Human Laryngeal Squamous Carcinoma Research Articles

Effect of DJ-1 overexpression on the proliferation, apoptosis, invasion and migration of laryngeal squamous cell carcinoma SNU-46 cells through PI3K/AKT/mTOR.

The aim of the present study was to explore the effect of DJ-1-mediated PI3K/AKT/mTOR pathway on the proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous cell SNU-46, through stable transfection and overexpression of the DJ-1 gene. Retrovirus carrying DJ-1 gene was used to stabilize transfected human laryngeal squamous carcinoma SNU-46 cell line, and monoclonal cell line of stably overexpressed DJ-1 protein was screened out by G418. DJ-1 protein expression was determined by western blotting, and changes of p-AKT, p-mTOR and PTEN protein content were detected, followed by the detection of changes in proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous carcinoma cell line with stably transfected DJ-1 protein overexpression by flow cytometry, CCK-8 method and Transwell. We successfully constructed a laryngeal squamous carcinoma cell line of stably overexpressed DJ-1 protein and termed it SNU-46-DJ-1. After overexpression of DJ-1 protein, the levels of PTEN expression in laryngeal squamous cell SNU-46 decreased and p-AKT and p-mTOR protein expression levels increased. Compared to the untreated SNU-46 cells, the proliferation rate of SNU-46-DJ-1 cells increased (0.834±0.336 vs. 0.676±0.112; p<0.001); invasiveness was enhanced (165.7±13.6 vs. 100.0±17.4; p=0.001), the migration ability was enhanced (207.3±13.1vs.175.3±13.3; p=0.036), and the apoptosis rate decreased (3.533±5.167 vs. 16.397±5.447%; p=0.019). The overexpression of DJ-1 protein in laryngeal squamous carcinoma SNU-46 cells can accelerate proliferation rate, increase the invasion and migration capacity, and reduce apoptosis, by activating the PI3K/AKT/mTOR pathway.

Clusterin silencing inhibits proliferation and reduces invasion in human laryngeal squamous carcinoma cells.

BackgroundClusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (sCLU), which plays important roles in cell survival and death. In laryngeal squamous cell carcinomas (LSCC), sCLU is up-regulated and its expression is related to the invasiveness of these tumors. The purpose of this study was to explore the inhibiting role of sCLU gene silence in the invasive ability and growth of Hep-2 human laryngeal squamous carcinoma cells (Hep-2) by transfection of short hairpin RNA expression plasmids against sCLU (sCLU-shRNA) (in vivo) or small interference RNA (sCLU-siRNA) (in vitro).MethodssCLU-siRNA and the control siRNA were transfected into Hep-2 cells using Lipofectamine 2000. RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression. The invasive activity of sCLU-siRNA-transfected Hep-2 cells was measured with the modified Boyden chamber assay and wound healing assay. The effects of sCLU-siRNA on cell proliferation were evaluated by MTT assay. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining methods. We next evaluated the effects of sCLU silencing by sCLU-shRNA transfection in vivo on tumor growth and metastatic properties to the lung. Terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe the apoptosis in the xenografts.ResultsIt showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro. Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability. In vivo, the average volume of tumors in the sCLU-shRNA transfected group was significantly lower than in the control group (P <0.01), and the significant apoptosis detected with TUNEL was indicated in the sCLU-shRNA transfected groups (P <0.05). Significantly, we found that sCLU-shRNA could exert marked inhibition of the lung metastasis of Hep-2 cells in nude mice in vivo.ConclusionssCLU gene silence can inhibit invasion and growth of LSCC. sCLU may provide a potential therapeutic target against human LSCC.

Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100–400μg/ml for 24h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP.

A polysaccharide from Boschniakia rossica induces apoptosis in Hep2 human laryngeal squamous carcinoma cells

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22kDa. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100-400μg/ml for 24h induced a significant apoptosis compared to untreated control cells. BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis

Expression and distribution of aggrecanases in human larynx: ADAMTS-5/aggrecanase-2 is the main aggrecanase in laryngeal carcinoma

