Laryngeal Hep-2 Research Articles

Synthesis of new peptide-based macrocycles: Evaluation of their in vitro anti-proliferative activity and molecular dynamic simulation studies

A simplified synthetic protocol was adopted for the preparation of the six novel cyclo-pentapeptide candidates of the structure cyclo-(m-phthaloyl)-bis-[L-Phe -Gly/Sar]-L-Lys–X, where X = COOMe (9a,b), COOH (10a,b) and NHNH2 (11a,b). The potential cytotoxicity (IC50, μg/mL) for cyclo-pentapeptides (9a,b), (10a,b), (11a,b) and doxorubicin® as reference drugs, is assayed against eight cell lines of human cancer namely; mamillary MCF-7 and T47D, hepatic HEPG2, colon HCT116, cell line of cervix HELA, larynx HEP2, Prostate PC3, and gut Caco. Cyclo-[m-phthaloyl chloride-bis-(L-phalanine-sar)-L-Lys]-COOH 10b was proved as cytotoxically more potent than doxorubicin against most of the screened cell lines. It can be inferred that the proposed molecular structural characteristics associated with cytotoxicity in these m-phthaloyl-bridged cyclopeptides appear noteworthy and hold promise as potential candidates for further exploration as novel anticancer agents. Molecular dynamics simulation studies of 10b into the binding site of Human Epidermal Growth Factor Receptor tyrosine kinase exhibited the highest MM-GBSA value (-16.59 kcal/mol). KEY WORDS: Anticancer, Cytotoxicity, Cyclopeptides, Cyclopeptides, m-phthaloyl-bis-peptides Bull. Chem. Soc. Ethiop. 2024, 38(4), 1129-1144. DOI: https://dx.doi.org/10.4314/bcse.v38i4.25

Gold Nanorods are Selective Cytotoxic Agents.

Gold nanorods (GNRs) are very promising agents with multiple applications in medicine and biology. However, the cytotoxic effects of GNRs have not been fully explored. Therefore, the main objective of this study was to determine the selective cytotoxic effect of GNRs towards several human tumor cell lines. To address this issue, three sizes of GNRs (10-nm, 25-nm, and 50-nm) were tested against two human tumor cell lines, namely, human hepatoma HepG2 and human prostate PC3 cancer cells. As GNRs are usually stored in soft tissues inside living bodies, we also tested the effect of GNRs on murine splenocyte viability. To determine if the GNRs displayed selective cytotoxicity towards cancer cells, active GNRs with the size showing the least cytotoxicity to splenocytes were then tested against a panel of 11 human tumor cell lines and two human non-tumor cell lines. Our results showed that the most cytotoxic size of GNRs is 10-nm, followed by the 25-nm GNRs, while the 50-nm GNRs did not show a significant effect. In addition, the 25-nm GNRs were the least cytotoxic to splenocytes when tested for 24 and 48 h. These GNRs showed a selective cytotoxic effect to prostate cancer PC3 cells with median inhibitory concentration (IC50) = 8.3 ± 0.37 μM, myeloblastic leukemia HL60 cells (IC50 = 19.7 ± 0.89 μM), cervical cancer HeLa cells (IC50 = 24.6 ± 0.37 μM), renal adenocarcinoma 786.0 cells (IC50 = 27.34 ± 0.6 μM), and hepatoma HepG2 cells (IC50 = 27.79 ± 0.03 μM) when compared to the effect on the non-tumor human cells; skin fibroblast BJ cell line (IC50 = 40.13 ± 0.7 μM) or epithelial breast MCF10A cells (IC50 = 33.2 ± 0.89 μM). High selectivity indices (SIs) were observed in GNRs-treated PC3 and HL60 cells with values ranging from 1.69 to 4.83, whereas moderate SIs were observed in GNRs-treated HeLa, 786.0, and HepG2 cells with values ranging from 1.19 to 1.63. Other cells did not show a similar selective effect, including human laryngeal HEp2 cells, colon HCT116, metastatic renal adenocarcinoma ACHN cells, and human breast cancer cells (MCF7, MDA-MB-231, and MDA-MB-468 cells). The effect of GNRs was confirmed using the colony formation assay and the effect was found to be cell cycle-specific. Finally, it was shown that laser treatment could potentiate the cytotoxic effect of the 25-nm GNRs. GNRs are selective cytotoxic agents and they have the potential to act as candidate anticancer agents.

