Human Laryngeal Cancer Hep-2 Cells Research Articles

Paclitaxel Promotes Oxidative Stress-Mediated Human Laryngeal Squamous Tumor Cell Death through the Stimulation of Calcium and Zinc Signaling Pathways: No Synergic Action of Melatonin.

The paclitaxel (PAX) and melatonin (MLT)-mediated mitochondria reactive free oxygen radical (miROS) generations via the influx of excessive Ca2+ and Zn2+ induce tumor cell death and apoptosis. However, a presence of resistance was demonstrated against thePAX treatment in the tumor cells. The stimulation of TRPM2 may increase the anticancer action of PAX after the treatment of MLT. We investigated the stimulating role of PAX with/without MLT on the excessive Ca2+ influx and miROS generation-mediated human laryngeal squamous cancer (Hep2) cell death through the stimulation of TRPM2. The Hep2 cells were divided into four groups as control, MLT (1mM for 2h), PAX (50μM for 24h), and PAX + MLT. In some experiments, we induced additional subgroups such as PAX+ACA and PAX+2APB.The stimulation of TRPM2 induced the increase of TRPM2 current densities, lipid peroxidation, cytosolic ROS, miROS, cytosolic Ca2+, and Zn2+ values in the Hep2 cells after the treatment of PAX, although their values were decreased by the treatment of MLT and TRPM2 antagonists (ACA and 2APB). In addition, the PAX induced apoptosis and cell death via upregulation of caspases and downregulation of antioxidant glutathione peroxidase and glutathione in the cells. The treatment of PAX increased protein band expression values of TRPM2, PARP-1, and caspase 3 and 9 in the Hep2. The increased expression, apoptotic, and cell death values were not affected by the treatment of MLT. In conclusion, PAX induced the increase of Hep2 cell death via upregulations of TRPM2andZn2+, although its downregulation via the treatment of MLT did not change the antitumor action of PAX.

A pair of new isocoumarin enantiomers of Ludwigia hyssopifolia

A pair of new isocoumarin derivative enantiomers, (S)-(-)-3-(3,3-dichloro-2-hydroxy-propyl)-4-chlorine-6,8-dihydroxy-isochromen-1- one (1a) and (R)-(+)-3-(3,3-dichloro-2-hydroxy-propyl)-4-chlorine-6,8-dihydroxy-isochromen-1- one (1 b), as well as seven known compounds (2–8) were isolated from Ludwigia hyssopifolia. Compounds 1a and 1 b were confirmed to be a pair of enantiomers by chiral HPLC-CD analysis, and the structure of compound 1 was determined by spectroscopic analyses including extensive 1 D (1H NMR, 13C NMR) and 2 D NMR spectra (COSY, HSQC and HMBC) and MS data. And the absolute configurations of compounds 1a and 1 b were determined by the quantum chemical ECD calculations. Compounds 2–8 are firstly reported from this plant. In the in vitro assays, compounds 5 and 8 can inhibit human laryngeal cancer Hep-2 cell line growth in a dose- and time-dependent manner. In addition, compounds 2 and 4 have effects on increasing glucose uptake in vitro. Compound 2 showed a strong glucose uptake in L6 cells, with enhancements by 1.8 folds.

Tetrandrine partially reverses multidrug resistance of human laryngeal cancer cells.

