Laryngeal Carcinoma Hep-2 Cells Research Articles

RNA interference targeting inhibition of S100A4 suppresses cell growth and promotes apoptosis in human laryngeal carcinoma Hep‑2 cells.

S100A4 is a small Ca2+ binding protein that belongs to the S100 family and is involved in a number of cellular functions, including cell cycle control, proliferation, apoptosis, and has a significant role in angiogenesis and neurite extension. However, the exact function and mechanism of S100A4 in laryngeal carcinogenesis remains to be elucidated. The present study was designed to investigate the potential use of RNA interference to inhibit S100A4 expression and activation, as well as the subsequent effect on human laryngeal cancer cell growth and apoptosis. The present study demonstrated that knockdown of S100A4 decreased the proliferation and growth of the human laryngeal cancer Hep‑2 cell line. The percentages of the apoptotic cells were 4.23±1.22, 4.92±1.85 and 11.70±4.02% in the control, negative control and S100A4 short hairpin RNA (shRNA) groups, respectively, indicating significant differences among the different groups. The S100A4‑mediated induction of apoptosis was demonstrated to be associated with the activation of caspase‑3, caspase‑8 and caspase‑9. Intratumoral injection of S100A4‑shRNA inhibited tumor growth in nude mice. Thus, knockdown of S100A4 inhibited the progression of laryngeal squamous carcinoma, decreased proliferation and promoted apoptosis. S100A4 is a potential candidate for therapeutic targeting of laryngeal carcinoma cells.

RNA interference (RNAi) mediated stable knockdown of protein casein kinase 2-alpha (CK2α) inhibits migration and invasion and enhances cisplatin-induced apoptosis in HEp-2 laryngeal carcinoma cells

Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial–mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α.

Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep-2 cells.

The aim of the present study was to assess the effect of quercetin and imperatorin administered separately and in combination on apoptosis and autophagy induction in human cervical carcinoma HeLa cells and laryngeal carcinoma Hep-2 cells cultured in vitro. Conducted MTT measurements proved that quercetin and imperatorin displayed a strong antiproliferative activity manifested in markedly reduction of HeLa and Hep-2 cells viability as a result of treatment with 50 μM of each compound. Further cell staining assays revealed that concentration mentioned above generated the highest percentage of apoptotic cells especially in the case of application of both drugs for 48 h. Simultaneous quercetin and imperatorin administration induced apoptosis remarkably stronger than treatment with single drugs. Experiments at the molecular level confirmed these results accompanied with the decreased Hsp27 and Hsp72 expression and, in addition, with increased caspases activity. Autophagy was not observed and no significant changes in the expression of beclin-1 were noticed. Additionally, experiments were performed on the above-mentioned cell lines with blocked Hsp27 and Hsp72 expression. In these cells, no significant changes in the sensitivity to apoptosis induction upon quercetin and imperatorin treatment were observed. The present study has provided evidence supporting the potential of the combination of quercetin and imperatorin drugs as a novel tool to be used in anticancer therapy. Our results have also demonstrated that blocking of the Hsp27 and Hsp72 gene expression is not enough to sensitize cancer cells to programmed cell death induction in HeLa and Hep-2 cells.

Overexpression, purification, molecular characterization and the effect on tumor growth of ribosomal protein L22 from the Giant Panda (Ailuropoda melanoleuca)

The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 μg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 μg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 μg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.

