharyngeal carcinoma: a systematic review and meta-analysis.

PURPOSE: The purpose of this systematic review was to assess the efficacy of traditional Chinese Medicine (TCM) as an adjunctive therapy to radiotherapy (RT) and/or chemotherapy (CT) for patients with nasopharyngeal carcinoma (NPC). METHODS: Randomized controlled trials (RCTs) with TCM to treat NPC were extensively searched in eight databases. Two researchers independently assessed the quality and validity of the included trials and extracted outcome data. Thirteen RCTs were included for analysis. RESULTS: Compared to using RT and/or CT, TCM combined with conventional cancer therapy had significantly improved Karnofsky performance status (KPS) [odds ratio (OR) 4.81, 95% confidence interval (CI) 3.06-7.56]. TCM as an adjunctive therapy significantly reduced the serious adverse effects of RT to the oral mucosa and skin so that grade I+II prevailed [OR 2.19, 95% CI 1.31-3.66; OR 8.63, 95% CI 3.28-22.70, respectively]. The combined therapy significantly enhanced immunoregulation, improving the levels of CD3, CD4 T cells (OR 10.08, 95% CI 1.38-18.78; OR 7.08, 95% CI 2.41-11.74, respectively). CONCLUSIONS: This systematic review suggests that TCM as a therapy adjunctive to RT and/or CT vs only RT and/ or CT has significant efficacy in terms of improvement of quality of life (QoL), alleviation of acute adverse effects, and enhancement of immunoregulation.

Exogenous interleukin-10 alleviates allergic inflammation but inhibits local interleukin-10 expression in a mouse allergic rhinitis model.

BACKGROUND: Interleukin-10 (IL-10) has an important anti-inflammatory and immunoregulatory function, and its expression is negatively correlated with the development and severity of allergic rhinitis (AR). However, the in vivo effects of exogenous IL-10 on AR have not been studied and the mechanisms underlying the effects of IL-10 have not been fully understood. Here, we investigated the effects of intranasal administration of recombinant mouse (rm) IL-10 on the expression of Th responses and local IL-10 in a mouse model of AR induced by ovalbumin. RESULTS: Administration of rmIL-10 during challenge significantly reduced the number of eosinophils and mast cells, as well as Type 2 helper T (Th2) and Th17 cell related cytokine and transcription factor levels in the nasal mucosa and nasal lavage fluid in AR mice. The rmIL-10 treatment significantly inhibited the number of IL-10-positive cells and IL-10 mRNA expression in the nasal mucosa in AR mice. CONCLUSION: Our results show that exogenous IL-10 administrated in challenge phase alleviates nasal allergic inflammation in AR mice, most likely by inhibiting Th2 and Th17 responses. It can also inhibit local IL-10 levels in the nasal mucosa. Our findings indicate that IL-10 may have the potential as an inhibitor of AR.

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.

Telomerase reverse transcriptase promotes the proliferation of human laryngeal carcinoma cells through activation of the activator protein 1.

TERT is the main functional unit of telomerase, which maintains telomere length and chromosome structure stability. TERT has been shown to act as a key factor in various biological processes, such as cell proliferation, via uncharacterized mechanisms. We transfected HEp-2 laryngeal carcinoma cells with a TERT overexpressing adenovirus (Ad-TERT) and TERT shRNA silencing adenovirus (Ad-sh-TERT), and examined the effect on TERT and the AP-1 transcription factor subunits c-Fos and c-Jun using RT-PCR and western blot analysis. TERT mRNA expression was quantified using RT-PCR in 24 human laryngeal carcinoma samples, and TERT protein co-expression with AP-1 was investigated in a human laryngeal carcinoma tissue microarray using quantum-dot based immunofluorescence. The effect of specific ERK and p38 inhibitors on ERK, p38, c-Jun and c-Fos phosphorylation was investigated in TERT-overexpressing HEp-2 cells. TERT overexpression led to increased TERT, c-Jun and c-Fos mRNA and protein expression and increased cell proliferation, while TERT silencing had the opposite effects. TERT mRNA expression levels were positively correlated with c-Fos and c-Jun mRNA in human laryngeal carcinoma tissue. TERT and AP-1 protein were expressed at high levels and positively correlated in laryngeal carcinoma tissues. Treatment of TERT-overexpressing HEp-2 cells with specific p38 and ERK inhibitors indicated that TERT modulates the expression and phosphorylation of the AP-1 subunits c-Jun and c-Fos through the p38 and ERK signaling pathways. In conclusion, the results of this study indicate that TERT is capable of promoting cell proliferation via activation of the AP-1 subunits, c-Jun and c-Fos, in laryngeal carcinoma cells.

