EZH2 promotes colorectal cancer stem-like cell expansion by activating p21cip1-Wnt/ß-catenin signaling.

Because colorectal cancer (CRC) stem-like cells (CCS-like cells) contribute to poor patient prognosis, these cells are a potential target for CRC therapy. However, the mechanism underlying the maintenance of CCS-like cell properties remains unclear. Here, we found that patients with advanced stage CRC expressed high levels of polycomb group protein enhancer of zeste homologue 2 (EZH2). High expression of EZH2 in tumor tissues correlated with poor patient prognosis. Conversely, silencing EZH2 reduced CRC cell proliferation. Surprisingly, EZH2 was more highly expressed in the CCS-like cell subpopulation than in the non-CCS-like cell subpopulation. EZH2 knockdown significantly reduced the CD133+/CD44+ subpopulation, suppressed mammosphere formation, and decreased the expression of self-renewal-related genes and strongly impaired tumor-initiating capacity in a re-implantation mouse model. Gene expression data from 433 human CRC specimens from TCGA database and in vitro results revealed that EZH2 helped maintain CCS-like cell properties by activating the Wnt/ß-catenin pathway. We further revealed that p21cip1-mediated arrest of the cell cycle at G1/S phase is required for EZH2 activation of the Wnt/ß-catenin pathway. Moreover, the specific EZH2 inhibitor EPZ-6438, a clinical trial drug, prevented CRC progression. Collectively, these findings revealed EZH2 maintaining CCS-like cell characteristics by arresting the cell cycle at the G1/S phase. These results indicate a new approach to CRC therapy.

Neuropilin-2 mediates lymphangiogenesis of colorectal carcinoma via a VEGFC/VEGFR3 independent signaling.

Lymphangiogenesis critically contributes to the lymphatic metastasis of colorectal carcinomas (CRCs), but the underlying mechanism of CRC lymphangiogenesis remains largely elusive. We have previously demonstrated that Semaphorin-3F (SEMA3F) is critically involved in CRC metastasis, and the receptor of SEMA3F, neuropilin-2 (NRP2), originally described as an axon guiding chemorepulsant implicated in nerve development, has been suggested in promoting lymphangiogenesis via acting as an obligate co-receptor of VEGFR3 cooperatively enhancing the activity of VEGF-C. Our present study revealed that in colorectal carcinomas, NRP2 expression levels of tumor-associated lymphatic endothelial cells (LECs) are significantly correlated with the density of tumor lymphatic vessels. In vitro, activation of NRP2 in LECs substantially facilitates their migration, sprouting, and tubulogenesis capacity via regulating the rearrangement of cytoskeleton polarity. In vivo model further showed that in the xenografts generated from SEMA3F knockdown CRC cells, NRP2 is substantially activated in tumor-associated LECs, resulting in a significantly increased tumor lymphangiogenesis. Further evidence demonstrated that CRC cell induces the activation of NRP2 in LECs to promote tumor lymphangiogenesis via integrinα9ß1/FAK/Erk pathway independent VEGF-C/VEGFR3 signaling. Our study for the first time revealed the novel molecular mechanism of NRP2-mediated-lymphangiogenesis in CRCs, suggesting NRP2 as a potential therapeutic target in preventing lymphatic metastasis of CRCs.

Expression of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa co-determines the prognosis of colon cancer patients.

Provirus integration site for Moloney murine leukemia virus (pim-1) is a proto-oncogene that is linked to the development and progression of several cancers. In this study, we evaluated pim-1 expression in tumors, tumor stroma and tumor-adjacent mucosa together as an independent prognostic factor for colon cancer patients. The study included 343 colon cancer patients. Immunohistochemical staining was used to detect pim-1. Multivariate cox regression for disease-free survival (DFS) were used to identify independent prognostic factors. Analytic hierarchy process (AHP) was used to calculate the weight of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa in order to obtain a Pim-1 total score (PTS) for recurrence and survival. Kaplan-Meier DFS curves and OS curves for patients with different pim-1 expression levels were compared using the log-rank test. In this study, four independent prognostic factors were identified for colon cancer patients: pim-1 expression in tumors, tumor stroma, tumor-adjacent mucosa, as well as tumor stage. It has been established that clinical stage is an important prognostic factor for colon cancer patients. However, PTS can identify the patients who are likely to recur not only in the whole radical excision group but also within each stage of this group. Based on the results of this study we can conclude that the PTS combined with clinical staging system may be a better predictor of colon cancer patients' prognosis than using the clinical stage system alone. ClinicalTrials.gov Number: ChiCTR-PRCH-12002842.

