Apoptosis Of Hep-2 Cells Research Articles

Citalopram, an antipsychotic agent, induces G1/G0 phase cell cycle arrest and promotes apoptosis in human laryngeal carcinoma HEP-2 cells

Human laryngeal squamous carcinoma (LSCC) is a common malignant tumor in the head and neck. Despite the recently developed therapies for the treatment of LSCC, patients’ overall survival rate still did not enhance remarkably; this highlights the need to formulate alternative strategies to develop novel treatments. The antitumor effects of antidepressant drugs such as citalopram have been reported on several cancer cells; however, they have yet to be investigated against LSCC. The current study was directed to explore the possible antitumor effects of citalopram on human laryngeal carcinoma cell lines (HEP-2). HEP-2 cells were cultured and treated with different doses of citalopram (50–400 µM) for 24, 48, and 72 h. The effects of citalopram on the viability of cancer cells were determined by the MTT assay. In addition, apoptosis and cell cycle analysis were performed by flow cytometry. Moreover, evaluation of the expression of proapoptotic and apoptotic proteins, such as cytochrome c, cleaved caspases 3 and 9, Bcl-2, and BAX, was performed by western blotting analysis. Our results revealed that citalopram significantly suppressed the proliferation of HEP-2 cells through the upregulation of p21 expression, resulting in the subsequent arrest of the cell cycle at the G0/G1 phase. Furthermore, citalopram treatment-induced HEP-2 cell apoptosis; this was indicated by the significant increase of cytochrome c, cleaved caspases 3 and 9, and BAX protein expression. On the contrary, Bcl-2 protein expression was significantly downregulated following treatment with citalopram. The ultrastructure studies were in accordance with the protein expression findings and showed clear signs of apoptosis with ring chromatin condensation upon treatment with citalopram. These findings suggest that citalopram’s anti-tumor activities on HEP-2 cells entailed stimulation of the intrinsic apoptotic pathway, which was mediated via Bcl-2 suppression.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

In Vitro Inhibition of Growth and Induction of Apoptosis by Mosinone-A in HEp-2 Oral Cancer Cells

The present study evaluated the influence of a Mosinone-A on the induction of apoptosis in HEp-2 human pharyngeal carcinoma cell lines as evidenced by cytotoxicity morphological changes, inhibition of cell proliferation in a time and dose dependent manner. Oral cancer is one of the leading causes of cancer worldwide. Many chemotherapeutics from plants have been tested in cancer, such as Mosinone-A, isolated from the bark of Annona squamous, acetogenin. further we analyzed anticancer activity by MTT assay, the generation of reactive oxygen species (ROS), the level of mitochondrial membrane potential and apoptotic morphological changes were analyzed by AO/EtBr. The results of an MTT assay demonstrated that Mosinone-A inhibited the growth of HEp2 cells in a dose-dependent manner. In addition, the induction of apoptosis was detected, as determined by morphological observation. These results indicated that Mosinone-A may induce apoptosis of HEp2 cells through the mitochondrial pathway.

Autophagic-Related Anticancer Effect of Grapes Extract and Tomatoes Extract: Ex-Vivo Study

Cells undergo autophagy to save themselves from injury, but progressive autophagy can cause cell death. This study characterized and compared the effect of grape (resveratrol) and tomato (lycopene) extracts and their combination on modulating autophagy-related miRNA and its target gene in squamous cell carcinoma cell line. Docking analysis for extracts and selected genes was performed. Methyl Thiazol Tetrazolium assays were used to assess the cytotoxicity of extracts and their combination toward HEp-2 cells. qRT-PCR was used to quantify changes in gene expression. Data were statistically analyzed. miRNA-20a was identified as a potential effector in laryngeal cancer, and sequestosome-1 (SQSTM1) was its target gene. Docking analysis showed that resveratrol interacted with miRNA-20a and showed less affinity toward SQSTM1. Hydrogen bonds and hydrophobic interactions were predicted. In contrast, lycopene showed less affinity toward miRNA-20a than resveratrol. Increasing doses of resveratrol, lycopene, and their combination induced a statistically significant reduction in mean percent viability and mean fold changes of miRNA-20a and SQSTM1 expression in treated HEp-2 cells. Pearson’s correlation showed a statistically significant positive correlation between miRNA-20a and SQSTM1 (R=0.812, p≤0.001). Grape and tomato extracts and their combination display promising cytotoxicity against HEp-2 cells in a dose- and time-dependent fashion. Both extracts reduce the expression of miRNA-20a and SQSTM1 with subsequent inhibition autophagy and promotion of apoptosis in HEp-2 cells.

