Human Laryngeal Cancer Research Articles

Andrographolide sensitizes Hep-2 human laryngeal cancer cells to carboplatin-induced apoptosis by increasing reactive oxygen species levels.

Andrographolide is a natural diterpenoid from Andrographis paniculata that has been proposed as an anticancer agent as well as a chemosensitizer for use in combination with anticancer drugs. Carboplatin is the first-line chemotherapeutic agent for advanced laryngeal carcinoma. However, the clinical efficacy of carboplatin is limited by drug resistance and side effects. The aim of this study was to investigate whether andrographolide has a synergistic antitumor effect with carboplatin on human laryngeal cancer cells. Hep-2 cells were exposed to andrographolide with or without carboplatin. The effects of indicated therapies were examined using the Cell Counting Kit-8 assay, the colony-forming assay, the Hoechst 33342/PI double staining, and flow cytometry analysis. The molecular mechanism was assessed by reactive oxygen species (ROS) detection and western blot. At the sublethal concentration, andrographolide increased carboplatin sensitivity of Hep-2 cells by increasing carboplatin-induced apoptosis and inhibiting cell viability. Moreover, we found that andrographolide sensitized carboplatin mainly through the induction of ROS generation and apoptotic signaling. Taken together, these results indicate that andrographolide, along with carboplatin, synergistically inhibited cell proliferation and induced mitochondrial apoptosis of Hep-2 cells by increasing the intracellular ROS, regulating the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3K/AKT) pathways, altering the BCL2/BAX ratio, and ultimately activating the cleavage of Caspase-3 and PARP. These results suggest that andrographolide sensitizes human laryngeal cancer cells to carboplatin-induced apoptosis by increasing ROS levels.

Berberis hispanica alkaloids extract induced cell death and apoptosis in human laryngeal cancer cells Hep-2

Berberis (Barberry) species contain an important amount of alkaloids and are commonly used in traditional medicine. Alkaloids are preponderantly characterized by their numerous properties including anticancer activity. The aims of the present study were to investigate the effect and the mechanism of action of the Algerian Berberis hispanica alkaloids extract (BHAE) on Hep-2 laryngeal cancer cells. In this purpose, total alkaloids were extracted from the roots bark of the plant, then, were submitted to HPLC analysis. In order to determine the cells inhibition concentration (IC50), we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were enumerated using Trypan blue exclusion assay and viability by cell cycle analysis. The morphology was assessed by coloration with May Grunwald Giemsa (MGG). The evaluation of such stress markers as malondialdehyde (MDA), cytochrome c, p38MAPK, NF-kB, Akt, Erk 1/ 2 and p53 was assessed by colorimetric assays and western blotting. Our results demonstrated that BHAE contains Berberine, an isoquinoline plant alkaloid. The IC50 was determined at 75 μg/mL and the treatment of Hep-2 cells with this dose showed anti-proliferative effect and increased SubG0 cell population (cells undergoing apoptosis) in treated cells (15.4%). Further, the coloration of fixed cells showed that BHAE induced cells density and morphological changes. Moreover, our results showed that the treatment induced elevation of MDA, cytochrome c, p38MAPK and NF-kB level, but did not affect Akt and Erk 1/2 rates. We have also observed that the treatment with BHAE enhanced the expression of p53. Taken together, our results suggest that the BHAE triggered cell death by activation of apoptosis through ROS induction.

Evaluation of the Expression of Matrix Metalloproteinase-1 of Laryngeal Squamous Cell Carcinoma by Ultrasound Molecular Imaging.

