Induction of Tca8113 tumor cell apoptosis by icotinib is associated with reactive oxygen species mediated p38-MAPK activation.

Icotinib, a selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has been shown to exhibit anti-tumor activity against several tumor cell lines. However, the exact molecular mechanism of icotinib's anti-tumor effect remains unknown. This study aims to examine the zytotoxic effect of icotinib on Tca8113 cells and its potential molecular mechanism. Icotinib significantly resulted in dose-dependent cell death as determined by MTT assay, accompanied by increased levels of Bax and DNA fragmentation. Icotinib could also induce Reactive Oxygen Species (ROS) generation. Further studies confirmed that scavenging of reactive oxygen species by N-acetyl-L-cysteine (NAC), and pharmacological inhibition of MAPK reversed icotinib-induced apoptosis in Tca8113 cells. Our data provide evidence that icotinib induces apoptosis, possibly via ROS-mediated MAPK pathway in Tca8113 cells.

[Extracranial/intracranial vascular bypass to control carotid artery blowout in postirradiated nasopharyngeal carcinoma patients].

Objective:To summarize and analyze the effect of extracranial/intracranial vascular bypass in the treatment of internal carotid artery burst hemorrhage after radiotherapy for nasopharyngeal carcinoma（NPC）. Methods:A retrospective analysis of the data of 9 patients with nasopharyngeal carcinoma and carotid artery blowout syndrome（CBS） who underwent extracranial/intracranial vascular bypass. Collected patient demographics, treatment course and dose of radiotherapy, analyze the effect of extracranial/intracranial vascular bypass on the prognosis of patients with internal carotid artery burst hemorrhage, including perioperative stroke and death, overall survival rate, and rebleeding rate. Results:Nine patients were included in the study. The average age is 53.5 years. The pathological types were all non-keratinizing squamous cell carcinoma, undifferentiated, stage Ⅳ; 7 cases of local NPC recurrence, 2 cases of skull base osteonecrosis; all 9 cases had internal carotid artery hemorrhage, including 7 cases of petrous carotid artery and 2 cases of cervical carotid artery; 3 cases of typeⅠthreatened CBS（33.3%）, 2 cases of type Ⅱ impending CBS（22.2%）, and 4 cases of type Ⅲ acute CBS（44.45%）. All patients underwent extracranial/intracranial vascular bypass surgery, and there were no perioperative deaths and strokes. The mean follow-up was 16.7 months. The median overall survival time of the patients was 22.1 months and the 90-day, 1-year and 2-year survival rates were 100.0%, 75.0% and 30.0%, respectively. Conclusion:Patients with internal carotid artery burst hemorrhage after radiotherapy for nasopharyngeal carcinoma can be safely treated by extracranial/intracranial vascular bypass surgery and obtain a longer survival rate.

Extracranial/Intracranial Vascular Bypass in the Treatment of Head and Neck Cancer - Related Carotid Blowout Syndrome.

OBJECTIVE/HYPOTHESIS: To investigate the endovascular intervention or extracranial/intracranial (EC/IC) vascular bypass in the management of patients with head and neck cancer-related carotid blowout syndrome (CBS). STUDY DESIGN: Retrospective case series. METHODS: Retrospective analysis of clinical data of patients with head and neck cancer-related CBS treated by endovascular intervention and/or EC/IC vascular bypass, analysis of its bleeding control, neurological complications, and survival results. RESULTS: Thrity-seven patients were included. Twenty-five were associated with external carotid artery (ECA); twelve were associated with internal or common carotid artery (ICA/CCA). All patients with ECA hemorrhage were treated with endovascular embolization. Of the 12 patients with ICA/CCA hemorrhage, 9 underwent EC/IC bypass, 1 underwent endovascular embolization, and 3 underwent endovascular stenting. For patients with ECA-related CBS, the median survival was 6 months, and the 90-day, 1-year, and 2-year survival rates were 67.1%, 44.7%, and 33.6%, respectively; the estimated rebleeding risk at 1-month, 6-month, and 2-year was 7.1%, 20.0%, and 31.6%, respectively. For patients with ICA/CCA-related CBS, the median survival was 22.5 months, and the 90-day, 1-year, and 2-year survival rates were 92.3%, 71.8%, and 41.0%, respectively; the estimated rebleeding risk at 1 month, 6 months, and 2 years is 7.7%,15.4%, and 15.4%, respectively. ICA/CCA-related CBS patients have significantly longer survival time and lower risk of rebleeding, which may be related to the more use of EC/IC vascular bypass as a definite treatment. CONCLUSIONS: For patients with ICA/CCA-related CBS, if there is more stable hemodynamics, longer expected survival, EC/IC vascular bypass is preferred. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:1548-1556, 2021.

Upregulation of p­Akt by glial cell line­derived neurotrophic factor ameliorates cell apoptosis in the hippocampus of rats with streptozotocin­induced diabetic encephalopathy.