ObjectiveStudies have demonstrated that tetrandrine reverses multidrug resistance (MDR) in animal models or cell lines derived from multiple cancer types. We examined the potential MDR reversal activity of tetrandrine in a multidrug-resistant variant of a human laryngeal cancer Hep-2 cell line and explored potential mechanisms involved.MethodsWe developed the multidrug-resistant variant cell line (Hep-2/v) by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (VCR). After Hep-2 or Hep-2/v cells were treated with tetrandrine (2.52 µg/mL), MDR was measured by MTT assay, rhodamine 123 retention was measured by flow cytometry, and mRNA and protein expression of multidrug resistance 1 (MDR1), regulator of G-protein signaling 10 (RGS10), high-temperature requirement protein A1 (HTRA1), and nuclear protein 1 (NUPR1) were detected by real-time reverse transcription-PCR and western blotting, respectively.ResultsTetrandrine significantly lowered the half-maximal inhibitory concentration (IC50) of VCR in Hep-2/v cells, resulting in a 2.22-fold reversal of MDR. Treatment with tetrandrine increased rhodamine 123 retention, downregulated the mRNA and protein expression of MDR1 and RGS10, and upregulated expression of HTRA1 in Hep-2/v cells.ConclusionWe showed that tetrandrine exerts anti-MDR activity in Hep-2/v cells, possibly by inhibiting MDR1 overexpression-mediated drug efflux and by altering expression of HTRA1 and RGS10.

Curcumin enhances cisplatin-induced human laryngeal squamous cancer cell death through activation of TRPM2 channel and mitochondrial oxidative stress

In this study, laryngeal tumor cells were killed through the production of excessive reactive oxygen species (ROS) and Ca2+ influx by cisplatin (CISP). Nevertheless, a resistance was determined against CISP treatment in the tumor cells. We have investigated the stimulating role of curcumin (CURC) on CISP-induced human laryngeal squamous cancer (Hep2) cell death through TRPM2 channel activation, and its protective role against the adverse effects of CISP in normal kidney (MPK) cells. Hep2 and MPK cells were divided into four groups as control group, CURC group (10μM for 24 hrs), CISP group (25 μM for 24 hrs), and CURC + CISP combination group. CISP-induced decrease of cell viability, cell count, glutathione peroxidase and glutathione level in Hep2 cells were further increased by CURC treatment, but the CISP-induced normal MPK cell death was reduced by the treatment. CISP-induced increase of apoptosis, Ca2+ fluorescence intensity, TRPM2 expression and current densities through the increase of lipid peroxidation, intracellular and mitochondrial oxidative stress were stimulated by CURC treatment. In conclusion, CISP-induced increases in mitochondrial ROS and cell death levels in Hep2 cells were further enhanced through the increase of TRPM2 activation with the effect of CURC treatment. CISP-induced drug resistance in Hep2 cells might be reduced by CURC treatment.

Cytotoxic Compounds From Ludwigia hyssopifolia

Purification of the petroleum ether and ethyl acetate fractions from Ludwigia hyssopifolia yielded 9 natural products, ethyl gallate (1), vanillin (2), trans- p-hydroxycinnamic acid (3), trans- p-hydroxy-ethyl cinnamate (4), ozoroalide (5), scopoletin (6), de- O-methyllasiodiplodin (7), syringaldehyde (8), and 3,3′-dimethoxy-4,4′-dihydroxy-stilbene (9). The structures of 1 to 9 were determined by spectroscopic analyses including 1D Nuclear Magnetic Resonance (NMR) (1H NMR and 13C NMR) and Mass Spectrum (MS) data. Four compounds displayed cytotoxic effects on the human laryngeal cancer Hep-2 cell line. Compounds 5 and 7 were also effective against TU212 cell line and significantly inhibited cancer cell growth in a dose- and time-dependent manner on Hep-2 cell line.

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

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

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

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

The role of autophagy in the curcumin induced proliferation in human laryngeal cancer Hep2 cell