Growth inhibitory effect of microRNA-519b-3p on larynx squamous Hep-2 cells

To investigate the effects of microRNA-519b-3p (miR-519b-3p) on laryngeal carcinoma Hep-2 cell growth, and to analyze the underlying molecular mechanisms. The effects of miR-519b-3p on the growth and cell cycle of Hep-2 cells transfected with miR-519b-3p mimic were tested by MTT assay and flow cytometry, respectively. The mRNA and protein expressions of the related genes were tested by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The expressions of miR-519b-3p were tested by real-time RT-PCR in 48 pairs of laryngeal carcinoma and adjacent tissue samples. The expression of miR-519b-3p in laryngeal carcinoma tissues was significant lower than that in adjacent non-cancerous tissues (S(ΔCt) = 2.989, t = 2.693, P < 0.01) . Increasing the level of miR-519b-3p inhibited significantly Hep-2 cell proliferation, arrested the cell cycle in the G2/M phase (10.29% ± 4.63%, t = 4.395, P < 0.05) , and decreased significantly the percentage of cells in the S phase (7.56% ± 2.05%, t = 3.555, P < 0.05) , with the increase in the expression of cyclin dependent kinase (CDK) 1 and the decrease in the expressions of CDK 2 and Cyclin A. RT-PCR and Western blot showed that miR-519b-3p down-regulated the protein but not mRNA expressions of HuR and cyclooxygenase-2(COX-2) genes. The expression of MiR-519b-3p as carcinoma suppressor gene is low in laryngeal carcinoma. The cell cycle of Hep-2 cells was arrested in the G2/M phase by MiR-519b-3p.

MicroRNA-34a/c function as tumor suppressors in Hep-2 laryngeal carcinoma cells and may reduce GALNT7 expression

A family of small non-coding RNAs, ~22 nt in length, known as microRNAs (miRNAs), regulating ~30% of all human gene expression, have been reported to be involved in the pathogenesis of a number of types of cancers, including laryngeal squamous cell carcinoma (LSCC). In the current study, miR-34a and miR-34c were observed to be downregulated in human LSCC tissues. Ectopic expression of miR-34a and miR-34c in Hep-2 cells significantly induced the cell proliferation and migration ability in vitro. UDP-N-acetyl-α-D-galactosamine:polypeptide-N-acetylgalactosaminyltransferase 7 (GALNT7), whose expression is negatively regulated by miR-34a and miR-34c in Hep-2 cells, is confirmed to be a novel direct target gene of miR-34a and miR-34c. In conclusion, the current results suggest that miR-34a and miR-34c may function as tumor suppressors in LSCC through downregulation of GALNT7. The study of miR-34a, miR-34c and its novel target, GALNT7, may serve as novel potential makers for LSCC therapy.

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Hexokinase 2 overexpression promotes the proliferation and survival of laryngeal squamous cell carcinoma

Proliferating cancer cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) is highly expressed in many malignant cells and is necessary for anaerobic glycolysis. The role of HK2 in laryngeal squamous cell carcinoma (LSCC) is unknown. In this study, the expression of HK2 in LSCC was investigated and the effect of inhibiting HK2 expression with small hairpin RNA (shRNA) on tumor growth was investigated. Using immunohistochemistry, HK2 expression was assessed in LSCC tissues. Human laryngeal carcinoma Hep-2 cells were stably transfected with a plasmid expressing HK2 shRNA (pGenesil-1.1-HK2) and were compared to control cells with respect to the cell cycle, cell viability, apoptosis, and their ability to form xenograft tumors. HK2 expression was significantly higher in LSCC than in papilloma or glottis polypus. Tumor samples of higher T, N, and TNM stage often had stronger HK2 staining. HK2 shRNA reduced HK2 mRNA, protein levels, and HK activity in Hep-2 cells. HK2 cells expressing shRNA demonstrated a higher G0-G1 ratio, increased apoptosis, and reduced viability. Xenograft tumors derived from cells expressing HK2 shRNA were smaller and had lower proliferation than those from untransfected or control-plasmid-transfected cells. In conclusion, depletion of HK2 expression resulted in reduced xenograft tumor development likely by reducing proliferation, altering the cell cycle, reducing cell viability and activating apoptosis. These data suggest that HK2 plays an important role in the development of LSCC and represents a potential therapeutic target for LSCC.

Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines.

