-Yuan Wei

Overexpression of E2F1 in human gastric carcinoma is involved in anti-cancer drug resistance.

BackgroundRoutine chemotherapy often cannot achieve good therapeutic effects because of multidrug resistance (MDR). MDR is frequently caused by the elevated expression of the MDR1 gene encoding P-glycoprotein (P-gp). E2F1 is a frequently overexpressed protein in human tumor cells that increases the activity of the MDR1 promoter, resulting in higher P-gp levels. The upregulation of P-gp might contribute to the survival of tumor cells during chemotherapy. E2F1 confers anticancer drug resistance; however, we speculate whether E2F1 affects MDR through other pathways. This study investigated the possible involvement of E2F1 in anticancer drug resistance of gastric carcinoma in vitro and in vivo.MethodsA cisplatin-resistant SGC7901/DDP gastric cancer cell line with stable overexpression of E2F1 was established. Protein expression levels of E2F1, MDR1, MRP, TAp73, GAX, ZEB1, and ZEB2 were detected by western blotting. The influence of overexpression of E2F1 on anticancer drug resistance was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, as well as the rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. We determined the in vivo effects of E2F1-overexpression on tumor size in nude mice, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining.ResultsThe SGC7901/DDP gastric cancer cell line stably overexpressing E2F1 exhibited significantly inhibited sensitivity to cisplatin, doxorubicin, and 5-fluorouracil. Flow cytometry confirmed that the percentage of apoptotic cells decreased after E2F1 upregulation, and that upregulation of E2F1 potentiated S phase arrest of the cell cycle. Furthermore, upregulation of E2F1 significantly decreased intracellular accumulation of doxorubicin. Western blot revealed that E2F1 upregulation suppressed expression of GAX, and increased the expression of MDR1, MRP, ZEB1, TAp73, and ZEB2.ConclusionsOverexpression of E2F1 promotes the development of MDR in gastric carcinoma, suggesting that E2F1 may represent an efficacious target for gastric cancer therapy.

MiR-1284 modulates multidrug resistance of gastric cancer cells by targeting EIF4A1.

Routine chemotherapy as an important treatment mode often can not be effective because of multidrug resistance (MDR). MicroRNA (miRNA) modulates the expression of a great number of genes, including MDR. In this study, the expression of miR-1284 was reduced in gastric cancer (GC) tissue specimens with metastasis and in vincristine-resistant (VCR) GC SGC7901 cells (SGC-7901/VCR) compared to that in the controls. Recombinant lentiviral vectors with miR-1284 led to the overexpression of miR-1284 mRNA and reversed the chemoresistance of SGC7901/VCR cells, promoted cell cycle arrested at the G0/G1 phase, accelerated drug-induced apoptosis, and decreased migration and invasiveness of SGC-7901/VCR. In addition, the overexpression of miR-1284 sensitized tumors to chemotherapy in vivo. Our data provide combined evidence that miR-1284 can heighten the expression of MYC and reduce the expression of JUN, MMP12, and EIF4A1 that was the direct target. In conclusion, miR-1284 can function as a new regulator to reduce GC MDR cells by targeting EIF4A1.

Reversal of Multidrug Resistance in Gastric Cancer Cells by E2F‐1 Downregulation In Vitro and In Vivo

Transcription Factor E2F‐1 plays a critical role in cell cycle regulation and other biological processes in cells. However whether or not it is involved in the multi‐drug resistance (MDR) process of gastric cancer has not been fully elucidated yet. To explore the role of E2F‐1 in the MDR process of gastric cancer in vitro and in vivo, a cisplatin‐resistant gastric cancer cell line with stable downregulation of E2F‐1 was established. E2F‐1 shRNA led to downregulation of endogenous E2F‐1 mRNA and protein. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin, doxorubicin, and fluorouracil. Flow cytometry confirmed that the percentage of apoptotic cells increased after E2F‐1 downregulation. This notion was further supported by the observation that downregulation of E2F‐1 blocked entry into the S‐phase of the cell cycle. Furthermore, downregulation of E2F‐1 significantly increased intracellular accumulation of doxorubicin. In addition, we determined the in vivo effects of E2F‐1 small interfering RNA (shRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling and hematoxylin and eosin staining. In molecular studies, semiquantitative RT‐PCR and western blotting revealed that E2F‐1 downregulation could inhibit expression of MDR1, MRP, Bcl‐2/Bax, c‐Myc, Skp2, Survivin, and Cyclin D1. In conclusion: E2F‐1 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that E2F‐1 may represent a novel target for gastric cancer therapy. J. Cell. Biochem. 115: 34–41, 2014.

