Ji-Wei Yu

Expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocacinoma

BackgroundTo study on expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocarcinoma (GC).MethodsExpressions of CD133 protein by immunostaining (99 cases) and CD133 mRNA by semi-quantitative RT-PCR (31 cases) were detected in primary lesion and in noncancerous gastric mucosa tissue (NCGT). Correlations of CD133 protein expression with clinicopathological parameters and post-operative survival were analyzed. Relations of CD133 mRNA level with Ki-67 labeling index (LI), and lymphatic metastasis were assessed too.ResultsBrown particles indicating CD133 protein positivity occurred in some parts of tumor cells and epithelium. Expressive percentage of CD133 protein positivity was significantly higher in subgroups with >5 cm diameter (P = 0.041), later TNM stage (P = 0.044), severer lymph node metastasis (P = 0.017), occurrences of lymphatic invasion (P = 0.000) and vascular invasion (P = 0.000) respectively. Severer invasion depth (P = 0.011), lymph node metastasis occurrence (P = 0.043) and later TNM stage (P = 0.049) were the independent risk factors for CD133 protein expression. Average brightness scale value (BSV) of CD133 mRNA was significantly higher in subgroups with >5 cm diameter (P = 0.041), lymph node metastasis occurrence (P = 0.004) and in lower Ki-67 LI (P = 0.02). Relative analysis revealed that BSV of CD133 mRNA related positively to metastatic lymphatic nodes ratio (P = 0.008) and metastatic lymph node number (P = 0.009), but negatively to Ki-67 LI (P = 0.009). Survival of positive subgroup of CD 133 protein was significantly poorer (P = 0.047). Lymph node metastasis occurrence (P = 0.042), later TNM stage (P = 0.046) and CD 133 protein positive expression (P = 0.046) were respectively the independent risk factors to survival.ConclusionHigher expressive level of CD133 mRNA is associated to lower Ki-67 LI and severer lymphatic metastasis. Therefore, the expressive level of CD133 mRNA can play an appropriate role to reflect the status of lymph node metastasis and proliferation of GC. CD133 protein expression is closely related with larger tumor, later TNM stage, lymphtic metastasis and survival of GC.

Expression and Significances of Contactin-1 in Human Gastric Cancer

Background. This study aimed at determining the relationship between vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), and contactin-1 (CNTN-1) expression in gastric cancer (GC). Methods. The expression level of CNTN-1 mRNA and CNTN-1 protein of 33 cases was determined using RT-PCR and Western Blot. And 105 cases were immunohistochemically examined for VEGF-C, VEGFR-3, and CNTN-1 expressions. Assessment of lymphatic vessel density (LVD) was also performed by D2-40 immunostaining. Then we analyzed the relationships between VEGF-C, VEGFR-3, and CNTN-1, as well as their correlations with clinicopathologic features, LVD, and survival time. Results. The positivity rate of VEGF-C, VEGFR-3, and CNTN-1 in primary tumor was 56.19%, 64.76%, and 58.09%. The expression of CNTN-1 significantly correlated with VEGF-C (P < 0.001) and VEGFR-3 (P < 0.001). All of them were closely related to TNM stage, lymphatic invasion, and lymph node involvement (P < 0.05). LVD was significantly correlated with VEGF-C (P = 0.001), VEGFR-3 (P = 0.011), and CNTN-1 expression (P < 0.001). VEGF-C, VEGFR-3, and CNTN-1 expression significantly associated with poorer prognosis (P < 0.001, P = 0.034, P = 0.012, resp.). Conclusion. CNTN-1 associated with VEGF-C and VEGFR-3 expression in GC. All of them correlated with lymphatic metastasis, which might play an important role in the lymphatic invasion via lymphangiogenesis pathway in GC.

