Proteomics and liquid biopsy characterization of human EMT-related metastasis in colorectal cancer.

Tumor cells undergo epithelial-mesenchymal transition (EMT), however, there is a room of disagreement in role of EMT heterogeneity to colorectal cancer metastasis (mCRC) evolution. To uncover new EMT-related metastasis proteins and pathways, we addressed the EMT status in colorectal cancer liver metastasis patient-derived CTCs to identify proteins that promote their distant metastasis. And then, we performed a comparative proteomic analysis in matched pairs of primary tumor tissues, adjacent mucosa tissues and liver metastatic tissues. By integrative analysis we show that, unstable Epithelial/Mesenchymal (E/M)-type CTCs had the strongest liver metastases formation ability and the proportion of E/M-type CTCs correlated with distant metastases. Using an optimized proteomic workflow including data independent acquisition (DIA) and parallel reaction monitoring (PRM), we identified novel EMT-related protein cluster (GNG2, COL6A1, COL6A2, DCN, COL6A3, LAMB2, TNXB, CAVIN1) and well-described (ERBB2) core protein level changes in EMT-related metastasis progression, and the proteomic data indicate ERBB2, COL6A1 and CAVIN1 are promising EMT-related metastatic biomarker candidates. This study contributes to our understanding of the role that EMT plays in CRC metastasis and identifies heterogeneous EMT phenotypes as a key piece for tumor progression and prognosis. We further propose that therapies targeting this aggressive subset (E/M-type) of CTCs and related protein may be worthy of exploration as potential suppressors of metastatic evolution.

MTUS1 is a promising diagnostic and prognostic biomarker for colorectal cancer.

BACKGROUND: The morbidity and mortality of colorectal cancer (CRC) remain high, posing a serious threat to human life and health. The early diagnosis and prognostic evaluation of CRC are two major challenges in clinical practice. MTUS1 is considered a tumour suppressor and can play an important role in inhibiting cell proliferation, migration, and tumour growth. Moreover, the expression of MTUS1 is decreased in different human cancers, including CRC. However, the biological functions and molecular mechanisms of MTUS1 in CRC remain unclear. METHODS: In the present study, data from The Cancer Genome Atlas (TCGA) database were analysed using R statistical software (version 3.6.3.) to evaluate the expression of MTUS1 in tumour tissues and adjacent normal tissues using public databases such as the TIMER and Oncomine databases. Then, 38 clinical samples were collected, and qPCR was performed to verify MTUS1 expression. We also investigated the relationship between MTUS1 expression and clinicopathological characteristics and elucidated the diagnostic and prognostic value of MTUS1 in CRC. In addition, the correlation between MTUS1 expression and immune infiltration levels was identified using the TIMER and GEPIA databases. Furthermore, we constructed and analysed a PPI network and coexpression modules of MTUS1 to explore its molecular functions and mechanisms. RESULTS: CRC tissues exhibited lower levels of MTUS1 than normal tissues. The logistic regression analysis indicated that the expression of MTUS1 was associated with N stage, TNM stage, and neoplasm type. Moreover, CRC patients with low MTUS1 expression had poor overall survival (OS). Multivariate analysis revealed that the downregulation of MTUS1 was an independent prognostic factor and was correlated with poor OS in CRC patients. MTUS1 expression had good diagnostic value based on ROC analysis. Furthermore, we identified a group of potential MTUS1-interacting proteins and coexpressed genes. GO and KEGG enrichment analyses showed that MTUS1 was involved in multiple cancer-related signalling pathways. Moreover, the expression of MTUS1 was significantly related to the infiltration levels of multiple cells. Finally, MTUS1 expression was strongly correlated with various immune marker sets. CONCLUSIONS: Our results indicated that MTUS1 is a promising biomarker for predicting the diagnosis and prognosis of CRC patients. MTUS1 can also become a new molecular target for tumour immunotherapy.

OX40 as a novel target for the reversal of immune escape in colorectal cancer.

