Rab11-FIP2 suppressed tumor growth via regulation of PGK1 ubiquitination in non-small cell lung cancer.

Mounting evidence has shown that the Rab11-FIP2 has critical roles in cancer cell growth. However, the clinical significance of Rab11-FIP2 in Non-small cell lung cancer (NSCLC) remains to be fully elucidated. In this study, we investigated the expression of Rab11-FIP2 using immunohistochemistry in 150 patients with NSCLC. We found that its expression level in NSCLC was much lower than that in the corresponding adjacent normal tissues. The DNA methylation data revealed that Rab11-FIP2 were significantly hypermethylated in NSCLC. The methylation level in the gene body was negatively correlated with the expression level of Rab11-FIP2 in NSCLC. Furthermore, enforced expression of Rab11-FIP2 dramatically reduced cancer cell proliferation and tumorigenesis, indicating a tumor suppressor role of PGK1 in NSCLC progression. Mechanistic investigations showed that Rab11-FIP2 interacted with the glycolytic kinase PGK1 and promoted its ubiquitination in NSCLC cells, leading to inactivation of the oncogenic AKT/mTOR signaling pathway. Overall, our data indicate that reduced expression of Rab11-FIP2 by DNA hypermethylation plays an important role in NSCLC tumor growth.

Overexpression of Rab11-FIP2 in colorectal cancer cells promotes tumor migration and angiogenesis through increasing secretion of PAI-1.

BACKGROUND: Rab11 family-interacting protein 2 (Rab11-FIP2) can interact with MYO5B and plays an important role in regulating plasma membrane recycling. However, little is known about the clinical significance of DUSP2 in colorectal cancer (CRC). METHODS: In this study, we investigated Rab11-FIP2 expression by immunohistochemistry in 125 patients with colorectal cancer. Conditioned media containing all secreted factors was harvested. Chemokine secretion and expression were analyzed by Chemi-array. RESULTS: We found that the expression level of Rab11-FIP2 was significantly increased in colorectal cancer tissues and high expression of Rab11-FIP2 was closely correlated with nodal metastasis in colorectal cancer patients. Rab11-FIP2 overexpression promoted colorectal cancer metastasis in vitro and in vivo. Finally, we demonstrated that Rab11-FIP2 overexpression may contribute to increased secretion of PAI-1 in human colorectal cancer cells. CONCLUSIONS: Our findings reveal a novel mechanism underlying the role of Rab11-FIP2 in colorectal cancer dissemination, suggesting that targeting Rab11-FIP2 might be a promising therapeutic strategy for CRC.

Differential expression of DUSP2 in left- and right-sided colon cancer is associated with poor prognosis in colorectal cancer.

Dual-specificity phosphatase-2 (DUSP2), a negative regulator of extracellular-regulated kinase activity, has been identified as an important kinase with emerging roles in cancer. However, the clinical significance of DUSP2 in colorectal cancer (CRC) remains to be fully elucidated. In the present study, the expression of DUSP2 was investigated using immunohistochemistry in 96 patients with CRC. Cell viability was estimated using a cell counting kit-8 assay, and cell apoptosis by flow cytometry. The relationship between DUSP2 expression and patient characteristics, including overall survival, were studied retrospectively in these patients. It was found that DUSP2 was differentially expressed between left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC). It was also found that decreased expression of DUSP2 was correlated with significantly shorter overall survival (P=0.001) and short distant-metastasis-free survival (P=0.002). In univariate comparisons, the decreased expression of DUSP2 was found to be an independent risk factor for poor survival rate (HR 3.55, CI 1.092-9.896; P=0.002). It was also found that the enforced overexpression of DUSP2 sensitized CRC cells to cetuximab. In conclusion, the findings demonstrated that DUSP2 was differentially expressed between RSCC and LSCC, and that the overexpression of DUSP2 increased the inhibitory effect of cetuximab in CRC, suggesting that DUSP2 may be a novel biomarker and therapeutic target in CRC therapy.

Application of Different Imaging Methods in the Early Diagnosis of Primary Hepatic Carcinoma.

