Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

BACKGROUND/AIM: Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. MATERIALS AND METHODS: The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. RESULTS: PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. CONCLUSION: PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3ß and FOXO1 phosphorylation.

Cytochrome P450 Family 46 Subfamily A Member 1 Promotes the Progression of Colorectal Cancer by Inducing Tumor Cell Proliferation and Angiogenesis.

BACKGROUND/AIM: Cytochrome P450 family 46 subfamily A member 1 (CYP46A1) has been implicated in the development and progression of various cancers. This study aimed to analyze the expression of CYP46A1, examining its relationship with oncogenic behaviors, and determining its prognostic implications in colorectal cancer (CRC). MATERIALS AND METHODS: A total of 225 patients with CRC who underwent curative surgical resection were examined using paraffin-embedded tissue blocks and subjected to tumor-specific survival analysis. The expression of CYP46A1 was assessed in CRC tissues through reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry. The CRC cells' apoptosis, proliferation, angiogenesis, and lymphangiogenesis were analyzed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, alongside immunohistochemical staining for Ki-67, CD34, and D2-40 antibodies. RESULTS: CYP46A1 expression was found to be up-regulated in CRC tissues compared to normal colorectal mucosa. Such expression was significantly associated with advanced stage, deeper tumor invasion, lymph node metastasis, distant metastasis, and decreased survival. Furthermore, the mean Ki-67 labeling index and microvessel density values in CYP46A1-positive tumors were significantly elevated compared to CYP46A1-negative tumors. However, there was no discernible correlation between CYP46A1 expression and either the apoptotic index or lymphatic vessel density value. CONCLUSION: CYP46A1 promotes CRC progression, specifically through the induction of tumor cell proliferation and angiogenesis. The insights provided may hold potential implications for future therapeutic interventions targeting CYP46A1.

Expression of an oxysterol-metabolizing enzyme in colorectal cancer and its relation to tumor cell behavior and prognosis.

Oxysterols and oxysterol-metabolizing enzymes have been implicated in the pathogenesis of various cancers. However, the distinct function of the oxysterol-metabolizing enzyme cytochrome P450 family 39 Subfamily A Member 1 (CYP39A1) in colorectal cancer (CRC) remains unclear. The aims of the current study were to evaluate whether CYP39A1 affects the oncogenic behaviors of CRC cells and to investigate the prognostic value of its expression in CRC. A CYP39A1 small-interfering RNA was used to block CYP39A1 gene expression in DLD1 and SW480 cells. The expression of CYP39A1 in CRC tissues was investigated by immunohistochemistry. Tumor angiogenesis and lymphangiogenesis were assessed by CD34 and D2-40 immunohistochemical staining, respectively. CYP39A1 knockdown inhibited tumor cell migration and invasion in DLD1 and SW480 cells. Angiogenesis was also inhibited through the decreased expression of vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor (HIF)-1α, and angiostatin and endostatin expression increased. In addition, CYP39A1 knockdown inhibited the lymphangiogenesis by decreasing the expression of VEGF-C. CYP39A1 expression was increased in CRC tissues compared with normal colorectal mucosa. CYP39A1 expression was associated with tumor stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival. The microvessel and lymphatic vessel density values of CYP39A1-positive tumors were significantly higher than those of CYP39A1-negative tumors. These results indicate that CYP39A1 is associated with tumor progression by influencing tumor cell angiogenesis and lymphangiogenesis in CRC.

Expression of Apurinic/Apyrimidinic Endonuclease 1 in Colorectal Cancer and its Relation to Tumor Progression and Prognosis.

BACKGROUND/AIM: Over-expression of apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated to be associated with cancer progression, chemo- and radioresistance in various cancers. This study examined the expression of APE1 and its relation to tumor progression and prognosis in patients with colorectal cancer (CRC). MATERIALS AND METHODS: We investigated 193 patients with CRC who received curative surgery for whom formalin-fixed and paraffin-embedded blocks were available, and long-term tumor-specific survival rate analysis was possible. The expression of APE1 was investigated by reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry in CRC and lymph node tissues. The apoptosis, proliferation, and angiogenesis of CRC cells were determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and immunohistochemical staining for Ki-67 and CD34 antibodies. RESULTS: APE1 was over-expressed in CRC and metastatic lymph node tissues compared with normal colorectal mucosa and non-metastatic lymph node tissues. Over-expression of APE1 was significantly associated with advanced stage, lymphovascular invasion, perineural invasion, deeper tumor invasion, lymph node metastasis, distant metastasis, and poor survival. Multivariate analysis demonstrated that APE1, perineural invasion, and lymph node metastasis were the independent prognostic factors associated with overall survival. The mean Ki-67 labeling index value of APE1-positive tumors was significantly higher than that of APE1-negative tumors. However, there was no significant association between APE1 expression and the apoptotic index or microvessel density. CONCLUSION: Over-expression of APE1 is significantly associated with tumor progression and poor survival in patients with CRC. Therefore, APE1 may be a novel biomarker and present a potential prognostic factor for CRC.