In the present study, we investigated the possible combined anticancer ability of bee venom (BV) and cisplatin towards two pairs of tumour cell lines: parental cervical carcinoma HeLa cells and their cisplatin-resistant HeLa CK subline,as well as laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CK2 subline. Additionally, we identified several peptides of BV in the BV sample used in the course of the study and determined the exact concentration of MEL. BV applied alone in concentrations of 30 to 60 μg ml(–1) displayed dose-dependent cytotoxicity against all cell lines tested. Cisplatin-resistant cervical carcinoma cells were more sensitive to BV than their parental cell lines (IC(50) values were 52.50 μg ml(–1) for HeLa vs.47.64 μg ml(–1) for HeLa CK cells), whereas opposite results were obtained for cisplatin-resistant laryngeal carcinoma cells (IC(50) values were 51.98 μg ml(–1) for HEp-2 vs. > 60.00 μg ml(–1) for CK2 cells). Treatment with BV alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium-bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. Combined treatment of BV and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, suggesting that BV could enhance the killing effect of selected cells when combined with cisplatin. Therefore, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. Our results suggest that combined treatment with BV could be useful from the point of minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance.

The impact on secreted VEGF of Hep-2 human laryngeal cancer cell induced by Rg3

Objective The aim of this study was to investigate the affecting of Rg3 to secreted VEGF of human laryngeal carcinoma Hep-2 cells and its mechanism of inhibition to tumor angiogenesis.

Investigation on biological characteristics of CD133+ cancer stem cells in human laryngeal carcinoma Hep-2 cell line

To investigate the biology characters of CD133+ cancer stem cells from Hep-2 cell line. The Flow cytometry was applied to purify CD133+ cells from Hep-2 cell line. The sorted CD133+ cells were cultured in RPMI1640. The ability of migration, invasion and clonality of CD133+ cells were performed to characterize the properties of the cells. CD133+ cells and CD133- control cells were treated with paclitaxel and exposed respectively to X-rays emitted by linear accelerator with a dose of 10 Gy. The surviving rates and growth inhibition ratio of cells in two groups were detected with MTT assay to observe the resistance to irradiation and chemotherapy in CD133+ cells. The percentage of CD133+ cells in the unsorted and the sorted cells were 3.1% +/- 0.2% and 90.2% +/- 5.5%, respectively. CD133+ cells showed the higher proliferation and colony ability than CD133- cells. The numbers of CD133+ and CD133- cells that passed the membrane of Transwell chamber were 526 +/- 39 and 220 +/- 20 respectively (t = 22.08, P < 0.001). The colony forming units of three passages were 30.0% +/- 4.7%, 32.2% + 3.6%, 32.7% + 3.4% in CD133 cells and 15.2% +/- 2.2%, 12.0% + 2.5%, 13.8% +/- 3.3% in CD133- cells. There were statistic difference between two groups (t = 8.99, t = 14.66, t = 12.69, P < 0.01). At 24, 48, and 72 hours of treatment with paclitaxel, the cell surviving rates of CD133+ cells were 90.1% +/- 5.9%, 85.1% +/- 7.1% and 70.3% +/- 6.4% and lower than those of CD133- cells, respectively (t = 5.24, t = 8.18, t = 8.14, P < 0.01) . After radiotherapy, growth inhibition ratio of CD133+ and CD133- cells were 30.0% +/- 7.1% and 55.0% +/- 6.3% (t = 8.30, P < 0.01). CD133+ cells exist in a small proportion in Hep-2 cell line and they show the properties of cancer stem cells, with the resistance to irradiation and chemotherapy.

Telomerase reverse transcriptase promotes the proliferation of human laryngeal carcinoma cells through activation of the activator protein 1

TERT is the main functional unit of telomerase, which maintains telomere length and chromosome structure stability. TERT has been shown to act as a key factor in various biological processes, such as cell proliferation, via uncharacterized mechanisms. We transfected HEp-2 laryngeal carcinoma cells with a TERT overexpressing adenovirus (Ad-TERT) and TERT shRNA silencing adenovirus (Ad-sh-TERT), and examined the effect on TERT and the AP-1 transcription factor subunits c-Fos and c-Jun using RT-PCR and western blot analysis. TERT mRNA expression was quantified using RT-PCR in 24 human laryngeal carcinoma samples, and TERT protein co-expression with AP-1 was investigated in a human laryngeal carcinoma tissue microarray using quantum-dot based immunofluorescence. The effect of specific ERK and p38 inhibitors on ERK, p38, c-Jun and c-Fos phosphorylation was investigated in TERT-overexpressing HEp-2 cells. TERT overexpression led to increased TERT, c-Jun and c-Fos mRNA and protein expression and increased cell proliferation, while TERT silencing had the opposite effects. TERT mRNA expression levels were positively correlated with c-Fos and c-Jun mRNA in human laryngeal carcinoma tissue. TERT and AP-1 protein were expressed at high levels and positively correlated in laryngeal carcinoma tissues. Treatment of TERT-overexpressing HEp-2 cells with specific p38 and ERK inhibitors indicated that TERT modulates the expression and phosphorylation of the AP-1 subunits c-Jun and c-Fos through the p38 and ERK signaling pathways. In conclusion, the results of this study indicate that TERT is capable of promoting cell proliferation via activation of the AP-1 subunits, c-Jun and c-Fos, in laryngeal carcinoma cells.