Purpose: The aims of this study were to evaluate the expression of matrix metalloproteinase-1 (MMP-1) on laryngeal squamous cell carcinoma (LSCC) and improve the early diagnosis rate via ultrasound molecular imaging (USMI). Methods: The microsized MMP-1-targeted microbubbles (MBMMP-1) and the control MBs (MBIgG) based on perfluorocarbon-filled lipid-shelled MBs were constructed and characterized. The in vitro binding experiment was performed with human epidermoid laryngeal cancer cells (HEp-2) and tested the binding efficiency of MBMMP-1 and MBIgG. In the in vivo study, the LSCC model was established in 10 mice. The MBMMP-1 and MBIgG were randomly injected into tumor-bearing mice via the tail vein at Day 7, Day 12, and Day 17 to dynamically evaluate the differential targeted enhancement (dTE) signals via USMI. Subsequent immunofluorescence analysis was used for confirmation of MMP-1 expression. Result: The effective adhesion rate of MBMMP-1 and MBIgG to HEp-2 was 298.42 ± 16.57 versus 12.38 ± 3.26 bubbles/per field in vitro experiment, which shows a significant difference (P < 0.01). The in vivo ultrasound molecular imaging (USMI) results demonstrated that dTE signal intensity from MBMMP-1 was significantly higher than that from the MBIgG at Day 7, Day 12, and Day 17 (Day 7, 41.21 ± 15.00 versus 2.25 ± 0.6 a.u., P < 0.05; Day 12, 124.64 ± 5.19 versus 11.13 ± 1.13 a.u., P < 0. 05; Day 17, 332.01 ± 64.88 versus 42.99 ± 11.9 a.u., P < 0.01). Moreover, immunofluorescence analysis further confirmed the expression of MMP-1 in LSCC with a gradual increase with the tumor growth. Conclusion: MBMMP-1 could be a potential probe that can be used in the early diagnosis of LSCC by USMI.

Cbl-b gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells

To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism. CD4+ T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA. The CD4+ T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals (P &lt; 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein (P &lt; 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (P&gt;0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment (P &lt; 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups (P &lt; 0.05). Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells in vitro probably as the result of enhanced IL-2 secretion and T lymphocyte activation.

Histone Deacetylase Inhibitors Enhance Cell Killing and Block Interferon-Beta Synthesis Elicited by Infection with an Oncolytic Parainfluenza Virus.

Previous results have shown that infection with the cytoplasmic-replicating parainfluenza virus 5 mutant P/V-CPI- sensitizes cells to DNA damaging agents, resulting in the enhanced killing of airway cancer cells. Here, we have tested the hypothesis that histone deacetylase (HDAC) inhibitors can also act with P/V-CPI- infection to enhance cancer cell killing. Using human small cell lung cancer and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid-mediated enhanced killing was eliminated in lung cancer cells that were engineered to express a protein which sequesters double stranded RNA. Scriptaid also enhanced cancer cell killing by two other negative strand RNA viruses – the La Crosse virus and vesicular stomatitis virus. Scriptaid treatment enhanced the spread of the P/V-CPI- virus through a population of cancer cells, and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). Taken together, these data support a role for combinations of a cytoplasmic-replicating RNA virus such as the P/V-CPI- mutant along with chemotherapeutic agents.

Validation of reference genes for the normalization of RT-qPCR expression studies on human laryngeal cancer and hypopharyngeal cancer.

Selecting stably expressed reference genes is crucial for evaluating real-time quantitative polymerase chain reaction (RT-qPCR) data via the relative quantification method. In the present-day study, our aim was to select optimal reference genes (RGs) for the investigation of target gene (TG) expression profiling in cancerous human laryngeal and hypopharyngeal tissues. 12 cancerous laryngeal tissues and 10 cancerous hypopharyngeal tissues were investigated. The expression characteristics of 11 reference genes (18S rRNA, GAPDH, B2M, ACTB, TBP, ALAS1, RPL29, HMBS, HPRT1, GUSB, and PUM1), which were commonly used in RT-qPCR for the analysis of gene expression, were investigated using the geNorm, NormFinder, and BestKeeper algorithm programs. HMBS, ALAS1, and B2M were suggested as optimal RGs for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself, respectively. If 2 or more reference genes are needed to achieve better standardization, 3 reference genes can optimally be used in combination to improve the accuracy of relative quantitation normalization. The recommended combinations for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself were HMBS + HPRT1 + GUSB, ALAS1 + GUSB + HMBS, and B2M + HPRT1 + TBP, respectively. The recommended reference genes could be used to improve the accuracy of gene expression studies on the molecular mechanisms of cancerous human laryngeal and hypopharyngeal tissues. The selected combination of reference genes can effectively improve the accuracy of the relative quantitative diagnosis of gene expression levels, such as messenger RNA, circular RNA, and long-noncoding RNA.