Objective:The aim of this study is to study the effect of 3-methyladenine (3-MA) on the autophagy and apoptosis of human laryngeal cancer Hep 2 cells induced by curcumin. Method:The proliferation of human laryngeal cancer Hep2 cells was observed by MTT assay. The autophagy level was detected by AO acridine orange staining. Annexin VFITC/PI double staining was used to detect the apoptosis of Hep2 cells. The expression of LC3, Beclin1, Bcl-2 and Bax protein were detected by Western blot. Result:MTT assay showed that curcumin inhibited the proliferation of Hep2 cells in a dose/time dependent manner. The apoptosis rate of curcumin combined with 3-MA increased (P<0.05). Acridine orange staining showed that 3-MA combined with curcumin could significantly reduce the autophagy level of laryngeal carcinoma Hep2 cells. The expression of Bcl-2, Bclin-1 and LC3 Ⅱ was decreased, while the expression of Bax protein was increased (P<0.05). Conclusion:Curcumin can induce apoptosis of Hep 2 cells and induce the development of protective autophagy. The inhibitory effect of curcumin on the apoptosis of laryngeal carcinoma Hep2 cell line was significantly enhanced by 3-MA.

Dioscin suppresses human laryngeal cancer cells growth via induction of cell-cycle arrest and MAPK-mediated mitochondrial-derived apoptosis and inhibition of tumor invasion

The anti-cancer effects of dioscin have been widely reported. However, its effect on laryngeal cancer remains unknown. In the present paper, our results showed that dioscin markedly caused cell apoptosis and DNA damage, increased reactive oxygen species (ROS) level, induced S-phase arrest, and inhibited invasion of human laryngeal cancer HEp-2 and TU212 cells. Mechanism investigation showed that dioscin markedly up-regulated p53 level, and down-regulated cyclin-dependent kinase 2 (CDK2) and Cyclin A levels. In addition, dioscin significantly down-regulated the levels of p-ERK, Bcl-2, up-regulated the levels of p-JNK, p-p38, Bax, cleaved caspase-3/-9, and caused Cytochrome c release. Furthermore, U0126, an ERK1/2 inhibitor, markedly down-regulated Bcl-2 level, up-regulated the levels of Bax, cleaved caspase-3/9, and enhanced Cytochrome c release inducted by dioscin. While, SP600125 (one JNK inhibitor) and SB203580 (one p38 inhibitor) markedly up-regulated Bcl-2 level, down-regulated the levels of Bax, cleaved caspase-3/9, and obviously boosted Cytochrome c release induced by dioscin. Interestingly, dioscin also markedly down-regulated the levels of MMP2 and MMP9 associated with tumor invasion. Taken together, our study indicated that dioscin suppressed laryngeal cancer cells growth via inducting cell-cycle arrest, MAPK-mediated mitochondrial- derived apoptosis and inhibiting tumor invasion, which could be used as one potential candidate for the treatment of laryngeal cancer in the future.

Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells.

Rabdosia rubescens, a commonly used traditional Chinese medicine, has increasingly gained attention for its use as an antitumor herb. Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to induce apoptosis in human laryngeal cancer HEp-2 cells by our group. Here, we made unexpected observations that the caspase-9 inhibitor (C9i) enhanced apoptosis in response to selected stimuli, and HEp-2 cells which were made deficient in caspase-9 using siRNA exhibited no resistance to apoptotic signals and actually demonstrated increased apoptotic sensitivity to oridonin. The results were reversed by the transfection of an exogenous caspase-9 expression vector. Caspase-9 reduced sensitivity to apoptotic stimuli through reactive oxygen species (ROS)-suppressing and autophagy-promoting methods. ROS triggered the progression of apoptosis through activation of both the caspase-9-independent mitochondrial pathway and death receptor pathways, and the autophagy had an anti-apoptotic function in oridonin-treated HEp-2 cells. These collective results suggest that oridonin targets caspase-9 to alter ROS production and autophagy situation to promote HEp-2 cell apoptosis. Therefore, oridonin has the potential to be developed as an anticancer agent, and the combination of oridonin with those agents leading to reduction of caspase-9 expression in tumor cells could represent a novel approach to human laryngeal cancer treatment.

