Human Laryngeal Carcinoma Cell Line Research Articles

Role of SATB1 in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanisms

Objective To investigate the role of special AT-rich sequence-binding protein 1 (SATB1) in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanism. Methods Hep-2 cells were cultured in vitro. The cell lines were divided into two groups. The cells in the experimental group was transfected with Lipofectamine 2000 liposome for SATB1 small interfering RNA (SATB1-siRNA), and the cells in the control group was transfected with Lipofectamine 2000 liposome for NC-siRNA. The before and after transfection SATB1, E-cadherin and Snail genes relative expression levels between the two groups were detected and compared using RT-PCR. Transwell cell invasion experiments were applied to evaluate the cells invasive ability of the two groups. The scratch experiments were applied to evaluate the 48 h cell migration ability of the two groups. Results The differences in the relative expression level of SATB1, E-cadherin and Snail genes in pre-transfected laryngeal squamous cell line Hep-2 between the two groups were not statistically significant (all P>0.05). Compared with the control group, the relative expression levels of the mRNA of SATB1 and Snail genes decreased in the experimental group after transfection, while the relative expression levels of the mRNA of E-cadherin gene increased, and the differences were statistically significant (all P<0.05). After the transfection, the number of transwell membrane cells and cell migration distances of the two groups of cells decreased. The values of cell number and migration distance in the experimental group were lower than those in the control group, and the differences were statistically significant (all P<0.05). Conclusions SATB1 can promote the invasion and metastasis of Hep-2 cells, and its mechanism may be related to the down-regulation of E-cadherin and the up-regulation of Snail gene expression to induce epithelial-mesenchymal transition. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; Neoplasm metastasis; Special AT-rich sequence-binding protein 1

Soyasapogenol B Attenuates Laryngeal Carcinoma Progression through Inducing Apoptotic and Autophagic Cell Death.

Soyasapogenol B (Soy B), a constituent of soybean, has been shown to exhibit antitumor activities against different types of cancers. However, to our knowledge, no studies so far have investigated the effect of Soy B in human laryngeal carcinoma. This study, therefore, aimed to determine the effect of Soy B in human laryngeal carcinoma cell lines HeP-2 and TU212 and to elucidate the possible underlying mechanisms by which Soy B can induce its antitumor effects. The results showed that Soy B effectively attenuated the cell growth by causing G0/G1 phase cell cycle arrest in laryngeal carcinoma cell lines. Moreover, the percentage of apoptotic and autophagic cells dramatically increased upon exposure to Soy B. Western blotting results confirmed that Soy B can alter the expression levels of established markers of apoptosis and autophagy. Interestingly, both apoptosis inhibitor (ZVAD-fmk) and autophagy inhibitor (3-MA) could partially reverse the effect of Soy B, while blocking autophagy did not cause obvious alteration in the percentage of apoptotic cells. Similarly, in vivo studies validated that Soy B could effectively reduce the size of the tumor and induce apoptosis and autophagy in tumor tissues. Collectively, these results suggested that Soy B can exert anticancer activities against laryngeal carcinoma through inducing apoptotic and autophagic cell death. Our study highlighted the potential role of Soy B as a chemotherapeutic agent for laryngeal carcinoma. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1851-1858, 2020. © 2019 American Association for Anatomy.

Pepsin promotes IL-8 signaling-induced epithelial\u2013mesenchymal transition in laryngeal carcinoma