Expression profile of stem cell markers and ABC transporters in 5-fluorouracil resistant Hep-2 cells.

Resistance of laryngeal squamous cell carcinoma cells to traditional therapeutic regimens still remains to be a major reason for therapeutic failure in patients. In this study, we aimed at investigating the expression profiles of ATP-binding cassette (ABC) transporters and stem cell markers in 5-fluorouracil (5-FU) resistant laryngeal Hep-2 cells. We treated parental Hep-2 cells, with stepwise increased doses of 5-FU for almost 1year to develop 5-FU resistant sub-lines with resistance against varying levels of 5-FU concentrations (4 sub-lines resistant to 1, 2, 4, and eightfold of 5-FU). Then, we measured the expression levels of 10 genes from ABC transporters family and 4 stem cell associated markers using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to find out a potential relationship between these markers and chemoresistance. We found that stemness-associated markers had elevated expressions from the beginning of 5-FU resistance acquisition. Their expressions elevated stepwise while parental Hep-2 cells got resistance to higher doses of 5-FU. Expressions of tested ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10 and ABCF2, and ABCG2) were also deregulated in 5-FU resistant Hep-2 cells. Although their expressions remained unaltered at the beginning of acquisition of resistance, expressions of ABC transporters except from ABCB6 increased significantly when cells became resistant to higher doses of 5-FU. Our results suggest that enrichment of cells with stemness characteristics and upregulation of ABC transporters might be amongst the crucial contributors of chemoresistance in laryngeal cancer cells.

Polyphenols-rich extract from Araucaria angustifolia: differential mechanisms on cancer and normal cells

Cancer cells present differential metabolism compared to normal cells. Multiple molecular mechanisms converge to alter cellular metabolism, and some of these include a process of metabolic reprogramming which provides advantages to tumor cells in energy generation, growth and proliferation. Tumor energy production is basically dependent on glucose driven to glycolysis (Warburg effect), but it also happens by means of fatty acids and glutamine metabolism. Among the current challenges in cancer therapy, the tumor cell resistance and the absence of selectivity of anti-cancer agents stand out. It has been already shown that polyphenols are able to exert differential effects on normal and tumor cells. However, the exact mechanisms of these actions are not fully understood. In our previous study, we showed that a polyphenols-rich extract (PE) from Araucaria angustifolia held a selective capacity to inhibit the proliferation of human larynx HEp-2 cancer cells with minimal cytotoxicity to normal epithelial cells. We hypothesized that the effect presented by PE have happened by reversing the “Warburg effect” on cancer cells and inhibiting the mitochondrial electron transport chain complex I activity along with ATP depletion on these cells. In this research highlight we will discuss the effects of the PE on mitochondrial metabolism and their possible role in the modulation of mitochondrial sirtuin 3 (SIRT3) on tumor (HEp-2) and normal (HEK-293) cells, which may help to clarify the tumor selectivity exhibited by polyphenols.

MicroRNA-10b Triggers the Epithelial-Mesenchymal Transition (EMT) of Laryngeal Carcinoma Hep-2 Cells by Directly Targeting the E-cadherin.

Laryngeal carcinoma is the second most common malignancy of the head and neck squamous cell carcinoma.Therefore, there is an urgent need to understand the molecular mechanism of its metastasis. The present study was designed to investigate effects of miR-10b on the invasion and migration of laryngeal Hep-2 cells. We found that miR-10b had limited effects on cell proliferation; however, it can significantly promote the migration and invasion of Hep-2 cells. Further studies revealed that overexpression of miR-10b can induce the epithelial-mesenchymal transition (EMT) of Hep-2 cells by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin (N-Cad) with a concomitant decrease of E-cadherin (E-Cad). However, the messenger RNA (mRNA) and protein level of transcription factors such as Snail, Slug, Twist and ZEB was not changed during this process. Bioinformatic analysis revealed that miR-10b can directly target CDH1 (E-Cad gene) at nucleotides 461 and 481 within the 3'-UTR. This was confirmed by the results that miR-10 downregulated the protein and mRNA levels of E-Cad via a time-dependent manner and luciferase analysis by use of four-nucleotide substitution in the core binding sites. The present study provided a better understanding of laryngeal carcinoma metastasis and the roles of miR-10b during this process.