In Vitro Effects of Ivermectin and Sulphadiazine on Toxoplasma gondii.

Ivermectin and sulphadiazine were tested individually to determine their in vitro effects on Toxoplasma gondii grown in human epidermoid larynx carcinoma (Hep-2) cell culture. In-vitro study. Toxoplasma growth was quantities by an enzyme immunoassay performed directly on the fixed cultures, using a rabbit anti-T. gondii immunoglobulin G as the first antibody and a phosphatase-labeled anti-rabbit immunoglobulin G as the second antibody. For each drug, regression models were used to quantify the relationship between optical density values and antimicrobial agent concentrations in the cultures. The 50% inhibitory concentrations (IC50) of ivermectin and sulphadiazine were found to be 0.2 μg/mL and 7.3 μg/mL after 48 h of exposure, respectively. None of the concentrations tested for each drugs demonstrated toxicity to Hep-2 cells after 72 h of incubation. These results indicate that ivermectin significantly inhibited replication of the tachyzoites of T. gondii RH strain.

Influencing of hep-2 cell function by RNAi silencing E-cadherin expression

To explore the function of human laryngeal carcinoma Hep-2 cell after down-regulating the expression of E-cadherin gene to provide theoretical rationales for gene therapy of laryngeal cancer. According to the GenBank database, 3 pairs of shRNA sequences of E-cadherin gene were designed and synthesized. shRNAs were transfected into the cell line Hep-2 by liposome. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the silencing effect of E-cadherin expression. The changed capacity of cell proliferation were detected in vitro by methyl thiazolyl tetrazolium (MTT) assay in the transfected Hep-2 cells and the cell proliferation rate (survival rate) was calculated. And Transwell was used to detect the migratory capacity of Hep-2 cells after siRNA transfection. The E-cadherin gene expression of RNAi transfected Hep-2 cells significantly decreased in interference group. And the proliferation of interference group became markedly enhanced. In Transwell test, the migrated cell numbers in interference group were significant higher than those in negative control group (262 ± 15, 288 ± 12, 292 ± 6 vs 74 ± 8, all P < 0.01). RNA interference silencing of E-cadherin gene expression can significantly enhance the proliferation and migratory capacity of Hep-2 cells. And E-cadherin may be considered as one of gene therapy targets for laryngeal cancer.

Anticancer Activity of Zea mays Leaf Extracts on Oxidative Stress-induced Hep2 Cells

Cancer is one of the leading causes of death in humans. It is believed that plants can provide potential bioactive compounds for the development of “new leads” to combat cancer and other diseases. The present study focuses on the ability of the different extracts (aqueous, methanol, and chloroform) of the leaves of Zea mays in influencing the process of apoptosis induced by hydrogen peroxide (H2O2) in Hep2 (laryngeal carcinoma) cells. Various apoptosis-related parameters, such as cell viability, morphological changes, nuclear changes, and apoptotic index were characterized. sulforhodamine B and MTT assays were used to quantify the extent of cell death in the group exposed to H2O2, plant extracts, and their combination. Treatment with H2O2 caused cytotoxicity in cancer cells. The administration of leaf extract also caused an increase in the death of cancer cells. Oxidatively stressed cancer cells co-treated with all the Z. mays leaf extracts (except the chloroform extract) demonstrated cytotoxicity on a par with the H2O2-treated groups. This indicated that the aqueous and methanol leaf extracts did not influence the cytotoxic action of H2O2 in the cancer cells. Thus, various apoptosis-related events in Hep2 cells exposed to leaf extract throw light on the potential anticancer activity of the Z. mays leaves. The maximum activity was exerted by the methanolic extract followed by the aqueous and chloroform extracts.