BackgroundLaryngopharyngeal reflux (LPR), with its increasing morbidity, is attracting considerable attention. In recent years, the causal role between LPR and laryngeal carcinoma has been debated. The main harmful component of LPR is pepsin, which has been shown to induce mucosal inflammation by damaging the mucous membrane. Thus, pepsin is linked to an increased risk of laryngeal carcinoma, although the potential mechanism remains largely unknown.MethodsThe human laryngeal carcinoma cell lines Hep-2 and Tu212 were exposed to different pepsin concentrations and the morphology, proliferation, migration, secretion of inflammatory cytokines, and epithelial–mesenchymal transition (EMT) of the cells were assessed. To evaluate whether interleukin-8 (IL-8) had a causal relationship with pepsin and EMT, an IL-8 inhibitor was used to suppress IL-8 secretion during pepsin exposure and the expression of EMT markers, cell proliferation, and migration were analyzed.ResultsPepsin promoted proliferation, colony formation, migration, and IL-8 secretion of Hep-2 and Tu212 cells in vitro. Furthermore, increased pepsin concentrations changed the morphology of Hep-2 and Tu212 cells; levels of the epithelial marker E-cadherin were reduced and those of mesenchymal markers vimentin and β-catenin and the transcription factors snail and slug were elevated. A similar effect was observed in laryngeal carcinoma tissues using immunohistochemistry. IL-8 level was reduced and EMT was restored when pepsin was inhibited by pepstatin. EMT was weakened after exposure to the IL-8 inhibitor, with significant reduction in pepsin-induced cell proliferation and migration.ConclusionsPepsin may induce EMT in laryngeal carcinoma through the IL-8 signaling pathway, which indicates that it has potential role in enhancing cell proliferation and metastasis of laryngeal carcinoma.

Luminescent Ag(I) scorpionates based on tris(2-pyridyl)phosphine oxide: Synthesis and cytotoxic activity evaluation

First representatives of Ag(I) scorpionates bearing tris(2-pyridyl)phosphine oxide (Py3PO), viz. [Ag(Py3PO)(PPh3)]BF4 (1) and [Ag(Py3PO)(PPh3)]ClO4 (2), have been synthesized and structurally characterized. X-ray diffraction analysis reveals that the silver atom of 1 is coordinated by Py3PO and Ph3P ligands in N,N′,N″-tripodal and P-monodentate fashions, respectively. The both complexes exhibit significant cytotoxic activity (IC50 being from 11 to 14 μM) screened in vitro against human larynx carcinoma (Hep-2) cell lines. The luminescent proprieties of the prepared scorpionates are also discussed.

Cytotoxic Serrulatane-Type Diterpenoids from the Gorgonian Euplexaura sp. and Their Absolute Configurations by Vibrational Circular Dichroism

Vibrational circular dichroism (VCD) method has become robust and reliable alternative for the stereochemical characterization of natural products. In this paper, three new serrulatane-type diterpenoids, euplexaurenes A–C (1–3), and a known metabolite, anthogorgiene P (4), were obtained from the South China Sea gorgonian Euplexaura sp. GXWZ-05. The absolute configuration of C-11 in 1–4, which was difficult to be determined by common means due to the high conformational flexibility of the eight-carbon aliphatic chain attached at C-4, was determined by VCD method, suggesting a new horizon to define the absolute configurations of natural products possessing chains. Compounds 1–4 were found to show selective cytotoxic activities against human laryngeal carcinoma (Hep-2) cell line with the IC50 values of 1.95, 7.80, 13.6 and 5.85 μM, respectively.

MiR-503 enhances the radiosensitivity of laryngeal carcinoma cells via the inhibition of WEE1.

Enhancing the sensitivity of laryngeal cells to radiation is crucial for improving the efficacy of laryngeal carcinoma. MicroRNAs are known to play a major role in regulating cellular radiosensitivity. This study was designed to explore the effect and the molecular basis of miR-503 in the radiosensitivity of laryngeal carcinoma cells. Quantitative real-time polymerase chain reaction analysis showed that miR-503 expression was decreased in human laryngeal carcinoma cell lines Hep-2 and TU212, and the downregulation of miR-503 was also observed after irradiation. Upregulation of miR-503 by pre-miR-503 transfection restrained proliferation, promoted progression of Hep-2 and TU212 cells through the cell cycle after irradiation, and sensitized cells to radiation. Dual-Luciferase Reporter Assay verified a direct interaction between miR-503 and the WEE1 messenger RNA 3'-untranslated region. The overexpression of miR-503 significantly decreased WEE1 expression at the messenger RNA and protein levels, whereas the inhibition of miR-503 upregulated the expression of WEE1. WEE1 knockdown by WEE1 small interfering RNA apparently abrogated the inhibitory effect of anti-miR-503 on radiosensitivity. In conclusion, miR-503 could function as an enhancer of radiation responses in laryngeal carcinoma cells by inhibiting WEE1, which may be a potential novel radiosensitizing strategy for laryngeal carcinoma.