Expression of human protection of telomere 1 correlates with telomere length and radiosensitivity in the human laryngeal cancer Hep-2 cell line

The close association between telomere length and radiosensitivity has been established by several studies. There is also a hypothesis that telomere length may be regulated by human protection of telomere 1 (hPOT1) in human carcinoma cells. In the present study, the hPOT1 level between the radioresistant Hep-2R cells and the wild-type were compared, and the results showed that the hPOT1 gene was upregulated in the radioresistant Hep-2R cell lines compared with the wild-type. This suggested that the expression level of hPOT1 correlates with radiosensitivity. Additionally, an hPOT1-directed short hairpin (sh)RNA plasmid was constructed and transferred into the Hep-2R cells, which lead to telomere shortening, an increase in apoptosis and markedly decreased growth of the RNAi-Hep-2R cell line. These results demonstrate that hPOT1-directed shRNAs are associated with telomere length and radiosensitivity, and maybe a potent sensitizer for laryngeal cancer radiotherapy.

Peptide GE11-Polyethylene Glycol-Polyethylenimine for targeted gene delivery in laryngeal cancer.

The objective of this study was to evaluate the possibility of using GE11-polyethylene glycol-polyethylenimine (GE11-PEG-PEI) for targeted gene delivery to treat epidermal growth factor receptor (EGFR)-overexpressing laryngeal cancer. This study described the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEG-PEI for gene delivery to treat laryngeal cancer. Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio, and these complexes were capable of binding plasmid DNA and condensing DNA into small positively charged nanoparticles. The results also revealed that GE11-PEG-PEI had a weaker effect on cell survival in vitro. Gene transfection was performed on human laryngeal cancer Hep-2 cells in vitro and in vivo. Both the in vitro and in vivo results demonstrated that GE11-PEG-PEI had greater transfection efficiency than mPEG-PEI. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p < 0.05) reduced tumor growth in nude mice with laryngeal cancer. Moreover, the GE11-PEG-PEI/pORF-hTRAIL-treated groups showed more apoptosis than the mPEG-PEI/pORF-hTRAIL-treated groups. Therefore, our results showed that the peptide GE11 conjugated to PEG-PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo, indicating that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer.

Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways.

Head and neck cancer is the sixth most frequently occurring cancer worldwide and accounts for about 2% of all cancer-related deaths annually. Curcumin is a well-known chemopreventive agent, and apoptosis induction by curcumin has been reported in many cancer cell types. We synthesized an ortho-hydroxy substituted analog of curcumin, bisdemethoxycurcumin analog (BDMC-A), and aimed to demarcate the apoptotic effects induced by BDMC-A on human laryngeal cancer Hep-2 cells and to compare these effects with those induced by curcumin. We evaluated the apoptotic effects of BDMC-A in comparison to those of curcumin on Hep-2 cells by performing Western blotting, RT-PCR, fluorescent staining and DNA fragmentation assays. In addition, we carried out an in silico molecular docking study on the EGFR kinase domain. We found that BDMC-A can induce apoptosis in Hep-2 cells by regulating the expression of both intrinsic and extrinsic apoptotic proteins, i.e., Bcl-2, Bax, apoptososme complex and death receptors, more efficiently than curcumin. We also observed increased nuclear fragmentation and chromatin condensation after BDMC-A treatment compared to curcumin treatment. Depolarized mitochondria and ROS generation was well pronounced in both BDMC-A and curcumin treated Hep-2 cells. Our in silico molecular docking study on the EGFR kinase domain revealed that BDMC-A may dock more efficiently than curcumin. From our results we conclude that BDMC-A can induce apoptosis in Hep-2 laryngeal carcinoma cells more effectively than curcumin, and that this activity can be attributed to the presence of a hydroxyl group at the ortho position within this compound.