First-generation immunological checkpoint inhibitors, such as CTLA-4, PD-L1 and PD-1 exhibit significant advantages over conventional cytotoxic drugs, such as oxaliplatin and 5-FU, for the treatment of colorectal cancer. However, these inhibitors are not ideal due to their low objective response rate and the vulnerability of these treatment methods when faced with emerging drug resistant cancers. This study summarizes the immunological characteristics of colorectal cancer treatment, and analyzes the ways in which OX40 may improve the efficacy of these treatments. Activation of the OX40 signaling pathway can enhance the activity of CD4+/CD8+ T cells and inhibit the function of Treg. Simultaneously, OX40 can directly inhibit the expression of Foxp3, affect the inhibitory function of Treg, and inhibit the immunosuppressive factors in the tumor microenvironment so as to reverse immune escape and reverse drug resistance. Therefore, OX40 is an important target for treating colorectal cancer in "cold tumors" with less immunogenicity.

Anlotinib suppresses metastasis and multidrug resistance via dual blockade of MET/ABCB1 in colorectal carcinoma cells.

Anlotinib, a highly selective multi-targeted tyrosine kinase inhibitor (TKI) has therapeutic effects on non-small-cell lung cancer (NSCLC). In this study, the anti-tumor activity and molecular mechanism of anlotinib in metastatic colorectal cancer (mCRC) was explored. The anti-angiogenesis, anti-metastasis, anti-proliferative, and anti-multidrug resistance efficacy of anlotinib were analyzed by using in vitro and in vivo models of human CRC cells. The results indicated that anlotinib boosted chemo-sensitivity of CRC cells, and restrained its proliferation. Besides the suppression of the MET signaling pathway, anlotinib also inhibited invasion and migration of CRC cells. Furthermore, anlotinib prevented VEGF-induced angiogenesis, N-cadherin (CDH2)-induced cell migration, and reversed ATP-binding cassette subfamily B member 1 (ABCB1) -mediated CRC multidrug resistance in CRC. The CRC liver metastasis and subcutaneously implanted xenograft model testified that anlotinib could inhibit proliferation and liver metastasis in CRC cells. Such an observation suggested that a combination of anlotinib with anti-cancer drugs could attenuate angiogenesis, metastasis, proliferative, and multidrug resistance, which constitutes a novel treatment strategy for CRC patients with metastasis.

Targeting snoRNAs as an emerging method of therapeutic development for cancer.

The relevance of the dysregulation of snoRNAs in human cancer has been widely investigated and has challenged the view that snoRNAs merely function as house-keeping genes for the posttranscriptional modification of rRNAs. Accumulating evidence has shown the intimate connection between snoRNAs and proliferation, apoptosis, invasion and migration of tumor cells via manual intervention patterns of snoRNA expression. In this review, we focused on how snoRNAs are dysregulated and its regulation of the formation and development of cancer. We summarized the non-classical functions of snoRNAs in the context of their regulations of the signaling pathways involving PI3K-AKT and K-Ras and p53-dependant manner. Under these novel functions and characteristics, snoRNAs can act as potential and feasible biomarkers for diagnosis. Simultaneously, these promising therapeutic strategies should be considered to counteract the perturbations of snoRNAs.

Prolonging Gastrointestinal-Stromal-Tumor-free life, an optimal suggestion of imatinib intervention ahead of operation.

Background: Imatinib has been regarded as the first successful synthetic small molecule targeting at blocking tyrosine kinase. Its high efficacy stabilized disease in above 80% of chronic myeloid leukemia (CML) patients over 10 years survival. Due to the similar canceration of gastrointestinal stromal tumor (GIST) as to CML, imatinib has been approved to be used as first-line treatment. Study design: Our retrospective study was proposed to enroll 191 GIST patients with larger tumor size (≥8 cm) who preoperative accepted imatinib from those with direct operation. Analysis included demographics, cancer specific survival and relationship of their risk factors. Results: Male patients and gastrointestinal (GI) tract location took higher proportion in total cases, detection of KIT mutant took 89.7% among all traceable genetic testing. Patients with preoperative imatinib can achieve higher cancer specific survival (CSS) after both in 1 year and 3 years duration than their counterpart. Tumor size above its threshold of 8 cm would be a hazardous factor for poor prognosis. Conclusion: In conclusion, as for regressing tumor progression and creating operative chance, preoperative imatinib should be considered for the patients with high risk, although the precise duration of this intervention needs further validation.