The tissue distribution and significance of B7-H4 in laryngeal carcinoma.

The costimulatory signals CD28 and B7 have been shown to control tumor invasion and metastasis by regulating T cell activation, whereas the distribution characteristics of B7-associated proteins in laryngeal carcinoma (LC) tissue are still unclear. Here, the expression of members of the B7 superfamily, including B7-H1 (PD-L1), B7-DC (PD-L2) and B7-H4, in fifty-two LC samples was determined by immunohistochemistry, and the relationship between B7-H4 and epithelial-mesenchymal transition (EMT)-associated markers was further assessed by immunofluorescence double staining. Furthermore, the human LC cell lines, Hep-2 and TU212 cells, were further transfected to overexpress B7-H4, and cell invasion and metastasis were analyzed. The results showed that B7-H1, B7-DC and B7-H4 were expressed in the tumor cells, and their expression was restricted to the cell membrane and the cytoplasm. The positive rates of these molecules in the tumor tissues were 57.7% (30/52), 32.7% (17/52) and 34.6% (18/52), respectively. Interestingly, double immunofluorescence staining showed that B7-H4 is coexpression with EMT-related markers, including p-Smad2/3, Snail and Vimentin, in carcinoma cells. Moreover, overexpression of B7-H4 in Hep-2 cells promotes the expression of pSmad2/3 and Snail by activating AKT-STAT3 signaling. Transwell and wound-healing assays demonstrated that B7-H4 enhanced both Hep-2 and TU212 cell invasion and metastasis. Our results suggest that B7-H4 transmits feedback signaling to tumor cells and promotes invasion and metastasis by promoting EMT progression. Therefore, blocking B7-H4 signaling might be a novel treatment strategy for LC.

In Vitro Cytotoxic Study for Purified Resveratrol Extracted from Grape Skin Fruit Vitis vinifera

&#x0D; This study was conducted with the aim to extract and purify a polyphenolic compound “ Resveratrol” from the skin of black grapes Vitis vinifera cultivated in Iraq. The purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis with 10% HCl solution then the aglycon moiety was taken with organic solvent&#x0D; ( chloroform). Using silica gel G60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid 20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). The study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma AMN-3 cell line, the human laryngeal carcinoma (Hep-2) cell line and the Rat embryo fibroblast (Ref) cell line, at different concentrations and different expousure time. The cytotoxic effect of the pure resveratrol was studied in comparison with trans- resveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations (0.05 , 0.1 , 0.2 and 0.4) µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. The cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (P&lt;0.01) was achieved after 24 hr of exposure on both AMN-3 and Ref cell lines. Hep-2 cell line responded to extracted resveratrol in different manners.&#x0D; Keywords: Resveratrol , grape skin , polyphenols, cytotoxic study

Supporting effect of polyethylenimine on hexarhenium hydroxo cluster complex for cellular imaging applications

Abstract The present work introduces an insight into pH- and concentration-dependent binding of branched polyethylenimine (PEI) with hexarhenium hydroxo cluster complex [{Re 6 S 8 }(OH) 6 ] 4− aimed at enhancing the stability, luminescence intensity and cellular uptake of the cluster complex in aqueous solutions at pH 7.4. Luminescence and conductivity measurements revealed pH 7.4 and 1:1 of PEI:cluster molar ratio as specific conditions ensuring multiple electrostatic interactions of protonated amino groups of PEI with the tetra-anionic cluster units. These interactions resulted in a significant supporting effect of PEI on stability and luminescence of the cluster complex, while no effect of the cluster complex on the acid-base and aggregation properties of PEI was detected from dynamic light scattering and 1 H NMR spectroscopy measurements. The cluster luminescence remained without degradation for at least two weeks due to the supporting effect of PEI, which can facilitate the application of the cluster complex in bioimaging. The cellular uptake of [{Re 6 S 8 }(OH) 6 ] 4− and [{Re 6 S 8 }(OH) 6 ] 4− @PEI was investigated. Confocal microscopic studies showed that the pristine cluster complex [{Re 6 S 8 }(OH) 6 ] 4− did not enter cells (the human larynx carcinoma cell line (Hep-2)), whereas the supramolecular assembly [{Re 6 S 8 }(OH) 6 ] 4− @PEI internalized into the cells.