BMI1 promotes the progression of laryngeal squamous cell carcinoma

BMI1 is highly expressed in several malignant tumors, and its expression level is associated with tumor progression, proliferation, and prognosis. However, no published studies have examined the role of BMI1 in laryngeal squamous cell carcinoma (SCC). Expression of BMI1 in primary tumors was analyzed by immunofluorescence staining, real-time PCR, and Western blotting. BMI1 was knocked down, and proliferation, apoptosis, and cell cycle assays were performed. Sensitivity to radiochemotherapy was evaluated, and tumorigenicity assays were performed in vivo. BMI1 was highly expressed in laryngeal SCCs. BMI1 promoted cell proliferation and tumor progression, and inhibited apoptosis due to influences on the cell cycle. More importantly, BMI1 suppressed the sensitization of laryngeal Hep2 cells to radiochemotherapy. BMI1 is essential to maintain the proliferation and progression of laryngeal SCCs. Therefore, depletion of BMI1 may be a potential therapeutic option for cancer management.

Enhanced Antitumoral Activity of Extracts Derived from Cultured Udotea flabellum (Chlorophyta)

Very few studies have been performed to evaluate the effect of culture conditions on the production or activity of active metabolites in algae. Previous studies suggest that the synthesis of bioactive compounds is strongly influenced by irradiance level. To investigate whether the antiproliferative activity of Udotea flabellum extracts is modified after cultivation, this green alga was cultured under four photon flux densities (PFD) for 30 days. After 10, 20, and 30 days, algae were extracted with dichloromethane: methanol and screened for antiproliferative activity against four human cancer cell lines (laryngeal—Hep-2, cervix—HeLa, cervix squamous—SiHa and nasopharynx—KB) by SRB assay. Lipid and phenol content were evaluated by standardized methods on algae organic extracts. After 10 days of cultivation, organic U. flabellum extracts showed a significant increase in antiproliferative activity on Hela and SiHa cells when compared to noncultured algae extracts. Extracts obtained after 10 and 20 days of culture were active on KB and Hep-2 cells. Total phenol and polyunsaturated fatty acid content in organic extracts changed with cultivation time but not by irradiance treatment. Extracts from U. flabellum obtained after 10 and 20 days of culture have been selected for fractionation and isolation of active compounds.

Anti-Adhesive Activities of Flavan-3-ols and Proanthocyanidins in the Interaction of Group A-Streptococci and Human Epithelial Cells

Bacterial adhesion to epithelial cells is a key step in infections, allowing subsequent colonization, invasion and internalization of pathogens into tissues. Anti-adhesive agents are therefore potential prophylactic tools against bacterial infections. The range of anti-adhesive compounds is largely confined to carbohydrate analogues. Tannins are generously recognized as potent antimicrobials, but little data exist on their anti-adherence potency. Using a model for mucosal pathogenesis with labeled group A-streptococci (GAS) and human laryngeal HEp-2 cells, a series of flavan-3-ols (epicatechin, epigallocatechin, epigallocatechin-3-O-gallate) and highly purified and chemically characterized proanthocyanidin samples including procyanidins based on epicatechin, catechin or ‘mixed’ constituent flavanyl units, prodelphinidins made up of (epi)gallocatechin monomeric unts as well as oligomers possessing A-type units in their molecules was evaluated for anti-adhesive effects. Reduced microbial adherence was observed exclusively for prodelphinidins, suggesting that pyrogallol-type elements, i.e., (epi)gallocatechin units are important structural features. This is the first report on structure-activity relationships regarding the anti-adhesive potency of proanthocyanidins. In addition, the structures of the first chemically defined proanthocyanidins from Pelargonium sidoides are disclosed.