Ononin Shows Anticancer Activity Against Laryngeal Cancer via the Inhibition of ERK/JNK/p38 Signaling Pathway.

BackgroundLaryngeal cancer is a type of head and neck tumor with a poor prognosis and survival rate. The new cases of laryngeal cancer increased rapidly with a higher mortality rate around the world.ObjectiveThe current research work was focused to unveil the in vitro antitumor effects of ononin against the laryngeal cancer Hep-2 cells.MethodologyThe cytotoxic effects of ononin against the laryngeal cancer Hep-2 cells and normal HuLa-PC laryngeal cells were studied using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The intracellular Reactive Oxygen Species (ROS) generation, apoptotic cell death, Mitochondrial Membrane Potential (MMP), and cell adhesion on the 25 and 50 µM ononin-treated Hep-2 cells were detected using respective staining assays. The levels of TBARS and antioxidants were assayed using specific kits. The expressions of c-Jun N-terminal kinase 1/2 (JNK1/2), Extracellular Signal-regulated Kinase 1/2 (ERK1/2), p38, Phosphatidylinositol-3 Kinase 1/2 (PI3K1/2), and protein kinase-B (Akt) in the ononin-treated Hep-2 cells were investigated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay.ResultsThe ononin treatment effectively inhibited the Hep-2 cell viability but did not affect the viability of HuLa-PC cells. Furthermore, the ononin treatment effectively improved the intracellular ROS accumulation, depleted the MMP, and triggered apoptosis in Hep-2 cells. The Thiobarbituric acid reactive substances (TBARS) were improved, and Glutathione (GSH) levels and Superoxide dismutase (SOD) were depleted in the ononin-administered Hep-2 cells. The ononin treatment substantially inhibited the JNK/ERK/p38 axis in the Hep-2 cells.ConclusionTogether, the outcomes of this exploration proved that the ononin has remarkable antitumor activity against laryngeal cancer Hep-2 cells.

Copper Oxide Nanoparticles Exhibit Cell Death Through Oxidative Stress Responses in Human Airway Epithelial Cells: a Mechanistic Study.

Copper oxide nanoparticles (CuONPs) are purposefully used to inhibit the growth of bacteria, algae, and fungi. Several studies on the beneficial and harmful effects of CuONPs have been conducted in vivo and in vitro, but there are a few studies that explain the toxicity of CuONPs in human airway epithelial cells (HEp-2). As a result, the purpose of this study is to look into the dose-dependent toxicity of CuONPs in HEp-2 cells. After 24h of exposure to 1-40µg/ml CuONPs, the MTT and neutral red assays were used to test for cytotoxicity. To determine the mechanism(s) of cytotoxicity in HEp-2 cells, additional oxidative stress assays (LPO and GSH), the amount of ROS produced, the loss of MMP, caspase enzyme activities, and apoptosis-related genes were performed using qRT-PCR. CuONPs exhibited dose-dependent cytotoxicity in HEp-2 cells, with an IC50 value of ~ 10μg/ml. The morphology of HEp-2 cells was also altered in a dose-dependent manner. The involvement of oxidative stress in CuONP-induced cytotoxicity was demonstrated by increased LPO levels and ROS generation, as well as decreased levels of GSH and MMP. Furthermore, activated caspase enzymes and altered apoptotic genes support CuONPs' ability to induce apoptosis in HEp-2 cells. Overall, this study demonstrated that CuONPs can cause apoptosis in HEp-2 cells via oxidative stress; therefore, CuONPs may pose a risk to human health and should be handled and used with caution.

L-ascorbic acid induces apoptosis in human laryngeal epidermoid Hep-2 cells by modulating the nuclear factor kappa-light-chain-enhancer of activated B cells/ mitogen-activated protein kinase/Akt signaling pathway

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl- 2 family and MAPK/Akt signaling pathways.