Central Role of CEMIP in Tumorigenesis and Its Potential as Therapeutic Target.

CEMIP (KIAA1199) was identified as migratory indicator protein which had been crudely studied in the last decade. Firstly its mutation site was reported to cause hearing loss due to the folding change of protein structure, meanwhile the over-expression of CEMIP referred to dreadful invasion and uncontrolled proliferation of tumor with distant metastasis, dedifferentiation, and limited survival opportunity of patients. Especially, over-expressed CEMIP also protected malignant tumor from strict microenvironment in hypoxia, low glucose and cracked barrier, leading to enhanced adaptability of tumor by stimulating the Wnt, EGFR, FGFR pathway. Here, we intend to elaborate the clinical function and dysregulation of CEMIP under the tumorous circumstance since CEMIP plays an important role in cytokine pathway and its over-expression in tumors provide a novel target for individual therapy. Targeting CEMIP would thereby dysregulate the cytokine pathway which would in turn, decide the growth and death of the vicious tumour cells.

E2F-1 promotes DAPK2-induced anti-tumor immunity of gastric cancer cells by targeting miR-34a.

Activation of the transcription factor E2F-1 gene is a negative event in dendritic cell (DC) maturation process. Down-regulation of E2F1 causes immaturity of DC thereby stopping antigen production which in turn leads to inhibition of immune responses. E2F-1-free stimulates the NF-kB signaling pathway, leading to activation of monocytes and several other transcription factor genes. In the study, we report that down-regulation of E2F-1 in DCs promote anti-tumor immune response in gastric cancer (GC) cells through a novel mechanism. DCs were isolated from peripheral blood mononuclear cells. E2F-1 small interfering RNA (E2F-1-shRNA) induced down-regulation of E2F-1 mRNA and protein expression in DCs. Furthermore, we identified the E2F-1-shRNA targeted the CD80, CD83, CD86, and MHC II molecules, promoted their expression, and induced T lymphocytes proliferation activity and up-regulation of IFN-I³ production and GC cell killing effect, which significantly correlated with the cytotoxic T lymphocytes activated by E2F-1-shRNA DCs. The higher expression of miR-34a was found which was significantly correlated with the DC enhancing anti-tumor immunity against gastric cancer cell, and miR-34a potently targeted DAPK2 and Sp1, both of which were involved in the deactivation of E2F-1. Moreover, in E2F-1-DC-down-regulation in mice, GC transplantation tumors displayed down-regulation of Sp1, DAPK2, Caspase3, and Caspase7 and progressed to anti-tumor immunity. Collectively, our data uncover an E2F-1-mediated mechanism for the control of DC anti-tumor immunity via miR-34a-dependent down-regulation of E2F-1 expression and suggest its contribution to GC immunotherapy.

miR-135a promotes gastric cancer progression and resistance to oxaliplatin.

Resistance to oxaliplatin (OXA)-based chemotherapy regimens continues to be a major cause of gastric cancer (GC) recurrence and metastasis. We analyzed GC samples and matched non-tumorous control stomach tissues from 280 patients and found that miR-135a was overexpressed in GC samples relative to control tissues. Tumors with high miR-135a expression were more likely to have aggressive characteristics (high levels of carcino-embryonic antigen, vascular invasion, lymphatic metastasis, and poor differentiation) than those with low levels. Patients with greater tumoral expression of miR-135a had shorter overall survival times and times to disease recurrence. Furthermore, miR-135a, which promotes the proliferation and invasion of OXA-resistant GC cells, inhibited E2F transcription factor 1 (E2F1)-induced apoptosis by downregulating E2F1 and Death-associated protein kinase 2 (DAPK2) expression. Our results indicate that higher levels of miR-135a in GC are associated with shorter survival times and reduced times to disease recurrence. The mechanism whereby miR-135a promotes GC pathogenesis appears to be the suppression of E2F1 expression and Sp1/DAPK2 pathway signaling.