Reversal of multidrug resistance in gastric cancer cells by CDX2 downregulation.

AIM To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo. METHODS A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was established. mRNA and protein expression levels of CDX2, survivin, cyclin D1, and c-Myc were detected by western blotting and semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of downregulation of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2 small interfering RNA (siRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining. RESULTS CDX2 siRNA led to downregulation of endogenous CDX2 mRNA (0.31 ± 0.05 vs 1.10 ± 0.51, 0.31 ± 0.05 vs 1.05 ± 0.21, P = 0.003) and protein (0.12 ± 0.08 vs 0.51 ± 0.07, 0.12 ± 0.08 vs 0.55 ± 0.16, P = 2.57 × 10(-4)) expression. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin (0.12 ± 0.05 vs 0.33 ± 0.08, 0.12 ± 0.05 vs 0.39 ± 0.15, P = 0.001), doxorubicin (0.52 ± 0.13 vs 4.11 ± 1.25, 0.52 ± 0.13 vs 4.05 ± 1.44, P = 2.81 × 10(-4)), and 5-fluorouracil (0.82 ± 0.13 vs 2.81 ± 0.51, 0.82 ± 0.13 vs 3.28 ± 1.03, P = 1.71 × 10(-4)). Flow cytometry confirmed that the percentage of apoptotic cells increased after CDX2 downregulation (32.15% ± 2.15% vs 17.63% ± 3.16%, 32.15% ± 2.15% vs 19.3% ± 2.25%, P = 1.73 × 10(-6)). This notion was further supported by the observation that downregulation of CDX2 blocked entry into the S-phase of the cell cycle (31.53% ± 3.78% vs 65.05% ± 7.25%, 31.53% ± 3.78% vs 62.27% ± 5.02%, P = 7.55 × 10(-7)). Furthermore, downregulation of CDX2 significantly increased intracellular accumulation of doxorubicin (0.21 ± 0.06 vs 0.41 ± 0.11, 0.21 ± 0.06 vs 0.40 ± 0.08, P = 0.003). In molecular studies, semiquantitative RT-PCR and western blotting revealed that CDX2 downregulation could inhibit expression of c-Myc, survivin and cyclin D1. CONCLUSION CDX2 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that CDX2 may represent a novel target for gastric cancer therapy.

Prognostic significance of Cdx2 immunohistochemical expression in gastric cancer: a meta-analysis of published literatures

Cdx2 is a homeobox domain-containing transcription factor that is important in the development and differentiation of the intestinal cells, and served as a potential biomarker of tumor progression in early intestinal-type gastric cancer. However, its prognostic value and significance in gastric cancer remain controversial. A meta-analysis based on published studies was performed to obtain an accurate evaluation of the association between the presence of Cdx2-positive in clinical samples and clinical outcome. A total of 13 eligible retrospective cohort studies with 1513 patients were included. Cdx2-positive cases were significantly associated with higher male-to-female ratio (RR=1.27, 95% CI: 1.17–1.38, P<0.00001 fixed-effect), lower (I+II) clinical stage (RR=1.63, 95% CI: 1.42–1.87, P<0.00001 fixed-effect), better histologic differentiation (RR=1.54, 95% CI: 1.34-1.76, P<0.00001 fixed-effect), and lower rate of vascular invasion (RR=1.23, 95% CI: 1.08-1.41, P=0.002 fixed-effect) and lymph node metastasis (RR=1.52, 95% CI: 1.33-1.73, P<0.00001 fixed-effect), as well as higher 5-year survival rate (HR=2.22, 95% CI: 1.78-2.75, P<0.00001 fixed-effect). However, the presence of Cdx2 was not associated with tumor size. In summary, Cdx2 is a prognostic factor in gastric cancer, which acts as a marker of good outcome in patients with gastric cancer. Further clinical studies are needed to confirm the role of Cdx2 in clinical practice.