MiR-221 affects the proliferation and apoptosis of laryngeal cancer cells through the PI3K/AKT signaling pathway

To investigate the effect of MiR-221 on proliferation and apoptosis of laryngeal carcinoma cells through the PI3K/AKT signaling pathway. LipofectamineTM 2000 liposomes were used to transfer MiR-221 analogue, MiR-221 NC into Hep-2 cells of laryngeal carcinoma. Real-time fluorescence quantitative polymerase chain reaction (PCR) method was used to detect the expression of MiR-221, MTT method was used to detect the proliferation of cells, flow cytometry was used to detect cell cycle, Western blotting was used to detect the expression of apoptosis proteinase-1 (Apaf-1) and cyclin-dependent kinase (CDK1, CDK2) protein and the activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT). Compared with MiR-221 NC group, the expression of MiR-221 in MiR-221 analogue group was up-regulated (p<0.01), the cell proliferation rate was decreased (p<0.01), the cell cycle was stagnated in the G1 phase (p<0.01), the expression levels of Cyclin A, CDK1, CDK2, PI3K, and p-AKT were significantly down-regulated (p<0.01), and the expression levels of Bax and Apaf-1 were significantly up-regulated (p<0.01). MiR-221 analogues can significantly inhibit the proliferation and induce apoptosis of Hep-2 cells in laryngeal cancer, and this is achieved by blocking the PI3K/AKT signaling pathway, which also indicates that MiR-221 affects the proliferation and apoptosis of laryngeal cancer cells through the PI3K/AKT signaling pathway.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells.

Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. MTT assay was used to detect the inhibitory rate of viability. Giemsa's staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. These results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

Mechanisms of IFNalpha-1a-Induced Apoptosis in a Laryngeal Cancer Cell Line.

BackgroundInterferon alpha (IFNalpha) exerts its anti-proliferative effect on many human cancers. Among the 13 subtypes of human IFNalpha, IFNalpha-1 subtype has 2 variants, named IFNalpha-1a and IFNalpha-1b, that differ from each other in only 1 amino acid, at residue 114. However, the mechanism by which IFNalpha-1a mediates growth inhibition is still unclear.Material/MethodsHuman laryngeal carcinoma HEp2 cells were treated with IFNalpha-1a by either transient transfection or exogenous delivery. Western blot and RT-PCR analysis were carried out to assess apoptotic pathways active in IFNalpha-1a-treated HEp2 cells. Microarray analysis was conducted to uncover the differential gene expressions after IFNalpha-1a treatment. KEGG pathway enrichment analysis was also performed.ResultsIFNalpha-1a markedly inhibited the proliferation and significantly promoted the apoptosis of HEp-2 cells. Mechanistic studies indicate that IFNalpha-1a-mediated cell apoptosis is directly linked to intrinsic and endoplasmic reticulum (ER) stress-related apoptosis, but is independent of extrinsic apoptosis. The top 40 differentially expressed genes discovered by microarray analysis included 20 upregulated genes (e.g., IFI6, IFI27, IFI44L, and MIR548X) and 20 downregulated genes (e.g., PRKDC, HIST1H3B, DYNC1H1, and HIST1H2AM). KEGG pathway enrichment analysis revealed that 4 out of 6 pathways are TP53-related.ConclusionsWe demonstrated a detailed mechanism involved in IFNalpha-1a-mediated anti-proliferation activity in human laryngeal carcinoma cells.

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.

Combining use of Phillyrin and autophagy blocker alleviates laryngeal squamous cell carcinoma via AMPK/mTOR/p70S6K signaling.

Phillyrin (PHN), one of the major active constituents of Forsythia suspensa and F. koreana, has been reported to produce antioxidant, antibacterial, anti-obesity and anti-inflammatory effects. However, no study has demonstrated the role of PHN in laryngeal squamous cell carcinoma (LSCC). We aimed to investigate the effects of PHN on the proliferation and apoptosis of HEp-2 cells. In the present study, PHN alone showed little effect on HEp-2 cell proliferation and apoptosis. Subsequent tests showed that PHN could largely enhance the level of autophagy on HEp-2 cells. Combining use of PHN and autophagy blockers including 3-methyladenine (3-MA) and chloroquine (CQ) significantly inhibited HEp-2 cell proliferation in a dose- and time-dependent manner and induced apoptosis after 24 h in a dose-dependent manner. Additionally, we found that the possible underlying molecular mechanism of PHN-induced autophagy might be through the AMPK/mTOR/p70S6K signaling pathway. Taken together, our study indicates that combining use of PHN and autophagy blockers may serve as a novel strategy in LSCC treatment.