The loss of neurotrophic factor support has been shown to contribute to the development of the central nervous system. Glial cell line­derived neurotrophic factor (GDNF), a potent neurotrophic factor, is closely associated with apoptosis and exerts neuroprotective effects on numerous populations of cells. However, the underlying mechanisms of these protective effects remain unknown. In the present study, a significant increase in Bax levels and DNA fragmentation was observed in the hippocampus obtained from the brains of diabetic rats 60 days after diabetes had been induced. The apoptotic changes were correlated with the loss of GDNF/Akt signaling. GDNF administration was found to reverse the diabetes­induced Bax and DNA fragmentation changes. This was associated with an improvement in the level of p­Akt/Akt. In addition, combination of GDNF with a specific inhibitor of the phosphoinositide 3­kinase (PI3K)/Akt pathway, Wortmannin, significantly abrogated the effects of GDNF on the levels of p­Akt/Akt, Bax and DNA fragmentation. However, a p38 mitogen­activated proten kinase (MAPK) inhibitor, SB203580, had no effect on the expression of p­Akt/Akt, Bax or DNA fragmentation. These results demonstrate the pivotal role of GDNF as well as the PI3K/Akt pathway, but not the MAPK pathway, in the prevention of diabetes­induced neuronal apoptosis in the hippocampus.

Effect of quercetin on the proliferation of the human ovarian cancer cell line SKOV-3 in vitro.

Quercetin is a hydrophobic agent that demonstrates potential anticancer activity. The aim of the present study was to observe the effects of quercetin on the proliferation and apoptosis of the ovarian cancer cell line SKOV-3, and to provide a foundation for the treatment of ovarian cancer using this agent. Ovarian cancer SKOV-3 cells were treated with quercetin at different doses. The inhibitory effect of quercetin on proliferation was detected using the MTT assay and the inhibition rate was calculated. Cell apoptosis was determined using Hoechst staining, and western blot analysis was used to analyze changes in the expression levels of survivin protein. The effects of quercetin on the cell cycle and apoptosis of the SKOV-3 cell line were analyzed using flow cytometry. Quercetin inhibited the proliferation of SKOV-3 cells in a time- and dose-dependent manner. Furthermore, Hoechst staining showed that quercetin induced SKOV-3 cell apoptosis. The protein expression levels of survivin were reduced as the concentration of quercetin increased. Flow cytometric analysis showed that quercetin caused ovarian cancer SKOV-3 cell cycle arrest in the G0/G1 phase and a significant decrease in the percentage of cells at the G2/M phase; furthermore, the apoptosis rate was observed to increase following quercetin treatment. The results in combination indicated that Quercetin could inhibit the proliferation of ovarian cancer SKOV-3 cells, inhibit cell cycle progression from G0/G1 to G2/M and induce cell apoptosis in vitro.

Sulindac attenuates valproic acid-induced oxidative stress levels in primary cultured cortical neurons and ameliorates repetitive/stereotypic-like movement disorders in Wistar rats prenatally exposed to valproic acid.

Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodevelopental disorders, such as autism. In the present study, the possible neuroprotective properties of sulindac, a non-steroidal anti-inflammatory drug (NSAID), were investigated in vitro using cultured cortical neurons with valproic acid (VPA)-induced neurotoxicity, as well as in vivo through the behavioral analysis of rats prenatally exposed to VPA as a model of autism. VPA induced 4-hydroxynonenal (4-HNE) expression, reactive oxygen species (ROS) generation and decreased cell viability in primary cultured cortical neurons established from timed-pregnant (embryonic day 18) Wistar rat pups. However, co-incubation of the neurons with VPA and sulindac reduced oxidative stress and increased cell viability. The rats were administered an intraperitoneal injection with one of the following: VPA, sulindac, VPA and sulindac, or physiological saline, and their offspring were subjected to the open field test. During the test trials, repetitive/stereotypic-like movements for each rat were recorded and analyzed. The results revealed that treatment with both sulindac and VPA reduced the VPA-induced repetitive/stereotypic-like activity and the sulindac and VPA-treated animals responded better in the open field test compared to the VPA-treated animals. The results from the present study demonstrate that the antioxidant properties of sulindac may prove to be beneficial in the treatment of autism, suggesting that the upregulation of the Wnt/ß-catenin signaling pathway disrupts oxidative homeostasis and facilitates susceptibility to autism.

Icotinib inhibits the invasion of Tca8113 cells via downregulation of nuclear factor κB-mediated matrix metalloproteinase expression.

Icotinib is an epidermal growth factor receptor tyrosine kinase inhibitor, which has been revealed to inhibit proliferation in tumor cells. However, the effect of icotinib on cancer cell metastasis remains to be explained. This study examines the effect of icotinib on the migration and invasion of squamous cells of tongue carcinoma (Tca8113 cells) in vitro. The results of the Boyden chamber invasion assay demonstrated that icotinib reduced cell invasion, suppressed the protein levels of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and increased the expression of tissue inhibitor of metalloproteinase-1. In addition, icotinib was found to significantly decrease the protein levels of nuclear factor κB (NF-κB) p65, which suggested that icotinib inhibits NF-κB activity. Furthermore, treatment with the NF-κB inhibitor, pyrrolidine dithiocarbamate, suppressed cell invasion and MMP-2 expression. These results suggested that icotinib inhibits the invasion of Tca8113 cells by downregulating MMP via the inactivation of the NF-κB signaling pathways.