EGCG induces apoptosis in human laryngeal epidermoid carcinoma Hep2 cells via mitochondria with the release of apoptosis-inducing factor and endonuclease G

(−)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, was tested for in vitro cytotoxicity against human laryngeal epidermoid carcinoma of the larynx Hep2 cells. EGCG-induced apoptotic cell death accompanied by a change in the cell cycle. However, EGCG did not result in caspase activation, nor did a caspase inhibitor block cell death. Furthermore, EGCG caused no change in the intracellular levels of reactive oxygen species (ROS). The levels of p53 were increased in the EGCG-treated cells, with a corresponding decrease in Bcl-2 and Bid protein levels as well as an increase in the Bax level. In addition, EGCG induced the cytoplasmic release of cytochrome c from the mitochondria accompanied by a decreased mitochondrial membrane potential, and subsequently upregulated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus during the apoptotic process. Taken together, these findings indicate that the p53-mediated mitochondrial pathway and the nuclear translocation of AIF and EndoG play a crucial role in EGCG-induced apoptosis of human laryngeal epidermoid carcinoma Hep2 cells, which proceeds through a caspase-independent pathway.

Bordetella bronchiseptica has a BvgAS-controlled cytotoxic effect upon interaction with epithelial cells.

The interaction between the respiratory pathogen Bordetella bronchiseptica and epithelial cells was studied. After 2-3 h, B. bronchiseptica strains exerted a strong cytotoxic effect on HeLa cells which was evident by rounding of the cells and detachment from the substrate and which ultimately resulted in total disintegration of the host cell. Production of this cytotoxic activity appeared to be regulated by the BvgAS sensory transduction system, which coordinately regulates many B. bronchiseptica virulence factors, since bacteria cultured in the presence of sulfate anions, inhibitors of the BvgAS response, did not exhibit this effect. Furthermore, spontaneous phase variants of B. bronchiseptica strains adhered to HeLa cells but were not cytotoxic. The cytotoxic component is presumably not secreted because the bacterial culture supernatant was not cytotoxic for HeLa cells. Besides HeLa cells other human epithelial cell lines such as Chang cells, larynx HEp-2 cells and lung NCI-H292 cells were sensitive to the cytotoxic activity of B. bronchiseptica. These results suggest the presence of a novel BvgAS-regulated virulence factor in B. bronchiseptica.

Molecular pathways in the chemosensitization of cisplatin by quercetin in human head and neck cancer

The aim of this study was to develop novel and less toxic therapy for human head and neck squamous cell carcinoma (HNSCCs) and to investigate the mechanism of quercetin-induced apoptosis in human laryngeal HeP2 cells and its effect on cisplatin induced apoptosis. Priming the cells with quercetin (40 ?M) increased the apoptosis induced by cisplatin alone from 18.7% to 42.2% in HeP2 cells. Quercetin induced apoptosis via inhibition of Akt/PKB phosphorylation, an upstream kinase of pro-survival protein kinase cascade. Inhibition of Akt phosphorylation was coupled with a significant decrease of anti-apoptotic Bcl-2 and Bcl-XL. Quercetin caused a downregulation of Cu-Zn Superoxide Dismutase which perhaps led to an increase of reactive oxidative stress (ROS). The decrease of Bcl-2 and Bcl-XL along with this oxidative stress caused release of mitochondrial cytochrome c into the cytosol and subsequent induction of pro-caspase-9 processing. Inhibition of heat shock proteins may be another mechanism for the pro-apoptotic activity of quercetin. Cisplatin induced apoptosis appears to be partly due to induction of JNK activity which leads to the activation of endonucleases. Increased JNK activity led to increased phosphorylation of c-Fos. Cisplatin additionally appears to induce apoptosis by downregulating the enzyme Nitric Oxide Synthase (NOS). Cisplatin also acts by increasing pro-apoptotic Bax concentration in the cells thereby leading to caspase-9 activation via the mitochondrial pathway. These results support the fact that quercetin and cisplatin act by separate pathways and demonstrate interactions between the pathways that result in synergistic actions. Possibly of greater potential value is the interaction of a conventional cytotoxic drug (cisplatin) and a non-toxic chemopreventive agent (quercetin) thereby allowing the use of less toxic doses of chemotherapy for treatment of HNSCCs.

Inhibitory effects of Celecoxib and Sc-58125 on proliferation of human carcinoma of larynx Hep-2 in vitro.

The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2 in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc-58125 and Celecoxib) at various concentrations for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis, and the cell cycle and apoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became rounded and detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G2 phase cells, whereas, Celecoxib rose G1 phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50 micromol/L and 100 micromol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G2 phase cells. However, Celecoxib had the same effect by changing the G1 phase cells and inducing apoptosis at higher concentration.

The role of intracellular zinc in modulation of life and death of Hep-2 cells.