Inhibition of Nemo-like Kinase Increases Taxol Sensitivity in Laryngeal Cancer

Several studies have shown that nemo-like kinase (NLK) plays a vital role in apoptosis of cancer cells. The present research concerned effects and mechanisms of Taxol on NLK knockdown human laryngeal cancerHep-2 cell lines in vitro. Using RNAi, methyl-thiazoltetrazolium (MTT) assays, real-time RT-PCR, Western blotting and flow cytometry analysis, growth and the cell cycle progression of NLK knockdown Hep-2 cells and expression of downstream molecules were observed. Cell growth was obviously suppressed in the Taxol treated group (P<0.001, 48 hours). Cell numbers of combined Taxol-based chemotherapy with lentivirus mediated RNAi treatment group (Lv-shNLK+Taxol goup) were significantly different from NLK-specific siRNA lentivirus infected group (Lv-shNLK group) (p<0.001). Flow cytometry analysis revealed that Lv-shNLK+Taxol caused the G0/G1-phase DNA content to decrease from 44.1 to 3.33% (p<0.001) and the S-phase DNA content to increase from 38.4 to 82.0% (p<0.001), in comparison with the Lv-shNLK+Taxol group. Immunoblot analysis showed that knockdown of NLK led to significant reduction in the levels of cyclin D1, PCNA and PARP, whereas cyclin B1 was elevated in. Cell growth was also obviously suppressed in the Hep-2 cell line, knockdown of NLK making them more sensitive to Taxol treatment. NLK is expected to become a target of new laryngeal cancer gene therapies.

Expression, purification and anticancer analysis of GST-tagged human perforin and granzyme B proteins in human laryngeal cancer Hep-2 cells

Granzyme B and perforin, two major effector molecules in the granule-mediated cytolytic pathway, are thought to be involved in suppression of tumor progression. In this study, the pGEX-4T-1 expression vector was used to express full-length human perforin or granzyme B as a GST-tagged fusion protein in Escherichia coli (E. coli). GST-tagged proteins were induced with IPTG and purified by GSTrap 4B columns. Purified fusion proteins migrated at the predicted molecular mass on SDS–PAGE and were recognized by specific antibodies. Moreover, the fusion proteins can induce apoptosis and directly inhibit the growth of human laryngeal cancer Hep-2 cells in vitro. These results suggest that active perforin and granzyme B fusion proteins can be produced in E. coli and exhibit anticancer potential in laryngeal cancer cells.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Identification of inositol polyphosphate 4-phosphatase type II as a novel tumor resistance biomarker in human laryngeal cancer HEp-2 cells

Although tumor resistance remains a significant impediment to successful radiotherapy, associated regulatory markers and detailed molecular mechanisms underlying this phenomenon are not well defined. In this study, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a novel marker of radioresistance by systematically analyzing Unigene libraries of laryngeal cancer. INPP4B was highly expressed in radioresistant laryngeal cancer cells and was induced by treatment with either radiation or anticancer drugs in various types of cancer cells. Ectopic INPP4B overexpression increased radioresistance and anticancer drug resistance by suppressing apoptosis in HEp-2 cells. Conversely, INPP4B depletion with small interfering RNA resensitized HEp-2 as well as A549 and H1299 cells to radiation- and anticancer drug-induced apoptosis. Furthermore, radiation-induced INPP4B expression was blocked by inhibition of extracellular signal-regulated kinase (ERK). INPP4B depletion significantly attenuated radiation-induced increases in Akt phosphorylation, indicating an association of INPP4B-mediated radioresistance with Akt survival signaling. Taken together, our data suggest that ERK-dependent induction of INPP4B triggers the development of a tumor-resistance phenotype via Akt signaling and identify INPP4B as a potentially important target molecule for resolving the radioresistance of cancer cells.

