Effects of sintilimab plus chemotherapy as first-line treatment on health-related quality of life in patients with advanced esophageal squamous cell carcinoma: results from the randomized phase 3 ORIENT-15 study.

Background: In ORIENT-15 study, sintilimab plus chemotherapy demonstrated significant improvement on overall survival (OS) versus placebo plus chemotherapy in first-line treatment of advanced esophageal squamous cell carcinoma (ESCC). Here, we report effect of sintilimab plus chemotherapy on health-related quality of life (HRQoL) in patients with advanced ESCC. Methods: From December 14, 2018 to August 28, 2022, HRQoL was evaluated in all randomized patients using European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core 30 items (QLQ-C30), EORTC Quality of Life Questionnaire Oesophageal Cancer Module 18 items (QLQ-OES18), and visual analogue scale (VAS) of the EuroQol five-dimensional five-level questionnaire (EQ-5D-5L). Mean scores of each scale were described by treatment group through week 60. Least-squares mean (LSM) score change from baseline through week 24 were analyzed using the mixed-model repeated-measures method. Time to the first onset of deterioration (TTD) and OS for each scale were estimated. Clinical Trials Registration: NCT03748134. Findings: As of August 28, 2022, 689 of 690 enrolled patients were assessed for HRQoL analysis (sintilimab group: 340, placebo group: 349). Median follow-up was 32.2 months. Differences in LSM favored sintilimab over placebo for QLQ-C30 social functioning (LSM difference: 3.06, 95% CI: 0.55 to 5.57; P = 0.0170), pain (-2.24, 95% CI: -4.30 to -0.17; P = 0.0337), fatigue (-2.24, 95% CI: -4.46 to -0.02; P = 0.0479), constipation (-3.27, 95% CI -5.49 to -1.05; P = 0.0039), QLQ-OES18 pain (-1.77, 95% CI -3.11 to -0.43; P = 0.0097), trouble swallowing saliva (-2.09, 95% CI: -3.77 to -0.42; P = 0.0146), and choked when swallowing (-3.23, 95% CI: -5.60 to -0.86; P = 0.0076). TTD favored sintilimab over placebo for QLQ-OES18 dysphagia (Hazard ratio [HR]: 0.76, 95% CI: 0.61-0.94, P = 0.0104), and trouble swallowing saliva (HR: 0.48, 95% CI: 0.35-0.67, P < 0.0001). Improved OS were observed in patients with better performance in several functioning and symptom scales of QLQ-C30 and QLQ-QES18. Interpretation: The statistically significant differences of several HRQoL scales and improvements in delayed deterioration observed in our study further support the use of sintilimab plus chemotherapy as first-line treatment for advanced ESCC. Funding: This study was funded by Innovent Biologics and was co-funded by Eli Lilly.

Heat-dependent postpollination limitations on maize pollen tube growth and kernel sterility.

Heat stress has a negative impact on pollen development in maize (Zea mays L.) but the postpollination events that determine kernel sterility are less well characterised. The impact of short-term (hours) heat exposure during postpollination was therefore assessed in silks and ovaries. The temperatures inside the kernels housed within the husks was significantly lower than the imposed heat stress. This protected the ovaries and possibly the later phase of pollen tube growth from the adverse effects of heat stress. Failure of maize kernel fertilization was observed within 6 h of heat stress exposure postpollination. This was accompanied by a significant restriction of early pollen tube growth rather than pollen germination. Limitations on early pollen tube growth were therefore a major factor contributing to heat stress-induced kernel sterility. Exposure to heat stress altered the sugar composition of silks, suggesting that hexose supply contributed to the limitations on pollen tube growth. Moreover, the activities of sucrose metabolising enzymes, the expression of sucrose degradation and trehalose biosynthesis genes were decreased following heat stress. Significant increases in reactive oxygen species, abscisic acid and auxin levels accompanied by altered expression of phytohormone-related genes may also be important in the heat-induced suppression of pollen tube growth.

Long noncoding RNA ZEB1-AS1 attenuates ferroptosis of gastric cancer cells through modulating miR-429/BGN axis.