Indole-3-carbinol inhibits cell proliferation and induces apoptosis in Hep-2 laryngeal cancer cells.

Indole-3-carbinol (I3C) is an active component of cruciferous vegetables and markedly inhibits the growth of a variety of tumors. However, its role in laryngeal cancer remains obscure. The aim of the present study was to elucidate the possible mechanisms whereby I3C influences Hep-2 laryngeal cancer cell proliferation and apoptosis. Treatment with I3C dose-dependently and significantly inhibited Hep-2 cell proliferation, and at doses of 100 and 150 µM, I3C induced cell morphological changes and promoted apoptosis. Following treatment of Hep-2 cells with I3C, we found that the protein expression of phosphatidylinositol-3-kinase (PI3K) p110α, PI3K p110ß, PI3K class III, p-PDK1, Akt, p-Akt and the downstream signaling proteins p-c-Raf and GSK3-ß were significantly downregulated. Additionally, tumor-bearing mouse models were constructed using BALB/c nude mice. The mice were subdivided into groups: pretreated with I3C, or treated with I3C or an untreated control group. After 8 weeks, mice pretreated or treated with IC3 developed smaller tumors compared to the untreated control group, and the protein expression of PI3K p110α, PI3K class III, Akt, p-Akt and the downstream signaling proteins p-c-Raf and GSK3-ß in the tumors were significantly downregulated. Furthermore, no harmful side effect were observed in the heart, liver and kidney of the I3C-treated nude mice. In conclusion, I3C inhibited proliferation and induced the apoptosis of laryngeal tumor cells both in vivo and in vitro, and exhibited low toxicity to normal cells. The inhibitory effects noted with I3C treatment may depend on decreased phosphatidylinositol-3 kinase/serine-threonine kinase (PI3K/Akt) expression. This approach may be applied to the clinical treatment of laryngeal tumors and in drug screening.

In vivo and in vitro study on the role of 3,3'-diindolylmethane in treatment and prevention of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is characterized by insidious progression and atypical early symptoms and mostly diagnosed in middle or late stages. The long-term prognosis is poor after conventional radiotherapy or chemotherapy, and the therapy often has strong toxic effects on normal tissue and organs. There were in vitro and in vivo preclinical evidences to support chemotherapeutic and chemopreventive effects of 3,3'-diindolylmethane (DIM), which was a natural compound extracted from cruciferous plants. The in vitro experiments showed that 100 µM DIM could induce remarkable apoptosis of NPC cells, and no obvious damage was observed in normal human bronchial epithelial cells and human liver cells (P < 0.01). DIM could simultaneously regulate several signaling pathways directly related to NPC, such as PI3K, MAPK, Akt and NF-κB. In animal model, the volume of transplanted tumor in animals raised by feed containing DIM was significantly less than that of control group (P < 0.01). The animals with 2 weeks of preventive feeding with feed containing DIM before inoculation had the smallest volume of transplanted tumor (P < 0.01). Intake of DIM had no toxic effects on vital organs such as heart, liver and kidney of experimental animals. In summary, the results of this study confirmed that the DIM effectively induced apoptosis of NPC cells, and had a preventive and curative role in the development and progression of NPC. The drug was safe and had no toxic effects on normal tissues and organs.

[Investigation of skin prick test on 2707 patients with allergic rhinitis in Wuhan area].