Primary hepatic carcinoma (PHC) is the one of the most common tumors and the common cause of cancer death in the world. Detecting PHC in its early stage by imaging methods may greatly increase survival rates of patients. Ultrasound, computed tomography, magnetic resonance imaging, and positron emission tomography/computed tomography are common imaging methods in the diagnosis of PHC. In this paper, the application of different imaging methods in diagnosing the primary hepatic carcinoma will be discussed.

[Arsenic trioxide restores ERα expression in ERα-negative human breast cancer cells and its treatment efficacy in combination with tamoxifen in xenografts in nude mice].

OBJECTIVE: To study the demethylation effect of arsenic trioxide (As2O3) on ERα-negative human breast cancer MDA-MB-435s cells and its possible mechanisms, and to observe its treatment efficacy in combination with tamoxifen (TAM) after ERα re-expression. METHODS: MTT assay was used to examine the inhibitory effect of As2O3 treatment alone or in combination with TAM on cell proliferation. A nude mouse xenograft model was used to further examine the treatment efficacy in vivo. MSP was used to detect the methylation status of ERα gene after treated with As2O3 in MDA-MB-435s cells and the transplanted tumor tissues. RT-PCR was used to detect the mRNA expression of DNMT1 and Erα. Western bolt was used to detect the DNMT1 and ERα protein expression. The diameter of xenograft tumors was measured weekly, and the tumor growth curve was drawn. RESULTS: The level of proliferation of the MDA-MB-435s cells was significantly suppressed after treatment with different concentration of As2O3 alone or As2O3 combined with TAM, and the 4 µmol/L As2O3 + TAM treatment for 72 h showed the highest inhibition rate (62.6%). 1, 2, 4 µmol/L As2O3 had demethylation effect on MDA-MB-435s cells, and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERα mRNA and protein re-expression. The unmethylation specific bands of ERα gene were enhanced after treated by As2O3 alone or As2O3 combined with TAM in the xenograft tumors. The expression of DNMT1 mRNA and protein was inhibited, and accompanied by ERα mRNA and protein re-expression. An significant decrease of volume and weight of the xenograft tumors in the As2O3 treated alone or combined with TAM groups was observed compared with those of the normal saline group or TAM alone group (P < 0.05), and the 4 mg/kg As2O3 + TAM group had the highest inhibition rate of tumor weight (79.5%) and volume (76.4%). CONCLUSIONS: ERα can be re-expressed in ERα-negative breast cancer MDA-MB-435s cells after treated with As2O3 by inhibiting the DNMT1 activity. MDA-MB-435s cells are re-sensitized to endocrine therapy after ERα re-expression. As2O3 combined with TAM may provide a new therapeutic approach for patients with ERα-negative breast cancer in the clinic.

Arsenic trioxide re-sensitizes ERα-negative breast cancer cells to endocrine therapy by restoring ERα expression in vitro and in vivo.

Approximately one-third of breast cancers lack estrogen receptor α (ERα) because of the hypermethylation of the CpG island in the receptor's promoter. These tumors are associated with poorer histological differentiation, a higher growth fraction, are rarely responsive to endocrine therapy and have a worse clinical outcome. Thus, re-expression of ERα in ERα-negative breast cancers may restore the sensitivity of antiestrogen therapy. The ERα-negative breast cancer cell line MDA-MB-435s was treated with different concentrations of arsenic trioxide (As2O3). MS-PCR was used to detect the change in the methylation status of ERα. RT-PCR, immunohistochemistry and Western blot analyses were used to detect changes in the mRNA and protein expression of DNA methyl-transferase-1 (DNMT1) and ERα. Cell proliferation was examined using the MTT assay. A xenograft model in nude mice was used to further examine the results we observed in vitro. The ERα gene was demethylated after As2O3 treatment of MDA-MB-435s cells. RT-PCR, immunohistochemistry and Western blot analyses revealed that DNMT1 expression was inhibited and ERα was re-expressed in a concentration-dependent manner after As2O3 treatment. The MTT assay showed that cell proliferation was significantly suppressed after exposure to different concentrations of As2O3. Addition of tamoxifen (TAM) further suppressed levels of cell proliferation. In vivo, the xenograft tumor volumes of As2O3-treated mice were smaller than those observed in untreated and TAM-treated mice. Treatment with a combination of As2O3+TAM resulted in further suppression. As2O3 can act as a demethylation agent to restore ERα expression in ERα-negative breast cancer cells and re-sensitize these cells to endocrine therapy in vitro and in vivo.

[Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid].

OBJECTIVE: To investigate the mechanism of apoptosis of EC9706 tumor-bearing nude mice induced by all-trans retinoic acid (ATRA). METHODS: Human esophageal carcinoma cell line EC9706 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were divided into the following groups: ATRA group, fluorouracil group, the two-drugs combination group, and with an equal volume fraction of solvent as the control group. The nude mice were sacrificed after 10 days of medication. TUNEL staining was used to detect cell apoptosis. RT-PCR was used to detect the expression level of mRNA and immunohistochemistry was used to detect the expression level of protein of caspase-3 and survivin, the apoptosis-related genes in the tumor tissue. RESULTS: The apoptosis rates of the ATRA group, 5-Fu group and ATRA + 5-Fu group were 44.3%, 39.7% and 91.0%, respectively. There was a significant difference in comparison with the control group (0.7%), and the ATRA group had no significant difference compared with that of the fluorouracil group (P > 0.05), but the apoptosis rate of the two-drugs combination group was significantly higher than that in the two single-drug groups (P < 0.05). The average gray value of caspase-3 protein expressed in the control group was 46.12 ± 0.33 and the relative expression of caspase-3 mRNA was 0.14 ± 0.03, both were significantly lower than that in the ATRA group, 5-Fu group and the two-drugs combination group (P < 0.05). The average gray value of survivin protein expressed in the control group was 96.07 ± 0.13 and the relative expression of survivin mRNA was 0.84 ± 0.04, both were significantly higher than those of other groups (P < 0.05). The ATRA group had no significant difference compared with the fluorouracil group (P > 0.05), but the two-drugs combination group was significantly different compared with the single-drug groups (P < 0.05). CONCLUSION: Apoptosis in the EC9706 tumor cells in nude mice can be induced by ATRA. The mechanism may be related with down-regulation of the level of survivin gene expression and up-regulation of the level of caspase-3 gene expression.

[Nedaplatin combined with tegafur in the treatment for advanced esophageal cancer].

OBJECTIVE: To investigate the efficacy and toxicity of nedaplatin combined with tegafur in the treatment for patients with advanced esophageal cancer. METHODS: Among the 65 patients with advanced esophageal cancer, 27 had no history of prior chemotherapy and the other 38 had ever received postoperative adjuvant chemotherapy before. The median age of those cases was 58.0 years. Nedaplatin was given daily by intravenous infusion at a dose of 20 mg/m(2) for 2 hours and tegafur at a dose of 500 mg/m(2) for 8 hours on D1 approximately D5, every 21 days as a cycle. RESULTS: 193 cycles of chemotherapy were accomplished in the 65 patients, and 63 patients were evaluable for response evaluation. Of 27 patients with no prior history of chemotherapy, 6 achieved complete response and 16 partial response, with a response rate (CR + PR) of 81.5%. Among the 36 patients who had ever received postoperative adjuvant chemotherapy, 6 obtained complete response and 10 partial response with a response rate (CR + PR) of 44.4%. The overall median time to tumor progression in this series was 5.6 months. The overall median actuarial survival was 9.3 months, and the one-year survival rate was 24.9%. Nausea and vomiting were the major toxicities, but were mild and well tolerable. Grade 3 to 4 neutropenia was only observed in two patients (3.2%). CONCLUSION: The regimen of nedaplatin combined with tegafur is effective and tolerable for the treatment of advanced esophageal cancer.

[Impact of RNA interference targeting hypoxia-inducible factor-1alpha on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia].