Long-term results of a randomized, double-blind, and placebo-controlled phase III trial: Endostar (rh-endostatin) versus placebo in combination with vinorelbine and cisplatin in advanced non-small cell lung cancer.

BACKGROUND: : Phase II-III trials in patients with untreated and previously treated locally advanced or non-small cell lung cancer (NSCLC) suggested that Endostar was able to enhance the effect of platinum-based chemotherapy (NP regimen) with tolerable adverse effects. METHODS: Four hundred and eighty six patients were randomized into two arms: study arm A: NP plus Endostar (n = 322; vinorelbine, cisplatin, Endostar), and study arm B: NP plus placebo (n = 164; vinorelbine, cisplatin, 0.9% sodium chloride). Patients were treated every third week for two to six cycles. RESULTS: : Overall response rates were 35.4% in arm A and 19.5% in arm B (P = 0.0003). The median time to progression was 6.3 months for arm A and 3.6 months for B, respectively (P < 0.001). The clinical benefit rates were 73.3% in arm A and 64.0% in arm B (P = 0.035). Grade 3/4 neutropenia, anemia, and nausea/vomiting were 28.5%, 3.4%, and 8.0%, respectively, in Arm A compared with 28.2%, 3.0%, and 6.6%, respectively, in Arm B (P > 0.05). There were two treatment related deaths in arm A and one in arm B (P > 0.05). The median overall survival was longer in arm A than in arm B (P < 0.0001). CONCLUSION: : Long-term follow-up revealed that the addition of Endostar to an NP regimen can result in a significant clinical and survival benefit in advanced NSCLC patients, compared with NP alone.

Interleukin-12 inhibits the survival of human colon cancer stem cells in vitro and their tumor initiating capacity in mice.

Interleukin-12 (IL-12) is a potent immunomodulatory cytokine with unknown direct effect on the property of cancer stem cells (CSCs). In this study, we investigated the capacity of IL-12 to regulate the self-renewal and differentiation of human colon CSCs in vitro, as well as the effect of IL-12 on the growth of tumors initiated by CSCs in mice. After over-expression of IL-12 with lentiviral transfection, CSCs exhibited reduced invasiveness and tumorsphere formation in association with increased apoptosis in vitro. After injection into NOD/SCID mice, tumors initiated by CSCs transfected with IL-12 showed markedly reduced rate of growth. Mechanistic studies revealed that over-expression of IL-12 reduced the expression of IL-4 and STAT6 in CSCs. Thus, our study demonstrates a potentially beneficial role of IL-12 in directly limiting the malignant phenotype of CSCs.

Multimodal therapy for adult Wilms' tumour: an experience from one centre.