Cytotoxicity and genotoxicity effects of arsenic trioxide on SQ20B human laryngeal carcinoma cells

This study investigates the cytotoxicity and the genotoxicity induced by arsenic trioxide As2O3in human laryngeal SQ20B carcinoma cell line. SQ20B cells were exposed to graded concentrations of arsenic trioxide (2 and 5μM) for 48h. Comet assay and γ-H2AX foci formation were used for measuring DNA damages, flow cytometry was used to identify cell cycle alterations and apoptosis, while cell morphology was visualized using transmission electron microscopy. The results show a dose-dependent induction of DNA damages and double strand breaks, alterations in cell cycle and morphologic alterations of cells. These results prove that As2O3 is highly cytotoxic and genotoxic at the micromolar range ina human laryngeal carcinoma cell line.

Identification and characterization of CD133+CD44+ cancer stem cells from human laryngeal squamous cell carcinoma cell lines.

Background: Laryngeal squamous cell carcinoma ranks second among head and neck squamous-cell carcinomas. Cancer stem cells can support cancer growth and malignant behavior. Therefore, cancer stem cells isolated from laryngeal squamous cell carcinoma tissue could be used to investigate the initiation, progression, and treatment strategies of this cancer. Methods: We isolated CD133-CD44-, CD133-CD44+, CD133+CD44- and CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep2 and TU-177 by magnetic-activated cell sorting. Sphere formation, cell proliferation, migration, invasion, colony formation, resistance to radio- and chemotherapy, and in vivo tumorigenicity of these populations were evaluated. Moreover, we investigated the expression of the stem-cell markers (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) in CD133-CD44-, CD133-CD44+, CD133+CD44-, CD133+CD44+ cell populations and parental Hep2 and TU-177 cells. Results: As compared with CD133-CD44-, CD133-CD44+, CD133+CD44- populations and parental cells, CD133+CD44+ cells showed higher cell viability, migration and invasive capability and colony formation ability as well as stronger resistance to cisplatin and irradiation. Moreover, levels of SOX2 and OCT4 and tumorigenicity in nude mice were greater in CD133+CD44+ Hep2 and TU-177 cells than other cell populations and parental cells.Conclusion: The CD133+CD44+ population of laryngeal squamous-cell carcinoma Hep2 and TU-177 cells have stem cell properties and showed more malignant features than CD133+CD44- and CD133-CD44+ cell populations. CD133+CD44+ cancer stem cells may be a promising target for developing anticancer drugs and treatment strategies for laryngeal squamous cell carcinoma.

AgNPs and Ag/C225 Exert Anticancerous Effects via Cell Cycle Regulation and Cytotoxicity Enhancement

Currently, cancer has become the leading cause of deaths and is a serious threat to human health. Nanotechnology-based medical applications are developing rapidly, especially in anticancer research, as nanomaterials can be retained in the cell after their entry across the cell membrane, making them potential candidates for application in the diagnosis and treatment of tumours. Here, we prepared silver nanoparticles (AgNPs) and silver/cetuximab (Ag/C225) particles of approximately 20 nm and investigated their inhibitory effect on proliferation of human nasopharyngeal carcinoma cell line CNE and laryngeal carcinoma cell line HEp-2. The MTT assay was used to determine their half-maximal inhibitory concentration (IC50) values. Electron microscopy revealed no obvious morphological differences between the prepared AgNPs and Ag/C225. Treatment of both cell lines with AgNPs and Ag/C225 increased the proportion of cells in the G0/G1 stage and decreased the proportion of cells in the S stage. AgNP and Ag/C225 treatments increased the expression of apoptotic proteins Bax and P21 and decreased the expression of Bcl-2 in CNE cells, compared to control cells. Ag/C225 is internalised by CNE cells, and it enhances X-irradiation-induced cytotoxicity. These results help increase the understanding of the anticancer effect of AgNPs and Ag/C225 and explore their application as radiosensitisers.