A disintegrin and metalloprotease 12 contributes to colorectal cancer metastasis by regulating epithelial­mesenchymal transition.

A disintegrin and metalloprotease 12 (ADAM12) and epithelial­mesenchymal transition (EMT) are linked in the metastasis of various types of cancer. The present study aimed to assess the ability of ADAM12 to induce EMT and its potential as a therapeutic target for colorectal cancer (CRC). ADAM12 expression in CRC cell lines, CRC tissues and a mouse model of peritoneal metastasis was assessed. The effect of ADAM12 on CRC EMT and metastasis was investigated using ADAM12­pcDNA6­myc and ADAM12­pGFP­C­shLenti constructs. ADAM12 overexpression enhanced the proliferation, migration, invasion and EMT of CRC cells. The phosphorylation levels of factors associated with the PI3K/Akt pathway were also increased by ADAM12 overexpression. The knockdown of ADAM12 reversed these effects. ADAM12 expression and the loss of E­cadherin expression were significantly associated with poorer survival compared with other expression statuses of both proteins. In a mouse model of peritoneal metastasis, overexpression of ADAM12 induced increased tumor weight and peritoneal carcinomatosis index compared with that in the negative control group. Conversely, knockdown of ADAM12 reversed these effects. Furthermore, E­cadherin expression was significantly decreased by overexpression of ADAM12 compared with in the negative control group. By contrast, E­cadherin expression was increased by knockdown of ADAM12 compared with in the negative control group. ADAM12 overexpression contributed to CRC metastasis by regulating EMT. In addition, in the mouse model of peritoneal metastasis, ADAM12 knockdown exhibited strong anti­metastatic action. Consequently, ADAM12 may be considered a therapeutic target for CRC metastasis.

Over-expression of Anterior Gradient 3 Is Associated With Tumor Progression and Poor Survival in Gastric Cancer.

BACKGROUND/AIM: Anterior gradient (AGR) proteins, including AGR1, AGR2, and AGR3, which are members of the protein disulfide isomerase family, have been reported as biomarkers for various carcinogenesis processes. Although AGR2 and AGR1 have been demonstrated to be associated with gastric cancer (GC) progression and poor survival, the effect of AGR3 on the progression and prognosis of GC remains unknown. Therefore, our study aimed to examine the expression and prognostic significance of AGR3 in patients with GC. PATIENTS AND METHODS: We investigated 271 GC patients receiving curative surgery. Formalin-fixed and paraffin-embedded tissue blocks were obtained, and long-term survival analysis was performed. The expression of AGR3 in GC tissues was investigated by quantitative reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry. RESULTS: AGR3 was over-expressed in GC tissue compared with paired normal tissue at the mRNA and protein levels. AGR3 over-expression was significantly associated with larger tumor size, deeper tumor invasion, lymph node metastasis, and advanced tumor stage. The overall survival of patients with positive AGR3 expression was significantly lower than that of patients without positive AGR3 expression. Multivariate analysis demonstrated that AGR3 and age were independent prognostic factors associated with overall survival. CONCLUSION: Over-expression of AGR3 was significantly associated with tumor progression and poor survival of GC patients. Therefore, AGR3 may be a novel biomarker and prognostic factor for GC.

CD47 mediates the progression of colorectal cancer by inducing tumor cell apoptosis and angiogenesis.