Aldehyde dehydrogenase 1 can be used as a new marker of cancer stem cells in laryngeal cancer cells in vitro

To detect the expression of aldehyde dehydrogenase 1 in human laryngeal cancer cells in vitro, and to explore whether it can be used as a marker of stem cells in human laryngeal cancer. Fluorescence staining and flow cytometry were used to detect the expression of ALDH1 in a human laryngeal cancer Hep-2 cell line, and fluorescence activated cells sorting was used to separate ALDH1(br) cells. ALDH1 tumor cells were cultured and their ability of proliferation and differentiation was observed in vitro. The expression of ALDH1 in Hep-2 cells was different. The number of cells highly expressing ALDH1 was 2.9% ± 0.6%. Compared with ALDH1(low) cells and unsorted cells, ALDH1(br) cells exhibited increased proliferation ability. In serum-containing RPM I1640 culture medium, the proportion of ALDH1(br) cells was decreasing as days passed. The percentage of ALDH1(br) cells decreased from 94.2% ± 3.8% to the level before sorting. The ALDH1(br) cells demonstrated enhanced tumorigenic ability in nude mice. In the laryngeal cancer Hep-2 cell line, the highly ALDH1-expressing cells show a strong ability of differentiation, proliferation and tumorigenesis. It indicates that ALDH1 can be used as a new marker of stem cells of laryngeal cancer cells.

In-vitro evaluation of paclitaxel-loaded MPEG–PLGA nanoparticles on laryngeal cancer cells

Induction chemotherapy or concurrent chemotherapy with radiotherapy can preserve laryngeal function without a detrimental effect on survival in patients with advanced laryngeal cancer. However, systemic chemotherapy with traditional injection often causes side effects. In this study, methoxy poly(ethylene glycol)-poly(lactide-co-glycolide) (MPEG-PLGA) nanoparticles (NPs) were developed as a carrier for paclitaxel, a potent cytotoxic agent. The NPs were prepared by an emulsification-solvent evaporation method with the particle size and zeta potential around 153.3+/-41.7 nm and -5.36 mV. Transmission electron microscopy showed that the NPs were homogeneous and spherical in shape. Differential scanning calorimetry and X-ray powder diffractometry did not detect any crystalline drug in the NP samples. High-performance liquid chromatography was used to measure the drug loading, encapsulation efficiency, and in-vitro drug release. The drug loading efficiency was (5.35+/-0.75)% and encapsulation efficiency was (75.56+/-2.61)%. In the in-vitro drug release study, paclitaxel was released from the NPs in a slow but time-dependent manner. In-vitro cytotoxicity of the paclitaxel-loaded NPs was investigated by using human laryngeal cancer Hep-2 cells. The anticancer activity of paclitaxel-loaded NPs was comparable with the free paclitaxel. No significant cytotoxicity was observed in blank MPEG-PLGA NPs. Laser scanning confocal microscopy was used to observe the uptake of fluorescent coumarin-6-loaded NPs by Hep-2 cells. We conclude that the formulation of NPs inhibits Hep-2 cell growth to a similar extent as free paclitaxel injection. These results suggest that MPEG-PLGA NPs may have potential as an alternative delivery system for paclitaxel.

Co-transfer of human wild-type p53 and granulocyte-macrophage colony-stimulating factor genes via recombinant adenovirus induces apoptosis and enhances immunogenicity in laryngeal cancer cells

Co-transfer of immunomodulatory and anti-proliferative genes may be the basis for new strategies to enhance tumor regression. The purpose of this study was to develop a combination gene therapy strategy for the treatment of laryngeal cancer. Human wild-type p53 and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes were transferred into human laryngeal cancer cells mediated by adenovirus type 5 vector co-expressing human wild-type p53 and GM-CSF (Ad-p53/GM-CSF). By the introduction of the wild-type p53 gene, the growth of human laryngeal cancer Hep-2 cells was inhibited and their apoptosis was induced. By the introduction of the GM-CSF gene, the immunogenicity of cancer cells was enhanced. Significant proliferation of tumor infiltrating lymphocytes and tumor-specific cytotoxicity of cytotoxic T lymphocytes were induced by Ad-p53/GM-CSF-infected cancer cells in vitro. The results suggest that the co-transfer of human wild-type p53 and GM-CSF genes into tumor cells via recombinant adenovirus may be further developed into an effective and practical combination gene therapy strategy for laryngeal cancer.