MTRR silencing inhibits growth and cisplatin resistance of ovarian carcinoma via inducing apoptosis and reducing autophagy.

Methionine synthase reductase (MTRR) is involved in the DNA synthesis and production of S-adenosylmethionine (SAM) and plays an important role in the carcinogenesis. However, the role of MTRR in the resistance of ovarian cancer (OC) to chemotherapy has yet to be elucidated. In order to investigate the clinical significance of MTRR in OC, MTRR expression was reduced by using the RNA interference technique, and therefore, and the tumor growth and cisplatin-resistance were evaluated in vitro and in vivo. Results showed MTRR expression increased orderly from normal tissues, benign ovarian tumor to OC tissue. MTRR over-expression in OC tissue was correlated with pathologic type (P=0.005), grade (P=0.037), FIGO stage (P=0.001), organ metastasis (P=0.009) and platinum resistance (P=0.038). MTRR silencing inhibited cell proliferation, cisplatin resistance and autophagy, and induced apoptosis of OC cells. In addition, MTRR silencing also affected the caspase expression as well as mTOR signaling pathway. Further, the tumor volume in MTRR-suppressed SKOV3/DDP mice treated with cisplatin significantly decreased when compared with controls (P<0.05). In summary, MTRR expression, which is increased in human OC, is related to the differentiation and cisplatin resistance of OC cells. MTRR silencing inhibits cell growth and cisplatin resistance by regulating caspase expression and mTOR signaling pathway in OC cells. It is suggested that MTRR may be a potential target for the therapy of OC.

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 CDX2 in gastric cancer cells promotes the development of multidrug resistance.

Modulator of multidrug resistance (MDR) gene is a direct transcriptional target of CDX2. However, we still speculate whether CDX2 affects MDR through other ways. In this study, a cisplatin-resistant (SGC7901/DDP) and a 5-fluoro-2, 4(1h,3h)pyrimidinedione-resistant (BGC823/5-FU) gastric cancer cell line with stable overexpression of CDX2 were established. The influence of overexpression of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP and BGC823/5-FU 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-overexpression lentiviral vector (LV-CDX2-GFP) 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 showed that LV-CDX2-GFP led to up-regulation of CDX2 mRNA and protein expression. It significantly inhibited the sensitivity of SGC7901/DDP and BGC823/5-FU cells to cisplatin, doxorubicin, and 5-fluorouracil. Flow cytometry confirmed that the percentage of apoptotic cells decreased after CDX2 up-regulation. This notion was further supported by the observation that up-regulation of CDX2 blocked entry into the M-phase of the cell cycle. Furthermore, up-regulation of CDX2 significantly decreased intracellular accumulation of doxorubicin. In molecular studies, quantitative reverse-transcriptase real-time polymerase chain reaction and western blotting revealed that CDX2 up-regulation could suppress expression of Caspase-3, Caspase-9 and PTEN, and increased the expression of MDR1, MRP, mTOR, HIF-1α.

Consumptive hypothyroidism due to a gastrointestinal stromal tumor expressing type 3 iodothyronine deiodinase.