Members of the ADAMTS family of proteases degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular functions. Aggrecanases are the main enzymes responsible for aggrecan degradation, due to their specific cleavage pattern. In this study, the expression status, the macromolecular organization and localization of ADAMTS-1, ADAMTS-4/aggrecanase-1 and ADAMTS-5/aggrecanase-2 in human normal larynx and laryngeal squamous cell carcinoma (LSCC) were investigated. On mRNA level, the results showed that ADAMTS-4 was the highest expressed enzyme in normal larynx, whereas ADAMTS-5 was the main aggrecanase in LSCC presenting a stage-related increase up to stage III (8-fold higher expression compared to normal), and thereafter decreased in stage IV. Accordingly, immunohistochemical analysis showed that ADAMTS-5, but not ADAMTS-4, was highly expressed by carcinoma cells. Sequential extraction revealed an altered distribution and organization of multiple molecular forms (latent, activated and fragmented forms) of the enzymes within the cancerous and their corresponding macroscopically normal laryngeal tissues, compared to the normal ones. Importantly, these analyses indicated that critical macromolecular changes occurred from the earliest LSCC stages not only in malignant parts of the tissue but also in areas that were not in proximity to carcinoma cells and appeared otherwise normal. Overall, the results of the present study show that ADAMTS-5/aggrecanase-2 is the main aggrecanase present in laryngeal carcinoma suggesting a critical role for the enzyme in aggrecan degradation and laryngeal tissue destruction during tumor progression.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

BackgroundUp-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.ResultsThe rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.ConclusionsThe polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

尼美舒利对人喉鳞癌Hep-2细胞祼鼠移植瘤CD44和MMP-7表达的影响

Objective To evaluate the inhibitory effect of Nimesulide on tumor growth of human laryngeal squamous carcinoma in nude mice and to explore the possible mechanism involved in the inhibition.Methods Nude mice model bearing human laryngeal squamous carcinoma cell line(Hep-2)was established.The volumes of the subcutaneous tumor were observed after Nimesulide treatment.The levels of COX-2,CD44 and MMP-7 protein and mRNA in transplantation tumor were examined by immunohistochemistry and RT-PCR,respectively.Results The growth of the carcinoma transplanted was significantly inhibited in the treatment group.Compared with the control group,the Nimesulide treatment group had significantly reduced weight and volume of tumor(all P0.05).There was no significantly difference in weights of mice increasing between the two groups.The levels of COX-2,CD44 and MMP-7 protein and mRNA of the treatment group were lower than those of the control group(all P0.05).Conclusion NIM has obvious inhibitory effect on the growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts in nude mice,through the down-regulation COX-2,CD44 and MMP-7.

CDNA Expression Analysis of a Human Radiosensitive-Radioresistant Cell Line Model Identifies Telomere Function as a Hallmark of Radioresistance

To achieve a more complete understanding of the molecular mechanisms underlying tumor radioresistance, we established a radioresistant cell line from the human larynx squamous cell carcinoma cell line Hep-2 after long-term radiation induction. The biological features of the resulting cell lines were characterized. cDNA microarray technology was used to measure the alterations of gene expression in the radioresistant cells. We found that certain genes associated with DNA repair, cell cycle, apoptosis, etc. were significantly changed. In particular, genes related to telomeres, such as POT1, were significantly altered. Radioresistant cells had higher telomerase activity and longer telomeres than their parental cells. Our research suggests that telomere function is a novel hallmark of cellular radiosensitivity, and the mechanistic link between telomere maintenance and radiosensitivity may involve the genes and pathways we implicated in this study.

Study of the efficacy of 5 ALA-mediated photodynamic therapy on human larynx squamous cell carcinoma (Hep2c) cell line

5-aminolevulanic acid (ALA), a precursor of Protoporphyrin IX, was evaluated as an inducer of photodamage on Hep2c, human larynx squamous cell carcinoma, cell line. Porphyrins are used as active cytotoxic antitumor agents in photodynamic therapy (PDT). The present study evaluates the effects of photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) using human larynx cells as experimental model. Hep2c cell line was irradiated with red light (a diode laser, λ = 635 nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of Hep2c cells were studied. The optimal uptake of photosensitizer ALA in Hep-2c cells was investigated by means of spectrometric measurement. Cells viability was determined by means of neutral red assay (NR). It was observed that sensitizer or light doses have no significant effect on cells viability when studied independently. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 7 h in vitro incubation. The photocytotoxic assay showed that light dose of 85 J/cm2 gives effective PDT outcome for Hep2c cell line incubated with 55 μg/ml of 5-ALA with a conclusion that Hep2c cell line is sensitive to ALA-mediated PDT.