Overexpression of CD133 enhances chemoresistance to 5-fluorouracil by activating the PI3K/Akt/p70S6K pathway in gastric cancer cells

CD133 has been reported to be associated with chemoresistance in various cancer cells. The efficacy of 5-fluorouracil (5-FU), an important chemotherapeutic agent for advanced gastric cancer (GC), is limited by 5-FU resistance. Hence, the present study investigated the function of CD133 in 5-FU resistance in human GC cells. We isolated CD133+ GC cells by immunomagnetic cell sorting and CD133 expression was modulated by transfection of CD133 gene or CD133 small interfering ribonucleic acid. To assess the 5-FU cytotoxicity, Cell Counting Kit-8 was used. Expression of CD133, P-glycoprotein (P-gp), B-cell lymphoma 2 (Bcl‑2), Bcl-2-associated X protein (Bax), phospho-Akt (p-Akt) and phospho-p70S6 kinase (p-p70S6K) were analyzed by western blotting. CD133, P-gp, Bcl-2 and Bax messenger ribonucleic acids were evaluated using semi-quantitative reverse transcriptase-polymerase chain reaction. Cell apoptosis was assessed by Hoechst 33258 staining. CD133+ cells were more resistant to 5-FU than CD133- cells, and showed higher expression of P-gp and Bcl-2 with lower expression of Bax. Furthermore, CD133 silencing enhanced 5-FU cytotoxicity and apoptotic characteristics, whereas CD133 overexpression increased 5-FU resistance. CD133 silencing and activation directly decreased and increased the expression of P-gp, Bcl‑2, p-Akt and p-p70S6K, respectively. Notably, Akt inhibition by LY294002 restored the 5-FU cytotoxicity suppressed by CD133 overexpression, while Akt activation by epidermal growth factor reversed the 5-FU cytotoxicity enhanced by CD133 silencing. Therefore, CD133 may inhibit 5-FU-induced apoptosis by regulating the expression of P-gp and Bcl-2 family mediated by phosphoinositide 3-kinase/Akt/p70S6K pathway in GC cells.

Influencing factors and clinical significance of the metastatic lymph nodes ratio in gastric adenocarcinoma

BackgroundTo investigate influencing factors of the metastatic lymph nodes ratio (MLR) and whether it is related to survival in patients with gastric adenocarcinoma.MethodsWe retrospectively evaluated the clinical features of 121 patients with gastric adenocarcinoma enrolled in our hospital between 2000 and 2007. The receiver operating characteristic (ROC) curve was used to determine the cutoff of the MLR, and CK20 immunohistochemical staining was used to detect micrometastasis of the lymph nodes.ResultsThe areas under the ROC curve of MLR used to predict the death of 3-year and 5-year postoperative patients were 0.826 ± 0.053 and 0.896 ± 0.046. Thus MLR = 30.95% and MLR = 3.15% were designated as cutoffs. The MLR was then classified into three groups: MLR1 (MLR<3.15%); MLR2(3.15% ≤ MLR ≤ 30.95%); and MLR3 (MLR>30.95%). We found that patients with a higher MLR demonstrated a much poorer survival period after radical operation than those patients with a lower MLR (P = 0.000). The COX model showed that MLR was an independent prognostic factor (P = 0.000). The MLR could also discriminate between subsets of patients with different 5-year survival periods within the same N stage (P < 0.05). The MLR has been shown to be 34.7% (242/697) by HE staining and 43.5% (303/697) by CK staining (P = 0.001). The clinicopathological characteristics of lymph vessel invasion and the depth of invasion could significantly affect the MLR.ConclusionMLR is an independent prognostic factor in gastric cancer. The combined ROC curve with MLR is an effective strategy to produce a curve to predict the 3-year and 5-year survival rates.

Contactin 1: A potential therapeutic target and biomarker in gastric cancer

Despite advances in diagnosis and treatment, gastric cancer remains one of the most common malignant tumors worldwide, and early diagnosis remains a challenge. The lack of effective methods to detect these tumors early is a major factor contributing to the high mortality in patients with gastric cancer, who are typically diagnosed at an advanced stage. Additionally, the early detection of metastases and the curative treatment of gastric cancer are difficult to achieve, and the detailed mechanisms remain to be fully elucidated. Thus, the identification of valuable predictive biomarkers and therapeutic targets to improve the prognosis of patients with gastric cancer is becoming increasingly important. Contactin 1 (CNTN1), a cell adhesion molecule, is a glycosylphosphatidylinositol-anchored neuronal membrane protein that plays an important role in cancer progression. The expression of CNTN1 is upregulated in primary lesions, and its expression level correlates with tumor metastasis in cancer patients. The current evidence reveals that the functions of CNTN1 in the development and progression of cancer likely promote the invasion and metastasis of cancer cells via the VEGFC/FLT4 axis, the RHOA-dependent pathway, the Notch signaling pathway and the epithelial-mesenchymal transition progression. Therefore, CNTN1 may be a novel biomarker and a possible therapeutic target in cancer treatment in the near future.