CONTEXT: Gastrointestinal stromal tumors (GISTs) are responsive to sunitinib (the tyrosine kinase inhibitor), this agent is widely used in prevention relapse of GISTs and neo-adjuvant chemotherapy in GIST patients without operation opportunity. The use of these agents has both advantages and disadvantages. On the one hand, it can improve the outcome for patient. On the other hand, it may lead to consumptive hypothyroidism, a rare syndrome caused by increased catabolism of T4 and T3 by increased type 3 iodothyronine deiodinase (D3) activity. D3 is the major physiologic inactivator of thyroid hormone, this selenoenzyme catalyzes the inner-ring deiodination of T(4) to reverse T(3) and T(3) to 3, 3;-diiodothyronine, both of which are biologically inactive [1]. Increased monitoring and supernormal thyroid hormone supplementation are required for affected patient. OBJECTIVE: The aim of the study was to report the first case of consumptive hypothyroidism in an athyreotic patient after surgical resection of gastrointestinal stromal tumor. DESIGN, SETTING, AND PATIENT: A 60-year-old athyreotic male was presented and he was euthyroid when receiving a stable therapeutic dose of thyroid hormone which was used to treat consumptive hypothyroidism resulting from the side effects of sunitinib, which is used for treatment of neo-adjuvant chemotherapy in gastrointestinal stromal tumor. With a discovery of large D3-expressing gastrointestinal stromal tumor, this patient suffered from marked Hyperthyrotropinemia, which instantly worsened after surgical resection of the gastrointestinal stromal tumor and then continued for 12 weeks after the surgical resection, in spite of further increases in levothyroxine therapy. The patient also had low serum T3 and elevated serum reverse T3 (rT3). INTERVENTION: The patient's consumptive hypothyroidism caused by marked overexpression of the thyroid hormone-inactivating D3 within the gastrointestinal stromal tumor and adjacent normal gastrointestinal tissue. MAIN OUTCOME MEASURES AND RESULTS: D3 immunostaining of the patient's gastrointestinal stromal tumor was positive, with no significant immunoreactivity in adjacent normal gastrointestinal tissue. The expression levels of CD34, CD117, and DOG1 in peri-tumor tissue samples was lower than that in tumor tissue. The mRNA expression level of KIT exon17 in peri-tumor tissue was higher than that in tumor tissue. CONCLUSIONS: This is the first case report of consumptive hypothyroidism in an adult after surgical partial resection of the gastrointestinal stromal tumor. This case demonstrates that hyperthyrotropinemia may worsen after surgical resection of the gastrointestinal stromal tumor.

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

BACKGROUND: Routine 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: Overexpression of E2F1 promotes the development of MDR in gastric carcinoma, suggesting that E2F1 may represent an efficacious target for gastric cancer therapy.

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.

Reversal of multidrug resistance in gastric cancer cells by E2F-1 downregulation in vitro and in vivo.

UNLABELLED: 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.

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.

[Effects of E2F-1 overexpression on apoptosis of gastric cancer cells and expressions of apoptosis-related genes].

BACKGROUND AND OBJECTIVE: E2F transcription factor 1 (E2F-1) is an important transcription factor in cell cycle. This study was to investigate the effects of E2F-1 overexpression on apoptosis of gastric cancer MGC-803 cells and expressions of the downstream genes. METHODS: The apoptotic rates were measured by flow cytometry in MGC-803/E2F-1 cells, MGC-803/EV cells or untransfected MGC-803 cells. The total RNA was extracted from MGC-803/E2F-1 cells or MGC-803 cells, and cDNA was obtained by RT-PCR. Fluorescent (fluorescence exchange clip) probes marked by Cy5 and Cy3 were hybridized with gene chips containing 21522 human genes. Subsequently, the two signal images were scanned by Lux Scan 10K/A dual pathways laser scanner and analyzed by LuxScan3.0 image analysis software. RT-PCR was used to verify the target genes. RESULTS: The apoptotic rate of MGC-803/E2F-1 cells [(8.40+/-0.91)%] was higher than that of MGC-803/EV [(4.53+/-0.61)%] and MGC-803 cells [(4.97+/-0.47)%]. Fifteen differentially expressed apoptosis-related genes were detected, 4 of which were up-expressed and 11 were down-expressed genes, and the same results were verified by RT-PCR. CONCLUSION: Overexpression of E2F-1 accelerates apoptosis of gastric carcinoma MGC-803 cells, which may be related to the 15 differentially expressed genes.