Expression and clinical significance of NY-ESO-1 protein in human laryngeal squamous carcinoma

Objective To detect the expression of Cancer-Testis antigen NY-ESO-1 in human laryngeal squamous carcinoma (LSC) and to explore its significance in immunotherapeutic application. Methods The expressions of NY-ESO-1 protein in the LSC and in the pathologically positive lymph nodes were detected by PV-9000 Immunohistochemistry. Western blotting was also employed to measure the expressions of NY-ESO-1 in the tumor core region(TC), the tissues at the sites of 0.5cm, 1.0cm away from LSC periphery and the distant normal larynx tissues. Results NY-ESO-1 protein expression was positive in 30 out of 69 (43.48 %) cases of LSC. The expression level of NY-ESO-1 protein were found to significantly decrease by tums in TC and corresponding adjacent tissues (P ＜0.01). None of the nine normal larynx tissues expressed NY-ESO-1 protein.It did not display an obvious correlation between the expression of NY-ESO-1 with T staging, pathological grading and lymph node metastasis (P ＞0.05). Its positive expression was found in pathologically positive cervical lymph nodes, which were significantly lower than that in the primary site (P ＜0.05). Conclusion NY-ESO-1 protein express at high level in human laryngeal squamous carcinoma, and they may play a role in genesis and development of tumors, which suggests that NY-ESO-1 gene might be used as target antigens for immunotherapy of LSC and the further research is necessary. Key words: Laryngeal neoplasm; Gene expression; Lymph nodes; NY-ESO-1 protein

Silencing STAT3 with short hairpin RNA enhances radiosensitivity of human laryngeal squamous cell carcinoma xenografts in vivo

Short hairpin RNA (shRNA) targeting signal transducer and activator of transcription 3 (STAT3) potentiate the radiosensitivity of human laryngeal squamous carcinoma cells in vitro. In the present study, we investigated the inhibitory effect of STAT3 shRNA plus radiotherapy on nude mouse laryngeal squamous cell carcinoma xenografts. The xenotransplanted tumors were treated with STAT3 shRNA, with or without radiation, following a planned scheme. The inhibition rate for tumor growth was calculated and the tumor growth curve was plotted. In addition, the expression of p-STAT3, B cell lymphoma 2 (Bcl-2), p53, vascular endothelial growth factor (VEGF) protein and intratumoral microvessel density (MVD) was determined by immunohistochemistry. Flow cytometry was used to detect the rate of cell apoptosis. The results revealed that STAT3 shRNA transfection plus radiotherapy significantly minimized tumor volume and increased the rate of tumor inhibition. p-STAT3 protein expression and intratumoral MVD were observed to be down-regulated, whereas apoptosis was increased. There was a positive correlation between the expression of p-STAT3 and Bcl-2, and also between the expression of p53 and VEGF, and MVD. These findings indicate that STAT3 shRNA potentiate the radiosensitivity of laryngeal carcinoma xenografts in vivo by regulating downstream signaling proteins in the STAT3 pathway.

STAT3 blockade with shRNA enhances radiosensitivity in Hep-2 human laryngeal squamous carcinoma cells

Signal transducer and activator of transcription 3 (STAT3) is an oncogene aberrantly activated in many human tumors. We studied whether radiation combined with STAT3 siRNA enhances radiosensitivity of Hep-2 human laryngeal squamous carcinoma cells (Hep-2 cells). Firstly, STAT3 targeting recombinant plasmid was constructed. Hep-2 cells were transfected with expression vector of STAT3 siRNA using Lipofectamine 2000. Semiquantitive RT-PCR detected effective STAT3 mRNA down-regulation by STAT3 siRNA. Secondly, Hep-2 cells were radiated with different doses of gamma-rays after transfection with STAT3 small interference RNA (siRNA). MTT assay showed cell proliferation decreased significantly (P<0.05) after STAT3 siRNA transfection combined with radiation. Thirdly, flow cytometry (FCM) demonstrated that cell apoptosis of combined treatment group increased significantly (P<0.05) and exhibited time dependency after 6 Gy irradiation (P<0.05). Simultaneously, STAT3, p-STAT3, Bcl-2, VEGF, p53 protein levels decreased in Hep-2 cells, with positive correlations between level of p-STAT3 and levels of Bcl-2, VEGF, p53, respectively (r=0.974, 0.988, 0.976, all P<0.01). Above all, specific siRNA targeting STAT3 gene is able to enhance the radiosensitivity in Hep-2 cells by regulating expression of Bcl-2, VEGF and p53 proteins.