Effect and Mechanism of Survivin on Hypoxia-Induced Multidrug Resistance of Human Laryngeal Carcinoma Cells.

This study aimed at clarifying the mechanism and role of survivin in hypoxia-induced multidrug resistance (MDR) of laryngeal carcinoma cells. Human laryngeal cancer cells were incubated under hypoxia or normoxia. The expression of survivin was silenced by performing RNA interference. Additionally, by Western blot and real-time quantitative RT-PCR, survivin expression was detected. The sensitivity of human laryngeal carcinoma cells to multiple drugs was measured by CCK-8 assay. Meanwhile, the apoptosis of cells induced by cisplatin or paclitaxel was assessed by Annexin-V/propidium iodide staining analysis. Under hypoxic conditions, the upregulation of survivin was abolished by RNA interference. Then, CCK-8 analysis demonstrated that the sensitivity to multiple agents of laryngeal carcinoma cells could be increased by inhibiting survivin expression (P < 0.05). Moreover, Annexin-V/propidium iodide staining analysis revealed that decreased expression of survivin could evidently increase the apoptosis rate of laryngeal carcinoma cells that were induced by cisplatin or paclitaxel evidently (P < 0.05). Our data suggests that hypoxia-elicited survivin may exert a pivotal role in regulating hypoxia-induced MDR of laryngeal cancer cells by preventing the apoptosis of cells induced by chemotherapeutic drug. Thus, blocking survivin expression in human laryngeal carcinoma cells may provide an avenue for gene therapy.

DGCR5 promotes cancer stem cell-like properties of radioresistant laryngeal carcinoma cells by sponging miR-506 via Wnt pathway.

Cancer stem cells (CSCs) have been recognized as the significant cause of tumor recurrence. Long noncoding RNAs (lncRNAs) are involved in various cancers, including human laryngeal cancer. So far the correlation between lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) and CSC-like properties in human laryngeal cancer remains barely known. In our current study, two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R) with different radio sensitivities were cultured. Interestingly, CSC-like phenotypes were much more enriched in Hep-2R cells. We found that DGCR5 was upregulated and microRNA-506 (miR-506) was downregulated in Hep-2R cells. In addition, silence of DGCR5 could inhibit the stemness and enhance the radiosensitivity of Hep-2R cells. Meanwhile, overexpression of miR-506 also suppressed the CSC-like traits and the radiosensitivity was increased significantly. In addition, miR-506 was predicted as target of DGCR5 and the correlation between them was validated in our study. Finally, we observed that Wnt pathway exerted a significant role in human laryngeal CSCs and DGCR5 inhibition could repress Wnt signaling activity by sponging miR-506. In vivo assays were performed and we found that DCGR5 depressed stemness of human laryngeal cancer cells through modulating miR-506 and Wnt signaling pathway. Taken these together, we reported that DGCR5 induced CSC-like properties by sponging miR-506 through activating Wnt in human laryngeal carcinoma cells.

Aspernolide A Inhibits the Proliferation of Human Laryngeal Carcinoma Cells through the Mitochondrial Apoptotic and STAT3 Signaling Pathways.

Aspernolide A, a butyrolactone secondary metabolite, was purified from the endophytic fungus Cladosporium cladosporioides derived from roots of Camptotheca acuminata Decne. In this study, the antitumor activity and mechanisms of aspernolide A on human laryngeal cancer Hep-2 and TU212 cells were studied by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, morphological observation and Western blotting. The results showed that aspernolide A significantly inhibited the proliferation of Hep-2 and TU212 cells in dose- and time-dependent manners. Morphological changes of apoptotic cells could be observed under an inverted microscope, such as irregular margins, decreased adherence ability and chromatin condensation. The expressions of Bax, Caspase-9, Caspase-3 and PARP (poly ADP-ribose polymerase) increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, the expression of the phosphorylation of STAT3 decreased with the increase of dosage, suggesting that the apoptotic mechanism might be related to the STAT3 signaling pathway. All these conclusions indicated that aspernolide A has the potential anti-laryngocarcinoma effects.