Indole-3-Carbinol Inhibits Laryngeal Cancer Growth Through Cell Cycle Arrest

The growth of a variety of tumors are inhibited by indole-3-carbinol (I3C) obviously. But, its role in laryngeal cancer is not clear. The goal of this study was to research the probable roles that laryngeal cancer cell apoptosis and proliferation Hep-2 was influenced by I3C. I3C dose-dependently therapy obviously inhibited Hep-2cell proliferation, and, I3C promoted apoptosis and induced cell morphological changes at 100, 200, 300, 400 μM doses. We discovered that I3C shows anticancer effect through various signal pathways after Hep-2 cells I3C therapy. In Hep-2laryngeal cancer cell line, through decreasing cell cycle-related proteins that include cyclin D1, CDK6, CDK4, and pRb, G1 arrest was induced by I3C. Apart from this, BALB/c nude mice constructed tumor-bearing mouse models. BALB/c nude mice were divided into three groups: treated with I3C, untreated control group and pretreated with I3C. After 8 weeks treatment, the untreated control group developed bigger tumors compared to mice treated or pretreated with I3C, and in the tumors such as cyclin D1, CDK6, CDK4 and pRb cell cycle-related proteins were obviously decreased. Further, the study result showed there was no harmful side effect in the heart, liver and kidney of the I3C-treated nude mice. In conclusion, both in vivo and in vitro I3C inhibited proliferation and induced the Hep-2 cells apoptosis, and showed low toxicity to normal cells. By suppressing the expression of cyclin families and CDK, we deduce that I3C can inhibit the Hep-2 cells growth in vitro. On normal organs and tissues, the I3C had no toxic effects and was safe.

TIMP-3 Increases the Chemosensitivity of Laryngeal Carcinoma to Cisplatin via Facilitating Mitochondria-Dependent Apoptosis.

Laryngeal carcinoma is a type of head and neck carcinoma with a high incidence and mortality. Chemotherapy treatments of human laryngeal carcinoma may fail due to the development of chemoresistance. Tissue inhibitor of metalloproteinase 3 (TIMP-3) has been shown to be implicated in a number of pathological processes typical for cancer. The present study aims to investigate the involvement of TIMP-3 in the chemoresistance of laryngeal carcinoma. We showed that TIMP-3 expression was significantly decreased in chemoresistant laryngeal carcinoma tissues compared with chemosensitivity tissues. Patients with low TIMP-3 expression exhibited poorer overall survival than those with high TIMP-3 expression. Moreover, cisplatin-resistant Hep-2 cells (Hep-2/R) were associated with the inhibition of mitochondrial membrane potential (MtMP) depolarization after cisplatin challenge. In addition, cisplatin resulted in a more pronounced mitochondrial cytochrome c release into the cytoplasm in Hep-2 cells than in their resistant variants. Overexpression of TIMP-3 by an adenovirus encoding TIMP-3 cDNA remarkably enhanced cisplatin-induced apoptosis, cytochrome c release, and caspase activation in Hep-2/R cells, thereby sensitizing cancer cells to cisplatin. On the other hand, downregulation of TIMP-3 markedly inhibited cisplatin-induced apoptosis in Hep-2 cells through attenuating mitochondria-dependent pathway activation. Taken together, these results demonstrate that decreased TIMP-3 expression may contribute to cisplatin resistance via inhibition of mitochondria-dependent apoptosis, indicating that forced TIMP-3 expression may be a useful strategy to improve the efficacy of cisplatin to treat laryngeal carcinoma.