Increased expression of telomere-related proteins correlates with resistance to radiation in human laryngeal cancer cell lines

Telomere-associated proteins function as survival factors in telomere maintenance, which are major contributors to radiosensitivity in human cancers. The aim of this study was to investigate the association of telomere-associated gene expression and radiation resistance in human larynx squamous carcinoma. The changes of telomere-associated gene expressions and bionomical characteristics that occur in two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were explored in the present study. Based on previous research, elevated POT1 and TPP1 expressions were detected by reverse transcription-PCR and Western blotting in Hep-2R cell lines. Furthermore, Hep-2R cells showed increased recovery ratio accompanied by a reduction of cell arrested in G2/S phase, suggesting that the radioresistance of Hep-2R cells was due to a faster growth in which telomere length had recently been demonstrated to be a powerful prognostic marker. These results manifest that radioresistant Hep-2R cell lines showed certain changes in gene expression and bionomical profiles that are different from the profile changes of the more-sensitive Hep-2 cell lines, and that evaluation of telomere-associated genes may be a prognostic marker for response to radiotherapy in larynx squamous cell carcinoma (LSCC).

Effects of elevating temperature on photodynamic reaction in laryngeal squamous carcinoma cells induced by delta-aminolevulinic acid

To investigate effect of temperature on delta-aminolevulinic acid (ALA) induced photodynamic reaction in laryngeal squamous carcinoma cells. Human laryngeal squamous carcinoma cells of the line Hep-2 cells were co-cultured with 2 mmol/L ALA (Group A) or without ALA (Group B) at the culturing temperature 19 - 46 degrees C. Three hours later cellular protoporphyrin IX (PpIX) level was determined by high performance liquid chromatography with fluorescent detection. Laser scanning confocal microscopy was used to observe the fluorescent strength in the Hep-2 cells. The ratio of cell death (including necrosis and apoptosis) was detected with flow cytometer before and after photodynamic reaction. In Group A the PpIX level at the temperature of 19 degrees C was (0.25 +/- 0.06) microg/L, and raised to (1.07 +/- 0.11) microg/L at 46 degrees C. There was no cellular PpIX detected in Group B at any temperature. Before laser radiation the cell death ratios of both groups were the same at the temperature 19 - 37 degrees C, and at the temperature 37 - 46 degrees C. After laser radiation the cell death ratio of Group A raised from 31.11% to 98.92% as the temperature went up steadily, but the results of Group B showed the same curve as before laser radiation. At any same temperatures the cell death ratios of Group A were all significantly higher than those of Group B (all P < 0.05), and as the temperature was elevated the difference between the 2 groups raised from 28.99% to 59.26%. Moderate higher temperature enhances the PpIX production and photodynamic reaction in human laryngeal squamous carcinoma induced by ALA in vitro.

Short hairpin RNA silencing of vascular endothelial growth factor, telomerase reverse transcriptase, and Bcl-xl expression in human laryngeal squamous carcinoma xenografted in nude mice

To investigate the biological impact of pEGFP-shVEGF-shTERT-shBcl-xl expression in human laryngeal squamous carcinomas xenografted in nude mice and the related antitumor mechanism. A recombinant plasmid vector containing 3 different short hairpin RNA (shRNA) segments including pEGFP-shVEGF-shTERT-shBcl-xl was constructed and directly injected into the grafted tumors of human laryngeal squamous carcinoma in nude mice. The mRNA and protein expressions were determined by real-time RT-PCR and Western blot respectively. Apoptosis was determined by TUNEL assay using a commercial kit. Intratumoral microvessel density (MVD) was assessed by immunhistochemistry. On the 14th days after the final treatment, mRNA and protein expression of VEGF, TERT, and Bcl-xl were markedly suppressed. The tumor sizes were significantly smaller than those in the other two group, with an overall tumor inhibition ratio of 91.2%. MVD counts in the pEGFP-shVEGF-shTERT-shBcl-xl treated group were significantly lower than those of the other two groups, along with increased apoptotic cells. The data showed that inhibition of VEGF, TERT, Bcl-xl expression by RNAi technique induces cellular apoptosis and suppresses the growth of laryngeal squamous carcinoma in vivo. VEGF, TERT and Bcl-xl may be involved in the development of laryngeal cancers. The findings suggest a synergistic tumor therapeutic effect through simultaneous inhibition of the three genes. Multi-target RNA interference may provide a powerful strategy against human laryngeal cancers.