CD47 is an immunoregulatory protein that is found on the cell surface and plays significant roles in cellular functions such as proliferation, apoptosis, migration, and immune homeostasis. CD47 is overexpressed in various human cancers and is associated with tumor development, progression, and poor prognosis. In this study, we analyzed the expression of CD47 to determine whether it affected the oncogenic behavior of colorectal cancer (CRC) and investigated the prognostic value of CD47 expression in patients with CRC. We investigated 468 patients with CRC who underwent curative surgery and examined the expression of CD47 in tumor and lymph node tissues by performing RT-PCR and immunohistochemistry. Apoptosis, proliferation, angiogenesis, and lymphangiogenesis were determined via a TUNEL assay and immunohistochemical staining for Ki-67, CD34, and D2-40. CD47 expression was increased in human CRC tumors and metastatic lymph nodes compared with normal colorectal mucosa and non-metastatic lymph node tissues. CD47 expression was significantly associated with perineural invasion, lymphovascular invasion, cell differentiation, cancer stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival. The mean apoptotic index and microvessel density value of CD47-positive tumors were significantly higher than those of CD47-negative tumors. However, no significant difference was observed between CD47 expression and Ki-67 labeling index or lymphatic vessel density. These results indicate that CD47 mediated the progression of CRC by inducing tumor cell apoptosis and angiogenesis.

Engulfment and Cell Motility 1 (ELMO1) Regulates Tumor Cell Behavior and Predicts Prognosis in Colorectal Cancer.

BACKGROUND/AIM: Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients. MATERIALS AND METHODS: We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients. RESULTS: ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3ß and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival. CONCLUSION: ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

Expression of ADAM12 in Gastric Cancer and its Relation to Tumor Cell Behavior and Prognosis.

BACKGROUND/AIM: A disintegrin and metalloprotease (ADAM) 12 expression has been found up-regulated in various cancer types. The aim of the study was to evaluate whether ADAM12 affects oncogenic behavior of gastric cancer (GC) cells and investigate its prognostic value. MATERIALS AND METHODS: The effect of ADAM12 on tumor cell behavior was examined using the small interfering RNA and pcDNA6-myc vector in human GC cell lines. Expression of ADAM12 in GC tissues was confirmed by immunohistochemistry. Apoptosis and proliferation were determined by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and immunohistochemical staining for Ki-67. RESULTS: ADAM12 overexpression enhanced tumor cell migration and invasion in AGS and SNU638 cells. Down-regulation of caspase-3 and PARP activity due to ADAM12 overexpression enhanced tumor cell proliferation and inhibited apoptosis. The expression of Snail and Vimentin increased and that of E-cadherin decreased following ADAM12 overexpression. In contrast, ADAM12 knockdown reversed these effects. ADAM12 overexpression increased the phosphorylation of Akt and GSK-3ß. The mean Ki-67 labeling index value of ADAM12-positive tumors was significantly higher compared to that of ADAM12-negative tumors. ADAM12 expression was associated with age, tumor size, cancer stage, depth of invasion, lymph node metastasis, and poor survival. CONCLUSION: ADAM12 enhances tumor progression by increasing cell mobility, enhancing cell proliferation, and inhibiting apoptosis in GC cells. Also, ADAM12 is associated with adverse clinicopathological features and poor survival. It may be used as a molecular marker for the prediction of clinical outcomes of patients with GC.

A Disintegrin and Metalloprotease 12 Promotes Tumor Progression by Inhibiting Apoptosis in Human Colorectal Cancer.

A disintegrin and metalloprotease 12 (ADAM12) has been implicated in cell growth, tumor formation, and metastasis. Therefore, we evaluated the role of ADAM12 in colorectal cancer (CRC) progression and prognosis, and elucidated whether targeted downregulation of ADAM12 could lead to therapeutic sensitization. The effect of ADAM12 on tumor cell behavior was assessed in CRC cell lines, CRC tissues, and a mouse xenograft model. ADAM12 overexpression enhanced proliferation, inhibited apoptosis, and acted as positive regulator of cell cycle progression in CRC cells. Phosphorylation of PTEN was decreased and that of Akt was increased by ADAM12 overexpression. These results were reversed upon ADAM12 knockdown. ADAM12 overexpression was significantly associated with the cancer stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival in CRC patients. In a mouse xenograft model, tumor area, volume, and weight were significantly greater for the ADAM12-pcDNA6-myc-transfected group than for the empty-pcDNA6-myc-transfected group, and significantly lower for the ADAM12-pGFP-C-shLenti-transfected group than for the scrambled pGFP-C-shLenti-transfected group. In conclusion, ADAM12 overexpression is essential for the growth and progression of CRC. Furthermore, ADAM12 knockdown reveals potent anti-tumor activity in a mouse xenograft model. Thus, ADAM12 may serve as a promising biomarker and/or therapeutic target in CRC.