A Study on the Correlations of the miR-31 Expression with the Pathogenesis and Prognosis of Head and Neck Squamous Cell Carcinoma.

To investigate the correlations of miR-31 expression with cell proliferation, invasion, and prognosis of patients with head and neck squamous cell carcinoma (HNSCC). The expression of miR-31 in human laryngeal cancer TU686 cells, human nasopharyngeal carcinoma CNE-2 cells, and normal human oral keratinocyte (NHOK) epithelial cells was detected via quantitative real-time polymerase chain reaction (qRT-PCR). The effects of miR-31 on the proliferation and invasion of HNSCC cells were explored through transfecting miR-31 analogs (miR-31 mimics) and miR-31 inhibitors (anti-miR-31). qRT-PCR was applied to detect the expressions of miR-31 in 56 cases of HNSCC tumor tissues and tumor-adjacent normal tissues. The correlation of miR-31 expression with pathological parameters and survival prognosis of HNSCC patients was also analyzed. The expressions of miR-31 in TU686 and CNE-2 cell lines were significantly higher than that in NHOK cells (p < 0.01). Compared with those in the negative control group, the proliferation and invasion abilities of cells transfected with miR-31 mimics were notably enhanced (p < 0.01), and those of cells transfected with anti-miR-31 were significantly reduced (p < 0.01). In addition, miR-31 mimics significantly reduced ARID1A expression (p < 0.01) and anti-miR-31 increased its expression (p < 0.05). The expression of miR-31 in tumor tissues of HNSCC patients was remarkably higher than that in tumor-adjacent normal tissues (p < 0.01). This, together with clinical data analysis, revealed that the expression of miR-31 was associated with tumor differentiation, metastasis, and staging of patients, and the survival period of patients with lowly expressed miR-31 was longer. The highly expressed miR-31 can stimulate the proliferation and invasion of HNSCC cells, closely correlated with tumor differentiation, metastasis, and staging of patients. Patients with lowly expressed miR-31 have a longer survival period. Therefore, miR-31 expression can be taken as a crucial reference indicator for the prognosis of HNSCC patients.

Knockdown of DGCR5 enhances the radiosensitivity of human laryngeal carcinoma cells via inducing miR-195.

Long noncoding RNAs (lncRNAs) exert critical roles in the development of various cancers, including human laryngeal cancer. Radioresistance contributes to the predominant causes of laryngeal cancer recurrence after radiotherapy. The aim of our study was to investigate the association of dysregulated lncRNA and radiation resistance in human larynx squamous carcinoma. Here, we investigated the biological roles of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in radioresistance of human laryngeal cancer. Two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were used in the present study. We observed that DGCR5 was significantly upregulated in Hep-2R cells. Inhibition of DGCR5 by LV-shDGCR5 transfection restrained Hep-2R cell proliferation and sensitized cells to radiation. Reversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. In addition, microRNA (miR)-195 was predicted as a direct downstream target of DGCR5. Dual-luciferase reporter and RNA immunoprecipitation assays verified the direct interaction between them. Meanwhile, miR-195 was observed to be reduced in Hep-2R cells and miR-195 mimics repressed Hep-2 cell growth. Moreover, radiosensitivity of Hep-2R cells was greatly enhanced by overexpression of miR-195, which could be reversed by upregulation of DGCR5. Finally, in vivo experiments were used to validate that knockdown of DGCR5 suppressed laryngeal carcinoma via targeting miR-195. In conclusion, we indicated that DGCR5 could contribute to the radioresistance of human laryngeal carcinoma cells via sponging miR-195.

Anticancer Activity of Mukonal Against Human Laryngeal Cancer Cells Involves Apoptosis, Cell Cycle Arrest, and Inhibition of PI3K/AKT and MEK/ERK Signalling Pathways.