Gastric cancer (GC) is the fifth utmost common malignant cancer type globally, in which ferroptosis acts a critical function in the progress of GC. Long noncoding RNA ZEB1-AS1 has been recognized in numerous cancers, but the role of ZEB1-AS1 in ferroptosis remains obscure. Hence, we investigated the efficacy of ZEB1-AS1 on ferroptosis of GC cells. The cell growth and viability were analyzed via cell counting kit assay and xenograft tumor model in vivo and in vitro, respectively. The RNA and protein expression were measured by qRT-PCR and western blot analysis assay, respectively. The levels of Fe2+ , malondialdehyde (MDA), and lipid reactive oxygen species (ROS) were tested to determine ferroptosis. The erastin and RSL3 were used to induce ferroptosis. The mechanism was analyzed via luciferase reporter gene and RIP assays. The treatment of ferroptosis inducer Erastin and RSL3 suppressed the viability of GC cells and the ZEB1-AS1 overexpression rescued the phenotype in the cells. The levels of Fe2+ , MDA, and ROS were enhanced through the depletion of ZEB1-AS1 in Erastin/RSL3 treated GC cells. ZEB1-AS1 directly sponged miR-429 in GC cells and miR-429 targeted BGN in GC cells, and the inhibition of miR-429 rescued ZEB1-AS1 depletion-inhibited BGN expression. We validated that miR-429 induced and BGN-repressed ferroptosis in cancer cells. The BGN overexpression and miR-429 suppression could reverse the efficacy of ZEB1-AS1 on proliferation and ferroptosis in cancer cells. The expression of ZEB1-AS1 and BGN was enhanced and miR-429 expression was decreased in clinical GC tissues. ZEB1-AS1 attenuated ferroptosis of cancer cells by modulating miR-429/BGN axis.

Identification of a potential competing endogenous RNA (ceRNA) network in gastric adenocarcinoma.

Background: Recently, a growing body of evidence has revealed the role of competing endogenous RNA (ceRNA) networks in various human cancers. However, there is still a lack of research on the systemic ceRNA network related to gastric adenocarcinoma. Methods: The intersection of differentially expressed genes (DEGs) was obtained by mining the GSE54129, GSE13861, and GSE118916 datasets from the Gene Expression Omnibus (GEO) website. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for the enrichment analysis. A protein-protein interaction (PPI) network was established with the STRING online database, and hub genes were identified by Cytoscape software. The prediction of key microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) was conducted by miRNet. The prognostic analysis, expression difference, and correlation analysis of messenger RNAs (mRNAs), lncRNAs, and miRNAs were carried out using the Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan-Meier plotter, and Encyclopedia of RNA Interactomes (ENCORI). Results: We identified180 significant DEGs. Extracellular matrix (ECM) receptor interaction, focal adhesion, ECM tissue, and collagen catabolic processes were the most significant pathways in the functional enrichment analysis. Nineteen upregulated hub genes and one downregulated hub gene were found to be significantly associated with the prognosis of gastric adenocarcinoma. Of the 18 miRNAs targeting 12 key genes, only six were associated with a promising prognosis in gastric adenocarcinoma. By comprehensive differential expression and survival analysis, 40 key lncRNAs were identified. Finally, we constructed a network of 24 ceRNAs associated with gastric adenocarcinoma. Conclusions: Potential mRNA-miRNA-lncRNA subnets were constructed, each RNA of which can be used as a prognostic biomarker for gastric adenocarcinoma.

A Retrospective Cohort Study Examining the Effects of Anti-PD-1 Antibody in Combination with Apatinib in Patients Previously Treated for Her2-Negative Advanced Gastric/Gastroesophageal Junction Cancer.