Study on the Biological Characteristics of CD133 (+) Cells Interfered by RNA Interference in Gastric Cancer.

Background. To detect the changes of biological characteristics in gastric cancer cells interfered by CD133-specific small interfering RNA (siRNA). Methods. First to select the siRNA which has the strongest interference effect among 3 siRNAs (i.e., siRNA1, siRNA2, and siRNA3) in KATO-III cells by RT-PCR and Western blotting assays. Then, CD133+ cells were sorted out from KATO-III cells using an immunomagnetic bead sorting method and transfected with the selected siRNA. Furthermore, the proliferating characteristics, the antichemotherapeutic assessment, Transwell invasion assay, monoclonal sphere formation assay, and subcutaneous transplanted tumor formation assay in nude mice were investigated. Results. siRNA3 showed the strongest interference effect in KATO-III cells. As compared to the uninterfered control group, the CD133+ cells treated by siRNA3 showed significant decreases in the abilities of proliferation, invasion, clone sphere formation, and resistance to antitumour drugs as well as the weight and size of the transplanted tumor, which was nearly similar to that of CD133− cells. Additionally, the protein expression level of the EMT factor E-cadherin increased while those of EMT-related Snail and N-cadherin decreased in CD133+ cells interfered by siRNA3. Conclusion. Inhibition of CD133 gene expression reduces the abilities of gastric cancer cells in proliferation, invasion, clonal sphere formation, and chemoresistance as well as tumor formation in nude mice.

Autophagy Facilitates Metadherin-Induced Chemotherapy Resistance Through the AMPK/ATG5 Pathway in Gastric Cancer

Background/Aims: Metadherin (MTDH) is overexpressed in some malignancies and enhances drug resistance; however, its role in gastric cancer (GC) and the underlying mechanisms remain largely unexplored. Here, we explore the mechanism by which MTDH induces drug resistance in GC. Methods: We analysed the level of MTDH in GC and adjacent normal gastric mucosal tissues by real-time quantitative PCR (q-PCR). We also analysed the level of autophagy by western blot analysis, confocal microscopy, and transmission electron microscopy after MTDH knockdown and overexpression, and examined fluorouracil (5-FU) resistance by Cell Counting Kit-8 at the same time. Finally, GC patient-derived xenograft tumours were used to demonstrate 5-FU resistance. An AMPK pathway inhibitor was applied to determine the molecular mechanisms of autophagy. Results: MTDH expression was significantly increased in the GC specimens compared with that in the adjacent normal gastric mucosal tissues. Further study showed a positive correlation between the expression level of MTDH and 5-FU resistance. MTDH overexpression in MKN45 cells increased the levels of P-glycoprotein (P-gp) and promoted 5-FU resistance, while inhibition of MTDH showed the opposite result. The simultaneous inhibition of autophagy and overexpression of MTDH decreased the levels of P-gp and inhibited 5-FU resistance. Moreover, MTDH induced AMPK phosphorylation, regulated ATG5 expression, and finally influenced autophagy, suggesting that MTDH may activate autophagy via the AMPK/ATG5 signalling pathway. Our findings reveal a unique mechanism by which MTDH promotes GC chemoresistance and show that MTDH is a potential target for improved chemotherapeutic sensitivity and GC patient survival. Conclusions: MTDH-stimulated cancer resistance to 5-FU may be mediated through autophagy activated by the AMPK/ATG5 pathway in GC.