BackgroundLaryngeal cancer is one of the major malignancies of the neck and head and is responsible for considerable mortality across the globe. The treatments for laryngeal cancer mainly involve surgical interventions followed by chemotherapy. However, due to unsatisfactory results, constant relapses and the adverse effects associated with the currently used drugs, there is pressing need to develop effective drug options for treatment of laryngeal cancer. Therefore, this study was undertaken to investigate the anticancer effects of a plant-derived alkaloid, Mukonal, against human AMC-HN-8 laryngeal cancer cells.Material/MethodsThe WST-1 and clonogenic assays were employed to determine the cell viability. Apoptosis was detected by Hoechst and AO/EB staining. Cell migration and cell cycle analysis was performed by Transwell assay and flow cytometry, respectively. Protein expression was examined by Western blotting.ResultsThe results revealed that Mukonal reduced the viability of laryngeal cancer cells dose-dependently. The IC50 of Mukonal was found to be 10 μM. However, the effects of Mukonal on the normal HuLa-PC cells was found to be 140 μM. The decrease in the viability of the AMC-HN-8 laryngeal cancer cells was found to be due to the induction of apoptosis and G2/M cell cycle arrest. Mukonal also suppressed the cell migration and of the AMC-HN-8 laryngeal cancer cells. Mukonal could also inhibit the PI3K/AKT and MEK/ERK signalling pathways in a concentration-dependent manner.ConclusionsTaken together, we conclude that Mukonal could prove a beneficial lead molecule for the treatment of laryngeal cancer.

Hesperidin inhibits angiogenesis, induces apoptosis, and suppresses laryngeal cancer cell metastasis

Introduction: Laryngeal carcinoma is the most common malignant head-and-neck tumor. Due to the low survival rate and the inadequate response to chemotherapy, effective therapy remains a challenge. Objectives: Therefore, the identification of new therapeutic options that preserve the larynx is needed. Hesperidin (Hsp) is a nontoxic plant flavanone that has proven effective against cancer. Materials and methods: Hence, the current in vivo and in vitro study was conducted to determine whether Hsp might suppress metastasis of cancer larynx. Results: In an in vivo mouse metastasis model, Hsp suppressed metastasis of human Hep2 laryngeal cancer cells to the livers and lungs. In vitro assays, Hsp significantly inhibited angiopoietin 1 secretion (an angiogenic promotor) and increased annexin-V (an apoptotic indicator) in Hep2 cell culture at relatively low levels (10 μM). Conclusions: These studies suggest that Hsp deserves further investigation as a possible treatment option for laryngeal cancer.

MicroRNA-1469, a p53-responsive microRNA promotes Genistein induced apoptosis by targeting Mcl1 in human laryngeal cancer cells

Genistein, a plant isoflavone, is reported to have therapeutic potentials in multiple cancers, However, the molecular mechanism underlying promoting cell apoptosis in laryngeal cancer remains unclear. In this study, we report that miR-1469 was induced by genistein in laryngeal cancer. Elevated miR-1469 promoted cell apoptosis and inhibited Mcl1 expression. In addition, we also observed that tumor suppressor p53 was increased under genistein treatment. Elevation of p53 promoted miR-1469 expression, leading to miR-1469 increase and Mcl1 decrease. Therefore, our findings suggest that genistein can suppress laryngeal cancer cell survival through p53 –miR-1469-Mcl1pathway.

Overexpression of Beclin1 inhibits proliferation and promotes apoptosis of human laryngeal squamous carcinoma cell Hep-2.