Combining immune checkpoint inhibitors with vascular endothelial growth factor/vascular endothelial growth factor receptor inhibitors is effective in treating a number of solid tumors; however, evidence in advanced gastric/gastroesophageal junction (G/GEJ) cancer is limited. This retrospective study included consecutive patients who received a programmed cell death protein 1 (PD-1) inhibitor plus the vascular endothelial growth factor receptor 2 inhibitor apatinib, second-line or later to treat unresectable advanced or metastatic, histologically proven, human epidermal growth factor receptor 2-negative G/GEJ cancer in a single center between November 1, 2018, and March 31, 2021. Treatment was continued until the disease progressed or the toxicity became intolerable. We examined data from 52 patients. The primary tumor site was the stomach in 29 patients and the GEJ in 23 patients. PD-1 inhibitors administered included camrelizumab (n = 28), sintilimab (n = 18), pembrolizumab (n = 3), and tislelizumab (n = 1), and all patients were given 200 mg every 3 weeks, and toripalimab (240 mg every 3 weeks) and nivolumab (200 mg every 2 weeks) were given to 1 patient each. For 28 days, apatinib 250 mg was administered orally once a day. The objective response rate was 15.4% (95% confidence interval [CI], 6.9-28.1), and the disease control rate was 61.5% (95%CI, 47.0-74.7). After 14.8 months of median follow-up, the median progression-free survival was 4.2 months (95%CI, 2.6-4.8), and the overall survival was 9.3 months (95%CI, 7.9-12.9). Twelve patients underwent grade 3-4 treatment-related adverse events (23.1%). There was no unexpected toxicity or death. This trial demonstrated combination therapy with an anti-PD-1 antibody and apatinib was effective and safe in patients with previously treated unresectable advanced or metastatic G/GEJ cancer.

Knockdown of kinesin family member 2C restricts cell proliferation and induces cell cycle arrest in gastric cancer.

Kinesin family member 2C (KIF2C) was reported to act as a vital player in several human cancers. However, the exact function of KIF2C in gastric cancer (GC) is poorly understood. In the present study, the potential role of KIF2C was studied in gastric cancer by bioinformatics analysis and proliferation assay. KIF2C expression was detected using reverse transcription-quantitative polymerase chain reaction and western blot. Our data showed that the expression of KIF2C was increased in different tumor tissues, including GC. KIF2C was overexpressed in GC tissues and might be used as a diagnostic and prognostic biomarker for GC. KIF2C expression was correlated with immune infiltration and the levels of cell cycle-related genes in GC. Moreover, silencing of KIF2C can cause cell cycle arrest, and inhibit the proliferative ability of GC cells. Thus, our studies revealed that KIF2C levels might serve as a promising biomarker for diagnosis and prediction of prognosis of GC, and targeting KIF2C might be an effective therapeutic strategy for GC.

Sintilimab versus placebo in combination with chemotherapy as first line treatment for locally advanced or metastatic oesophageal squamous cell carcinoma (ORIENT-15): multicentre, randomised, double blind, phase 3 trial.

OBJECTIVE: To evaluate sintilimab versus placebo in combination with chemotherapy (cisplatin plus paclitaxel or cisplatin plus 5-fluorouracil) as first line treatment of unresectable locally advanced, recurrent, or metastatic oesophageal squamous cell carcinoma. DESIGN: Multicentre, randomised, double blind, phase 3 trial. SETTING: 66 sites in China and 13 sites outside of China between 14 December 2018 and 9 April 2021. PARTICIPANTS: 659 adults (aged ≥18 years) with advanced or metastatic oesophageal squamous cell carcinoma who had not received systemic treatment. INTERVENTION: Participants were randomised 1:1 to receive sintilimab or placebo (3 mg/kg in patients weighing <60 kg or 200 mg in patients weighing ≥60 kg) in combination with cisplatin 75 mg/m2 plus paclitaxel 175 mg/m2 every three weeks. The trial was amended to allow investigators to choose the chemotherapy regimen: cisplatin plus paclitaxel or cisplatin plus 5-fluorouracil (800 mg/m2 continuous infusion on days 1-5). MAIN OUTCOME MEASURES: Overall survival in all patients and in patients with combined positive scores of ≥10 for expression of programmed cell death ligand 1. RESULTS: 659 patients were randomly assigned to sintilimab (n=327) or placebo (n=332) with chemotherapy. 616 of 659 patients (93%) received sintilimab or placebo in combination with cisplatin plus paclitaxel and 43 of 659 patients (7%) received sintilimab or placebo in combination with cisplatin plus 5-fluorouracil. At the interim analysis, sintilimab with chemotherapy showed better overall survival compared with placebo and chemotherapy in all patients (median 16.7 v 12.5 months, hazard ratio 0.63, 95% confidence interval 0.51 to 0.78, P<0.001) and in patients with combined positive scores of ≥10 (17.2 v 13.6 months, 0.64, 0.48 to 0.85, P=0.002). Sintilimab and chemotherapy significantly improved progression free survival compared with placebo and chemotherapy in all patients (7.2 v 5.7 months, 0.56, 0.46 to 0.68, P<0.001) and in patients with combined positive scores of ≥10 (8.3 v 6.4 months, 0.58, 0.45 to 0.75, P<0.001). Adverse events related to treatment occurred in 321 of 327 patients (98%) in the sintilimab-chemotherapy group versus 326 of 332 (98%) patients in the placebo-chemotherapy group. Rates of adverse events related to treatment, grade ≥3, were 60% (196/327) and 55% (181/332) in the sintilimab-chemotherapy and placebo-chemotherapy groups, respectively. CONCLUSIONS: Compared with placebo, sintilimab in combination with cisplatin plus paclitaxel showed significant benefits in overall survival and progression free survival as first line treatment in patients with advanced or metastatic oesophageal squamous cell carcinoma. Similar benefits of sintilimab with cisplatin plus 5-fluorouracil seem promising. TRIAL REGISTRATION: ClinicalTrials.gov NCT03748134.