Metadherin regulates actin cytoskeletal remodeling and enhances human gastric cancer metastasis via epithelial-mesenchymal transition

Metadherin (MTDH) can be recruited to mature tight junction complexes, and it regulates mesenchymal marker protein expression in many tumors and promote cancer metastasis. This study investigated the influence of MTDH expression on gastric cancer and to elucidate the potential mechanisms by which MTDH regulates actin cytoskeletal remodeling and enhances human gastric cancer metastasis via epithelial-mesenchymal transition (EMT). Relative MTDH mRNA expression levels were assessed by quantitative real-time PCR (Q-PCR), and MTDH protein expression levels and localization were evaluated via immunohistochemical (ICH) staining. We studied the role of MTDH in cancer cell migration and invasion by modulating MTDH expression in the gastric cancer cell lines MKN45 and AGS. We also confirmed the functions of MTDH through in vivo experiments. We found that MTDH expression levels were correlated with lymph node metastasis, TNM stages and decreased OS (P=0.002, <0.001 and 0.010, respectively) in human gastric cancer and that MTDH upregulation promoted EMT in vitro. Consistent with this finding, MTDH downregulation inhibited cell migration and invasion in vitro and suppressed tumor growth and metastasis in vivo. Furthermore, MTDH knockdown regulated actin cytoskeletal remodeling and inhibited EMT. Overall, our results provide a novel role for MTDH in regulating gastric cancer metastasis.

Bone marrow-derived mesenchymal stem cells increase drug resistance in CD133-expressing gastric cancer cells by regulating the PI3K/AKT pathway

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited to primary tumours to compose the tumour microenvironment. In various cancers, CD133-positive cells have been shown to possess cancer stem cell properties that confer chemoresistance. This study aimed to investigate the role of BM-MSCs in the anti-tumour drug resistance of CD133-expressing gastric cancer cells and explore the underlying mechanisms that governing this role. We found that CD133+ gastric cancer cells displayed more resistance to chemotherapeutics than CD133− cells. In addition, BM-MSCs increased the anti-apoptotic abilities and chemoresistance of CD133+ cells via upregulation of Bcl-2 and downregulation of BAX. Mechanistically, BM-MSCs triggered activation of the PI3K/Akt signalling cascade in CD133+ cells. Blocking the PI3K/Akt pathway inhibited the promotion of chemoresistance. Furthermore, BM-MSCs enhanced the drug resistance of CD133-overexpressing cells in vitro and in vivo, but not that of CD133-knockdown cells, which demonstrated the contribution of CD133 to this process. In conclusion, we demonstrated that BM-MSCs increased the anti-apoptotic abilities and drug resistance of CD133-expressing cells via activation of the PI3K/Akt pathway following Bcl-2 upregulation and BAX downregulation, in which CD133 played a significant role. Targeting this route may help improve the efficacy of chemotherapy in gastric cancer.

Preliminary Study on the Expression and the Clinical Significance of CD133 in Peripheral Blood of Patients with Gastric Adenocarcinoma

Background. Significances of CD133 mRNA in peripheral blood mononuclear cells (PBMCs) of gastric adenocarcinoma (GC) patients were investigated. Methods. Correlations of CD133 mRNA expression in PBMCs on clinicopathological parameters or CD133 protein expression were analyzed. Receiver operating characteristic curve according to bright scale value (BSV) of CD133 mRNA was used to group patients for prognosis analysis. Results. BSV of preoperative CD133 mRNA in PBMCs in GC was significantly higher than that in volunteers or in GU. Invasive depth or metastatic lymph node number for higher BSV of preoperative CD133 mRNA and invasive depth or lymphatic vessel invasion for higher BSV of postoperative CD133 mRNA in the PBMCs were identified. Patients with CD133+ expression in primary lesion had a significantly higher expression of preoperative CD133 mRNA in the PBMCs. The expression of preoperative or postoperative CD133 mRNA in PBMCs related positively to CD133 mRNA expression in primary lesion. Patients with higher expression of preoperative or postoperative CD133 mRNA shared significantly shorter survival compared with that in lower expression group. Conclusion. Higher levels of preoperative or postoperative CD133 mRNA in PBMCs of GC correlated positively to the lymphatic metastasis and the BSV of CD133 mRNA in primary lesion, indicating the poorer survival.