MicroRNA-133a suppresses the proliferation, migration, and invasion of laryngeal carcinoma cells by targeting CD47.

The study aims to investigate the possible mechanisms of microRNA-133a (miR-133a) targeting CD47 on cell proliferation, apoptosis, migration, and invasion in laryngeal carcinoma. Forty-two laryngeal carcinoma tissue specimens confirmed by pathological examination from laryngeal carcinoma patients as the case group were collected, and 20 chronic laryngitis tissues were gathered as the control group. The human laryngeal carcinoma cell line Hep-2 was marked as the miR-133a mimic, negative control (NC), miR-133a inhibitor, CD47-siRNA, miR-133a inhibitor + CD47-siRNA, and Mock groups. The expression of CD47 protein and miR-133a was detected by immunohistochemistry (IHC) and qRT-PCR. Dual luciferase assay system was used to determine the relationship between CD47 and miR-133a. Western blotting was used to measure the protein expression of CD47 and miR-133a. 5-Ethynyl-2'-deoxyuridine (EDU) method was used to detect the cell proliferation, and flow cytometry and Transwell were used to measure the cell apoptosis and migration and invasion, respectively. The miR-133a expression in laryngeal carcinoma tissues was significantly lower, while the CD47 expression was higher than that in chronic laryngitis tissues (both P<0.01). The expression of miR-133a in the miR-133a mimic group was significantly higher than that in other groups (P<0.05), and the messenger RNA (mRNA) and protein expression of CD47 in the CD47-siRNA and miR-133a mimic groups were significantly lower than those in the Mock and NC group (all P<0.05), while the mRNA and protein expression of CD47 in the miR-133a inhibitor group were higher than in other groups (all P<0.05). After transfection, the CD47-siRNA group had the strongest inhibitory activity, while the number of living cells in the miR-133a inhibitor group was significantly higher than that in other groups (all P<0.05). The apoptosis rates in the miR-133a mimic and CD47-siRNA groups were significantly higher than that in the Mock and NC groups (all P<0.05). The cell numbers that penetrated membrane in the miR-133a mimic and CD47-siRNA groups were less than in the Mock and NC groups (all P<0.05). Upregulated miR-133a could inhibit proliferation, invasion, and migration and promote cell apoptosis in laryngeal carcinoma by targeting CD47. miR-133a targeting CD47 could be a new direction in the diagnosis and treatment of laryngeal carcinoma.

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

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

Studies on Biosurfactant producing bacteria from Jeppiaar salt pan with special reference to anticancer activity

Biosurfactants are surface-active compounds produced by microorganisms. These molecules reduce surface tension between aqueous solutions and hydrocarbon mixtures. In this study, we collected water samples from Jeppiaar salt pan in Chennai, Tamilnadu, India. Ten different microorganisms were isolated from salt pan water samples. Screening of biosurfactants producing potential strain was done using Hemolytic activity, Oil displacement test, Drop Collapse assay. Four isolates were positive for biosuractant production among that one isolate was found as potential biosurfactant producer. Both biochemical and 16S rRNA sequencing methods one of the potential strain was identified as Bacillus cereus JS06. The biosurfactant was extracted from cell-free broth at 72-h grown cells by step-by-step purification of acid precipitation method. The dried surfactant was used for further characterization by Fourier transmission Infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and anticancer studies. On FTIR-ATR analysis characteristic absorbance bands were observed for the extracted biosurfactant. The crude biosurfactant was screened for its anticancer activity against human laryngeal carcinoma (HEP-2) cell line and transformed aneuploid immortal keratinocyte (HeCAT) cell line. The results obtained showed that biosurfactants have better interfacial-activity and anticancer property, thus being more attractive to be applied in pharmaceuticals and medical field.