Engulfment and cell motility 1 promotes tumor progression via the modulation of tumor cell survival in gastric cancer.

BACKGROUND/AIM: Engulfment and cell motility 1 (ELMO1) protein has been implicated in phagocytosis of apoptotic cells, cell migration, neurite outgrowth, cancer cell invasion and metastasis, and poor prognosis in various cancers. We investigated the role of ELMO1 in mediating the oncogenic behavior of gastric cancer (GC) cells. We also investigated the correlation between expression of ELMO1 in GC tissues and various clinicopathological parameters. METHODS: We studied the impact of ELMO1 on tumor cell behavior using the pcDNA-myc vector and small interfering RNA in AGS and SNU1750 GC cell lines. We performed western blotting and immunohistochemistry to investigate the expression of ELMO1 in GC cells and tissues. RESULTS: ELMO1 overexpression inhibited apoptosis via the modulation of PARP, caspase-3 and caspase-7 in GC cells. ELMO1 overexpression led to significant increase in the number of migrating and invading GC cells. The expression of E-cadherin decreased and that of Snail increased in GC cells upon ELMO1 overexpression. Phosphorylation of PI3K/Akt and GSK-3ß was increased and that of ß-catenin was decreased upon ELMO1 overexpression in GC cells. These results were reversed after ELMO1 knockdown. ELMO1 expression was significantly associated with tumor size, cancer stage, lymph node metastasis and survival. ELMO1-positive tumors had significantly higher mean of Ki-67 labeling index than ELMO1-negative tumors. There was no significant relationship between ELMO1 expression and the mean value of the apoptotic index. CONCLUSIONS: Our results indicate that ELMO1 promotes tumor progression by modulating tumor cell survival in human GC.

Forkhead­box A1 regulates tumor cell growth and predicts prognosis in colorectal cancer.

Forkhead box A1 (FOXA1) functions as a tumor suppressor gene or an oncogene in various types of cancer; however, the distinct function of FOXA1 in colorectal cancer is unclear. The present study aimed to evaluate whether FOXA1 affects the oncogenic behavior of colorectal cancer cells, and to investigate its prognostic value in colorectal cancer. The impact of FOXA1 on tumor cell behavior was investigated using small interfering RNA and the pcDNA6­myc vector in human colorectal cancer cell lines. To investigate the role of FOXA1 in the progression of human colorectal cancer, an immunohistochemical technique was used to localize FOXA1 protein in paraffin­embedded tissue blocks obtained from 403 patients with colorectal cancer. Tumor cell apoptosis and proliferation were evaluated using a terminal deoxynucleotidyl transferase­mediated dUTP nick­end labeling assay and Ki­67 immunohistochemical staining, respectively. FOXA1 knockdown inhibited tumor cell invasion in colorectal cancer cells, and induced apoptosis and cell cycle arrest. FOXA1 knockdown activated cleaved caspase­poly (ADP­ribose) polymerase, upregulated the expression of p53 upregulated modulator of apoptosis, and downregulated BH3 interacting domain death agonist and myeloid cell leukemia­1, leading to the induction of apoptosis. FOXA1 knockdown increased the phosphorylation level of signal transducer and activator of tran-scription­3. By contrast, these results were reversed following the overexpression of FOXA1. The overexpression of FOXA1 was associated with differentiation, lymphovascular invasion, advanced tumor stage, depth of invasion, lymph node metastasis and poor survival rate. The mean Ki­67 labeling index value of FOXA1­positive tumors was significantly higher than that of FOXA1­negative tumors. However, no significant association was observed between the expression of FOXA1 and the mean apoptotic index value. These results indicate that FOXA1 is associated with tumor progression via the modulation of tumor cell survival in human colorectal cancer.

Reversine induces cell cycle arrest and apoptosis via upregulation of the Fas and DR5 signaling pathways in human colorectal cancer cells.