OBJECTIVE: To investigate the impact of RNA interference (RNAi) targeting hypoxia-inducible factor 1alpha (HIF-1 alpha) on chemosensitivity of esophageal squamous cell carcinoma cells under hypoxia. METHODS: Human esophageal squamous cell carcinoma cells of the line EC9706 were cultured and divided into 3 groups: untransfected group, added with cobalt chloride (CoCl(2)), a chemical hypoxia inducer, for 8 h so as to establish a hypoxia model; control siRNA transfected group, transfected with control siRNA, and 30 h after the transfection exposed to CoCl(2) for 8 h; and HIF-1 alpha siRNA-transfected group, transfected with HIF-1 alpha siRNA, and 30 h later exposed to CoCl(2) for 8 h. Western blotting was used to detect the protein expression of HIF-1 alpha. Another EC9706 were cultured and divided into 3 groups to be treated as mentioned above, and then exposed to cisplantin or platixal under normoxic or hypoxic condition. 24 hours later 3-(4, 5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay was used to detect the inhibition rates of the cells. Further another EC9706 cells were cultured and then divided into 5 groups: cultured under normoxic condition, cultured under hypoxic condition for 8 h, transfected with control siRNA for 30 h and then under hypoxic condition for 8 h, transfected with HIF-1 alpha siRNA for 30 h and then under hypoxic condition for 8 h. The cell cycle was measured by flow cytometry. RESULTS: The HIF-1alpha protein expression of the HIF-1alpha siRNA group was significantly lower than those of the untransfected and control siRNA transfected groups. The inhibition rates of the EC9706 cells of the groups treated by cisplatin of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.01). The inhibition rates of the EC9706 cells of the groups treated by platixal of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.05) Under hypoxic condition, the inhibition rates of the HIF-1alpha siRNA transfected EC9706 cells treated by cisplatin and platixal of different concentrations were all significantly higher than those of the control siRNA transfected and untransfected EC9706 cells (all P < 0.05). Flow cytometry showed that under hypoxic condition the proportion of cells in G(1)-phase of the EC9706 cells was significantly higher, and the proportion of S-phase cells was significantly lower than those of the normoxic group (both P < 0.05), and under the same hypoxic condition the proportion of the EC9706 cells in G(1)-phase was significantly lower, and the proportion the EC9706 cells in S-phase was significantly higher than those of the normoxic group (all P < 0.05). CONCLUSION: The cell cycle arrest induced by HIF-1alpha may be the mechanism of the resistance to anticancer drugs of the esophageal squamous cell carcinoma cells under hypoxic condition. Blocking HIF-1alpha in esophageal squamous cell carcinoma cells may reverse the multidrug resistance of the tumor cells, so it may offer an avenue for gene therapy.

[Relationship between the expression of hypoxia-inducible factor-1alpha and chemotherapy response in esophageal squamous cell carcinoma].

OBJECTIVE: To observe the relationship between expression of hypoxia-inducible factor (HIF)-1alpha and chemotherapy response and clinical outcome. METHODS: Platixal in combination with cisplatin was used in 48 patients with ESCC at advanced stage. platixal 175 mg/m(2), d1; cisplatin 80 mg/m(2), d 2, d 3, d 4, 21 days was one cycle. Chemotherapy response was evaluated. Specimens mentioned above comes from pathology department, the first affiliated hospital of Zhengzhou university. Immuno-histochemistry was used to examine the expression of HIF-1alpha protein in esophageal squamous cell carcinoma. 20 cases normal tissue of esophago was used as control group. chi(2) test and Kaplan-Merier was used to analysis data. RESULTS: HIF-1alpha immunoreactivity was recognized in both cytoplasm and nuclei. HIF-1alpha protein positive expression rate was 43.75% (21/48) in tumor samples, and was negative expression in normal tissue. Among 21 cases with positive expression of HIF-1alpha, there was none complete response case and 5 cases of partial responses, and the response rate was 23.81%; whereas in 27 cases with negative expression of HIF-1alpha, there were 8 cases of complete response and 11 cases of partial responses, and the response rate was 70.37%. There was significant difference between two groups (P < 0.05). In patients of HIF-1alpha positive expression, the median time to tumor progression was 2.0 months, and the median actuarial survival was 6 months, and survival rate was 13.3% in two years; Whereas in patients of HIF-1alpha negative expression, the median time to tumor progression was 5.0 months, and the median actuarial survival was 11.0 months, and survival rate was 42.2% in two years. There was significant difference between two groups (P < 0.05). CONCLUSION: HIF-1alpha protein expression by immunohistochemistry may be a useful indicator to prediction the chemotherapy response and clinical outcome for esophageal squamous cell carcinoma.