Varying intracellular concentrations of zinc in laryngeal Hep-2 cells in relation to changing cultivation conditions in vitro were determined by atomic absorption spectrophotometry. Upon standard cultivation in DMEM with 10% serum, the mean concentration of zinc was determined at 0.88 +/- 0.09 microg/mg protein, with substantially decreased values in the cells exposed to a low-serum medium. Next, the study of the effects of a series of physiological and supraphysiological concentrations of ZnSO4 on laryngeal cells and their correlation with determined intracellular concentrations of zinc was performed. It was found that zinc concentrations above 100 microM were toxic to Hep-2 cells, inducing cell death in the interval of 96 h as determined by videomicroscopy, selective nuclear staining, and immunofluorescence detection of caspase-3 and specific cytokeratin 18 fragment. Both types of cell death were observed, with apoptosis being induced at moderately toxic zinc concentration of 150 microM and necrosis at higher zinc concentrations of 300 microM and 750 microM, respectively. Lower concentrations (1.5-100 microM), on the other hand, did not produce any measurable changes in cell morphology and function in the same time interval. Zinc at concentration of 1.5 microM was found to slightly enhance proliferation of Hep-2 cells up to the certain time point, which seemed to correlate with maximal tolerable momentary intracellular level of zinc. These results illustrate the importance of determining the intracellular levels of zinc when trying to characterize the effect of exogenous zinc on life and death of laryngeal cells.

Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response.

Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium. Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3, fimX, and fimA. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus. The fimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim(-) B. bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of DeltafimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B. bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of B. bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B. bronchiseptica at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

Bordetella bronchiseptica has a BvgAS-controlled cytotoxic effect upon interaction with epithelial cells

The interaction between the respiratory pathogen Bordetella bronchiseptica and epithelial cells was studied. After 2–3 h, B. bronchiseptica strains exerted a strong cytotoxic effect on HeLa cells which was evident by rounding of the cells and detachment from the substrate and which ultimately resulted in total disintegration of the host cell. Production of this cytotoxic activity appeared to be regulated by the BvgAS sensory transduction system, which coordinately regulates many B. bronchiseptica virulence factors, since bacteria cultured in the presence of sulfate anions, inhibitors of the BvgAS response, did not exhibit this effect. Furthermore, spontaneous phase variants of B. bronchiseptica strains adhered to HeLa cells but were not cytotoxic. The cytotoxic component is presumably not secreted because the bacterial culture supernatant was not cytotoxic for HeLa cells. Besides HeLa cells other human epithelial cell lines such as Chang cells, larynx HEp-2 cells and lung NCI-H292 cells were sensitive to the cytotoxic activity of B. bronchiseptica. These results suggest the presence of a novel BvgAS-regulated virulence factor in B. bronchiseptica.

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of M r 20–21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein: (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester. TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Novel red absorbing benzo[a]phenoxazinium and benzo[a]phenothiazinium photosensitizers: in vitro evaluation.

Abstract Taking advantage of the‘heavy atom’effect, we have recently prepared a series of nine novel halogenated and sulfur‐substituted benzophenoxazines which have enhanced intersystem crossing to the triplet state as measured by singlet oxygen photobleaching of 1,3‐diphenylisobenzofuran. The dyes were evaluated for their dark and light‐induced toxicities towards several carcinomata and normal primary cell lines. One of these days, 5‐amino‐6‐iodo‐9‐diethylaminobenzol[a]phenoxazinium chloride, was found to be a potent photosensitizer for a murine sarcoma 180 and four human carcinomata cell lines (larynx Hep2, cervical HeLa, rectal tumor cell HRT, and transitional‐cell bladder BTC). Several dyes caused marked light dependent killing of two tumor cell lines (Hep2 and HRT) but were minimally toxic to a normal bovine fetal kidney (BFK) line. Sulfur substitutuion into the benzophenoxazine nucleus results, under the conditions studied, in the largest enhancement of phototoxicity both to normal and cancer cells. Comparisons between appropriate dyes show a correlation between the efficiency of singlet oxygen generation and phototoxicity. These results suggest that the photosensitizing efficacy of certain cationic benzophenoxazinium chromophores, such as Nile Blue A, can be greatly increased by appropriate structural modification. The demonstrated selectivity for carcinoma cells by some of these new photosensitizers may be useful therapeutically.