MiR-196b affects the progression and prognosis of human LSCC through targeting PCDH-17

ObjectiveTo explore the effect of miR-196bon the biological features of human laryngeal squamous cell carcinoma (LSCC) through targeting PCDH-17. MethodsmiR-196b and PCDH-17 expressions were determined in tissues, and the targeting relation of miR-196b and PCDH-17 was verified through dual-luciferase reporter system. In vitro, Hep-2 cells were divided into the Control, miR-196b inhibitors, miR-NC, PCDH-17, and miR-196b mimics+PCDH-17 groups. The miR-196b and PCDH-17 expressions were determined by qRT-PCR or/and Western blot, and the biological features by MTT, Annexin V-FITC/PI, wound-healing and Transwell assays. ResultsMiR-196b was found to be up-regulated, while PCDH-17 was down-regulated in a negative correlation in LSCC patients, which was related to histological grade and TNM stage. And low expression of miR-196b and high expression of PCDH-17 contributed to an increase in the 5-year-survival rate of LSCC patients. Besides, miR-196b directly targeted PCDH-17, while miR-196b inhibitors could up-regulate the PCDH-17 in Hep-2 cells. Moreover, miR-196b inhibitors and PCDH-17 curbed Hep-2 cell proliferation but facilitated the apoptosis, with decreases in cell invasion and migration. In addition, no statistical significance was found in cell proliferation, apoptosis, invasion and migration between Control group and miR-196b mimics+PCDH-17 group. ConclusionLSCC patients exhibited the up-regulated miR-196b and down-regulated PCDH-17, which are correlated with the major clinical features and prognosis. Inhibiting miR-196b may suppress proliferation, migration and invasion abilities, and promote apoptosis of Hep-2 cells via targeting PCDH-17.

Antiproliferative potential of miR-33a in laryngeal cancer Hep-2 cells via targeting PIM1.

Laryngeal cancer is a frequent cause of cancer-associated mortality worldwide with an overall poor prognosis along with high mortality rates. Therefore, comprehensive investigation of underlying molecular mechanisms of laryngeal carcinogenesis remains an important problem. In this study, proliferative and apoptotic features of Hep-2 cells overexpressing microRNA-33a (miR-33a) were evaluated and in silico analysis along with literature search was used to find putative targets of miR-33a. The potential of PIM1 (pim-1 oncogene) as a direct target of miR-33a was tested using quantitative real-time polymerase chain reaction, Western blot, and luciferase assay. Induced miR-33a expression significantly inhibited proliferation through inducing apoptosis of Hep-2 cells. Further in vitro tests showed downregulation of PIM1 in messenger ribonucleic acid (mRNA) and protein level upon miR-33a overexpression and confirmed PIM1 as a direct target of miR-33a. Mir-33a was demonstrated to act as a tumor suppressor in larnygeal cancer via directly targeting the 3' untranslated region of PIM1.

Knockdown of the prolyl isomerase Pin1 inhibits Hep-2 cell growth, migration, and invasion by targeting the β-catenin signaling pathway.

There is increasing evidence indicating that peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) plays a decisive role in a variety of cancers. Nevertheless, its function in laryngeal squamous cell carcinoma (LSCC) has not been elaborated. The aim of this study is to determine the role of Pin1 in LSCC. Here, we established stably transfected Hep-2 cells with low expression of Pin1. Intriguingly, cell proliferation, migration, and invasion was significantly inhibited in Pin1-silenced Hep-2 cells. Similarly, knockdown of Pin1 induced apoptosis of Hep-2 cells, as evidenced by increased expression of cleaved-caspase-3, cleaved-PARP, and bax, and decreased expression of bcl2. We also demonstrated that silencing of Pin1 down-regulated β-catenin and cyclin D1 expression. Inversely, over-expression of β-catenin reversed the inhibiting effect of Pin1 silencing on Hep-2 cells. Moreover, we proved that knockdown of Pin1 inhibited tumorigenesis of Hep-2 cells in vivo. Taken together, we demonstrate that silencing of Pin1 effectively suppresses the growth of Hep-2 cells through β-catenin, indicating that Pin1 possess the potential to serve as a therapeutic target for the treatment of LSCC.