Vertical distribution and seasonal variation of soil moisture after drip-irrigation affects greenhouse gas emissions and maize production during the growth season.

Providing enough food for the increasing global population is difficult due to water shortages, which can be partially resolved by regulating soil moisture. Soil moisture influences soluble nutrient uptake and microbial activity, which determine crop growth, but also affects greenhouse gas (GHG) emissions. Farming is increasingly contributing to GHG emission, but little is known about the effects of the vertical soil moisture distribution on GHG or maize (Zea mays L.) yield over the growth season. In this study, there were five irrigation treatments: no irrigation (NI), and irrigation of the top (0-30 cm) (TI), middle (30-60 cm) (MI), bottom (60-90 cm) (BI), and all (0-90 cm) (AI) soil layers. The results showed that TI, MI, BI, and AI increased CO2 (25-60%), CH4 (80-270%), and N2O (17-96%) emissions, and the global warming potential (25-63%), while also increasing grain yield (13-119%) and reducing GHG intensity by 12-27%. While higher soil moisture in the shallow soil layer increased grain yield and GHG emissions, GHG intensity was lowest. Subsurface irrigation or control of the "drip irrigation interval" improve grain yield and resource use efficiency with lower environmental costs contributing agricultural sustainable development.

LncRNA MCF2L-AS1 aggravates proliferation, invasion and glycolysis of colorectal cancer cells via the crosstalk with miR-874-3p/FOXM1 signaling axis.

Long non-coding RNAs (lncRNAs) regulate a series of biological processes, and their anomalous expression exerts critical roles in progression of multiple malignancies, including colorectal cancer (CRC). The present study was designed to provide new ideas and perspectives for the role of lncRNA MCF2L-AS1 and disclose the underlying mechanism in CRC. Herein, we observed that MCF2L-AS1 expression was enriched in CRC tissues and cell lines. Additionally, silencing of MCF2L-AS1 dramatically impeded cell proliferation, invasion and migration capacities of CRC, and distinctly attenuated the expression of invasion associated targets MMP-2 and MMP-9. Moreover, depletion of MCF2L-AS1 apparently restricted the glucose consumption and lactate production, and downregulated GLUT1 and LDHA expression. More importantly, we predicted and verified that MCF2L-AS1 acted as a molecular sponge for miR-874-3p and inversely regulated miR-874-3p expression. Interesting, FOXM1 was identified as direct target of miR-874-3p, and positively modulated by MCF2L-AS1 through sponging miR-874-3p. Mechanistically, MCF2L-AS1 accelerated cell proliferation, invasion and glycolysis through competitively binding to miR-874-3p, leading to enhance FOXM1 expression. Collectively, these outcomes highlighted that MCF2L-AS1 acted as a motivator by modulating the miR-874-3p/FOXM1 axis, thereby aggravating tumorigenesis and glycolysis progress of CRC, disclosing that MCF2L-AS1 may serve as a valuable and promising therapeutic strategy for CRC.