Reversine, a 2,6­diamino­substituted purine analogue, has been reported to be effective in tumor suppression via induction of cell growth arrest and apoptosis of cancer cells. However, it remains unclear whether reversine exerts anticancer effects on human colorectal cancer cells. In the present study, in vitro experiments were conducted to investigate the anticancer properties of reversine in human colorectal cancer cells. The effect of reversine on human colorectal cancer cell lines, SW480 and HCT­116, was examined using a WST­1 cell viability assay, fluorescence microscopy, flow cytometry, DNA fragmentation, small interfering RNA (siRNA) and western blotting. Reversine treatment demonstrated cytotoxic activity in human colorectal cancer cells. It also induced apoptosis by activating poly(ADP­ribose) polymerase, caspase­3, ­7 and ­8, and increasing the levels of the pro­apoptotic protein second mitochondria­derived activator of caspase/direct inhibitor of apoptosis­binding protein with low pI. The pan­caspase inhibitor Z­VAD­FMK attenuated these reversine­induced apoptotic effects on human colorectal cancer cells. Additionally, reversine treatment induced cell cycle arrest in the subG1 and G2/M phases via increase in levels of p21, p27 and p57, and decrease in cyclin D1 levels. The expression of Fas and death receptor 5 (DR5) signaling proteins in SW480 and HCT116 cells was upregulated by reversine treatment. Reversine­induced apoptosis and cell cycle arrest were suppressed by inhibition of Fas and DR5 expression via siRNA. In conclusion, Reversine treatment suppressed tumor progression by the inhibition of cell proliferation, induction of cell cycle arrest and induction of apoptosis via upregulation of the Fas and DR5 signaling pathways in human colorectal cancer cells. The present study indicated that reversine may be used as a novel anticancer agent in human colorectal cancer.

Chlorogenic acid suppresses lipopolysaccharide­induced nitric oxide and interleukin­1ß expression by inhibiting JAK2/STAT3 activation in RAW264.7 cells.

Chlorogenic acid (CA) is a phenolic compound purified from coffee, fruits and their associated beverages, which possess various biological properties, such as antioxidant and anticarcinogenic activities. The present study evaluated the effects of CA on lipopolysaccharide (LPS)­induced inflammation in RAW264.7 cells and the associated intracellular signaling pathways using reverse transcription­quantitative polymerase chain reaction, western blotting and enzyme­linked immunosorbent assays. CA pretreatment inhibited LPS­induced expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and pro­inflammatory mediators including interleukin (IL)­6, tumor necrosis factor­α (TNF­α), macrophage inflammatory protein­2 (MIP­2) and IL­1ß in RAW264.7 cells. In addition, phosphorylation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) with LPS was inhibited by CA pretreatment. CA and STAT3 inhibitor (STAT3i) pretreatment inhibited LPS­induced nuclear translocation of phosphorylated STAT3. In addition, STAT3i inhibited the LPS­induced expression of iNOS, NO and IL­1ß similar to the results of CA pretreatment. By contrast, STAT3i did not inhibit the LPS­induced increase in IL­6, TNF­α and MIP­2 expression. These results indicate that CA may suppress LPS­induced NO and IL­1ß expression by inhibiting JAK2/STAT3 activation in RAW264.7 cells.

Malformin A1 treatment alters invasive and oncogenic phenotypes of human colorectal cancer cells through stimulation of the p38 signaling pathway.

Malformin A1 (MA1), a cyclic pentapeptide isolated from Aspergillus niger, has been found to possess a range of bioactive properties including antibacterial activity. However, it is unclear whether MA1 exerts an anticancer effect or not. In this study, we conducted in vitro experiments to investigate its anticancer properties in human colorectal cancer cells. The effect of MA1 on human colorectal cancer cells, SW480 and DKO1, was examined by the WST-1 cell viability assay, inverted microscopy, 5-bromo-2-deoxyuridine (BrdU) incorporation, flow cytometry, DNA fragmentation, wound healing, Transwell assays, and western blotting. MA1 treatment showed potent cytotoxic activities on human colorectal cancer cells. MA1 treatment induced apoptosis by activating the poly(ADP-ribose) polymerase (PARP), caspase­3, -7, and -9. MA1 treatment led to the increase in p53 upregulated modulator of apoptosis (PUMA) and the decrease in X-linked inhibitor of apoptosis protein (XIAP) and Survivin. In addition, MA1 treatment induced cell cycle arrest in the sub-G1 phase. The pan-caspase inhibitor, Z­VAD­FMK, attenuated these MA1-induced apoptotic effects on human colorectal cancer cells. Moreover, MA1 treatment suppressed tumor cell migration and invasion. The phosphorylation level of p38 was upregulated by MA1 treatment, and the inhibitor of p38, SB203580, attenuated the MA1-induced p38 phosphorylation as well as caspase­3 and PARP activation. These results indicate that MA1 treatment alters invasive and oncogenic phenotypes of human colorectal cancer cells through the stimulation of the p38 signaling pathway.