ObjectiveBeclin1 was previously found to be downregulated in human laryngeal cancer (LC) tissues, and it results in poor prognosis. This study aimed to further confirm the antitumor effects of Beclin1 in LC cell line Hep-2.Materials and methodsBeclin 1 was overexpressed in Hep-2 cells using liposomal transfection and confirmed using reverse transcription polymerase chain reaction and Western blotting. Then, cell proliferation and apoptosis were determined in control (untransfected), empty vector transfected, and Beclin1 overexpressed groups using MTT and flow cytometry procedure, respectively.ResultsThe expression of the Beclin1 gene in Hep-2 cells was significantly increased after vector transfection compared with control (1.173±0.046 vs 0.453±0.016, P<0.01) and empty vector (1.173±0.046 vs 0.440±0.021, P<0.01). Overexpression of Beclin1 inhibited proliferation at 4 days (0.619±0.051 vs 0.891±0.081 and 0.619±0.051 vs 0.878±0.105, P<0.01), 5 days (0.684±0.078 vs 1.127±0.094 and 0.684±0.078 vs 1.162±0.117, P<0.01), and 6 days (0.725±0.069 vs 1.168±0.103 and 0.725±0.069 vs 1.194±0.097, P<0.01) and promoted apoptosis (14.48%±1.42% vs 4.07%±0.66% and 14.48%±1.42% vs 4.39%±0.80%, P<0.01) in Hep-2 cells in comparison with the control and empty vector groups, respectively.ConclusionBeclin1 may be an underlying target for the treatment of LC. This study has provided some experimental basis for the gene therapy of LC.

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI-) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI- infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI- as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors.IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors.

Identification of novel enriched recurrent chimeric COL7A1-UCN2 in human laryngeal cancer samples using deep sequencing

BackgroundAs hybrid RNAs, transcription-induced chimeras (TICs) may have tumor-promoting properties, and some specific chimeras have become important diagnostic markers and therapeutic targets for cancer.MethodsWe examined 23 paired laryngeal cancer (LC) tissues and adjacent normal mucous membrane tissue samples (ANMMTs). Three of these pairs were used for comparative transcriptomic analysis using high-throughput sequencing. Furthermore, we used real-time polymerase chain reaction (RT-PCR) for further validation in 20 samples. The Kaplan-Meier method and Cox regression model were used for the survival analysis.ResultsWe identified 87 tumor-related TICs and found that COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%), whereas none of the ANMMTs were positive (0/23; p < 0.0001). COL7A1-UCN2, generated via alternative splicing in LC tissue cancer cells, had disrupted coding regions, but it down-regulated the mRNA expression of COL7A1 and UCN2. Both COL7A1 and UCN2 were down-expressed in LC tissues as compared to their paired ANMMTs. The COL7A1:β-actin ratio in COL7A1-UCN2-positive LC samples was significantly lower than that in COL7A1-UCN2-negative samples (p = 0.019). Likewise, the UCN2:β-actin ratio was also decreased (p = 0.21). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients (p = 0.032; HR, 13.2 [95%CI, 1.2–149.5]).ConclusionLC cells were enriched in the recurrent chimera COL7A1-UCN2, which potentially affected cancer stem cell transition, promoted epithelial-mesenchymal transition in LC, and resulted in poorer prognoses.

Small interference of transcription factor Snail contribute to enhanced cisplatin sensitivity on human laryngeal resistant cancer cells

Objective:To study the relationship between transcription factor Snail and the sensitivity of cisplatin on human laryngeal resistant cancer cells.Method:siRNA interference of Snail was transfected by small RNA interference technology. The interference efficiency on mRNA level were detected by RT-qPCR assay; the expression of Snail protein level was assessed by immunofluorescence. The inhibition ratio of different cisplatin concentration (0, 1, 2, 4, 8, 16 μg/ml) was detected by CCK-8 assay; the protein level of Snail, E-cadherin, MDR1were detected by Western blot assay.Result:RT-qPCR assay show the expression of Snail on mRNA level was decreased to (67.85±9.50)% after transfection in Hep-2/CDDP cell(P<0.05). Immunofluorescence show fluorescence intensity of si-Hep-2/CDDP group was reduced both in nucleus and cytoplasm; CCK-8 assay show the inhibitory ratio of transfected group was increased compared to negative control and Hep-2/CDDP group in different cisplatin concentration (0, 1, 2, 4, 8, 16 μg/ml) (P<0.05). Western blot assay show the protein expression of Snail and MDR1 were down-regulated in transfected Hep-2/CDDP cells (allP<0.05), while epithelial marker E-cadherin was up-regulated in protein level (P<0.05).Conclusion:Small interference of transcription factor Snail could increase the expression of E-cadherin while decrease the expression of MDR1, and it was confirmed that interference Snail contribute to enhanced cisplatin sensitivity on human laryngeal resistant cancer cells.