MiR-645 promotes invasiveness, metastasis and tumor growth in colorectal cancer by targeting EFNA5.

MicroRNA-645 (miR-645) has been implicated in numerous types of human cancers including colon cancer. However, the effects and mechanisms of action of miR-645 dysregulation on the growth and malignancy of colorectal cancer (CRC) remain unclear. In this study, we demonstrated that miR-645 knockdown significantly diminished CRC cell migration and invasion and repressed epithelial-mesenchymal transition (EMT). Conversely, miR-645 overexpression enhanced CRC cell migration, invasion, and EMT. In vivo assays confirmed that miR-645 knockdown substantially reduced CRC growth and metastasis. Regarding the mechanism, ephrin-A5 (EFNA5) was identified as a direct target gene of miR-645. MiR-645 specifically targeted the 3'-untranslated region of EFNA5 mRNA and hindered its expression. EFNA5 knockdown attenuated the effects of miR-645 knockdown on CRC cell migration and invasion. Additionally, we noted a statistically significant inverse correlation between EFNA5 mRNA and miR-645 levels in tumors from 28 patients with CRC. Hence, miR-645 acts as an oncogenic miRNA that may increase CRC cell migration, invasiveness, and metastasis by targeting EFNA5.

Reduction in seed set upon exposure to high night temperature during flowering in maize.

High temperature reduces crop production; however, little is known about the effects of high night temperature (HNT) on the development of male and female reproductive organs, pollination, kernel formation and grain yield in maize (Zea mays L.). Therefore, a temperature-controlled experiment was carried out using heat-sensitive maize hybrid and including three temperature treatments of 32/22°C (day/night; control), 32/26°C and 32/30°C during 14 consecutive days encompassing the flowering stage. When exposed to 30°C night temperature, grain yield and kernel number reduced by 23.8 and 25.1%, respectively, compared with the control. The decrease in grain yield was mainly because of the lower kernel number rather than change in kernel weight under HNT exposure around flowering. No significant differences in grain yield and kernel number were found between 22 and 26°C night temperatures. HNT had no significant effects on the onset of flowering time and anthesis-silking interval but significantly reduced time period of pollen shedding duration and pollen viability, and increased leaf night respiration. Different from high daytime temperature, HNT had no lasting effects on daytime leaf photosynthesis, biomass production and assimilate transportation. From the perspective of source-flow-sink relationship, the unchanged source and flow capacities during daytime are supposed to alleviate the adverse effects on sink strength caused by HNT compared with daytime heat stress. These new findings commendably filled the knowledge gaps concerning heat stress in maize.

Anoctamin 5 regulates cell proliferation and migration in pancreatic cancer.

Pancreatic cancer (PC) has one of the worst survival rates of all cancers. Anoctamin (ANO) inlcudes a family of Ca2+-activated Cl- channels (CaCCs) that participate in tumorigenesis and progression. However, the exact role of ANO5 in PC has not yet been clarified. Our previous study showed that ANO5 is highly expressed in the epithelial cells of the digestive tract, but there have been no reports of ANO5 expression in normal pancreatic tissue and in PC tissue. In the present study, we investigated the expression of ANO5 in normal pancreatic tissues and PC tissues using immunohistochemistry. The results indicated that ANO5 expression was significantly elevated in PC tissues compared with normal pancreatic tissues. Next, we investigated the expression of ANO5 in pancreatic ductal epithelial cells and PC cells using western blotting and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). The results indicated that ANO5 expression was significantly elevated in PC cells compared with pancreatic ductal epithelial cells. The impact of siRNA-mediated ANO5 knockdown on PC cell proliferation and migration was detected using CCK-8 assays and scratch experiments. The results indicated that ANO5 siRNA reduced the proliferation and migration of PC cells. Collectively, the results suggest that downregulation of ANO5 inhibits the proliferation and migration of PC cells. Therefore, ANO5 is potentially a novel therapeutic target for PC treatment.

Effects of obatoclax combined with gemcitabine on the biological activity of pancreatic cancer cells under hypoxic conditions.