OBJECTIVE: To investigate the distribution of common allergens in patients with allergic rhinitis (AR) from 2006 to 2010 in Wuhan area, and provide the objective evidence for the prevention and treatment of AR. METHODS: The medical records of skin prick test (SPT) performed on 2707 AR patients from 2006 to 2010 were retrospectively analysed, and the positive rate of different allergens and changing trends in this time were compared. SPSS 17.0 software was used to analyse the data. RESULTS: There were significant differences among the Dermatophagoides pteronyssinus positive rate (χ(2) = 12.11, P < 0.05) and Dermatophagoides farinae positive rate (χ(2) = 11.11, P < 0.05) in the past 5 years. Meanwhile, there was an upward trend in the positive rate of dust mite, which the positive rate of Dermatophagoides pteronyssinus increased from 84.5% in 2006 to 90.5% in 2010 (χ(2) = 6.88, P < 0.05), positive rate of Dermatophagoides farinae increased from 81.5% in 2006 to 89.0% in 2010 (χ(2) = 9.68, P < 0.05); There were significant differences among the Mugwort and Ragweed positive rate of 5 years (χ(2) = 194.10, P < 0.05; χ(2) = 67.06, P < 0.05). There were significant differences among the mold I and mold II positive rate of 5 years between (χ(2) = 18.95, P < 0.05; χ(2) = 36.62, P < 0.05). Meanwhile, there was an upward trend in the positive rate of mold and fluctuant trend in the positive rate of spring-pollen. CONCLUSIONS: Nearly five years, dust mites is still the most common allergens in AR patients, presenting upward trend; the positive rate of mold presenting upward trend; the positive rate of wormwood and guinea wood presenting downward trend; the positive rate of pollen presenting fluctuant trend.

γ-secretase inhibition combined with cisplatin enhances apoptosis of nasopharyngeal carcinoma cells.

The Notch signaling pathway plays an important role in the proliferation and differentiation of cells. Although recent studies have shown that Notch plays a role in the mechanisms of cisplatin resistance, the mechanism by which Notch plays roles in intrinsic or acquired cisplatin resistance remains unclear. In the present study, poorly differentiated nasopharyngeal carcinoma cells were treated with a γ-secretase inhibitor (DAPT), which led to a decrease in the Notch intracellular domain and inhibition of Notch signaling. Treatment was not sufficient to induce pronounced apoptosis of CNE-2 cells, but did result in the down-regulation of the P-glycoprotein and ERCC1 protein. In contrast, the combined treatment of DAPT and cisplatin induced substantial cell apoptosis compared to cisplatin treatment alone.

Deletion of Forkhead Box M1 transcription factor reduces malignancy in laryngeal squamous carcinoma cells.

The transcription factor, Forkhead Box M1 (FoxM1), has a specific expression pattern during the cell cycle. It also plays an important role in cellular developmental pathways and in the maintenance of homeostasis between cell proliferation and apoptosis. However, the precise role and molecular mechanisms associated with FoxM1 in laryngeal squamous carcinoma remain unclear. Therefore, laryngeal squamous carcinoma cells were transfected with FoxM1-targeted small interfering RNA (siRNA) and compared with cells transfected with a control siRNA. Assays of these two treatment groups detected a decrease in cell viability associated with down-regulation of FoxM1 expression, and resulted in an inhibition of cell proliferation, migration, and invasion. These phenotypes were also associated changes in expression of VEGF and MMP-2, a decrease in expression of cyclin B, and an increase in expression of p27. These findings suggest that deregulation of FoxM1 protein signaling is sufficient to affect tumorigenesis and cancer progression. These results also indicate that inhibition of FoxM1 represents an attractive target for cancer therapy.

Adenovirus-mediated transfer of tris-shRNAs induced apoptosis of nasopharyngeal carcinoma cell in vitro and in vivo.

RNA interference (RNAi) is an evolutionary conserved mechanism for specific gene silencing. There are currently numerous cancer therapy clinical trials based on RNAi technology. Using an adenoviral system as a delivery mediator of RNAi, we investigated the therapeutic effects of targeting three genes simultaneously in vitro and in vivo. In this study, we constructed an recombinant adenoviral shRNA expression system as Adv-pEGFP-shVEGF-shTERT-shBcl-xl for multi-genes silencing. Our results showed that the adenoviral vector can achieve above 90% of transfection efficiency and induced obvious apoptosis in CNE-2 cell both in vitro and in vivo compared with targeting the TERT alone or controlled group.

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.