Effect of ECRG2 in combination with cisplatin on the proliferation and apoptosis of EC9706 cells.

The aim of the present study was to explore the effect of esophageal cancer-related gene 2 (ECRG2) protein in combination with cisplatin (DDP) on the proliferation and apoptosis of esophageal cancer cells. A 3-(4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the effects of ECRG2 alone and ECRG2 in combination with DDP on the proliferation of EC9706 esophageal cancer cells. Hoechst 33258 staining was performed to analyze the effects of ECRG2 alone and ECRG2 in combination with DDP on apoptosis in the EC9706 cells. The expression levels of Bcl-2-associated X protein (Bax) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results from the MTT assay revealed that ECRG2 inhibited the proliferation of EC9706 cells and that ECRG2 in combination with DDP had a greater inhibitory effect on cell proliferation. The antiproliferative effects were time- and concentration-dependent, within a certain range of concentrations. The Hoechst 33258 staining results demonstrated that the number of apoptotic cells following treatment with ECRG2 in combination with DDP for 24 h was higher than that following treatment with ECRG2 alone for the same duration. Western blot analysis and RT-PCR results revealed that the expression levels of Bax mRNA and protein were upregulated in cells treated with ECRG2 in combination with DDP compared with those in cells treated with ECRG2 alone. Thus, ECRG2 in combination with DDP had an enhanced inhibitory effect on EC9706 cell proliferation compared with that of ECRG2 alone, and an increased inductive effect on EC9706 cell apoptosis, possibly due to the upregulation of the expression of Bax.

Expression of bcl-2 and p53 in induction of esophageal cancer cell apoptosis by ECRG2 in combination with cisplatin.

AIM: To investigate the mechanisms of induction of apoptosis of esophageal cancer cells by esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP). METHODS: Hoechest staining was performed to analyze the effects of single ECRG2 and ECRG2 in combination with DDP on apoptosis of EC9706 cells. The expression levels of p53 and bcl-2 mRNA and protein were determined by RT-PCR and Western blotting, respectively. RESULTS: The number of apoptotic cells after the treatment with ECRG2 in combination with DDP for 24 hours was more than that after the treatment with single ECRG2. RT-PCR and Western blotting showed that the expression levels of bcl-2 mRNA and protein were both down-regulated, while p53 mRNA and protein were both up-regulated in the cells treated with ECRG2 in combination with DDP compared with those given ECRG2 alone. CONCLUSION: ECRG2 in combination with DDP can enhance the apoptosis of EC9706 cells, possibly by down-regulating bcl-2 expression and up-regulating p53.

Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously.

The present study aimed to explore the factors that affect polymerase chain reaction using sequence-specific primers (PCR-SSP) and to establish an optimized PCR-SSP method for detecting multiple gene polymorphisms simultaneously. The amplification system parameters, including the concentrations of Mg2+, dNTPs, pfu Taq, primers and control primers, were optimized using the designed PCR-SSP reactions. The resulting optimized reaction system was used to determine the melting temperature of the genomic DNA and the cycling parameters. The optimized PCR-SSP method was used to analyze the polymorphisms of the following genes: mutations -308A/G and -238G/A in TNF-α, -174G/C in IL-6 and C/T mutation at exon 188 of CYP2D6 *10B. The PCR-SSP amplification system was optimized; in a 20 µl reaction system, the quantities of Mg2+, dNTPs, pfu Taq, primers, control primers and genomic DNA were 3.25 µM, 0.5 mM, 2.5 units, 0.5 µM, 0.2 µM and 0.15 µg, respectively. The cycling system comprised 5 start cycles and took 15 min to melt a genomic DNA sample using a touchdown protocol. The optimized PCR-SSP is suitable for polymorphism analysis of polygenic SNPs in large genomic DNA samples and a number of different genes.

Effects of quercetin on the proliferation of breast cancer cells and expression of survivin in vitro.

Quercetin is a hydrophobic agent with potential anticancer activity. The aim of the present study was to observe the effects of quercetin on the proliferation of the breast cancer cell line MCF-7 and the gene expression of survivin. The molecular mechanism underlying the antiproliferative effect of quercetin was also investigated. MCF-7 breast cancer cells were treated with various concentrations of quercetin. The inhibitory effect of quercetin on proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and the inhibition rate was calculated. Cellular apoptosis was detected by immunocytochemistry and survivin mRNA expression levels were observed using reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis was used to analyze changes in the expression levels of survivin protein. Quercetin induced the apoptosis of MCF-7 cells and inhibited the proliferation of the MCF-7 breast cancer cells in a time- and concentration-dependent manner. The mRNA and protein expression levels of survivin were reduced as the concentration of quercetin increased. Quercetin inhibited the growth of MCF-7 cells and promoted apoptosis by inducing G0/ G1 phase arrest. It also regulated the expression of survivin mRNA in MCF-7 cells, which may be the mechanism underlying its antitumor effect.