Livin enhances chemoresistance in head and neck squamous cell carcinoma.

The responsiveness of head and neck squamous cell carcinoma (HNSCC) to chemotherapy widely affects prognosis. Overcoming chemoresistance is necessary to improve prognoses in patients with advanced HNSCC. Evasion of apoptosis by cancer cells is a major cause of chemoresistance. Livin, a member of the human inhibitors of apoptosis protein family, is highly expressed in various human cancer tissues and is associated with tumor progression and poor prognosis in human cancers. The aim of the present study was to evaluate the role of Livin in the susceptibility to popularly used chemotherapeutic drugs such as cisplatin, 5-fluorouracil (FU) and docetaxel in human HNSCC cell lines (SNU1041, PCI1 and PCI50 cells). Reverse transcription polymerase chain reaction and western blotting were performed to determine mRNA and protein expression levels. Cell viability and apoptosis assays were used to assess the functional effects of small-interfering RNA-mediated knockdown of Livin. Each HNSCC cell line had different sensitivity to chemotherapeutic drugs. Livin knockdown significantly enhanced cytotoxicity to cisplatin, 5-FU and docetaxel in human HNSCC cells. Livin knockdown induced apoptosis and enhanced chemotherapy-induced apoptosis to cisplatin, 5-FU and docetaxel. Consistent with this, Livin-knockdown cells showed greater expression of cleaved caspases-3 and -7 and poly(ADP-ribose)polymerase compared with that in control cells after cisplatin, 5-FU, or docetaxel treatment. In conclusion, our results suggest that siRNA-mediated Livin knockdown enhanced the chemosensitivity of the three HNSCC cell lines to cisplatin, 5-FU and docetaxel. Although further investigations are required to support these findings, our results demonstrated that novel therapeutic strategies with combined use of siRNA targeting Livin and chemotherapeutic agents may have applications in the treatment of advanced HNSCC.

Prospero homeobox 1 mediates the progression of gastric cancer by inducing tumor cell proliferation and lymphangiogenesis.

BACKGROUND: Prospero homeobox 1 (PROX1) functions as a tumor suppressor gene or an oncogene in various cancer types. However, the distinct function of PROX1 in gastric cancer is unclear. We determined whether PROX1 affected the oncogenic behavior of gastric cancer cells and investigated its prognostic value in patients with gastric cancer. METHODS: A small interfering RNA against PROX1 was used to silence PROX1 expression in gastric cancer cell lines AGS and SNU638. Expression of PROX1 in gastric cancer tissues was investigated by performing immunohistochemistry. Apoptosis, proliferation, angiogenesis, and lymphangiogenesis were determined by performing the TUNEL assay and immunohistochemical staining for Ki-67, CD34, and D2-40. RESULTS: PROX1 knockdown induced apoptosis by activating cleaved caspase-3, caspase-7, caspase-9, and poly(ADP-ribose) polymerase, and by decreasing the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. PROX1 knockdown also suppressed tumor cell proliferation. In addition, PROX1 knockdown decreased lymphatic endothelial cell invasion and tube formation and the expression of vascular endothelial growth factor (VEGF)-C and -D and cyclooxygenase (COX)-2. However, PROX1 knockdown only decreased umbilical vein endothelial cell invasion, not tube formation. The mean Ki-67 labeling index and lymphatic vessel density value of PROX1-positive tumors were significantly higher than those of PROX1-negative tumors. However, no significant difference was observed between PROX1 expression and apoptotic index or microvessel density. PROX1 expression was significantly associated with age, cell differentiation, lymph node metastasis, cancer stage, and poor survival. CONCLUSIONS: These results indicate that PROX1 mediates the progression of gastric cancer by inducing tumor cell proliferation and lymphangiogenesis.

Filamin A interacting protein 1-like expression inhibits progression in colorectal cancer.