The present study aimed to investigate the effects of obatoclax (OBX) combined with gemcitabine (GEM) treatment on the proliferation, migration, invasion and epithelial­mesenchymal transition (EMT) related proteins of pancreatic cancer cell line BxPC­3 under hypoxic conditions. Protein expression levels of hypoxia­inducible factor 1α (HIF­1α) in BxPC­3 pancreatic cancer cells under normoxic and hypoxic conditions were detected by western blotting. Cells were divided into four groups: Normoxia group, hypoxia group, OBX group and OBX + GEM group. The proliferation activity of BxPC­3 cells was detected by Cell Counting kit­8. The migratory and invasive abilities of BxPC­3 cells were detected by the scratch test and Matrigel assay, respectively. The protein expression levels of vimentin, E­cadherin and p53 in BxPC­3 cells were also detected by western blotting. HIF­1α expression under hypoxic conditions was significantly increased compared with expression under normoxic conditions. Under hypoxic conditions, OBX treatment reduced cell activity, decreased cell migration and invasion, promoted the expression of E­cadherin and p53. In the OBX + GEM group, BxPC­3 cell activity decreased significantly, cell migration and invasion decreased significantly, the expression of vimentin was significantly reduced and the expression of E­cadherin and p53 further increased. In conclusion, the present results demonstrated that under hypoxic conditions, OBX combined with a small dose of GEM may be able to inhibit the growth, migration and invasion of pancreatic cancer cells, possibly via inhibition of EMT process. These results may provide a promising strategy for pancreatic cancer therapy.

ECRG2 enhances the anti-cancer effects of cisplatin in cisplatin-resistant esophageal cancer cells via upregulation of p53 and downregulation of PCNA.

AIM: To explore the anti-tumor effects of esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP) in DDP-resistant esophageal cancer cells (EC9706/DDP). METHODS: A drug-resistant cell model was established, with EC9706/DDP cells being treated with ECRG2 and/or DDP. Cell viability was examined by MTT assay. The rate of cell apoptosis was determined by flow cytometry. The mRNA expression levels of proliferating cell nuclear antigen (PCNA), metallothionein (MT), and p53 were determined by RT-PCR and PCNA, while MT and p53 protein expression levels were determined by western blotting. RESULTS: The anti-proliferative effect of ECRG2 in combination with DDP was superior when compared to ECRG2 or DDP alone. The inhibition rate for the combination reached its peak (51.33%) at 96 h. The early apoptotic rates of the control, ECRG2 alone, DDP alone, and ECRG2 plus DDP groups were 5.71% ± 0.27%, 12.68% ± 0.61%, 14.15% ± 0.87%, and 27.96% ± 0.36%, respectively. Although all treatment groups were significantly different from the control group (P < 0.05), the combination treatment of ECRG2 plus DDP performed significantly better when compared to either ECRG2 or DDP alone (P < 0.05). The combination of ECRG2 and DDP significantly upregulated p53 mRNA and protein levels and downregulated PCNA mRNA and protein levels compared to ECRG2 or DDP alone (P < 0.05). However, no changes were seen in the expression of MT mRNA or protein. CONCLUSION: ECRG2 in combination with DDP can inhibit viability and induce apoptosis in esophageal cancer DDP-resistant cells, possibly via upregulation of p53 expression and downregulation of PCNA expression. These findings suggest that the combination of ECRG2 and DDP may be a promising strategy for the clinical treatment of esophageal cancers that are resistant to DDP.

MiR-185 acts as a tumor suppressor by targeting AKT1 in non-small cell lung cancer cells.

Increasing evidence has shown that microRNAs play critical roles in the initiation and progression of non-small cell lung cancer (NSCLC). miR-185 is deregulated in various cancers, whereas its functional mechanism in NSCLC is still unclear. Here, we confirmed that the expression of miR-185 was significantly down-regulated in NSCLC tissues and cell lines. miR-185 over-expression caused significant suppression of in vitro cell proliferation, migration and invasion, and in vivo tumor growth. We subsequently identified that AKT1 was a target gene of miR-185. Re-expression of AKT1 could partially rescue the inhibitory effects of miR-185 on the capacity of NSCLC cell proliferation and motility. Collectively, we conclude that miR-185 has a critical function by blocking AKT1 in NSCLC cells, and it may be a novel therapeutic agent for miRNA based NSCLC therapy.

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.