Cellular internalisation, bioimaging and dark and photodynamic cytotoxicity of silica nanoparticles doped by {Mo6I8}4+ metal clusters.

Silica nanoparticles (SNPs) doped by hexanuclear molybdenum cluster complexes [{Mo6X8}L6]n (X = Cl, Br, or I; L = various inorganic or organic ligands) have been recently suggested as materials with high potential for biomedical applications due to both their outstanding photoluminescence properties and their ability to efficiently generate singlet oxygen upon photoirradiation. However, no studies were undertaken so far to prove this concept. Therefore, here we examined the potential of photoluminescent SNPs doped by {Mo6I8}4+ for applications such as bioimaging and photodynamic therapy using the human epidermoid larynx carcinoma (Hep-2) cell line as a model. Our results demonstrated both: (i) significant luminescence from cells with internalised molybdenum cluster-doped SNPs combined with the low cytotoxicity of particles in the darkness and (ii) significant cytotoxicity of the particles upon photoirradiation. Thus, this research provides strong experimental evidence for high potential of molybdenum-cluster-doped materials in biomedical applications such as optical bioimaging, biolabeling and photodynamic therapy.

Consumer acceptability of liquorice root (Glycyrrhiza glabra L.) as an alternative sweetener and correlation with its bioactive content and biological activity

Consumer acceptability and sensory properties of liquorice root (Glycyrrhiza glabra L.) were evaluated. Quantitation of total polyphenolics and glycyrrhizic acid (GA), as well as the antioxidant capacity of liquorice extracts, was conducted and their biological effects (cytotoxic, antioxidative/pro-oxidative activity, lipid peroxidation on human laryngeal carcinoma cell line) compared to the ones of their predominant bioactive compound – GA. Conducted consumer survey revealed poor familiarity with liquorice (12.37% of correspondents), but willingness towards its use as an alternative sweetener (77.32% of consumers). Polyphenolic content of evaluated extracts ranged from 1018.18 to 1277.27 mg gallic acid equivalents (GAE)/l while GA content varied between 2179.53 and 2944.13 mg/l. The most pronounced cytotoxic effect (60%) and lipid peroxidation were exerted by treatment with the highest applied extract concentrations (10 mg/ml). Pure GA exhibited cytotoxic and pro-oxidative effects at concentrations of 0.12–0.6 mg/ml. Due to high GA content, coupled with its pronounced cytotoxic activity, the intake of liquorice root should be limited.

Comparative evaluation of PLGA nanoparticle delivery system for 5-fluorouracil and curcumin on squamous cell carcinoma

PurposeThe purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. Material and methodsPLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. ResultsThe drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. ConclusionCurcumin nanoparticles could be more active in inducing apoptosis in short term assays (24h) than long term assays (48h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48h) compared to curcumin group.

Haemophilus influenzae triggers autophagy in HEp-2 cells.

The MAP-LC3 system regulates the intracellular formation of autophagy-associated vacuoles. These vacuoles contain the LC3 protein; thus it has been utilized as a marker to identify autophagosomes. The aim of our study was to investigate whether Haemophilus influenzae strains and their supernatants could activate autophagy in human larynx carcinoma cell line (HEp-2). We demonstrate that higher expression of the LC3B-II protein was induced, particularly by nontypeable Haemophilus influenzae (NTHi) 49766 and by supernatants, containing <50 kDa proteins, of both strains. Ultrastructural studies demonstrate vacuoles with a double membrane and/or membrane material inside, showing similar features to those of autophagic vacuoles. Together, our findings demonstrate that H. influenzae strains and their supernatants trigger an autophagic process.

A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Human esophageal cancer-related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep-2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1-ECRG4 was constructed and introduced into Hep-2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high-level expression of ECRG4. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 over-expression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpres-sion of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl-2-associated X protein, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase were upregulated in the apoptotic process, whereas B-cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.