Preparation and in Vitro Anti-Laryngeal Cancer Evaluation of Protopanaxadiol-Loaded Hollow Gold Nanoparticles

Abstract Protopanaxadiol (PPD) has inhibitory effects on a variety of tumor cells, but the poor water solubility and low availability limit its clinical application. By using polyethylene glycol (PEG)-modified hollow gold nanoparticles (HAuNs-PEG) as the transport carriers, we prepared here the PPD-loaded HAuNs-PEG (HAuNs-PEG-PPD). And then we conducted a number of experiments to investigate the in vitro anti-laryngeal cancer effect (Hep-2 cell as a model) of HAuNs-PEG-PPD, such as using high performance liquid chromatography (HPLC) to detect the sustained release effect, MTT assay to detect the inhibitory effect on the proliferation of Hep-2 cells, and flow cytometry to determine the apoptosis of Hep-2 cells. The results showed that HAuNs-PEG-PPD had an obvious sustained release effect. Compared with those in the control group, HAuNs-PEG group and PPD group, the survival rate of Hep-2 cells decreased and the apoptosis rate increased significantly (p

Effects of gold nanorods modified with antiepidermal growth factor receptor monoclonal antibody on laryngeal cancer cells.

We aimed to evaluate the photothermal effects of gold nanorods (GNRs) modified with antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb) on laryngeal cancer cells. EGFR protein expressions in HEP-2 cells, and normal laryngeal epithelial and laryngeal cancer tissues were detected by western blot. The cytotoxicity against HEP-2 and BEAS-2B cells was tested by MTT assay, and the photothermal effects were assessed by near-infrared (NIR) irradiation. The apoptosis of HEP-2 cells was detected by flow cytometry. EGFR expression in laryngeal cancer tissues was significantly higher than that in normal tissues (P < 0.05). The inhibitory effects of GNRs and anti-EGFR mAb/GNRs on the apoptosis of HEP-2 and BEAS-2B cells were enhanced with increasing dose. AntiEGFR mAb/GNRs were more cytotoxic to HEP-2 cells and less cytotoxic to BEAS-2B cells than GNRs. NIR irradiation inhibited cell proliferation, which was enhanced with rising power, accompanied by continuously dropping survival rate. The apoptosis rate of the anti-EGFR mAb/GNRs group was significantly higher than that of the GNRs group, and the apoptosis rate of the irradiation + antiEGFR mAb/GNRs group also significantly exceeded that of the irradiation + GNRs group (P < 0.05). Anti-EGFR mAb/GNRs promoted HEP-2 cell apoptosis more evidently than GNRs did. Functional modification of GNRs augmented the targeted specificity to cancer cells, biocompatibility, and stability. Anti-EGFR mAb/GNRs have great potential in biomedical fields.

Effects of aspirin on proliferation, invasion and apoptosis of Hep-2 cells via the PTEN/AKT/NF-κB/survivin signaling pathway.

Aspirin may exhibit antitumor activities, as it is able to inhibit cell proliferation. However, the ability of aspirin to inhibit cellular proliferation in Hep-2 cells and its underlying molecular mechanisms have been poorly determined. The aim of the present study was to investigate whether aspirin may induce cell apoptosis in the neoplastic cell line Hep-2. The effects of aspirin on the migratory and invasive abilities of Hep-2 cells were also investigated using Transwell assays. In the present study, it was demonstrated that aspirin induced apoptosis and inhibited proliferation, migration and invasion in Hep-2 cells. Aspirin also significantly decreased the expression of B-cell lymphoma 2 (Bcl-2) and caspase-3, and increased the expression of Bcl-2-associated X protein, suggesting that aspirin induced apoptosis through the intrinsic apoptotic pathway. Hep-2 cells treated with aspirin exhibited a significant upregulation of phosphatase and tensin homolog (PTEN) and decreased levels of phosphorylated protein kinase B (AKT). However, the total amount of AKT protein was not altered in response to aspirin treatment. Furthermore, the expression of nuclear factor (NF)-κB and survivin, which are the downstream targets of the PTEN/AKT signaling pathway, was inhibited. These results indicated that the molecular mechanism underlying the antitumor effects of aspirin may be associated with the inhibition of tumor invasion and induction of apoptosis by regulating the activity of the PTEN/AKT/NF-κB/survivin signaling pathway.