Role of miR-647 in human gastric cancer suppression

MicroRNAs (miRNAs) regulate various oncogenes concomitantly, resulting in tumor suppression. They regulate proliferation and migration pathways in tumor development, suggesting a potential therapeutic role. In the present study, we found that miR-647 was markedly downregulated in gastric cancer (GC), and was significantly correlated with reduced tumor size and metastasis. In addition, miR-647 was also reduced in GC cell lines. Furthermore, overexpression of miR-647 in the GC cell lines inhibited cell proliferation, promoted cell cycle arrest at the G0/G1 phase and induced cell apoptosis. miR-647 also significantly inhibited tumor growth in vivo. Notably, we found that miR-647 overexpression suppressed the migration and invasion of the cancer cells, particularly liver metastasis in nude mice. miR-647 also reduced the expression levels of genes associated with proliferation and metastasis in tumors, including ANK2, FAK, MMP2, MMP12, CD44 and SNAIL1. Overall, our findings demonstrated that miR-647 exerts powerful antitumorigenic effects in vitro and in vivo, and may represent a promising therapeutic agent against GC.

New insights into the functions and localization of the homeotic gene CDX2 in gastric cancer

Gastric cancer is one of the most frequent cancers, and it ranks the third most common cancer in China. The most recently caudal-related homeobox transcription factor 2 (CDX2) is expressed in a large number of human gastrointestinal cancers. In addition, gastric epithelial cell mutations in CDX2 result in tumor promotion, which is characterized by cellular drug resistance and a high proclivity for developing cancer. A series of publications over the past years suggests a mechanism by which CDX2 overexpression results in multidrug resistance. CDX2 appears to forward control regenerating IV and the multidrug resistance 1 expression signaling pathway for regulation of cell drug resistance.

Regulation of drug resistance and metastasis of gastric cancer cells via the microRNA647-ANK2 axis

Due to a lack of effective methods for early diagnosis, the majority of patients with gastric cancer (GC) are diagnosed during the late stages of the disease, which are often accompanied by metastasis. For these patients, despite being considered an important therapeutic modality in the treatment of cancer, chemotherapy is usually not effective due to multidrug resistance (MDR). The expression levels of MDR/metastasis-associated genes are regulated by numerous microRNAs (miRNAs/miRs). The expression of miR-647 in GC tissues and SGC7901/VCR cell line (drug resistance to vincristine) was detected by qRT-PCR. The effect of overexpression of miR-647 on drug resistance was evaluated by measuring the half maximal inhibitory concentration (IC50) value of SGC-7901/VCR to vincristine and tumor growth in vivo. Moreover, drug-induced cell apoptosis and cell cycle were evaluated by flow cytometry, as well as the ability of cell migration and invasiveness detected by wound healing and transwell assay. Furthermore, underlying targets of miR-647 were predicted by TargetScan and MicroRNA; meanwhile, the expression of ANK2, FAK, MMP2, MMP12,CD44,SNAIL1 were observed by qRT-PCR and western blot analysis. The present study established that the expression levels of miR-647 were downregulated in GC tissues from patients with metastasis and in the vincristine-resistant SGC7901 (SGC-7901/VCR) GC cell line. The IC50 value for vincristine was significantly decreased, whereas the proportion of cells in G0/G1 phase and the drug-induced apoptotic rate were significantly increased following upregulation of miR-647. Furthermore, the results demonstrated that miR-647 overexpression led to decreased migration and invasion of SGC-7901/VCR cells. Overexpression of miR-647 was also demonstrated to sensitize tumors to chemotherapy in vivo. In addition, miR-647 overexpression was able to reduce the expression levels of ankyrin-B, focal adhesion kinase, matrix metalloproteinase (MMP)2, MMP12, cluster of differentiation 44 and snail family transcriptional repressor 1. In conclusion, these findings demonstrated that miR-647 may function as a novel target to ameliorate drug resistance and metastasis of GC cells.