A dual role of p21waf1/cip1 gene in apoptosis of HEp-2 treated with cisplatin or methotrexate

We investigated the effects of p21(waf1/cip1) gene overexpression in human laryngeal squamous carcinoma cells HEp-2 lacking p53 protein expression on apoptosis induction upon the treatment with two commonly used chemotherapeutic agents, cisplatin and methotrexate. For that purpose, we employed cDNA arrays and qPCR to monitor gene expression upon treatment with AdCMV-p21 alone or in combination with the chemotherapeutic compounds. We found that p21(waf1/cip1) gene overexpression provoked apoptosis of HEp-2 through the induction of the TNFRSF9 gene and activation of caspase 7. In addition, we have proved that p21(waf1/cip1) can assume a dual role in apoptosis in the same cell system depending on the chemotherapeutic agent: its overexpression enhances apoptosis in cisplatin-treated cells and attenuates apoptotic signals in methotrexate-treated cells. The observed dual role of p21(waf1/cip1) was in direct correlation with the modulation of caspases 3 and 7 activation and changes in the expression of GADD45a gene. The results presented herein encourage future use of targeted p21(waf1/cip1) gene therapy in cancer treatment in a well-defined therapeutic and genetic context.

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.

Vitamin D3 analogue EB1089 inhibits the proliferation of human laryngeal squamous carcinoma cells via p57

The objective of this study is to evaluate the role of the cyclin-dependent kinase inhibitor p57 in EB1089-inhibited proliferation of human laryngeal squamous carcinoma cells (HEp-2). HEp-2 cells were treated with the vitamin D3 analogue EB1089 for 48 h and total RNA was extracted for reverse transcription-PCR amplification using primers for the p57 coding sequence. Proteins were detected by Western blot analysis. For interference using silencing RNA (siRNA), HEp-2 cells were transfected with siRNA specific for p57 (siRNA-p57) or a negative control sequence (siRNA-con) followed by treatment with 10 nmol/L EB1089. The effects of EB1089 on cell proliferation were evaluated by 5-bromo-2'-deoxyuridine incorporation and '3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Cell death and cell cycle dynamics were monitored using flow cytometry. EB1089 significantly inhibited HEp-2 cell proliferation and increased p57 mRNA and protein levels; this was blocked by siRNA-p57 but not by siRNA-con. The EB1089-induced suppression of HEp-2 cell proliferation recovered to near-normal levels with siRNA-p57 transfection. EB1089 inhibits the proliferation of HEp-2 cells and p57 plays an important role in this.

NKG2D Ligands Expression and NKG2D‐Mediated Cytotoxicity in Human Laryngeal Squamous Carcinoma Cells

The NKG2D is an activating immunoreceptor expressed by NK cells and CD8(+) T cells. Engagement of NKG2D by its ligands is critical for both innate and adoptive immunity. While the overexpression of NKG2D ligands on certain tumour cells has previously been demonstrated, little is known about NKG2D ligand expression on human laryngeal tumour cells. In this study, we first verified that the interaction between NKG2D and its ligands was critical for NK cell-based immune response to human laryngeal squamous carcinoma cells Hep-2. This NKG2D-mediated effect was observed by transfecting the recombinant eukaryotic expression vector pEGFP-N1/NKG2D as well as the NKG2D blockade. The mRNA and protein expression of NKG2D ligands, MHC class I-related chain molecules A (MICA) and UL16-binding proteins (ULBPs), in human laryngeal carcinoma cell line Hep-2 and fresh tumour tissues were evaluated. Compared with non-tumour tissues of vocal cords polyps, MICA and ULBP-3 were strongly overexpressed on both the human laryngeal carcinoma cell line Hep-2 and fresh human laryngeal carcinoma tissues. The mechanism and impact of NKG2D ligands overexpression on NK cell-mediated anti-laryngeal cancer immune response would require further investigation.