Fibroblast growth factor receptor 1 and 3 expression is associated with regulatory PI3K/AKT kinase activity, as well as invasion and prognosis, in human laryngeal cancer.

Aberrant fibroblast growth factor receptor (FGFR) expression is thought to contribute to the development of many types of cancer. As yet, however, their impact on the course and prognosis of head and neck cancer remains to be determined. Here, we aimed to investigate the effects of expression of the FGFR family members FGFR1 and FGFR3, as well as their downstream PI3K/AKT signal-regulated kinases, on the aggressiveness and prognosis of laryngeal cancer. In total 137 surgically removed squamous cell laryngeal cancer (SCLC) and 100 matched non-cancerous laryngeal mucosa (NCLM) samples were assessed for mRNA expression using quantitative real-time PCR. The corresponding proteins were analyzed by Western blotting. SLUG expression was assessed by immunohistochemistry. The expression data were subsequently related to tumor front grading (TFG), local/nodal recurrences, prognosis and overall survival. The FGFR1, FGFR3 and PI3K/AKT kinase mRNA and protein levels were found to be significantly higher in the SCLC than the NCLM samples (p < 0.05). A high FGFR1 mRNA/protein expression level was found to be associated with an increased invasion rate, according to TFG scale and SLUG level, a high local/nodal recurrence rate and a poor prognosis (p < 0.05). Similarly, we found that a high FGFR3 mRNA/protein expression level was associated with a shorter survival time (p < 0.05). In addition, we found that high PI3K/AKT kinase mRNA/protein levels were associated with a high TFG (p < 0.05). We also found that FGFR1/3 mRNA and FGFR1 protein levels were inversely associated with overall survival (log-rank test: FGFR1 mRNA p = 0.03, FGFR3 mRNA p = 0.04, FGFR1 protein p = 0.03). Subsequent multivariate analyses revealed that high FGFR3 mRNA expression may serve as an independent poor prognostic factor (HR 2.32, 95% CI 1.03-6.59; p = 0.04). We also found that the p-PI3K regulatory kinase protein level was significantly associated with survival in the cohort studied (HR 1.78, 95% CI 0.64-8.53; p = 0.03). From our data we conclude that FGFR1 and FGFR3, as well as its downstream regulatory PI3K/AKT kinases, may serve as potential biomarkers for the invasiveness and prognosis of laryngeal cancer. The expression of FGFR1/3-PI3K/AKT regulatory pathway members may be instrumental for the identification of patients at risk for an unfavorable clinical outcome.

Introducing Potential Key Proteins and Pathways in Human Laryngeal Cancer: A System Biology Approach.

The most common malignant neoplasm of the head and neck region is laryngeal cancer which presents a significant international health problem. The present study aims to screen potential proteins related to laryngeal cancer by network analysis to further understanding disease pathogenesis and biomarker discovery. Differentially expressed proteins were extracted from literatures of laryngeal cancer that compare proteome profiling of patient›s tissue with healthy controls. The PPI network analyzed for up and down regulated proteins with Cytoscape Version 3.4. After PPI construction, topological properties of the two networks have been analyzed. Besides, by using MCODE. the Gene Ontology (GO) analysis, the related modules and pathways were examined. Our study screened 275 differentially changed proteins, including 136 up- and 139 down-regulated proteins. For each network, it has been considered 20 key proteins as hub and 20 as bottleneck. A number of 26 hub-bottleneck nodes is introduced for the two networks. A total of 11 modules including 6 downregulated and 5 upregulated network modules were obtained. The most significant GO function in the significant upregulated module was the RNA processing, and the most significant one in the downregulated module with highest score was the respiratory electron transport chain. Among 275 investigated proteins, 12 crucial proteins are determined that 4 of them can be introduce as a possible biomarker panel including YWHAZ, PPP2R1A, HSP90AA1, and CALM3 for human laryngeal cancer.