E2F-1 overexpression inhibits human gastric cancer MGC-803 cell growth in vivo

AIM To evaluate the influence of E2F-1 on the growth of human gastric cancer (GC) cells in vivo and the mechanism involved. METHODS E2F-1 recombinant lentiviral vectors were injected into xenograft tumors of MGC-803 cells in nude mice, and then tumor growth was investigated. Overexpression of transcription factor E2F-1 was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis. Apoptosis rates were determined using a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Expression levels of certain cell cycle regulators and apoptosis-related proteins, such as Bax, survivin, Bcl-2, cyclin D1, S-phase kinase-associated protein 2, and c-Myc were examined by Western blotting and RT-PCR. RESULTS Xenograft tumors of MGC-803 cells in nude mice injected with E2F-1 recombinant lentiviral vectors stably overexpressed the E2F-1 gene as measured by semi-quantitative RT-PCR (relative mRNA expression: 0.10 ± 0.02 vs 0.05 ± 0.02 for control vector and 0.06 ± 0.03 for no infection; both P < 0.01) and Western blotting (relative protein expression: 1.90 ± 0.05 vs 1.10 ± 0.03 in control vector infected and 1.11 ± 0.02 for no infection; both P < 0.01). The growth-curve of tumor volumes revealed that infection with E2F-1 recombinant lentiviral vectors significantly inhibited the growth of human GC xenografts (2.81 ± 1.02 vs 6.18 ± 1.15 in control vector infected and 5.87 ± 1.23 with no infection; both P < 0.05) at 15 d after treatment. TUNEL analysis demonstrated that E2F-1 overexpression promoted tumor cell apoptosis (18.6% ± 2.3% vs 6.7% ± 1.2% in control vector infected 6.3% ± 1.2% for no infection; both P < 0.05). Furthermore, lentiviral vector-mediated E2F-1 overexpression increased the expression of Bax and suppressed survivin, Bcl-2, cyclin D1, Skp2, and c-Myc expression in tumor tissue. CONCLUSION E2F-1 inhibits growth of GC cells via regulating multiple signaling pathways, and may play an important role in targeted therapy for GC.

Overexpression of caudal type homeobox transcription factor 2 inhibits the growth of the MGC‑803 human gastric cancer cell line in vivo

Caudal type homeobox transcription factor 2 (CDX2) is important in intestinal cell fate specification and multiple lines of evidence have substantiated that CDX2 is important in carcinogenesis of the digestive tract. The CDX2 regulatory network is intricate and remains to be fully elucidated in gastric cancer. The aim of the present study was to examine the effects of CDX2 on the growth of the MGC-803 human gastric cancer cell line in vivo, and to elucidate the mechanism involved. The effects of the overexpression of CDX2 in xenograft tumors of MGC-803 cells was investigated in nude mice through the injection of CDX2 recombinant lentiviral vectors. The tumor size was measured using vernier callipers. The expression levels of CDX2, survivin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1, s-phase kinase-associated protein 2 (Skp2) and c-Myc in the tumor cells were analyzed by western blotting and semi-quantitative reverse transcription polymerase chain reaction. The apoptotic rates were determined using a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The overexpression of CDX2 was observed in the group subjected to the injection of CDX2 recombinant lentiviral vectors. CDX2 had an inhibitory effect on the MGC-803 human gastric cancer cell line and promoted tumor cell apoptosis in vivo. Furthermore, the overexpression of CDX2 upregulated the expression of Bax and downregulated the expression levels of survivin, Bcl-2, cyclin D1, Skp2 and c-Myc in the tumor tissues. These results indicated that CDX2 may serve as a tumor suppressor in gastric cancer, and inhibits gastric cancer cell growth by suppressing the nuclear factor-κB signaling pathway.