Evaluation of naproxen and cromolyn activities against cancer cells viability, proliferation, apoptosis, p53 and gene expression of survivin and caspase-3

We previously reported the inhibitory profiles of naproxen and cromolyn against glycogen synthase kinase-3β, which partly explain the molecular mechanisms of their anti-cancer properties. In this study, we performed a detailed biochemical evaluation of the two drugs against colorectal adenocarcinoma (Caco2), hepatocellular carcinoma (HepG2), mammary gland carcinoma (MCF7), epitheloid cervix carcinoma (Hela), lung carcinoma (A5W9) and epidermoid larynx carcinoma (Hep2) cell lines. Additionally, we performed cellular viability tests using trypan blue, proliferation MTT assay, apoptosis, p53 and real-time polymerase chain reaction for gene expression of survivin and caspase-3. Not only the two drugs were found to significantly reduce the viability of different cell lines, but they also were shown to have potent dose-dependent reduction of cellular proliferation. They exhibited cytotoxicity IC50 values of 3.69 and 4.16 μM for naproxen and cromolyn, respectively. Viability and proliferation results clearly correlated with apoptosis and p53 experiments in showing that both drugs significantly raised apoptotic percentages. Furthermore, we observed a significant reduction in survivin and elevation of caspase-3 gene expression upon exposure to the two drugs. It can be concluded that both naproxen and cromolyn have significant anti-cancer properties.

S100A11 Is a Migration-Related Protein in Laryngeal Squamous Cell Carcinoma

Objective: As a member of the S100 proteins family, the involvement of S100A11 has been suggested in a wide range of biological processes such as cell growth and motility, cell-cycle progression, transcription, differentiation and smooth muscle cell migration. However, the expression of S100A11 and its exact function in laryngeal squamous cell carcinoma (LSCC) have not been elucidated.Methods: The protein and mRNA expression levels of S100A11 were analyzed in primary tumors and matched tumor-adjacent tissues of LSCC by western blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) or quantitative real time PCR (Q-RT-PCR), respectively. Cell proliferation, colony formation, migration and wound-healing assays were performed to assess whether the knockdown of S100A11 by small interfering RNA (siRNA) could influence the biological behavior of human laryngeal carcinoma Hep-2 cells in vitro.Results: We found that both protein and mRNA levels of S100A11 were overexpressed in laryngeal tumor tissues when compared to the corresponding noncancerous tissues. Further, it was demonstrated that the expression of S100A11 could induce migration but not proliferation of Hep-2 cells. Additionally, S100A11 altered a series of intracellular events, including the down-regulation of epidermal growth factor receptor (EGFR), CD44 and MMP2.Conclusions: These results highlight the significance of S100A11 in LSCC progression and suggest that the dysregulation of S100A11 might contribute to the metastatic progression of LSCC.

Overexpression, purification, and pharmacologic evaluation of anticancer activity of ribosomal protein L24 from the giant panda (Ailuropoda melanoleuca)