Filamin A interacting protein 1-like (FILIP1L) expression, which is decreased in various cancers, may inhibit carcinogenesis. In this study, we evaluated the effects of FILIP1L on oncogenic behavior and prognosis in colorectal cancer. siRNA-mediated FILIP1L knockdown enhanced tumor cell migration and invasion and inhibited apoptosis and cell cycle arrest in COLO205 cells. pcDNA-myc vector-mediated FILIP1L overexpression suppressed tumor cell migration and invasion and induced apoptosis and cell cycle arrest in HCT116 cells. FILIP1L knockdown enhanced angiogenesis by increasing VEGF-A and HIF-1α levels and decreasing angiostatin level. FILIP1L overexpression suppressed angiogenesis by decreasing VEGF-A and -D l level and increasing angiostatin and endostatin levels. Phosphorylated ß-catenin levels decreased and phosphorylated Akt and GSK-3ß levels increased following FILIP1L knockdown. FILIP1L overexpression had the opposite effects. FILIP1L expression was associated with reductions in tumor size, cell differentiation, lymphovascular invasion, stage, invasion depth and lymph node metastasis, and with longer overall survival. Mean Ki-67 labeling indexes and microvessel density values were lower in FILIP1L-positive tumors than in FILIP1L-negative tumors. These results indicate that FILIP1L suppresses tumor progression by inhibiting cell proliferation and angiogenesis in colorectal cancer.

Livin enhances tumorigenesis by regulating the mitogen-activated protein kinase signaling pathway in human hypopharyngeal squamous cell carcinoma.

Livin, a member of the human inhibitor of apoptosis protein (IAP) family, is expressed at high levels in various human cancer tissues and may have prognostic significance. The aim of the present study was to evaluate the effect of Livin on tumor cell behavior and oncogenic signaling pathways in human hypopharyngeal squamous cell carcinoma (HSCC). Reverse transcription­quantitative polymerase chain reaction and western blot analyses were used to determine the mRNA and protein expression levels, respectively. A cell proliferation assay and cell cycle analysis were used to assess the functional effects of small interfering RNA­mediated Livin knockdown. Livin was overexpressed in fresh HSCC tissues, compared with the adjacent normal mucosa. Livin knockdown led to significantly reduced cell proliferation and cell cycle arrest in the G1 phase of the human HSCC cells. The expression levels of c­myc, cyclin D1, cyclin D3, cyclin­dependent kinase (CDK)4 and CDK6 were decreased. The phosphorylation levels of extracellular signal­regulated kinase 1/2, p38, c­Jun N-terminal kinase and Akt were also decreased by Livin knockdown in the HSCC cells. Taken together, the results of the present study suggested that Livin may enhance tumorigenesis by modulating the mitogen­activated/Akt signaling pathways in human HSCC.

Effect of Recepteur d'Origine Nantais expression on chemosensitivity and tumor cell behavior in colorectal cancer.

Recepteur d'Origine Nantais (RON) expression is known to induce oncogenic properties including tumor cell growth, survival, motility, angiogenesis and chemoresistance. In the present study, we evaluated whether RON affects chemosensitivity and oncogenic behavior of colorectal cancer cells and investigated its prognostic value in colorectal cancer. To evaluate the impact of RON on chemosensitivity and tumor cell behavior, we treated colorectal cancer cells with small interfering RNAs specific to RON. This was followed by flow cytometric analyses and migration, Matrigel invasion and endothelial tube formation assays. The expression of RON was investigated by immunohistochemistry in colorectal cancer tissues. TUNEL assay and immunohistochemical staining for CD34 and D2-40 were deployed to determine apoptosis, angiogenesis and lymphangiogenesis. RON knockdown enhanced 5-fluorouracil (FU)-induced apoptosis by upregulating the activities of caspases and expression of proapoptotic genes. Moreover, it enhanced 5-FU-induced cell cycle arrest by decreasing the expression of cyclins and cyclin­dependent kinases and inducing that of p21. Furthermore, RON knockdown augmented the 5-FU-induced inhibition of invasion and migration of colorectal cancer cells. The ß-catenin signaling cascade was blocked by RON knockdown upon 5-FU treatment. RON knockdown also decreased endothelial tube formation and expression of VEGF-A and HIF-1α and increased angiostatin expression. Furthermore, it inhibited lymphatic endothelial cell tube formation and the expression of VEGF-C and COX-2. RON expression was observed to be associated with age, tumor size, lymphovascular and perineural invasion, tumor stage, lymph node and distant metastasis, and poor survival rate. The mean microvessel density value of RON-positive tumors was significantly higher than that of RON-negative ones. These results indicate that RON is associated with tumor progression by inhibiting chemosensitivity and enhancing angiogenesis in colorectal cancer.