The ribosomal protein L24 (RPL24) belongs to the L24E family of ribosomal proteins and is located in the cytoplasm. The purpose of this study was to investigate the structure and anti-cancer function of RPL24 of the giant panda (Ailuropoda melanoleuca). The complementary DNA of RPL24 was cloned successfully using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing RPL24 complementary DNA and overexpressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified using Ni-chelating affinity chromatography. The results indicated that the length of the fragment cloned is 509 bp, and it contains an open-reading frame of 474 bp encoding 157 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL24 protein is 17.78 kDa with a theoretical isoelectric point of 11.86. The RPL24 gene is readily expressed in E. coli, and the RPL24 fused with the N-terminal histidine-tagged protein to give rise to the accumulation of an expected 23.51-kDa polypeptide. The inhibitory rate in mice treated with 0.1 mg/mL RPL24, the highest of 3 doses administered, can reach 67.662%, which may be comparable to the response to mannatide. The histology of organs with tumors showed that the tissues in the RPL24 group displayed a looser arrangement compared with that in the control group. Furthermore, no obvious damage was apparent in other organs, such as heart, lung, and kidney. The data showed that the recombinant RPL24 had time and dose dependency on the cell growth inhibition rate. Human laryngeal carcinoma Hep-2 cells treated with 0.3125-10 µg/mL RPL24 for 24 h displayed significant cell growth inhibition (P < 0.05; N = 6) in assays using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide compared with that in control (untreated) cells. By contrast, human hepatoma Hep G-2 cells displayed no significant change (P > 0.05; N = 6) from control (untreated) cells. RPL24 has time and dose dependency on Hep-2 cell growth inhibition. The data indicate that the effect at low concentrations is better than that at high concentrations, and the concentration of 0.625 µg/mL provides the best rate of growth inhibition. Further research is ongoing to determine the bioactive principles of recombinant RPL24 protein that are responsible for its anticancer activity.

Iron-Cobalt Ferrite Nanoparticles—Biocompatibility and Distribution After Intravenous Administration to Rat

The biocompatibility of iron-cobalt ferrite magnetic nanoparticles (MNP) in vitro and in vivo and their distribution under an external magnetic field after intravenous administration to tumor-bearing rats were studied. In vitro rat mesenchymal stem cells and human larynx carcinoma HEp-2 cells accumulated the magnetic material intracellularly. The proliferation of HEp-2 cells was inhibited moderately by the MNP containing ca. 1.5% Co (either uncoated or polysiloxane-coated) and effectively-by the MNP containing ca. 25% Co. In vivo the most MNP-accumulating organs were the spleen, the kidneys, and the liver. No accumulation of the MNP was observed in the brain. It was found that the differences in the MNP distribution caused by topical application of a magnet were more pronounced at 24 h post injection than at 0.5-3.0 h-while the expected plasma life of the MNP doesn't exceed 10 min. E.g., application of the magnet caused a 2.4-fold MNP-attributed increase in iron concentration in M-1 sarcoma model at 24 h post injection. Similarities were found in lymph tissue tropism between our particles that readily agglutinate in plasma-containing media and nonagglutinating, long-circulating MNP reported by other authors.

Inhibition of p21-activated kinase 4 expression suppresses the proliferation of Hep-2 laryngeal carcinoma cells via activation of the ATM/Chk1/2/p53 pathway

In the present study, we investigated the effects of the p21-activated kinase 4 (Pak4) gene on Hep-2 laryngeal carcinoma cells in vivo and in vitro. The expression of Pak4 was downregulated using small interfering RNA (siRNA). The downregulation of Pak4 decreased the proliferation and increased apoptosis and S phase arrest in Hep-2 cells in vitro. In further experiments, we determined that the S/G(2) transition was obstructed by the downregulation of Pak4 using 5‑chloro-2'‑deoxyuridine (CldU) and 5‑iodo‑2'‑deoxyuridine (IdU) double staining. A xenografted Hep-2 tumor mouse model was created by inducing human tumors with a subcutaneous (s.c.) injection of 5x10(6) Hep-2 cells into the dorsal flank region of nu/nu mice. The downregulation of Pak4 in established xenografted tumors decreased tumor size and weight. The survival rate of the mice with tumors that did not express Pak4 was significantly higher compared to the mice with tumors expressing Pak4. These results confirm the role of Pak4 as an oncogene in laryngeal carcinoma cells. To identify the mechanism of the cell cycle arrest induced by Pak4, immunohistochemical staining was performed to detect changes in cell cycle‑related proteins. The results demonstrated that p53 was activated following the downregulation of Pak4. The levels of ataxia telangiectasia mutated (ATM), the upstream protein of checkpoint kinase (Chk)1 and Chk2, also increased. Therefore, we confirmed that the mechanisms of the Pak4-induced cell cycle arrest invovlve the activation of the ATM/Chk1/2/p53 pathway. These results may prove helpful for the development of novel therapies for the treatment of laryngeal carcinoma.