INTRODUCTION: Wilms' tumour (WT) is very rare in adults but very common in children. Treatment guidelines for adult patients with WT are still insufficient. Some study groups recommend that therapeutic protocols for adults with WT (AWT) should follow the guidelines that have been established for children. OBJECTIVE: To describe the clinical and pathological characteristics of AWT as well as the treatment protocols and outcomes for AWT at our treatment centre. MATERIAL AND METHODS: Seven patients (5 females and 2 males) were diagnosed with AWT in our hospital between 2002 and 2009. The tumours were staged and the patients were treated according to the paediatric regimen recommended by the National Wilms' Tumor Study Group. RESULTS: The median patient age at the time of diagnosis was 29 years (range, 16-37 years). Flank pain was the most common clinical presentation. One patient was in Stage I of disease development, two were in Stage II, two were in Stage III and two were in Stage IV. Anaplasia was present in 3 patients with Stage III or Stage IV disease. All of the patients but one underwent nephrectomy and 2 incomplete surgeries were performed. Seven patients received 2-drug or 3-drug chemotherapy (dactinomycin and vincristine and/or doxorubicin). Two patients with Stage III disease also received radiation therapy (a total dose of 3600 or 3960 cGy). Complete remission was achieved in 4 patients. Three patients (one with Stage III disease, 2 patients with Stage IV disease) died of their disease and those patients were all classified with an unfavourable histological type called anaplasia. With a median follow-up of 53.5 months (range, 40-102 months), the 3-year and 5-year overall survival rates were 57.1% (95% confidence interval, 20.4-93.8%). CONCLUSIONS: The results of this report suggest that histological anaplasia might be an adverse prognostic factor for AWT. Proper application of the diagnostic and therapeutic regimens established for children may improve the prognosis of adult patients with WT.

Highly enriched CD133(+)CD44(+) stem-like cells with CD133(+)CD44(high) metastatic subset in HCT116 colon cancer cells.

Stem-like cancer cells (SLCCs) are distinct cellular subpopulation in colon cancer that is essential for tumor maintenance. Previous studies indicated that SLCCs accounted for only a minor subset in a given cancer model. However, we found that SLCCs frequency varied among a panel of colon cancer cell lines, with HCT116 cells composed mainly of SLCCs, as demonstrated by colonosphere forming capability and CD133 expression. Indeed, flow cytometric analysis revealed more than 60% HCT116 cells co-expressed the putative SLCCs markers CD133 and CD44. Compared with non-CD133(+)CD44(+) cells, FACS sorted CD133(+)CD44(+) cells were undifferentiated, endowed with extensive self-renewal and epithelial lineage differentiation capacity in vitro. CD133(+)CD44(+) exhibited enhanced tumorigeneicity in NOD/SCID mice. One thousand CD133(+)CD44(+) cells initiated xenograft tumors efficiently (3/6) while 1 × 10(5) non-CD133(+)CD44(+) cells could only form palpable nodule with much slower growth rate (1/6). More interestingly, long-term cultured self-renewing CD133(+)CD44(+) cells enriched CD133(+)CD44(high) subset, which expressed epithelial to mesenchymal transition marker, were more invasive in vitro and responsible solely for liver metastasis in vivo. In conclusion, these data demonstrated for the first time that CD133(+)CD44(+) SLCCs were highly enriched in HCT116 cells and that metastatic SLCCs resided exclusively in a CD133(+)CD44(high) subpopulation.

Colorectal tumor derived fibronectin alternatively spliced EDA domain exserts lymphangiogenic effect on human lymphatic endothelial cells.

The objective of this study was to investigate whether tumor derived fibronectin alternatively spliced EDA domain has a lymphangiogenic potency on human lymphatic endothelial cells (LECs) in tumor generation to facilitate tumor lymphatic metastasis. LECs were cultured in three-dimentional culture system and treated with SW480 supernant which was highly rich in EDA, the result demonstrated that SW480 supernant could facilitate tube-like formations of LECs evidently when compared with controls. Integrinalpha9 was identified by immunofluorescence to be a specific receptor for EDA because we found co-locozation of EDA and integrinalpha9 on LECs as well as significant upregulation of integrinalpha9 in SW480 supernant treated group. Western blot and immunofluorescence revealed that EDA also had important roles accommodating the expressions of some key regulators of lymphangiogenesis such as Prox1 and F-actin so as to facilitate motility and sprouting of LECs. In addition, it had been confirmed that all of these effects could be inhibited markedly by EDA antibody (IST-9). Based on these findings, we assert that EDA derived from tumor cells has an important role in facilitating lymphangiogenesis of malignant tumor. Furthermore, EDA pathway may provide a potent target for tumor lymphatic metastasis therapy.

Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance.

Chemotherapy resistance in solid tumors is broad and encompasses diverse unrelated drugs. Three-dimensional multicellular spheroids (MCSs) are a good model for studying in vitro drug resistance. In the current study, we investigated the role of focal adhesion kinase (FAK) in 5-fluorouracil (5-FU) chemoresistance in colon carcinoma MCS culture cells. The expression of FAK was inhibited significantly by specific small hairpin RNA targeting FAK. The suppression of FAK expression did not affect the growth of spheroid cells. However, silencing of FAK combined with 5-FU treatment significantly decreased the 50% inhibitory concentration (IC(50)) of 5-FU and markedly increased the population of apoptosis cells, which was associated with the reduction of the levels of Akt and nuclear factor-kappa B (NF-kappaB). Moreover, knockdown of FAK could inhibit tumor growth and increase the sensitivity of the tumor to 5-FU in the nude mouse xenograft. These results indicate that while not affecting cellular proliferation in the absence of 5-FU, RNA interference targeting FAK potentiated 5-FU-induced cytotoxicity in vitro and in vivo, and partially reversed multicellular resistance, which may contribute to its chemosensitizing effect through efficiently suppressing Akt/NF-kappaB activity.

Down-regulation of alphav integrin by retroviral delivery of small interfering RNA reduces multicellular resistance of HT29.

Since multicellular resistance (MCR) has been shown to be as adhesion-dependent, the role of alphav integrin in MCR of HT29 was investigated in this paper. Down-regulation of alphav integrin reduced MCR to oxaliplatin, but did not detectably change the drug sensitivity of monolayers. Down-regulation of alphav integrin decreased phosphorylated NF-kappaB p65 and increased phosphorylated JNK2 in multicellular spheroids. Cell-cell adhesion and cell-cell junctions in multicellular spheroids resembled the in vivo situation. Since force, including adhesion, can activate alphav integrin, cell-cell contact may contribute to activation of alphav integrin, through which increasing phosphorylated p65 and decreasing phosphorylated JNK2 takes part in MCR.

Downregulation of the hexokinase II gene sensitizes human colon cancer cells to 5-fluorouracil.

BACKGROUND: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proven that specific inhibitors of hexokinase (HK) II, a key glycolytic enzyme, may enhance the activity of anticancer drugs. The aim of this study was to investigate the effect and mechanisms of HK II on chemosensitivity of a colon cancer cell line (LoVo) to 5-fluorouracil (5-FU). METHODS: HK II gene expression was downregulated by RNA interference in the colon cancer cell line LoVo, which was detected by Western blot analysis. Then the IC(50) value of 5-FU was determined in LoVo cells via MTT assay. In addition, cell apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry and caspase-3 activity by its substrate color reaction. RESULTS: In LoVo cells, HK II downregulation resulted in a decreased IC(50) value of 5-FU and increased apoptosis. Furthermore, HK II downregulation resulted in a decreased MPP and activation of caspase-3. CONCLUSION: Our findings suggest that targeting HK II may be beneficial for patients with colon cancer treated with 5-FU.

[Expression of hexokinase-II gene in human colon cancer cells and the therapeutic significance of inhibition thereof].

OBJECTIVE: To investigate the expression of hexokinase (HK)-II gene in human colon cancer cells and the therapeutic significance of inhibition of HK-II gene. METHODS: Human colon cancer cells of the lines HCT-116, LOVO, HT-19, and SW480 were cultured. The mRNA expression and protein expression of HK-II in these cells were detected by RT-PCR and Western blotting respectively. 3-Bromopyruvic acid (3-BrPA), a HK-II specific inhibitor, of different concentrations was added into the culture fluid of the colon cancer cells for 48 h, then MTT method was used to examine the proliferation of the cells. 3-BrPA combined with adriamycin (ADM) of the concentrations of 0.05 and 0.1 microg/ml, or with oxaliplatin (L-OHP) of the concentration of 0.5 and 1.0 microg/ml was added into culture fluid for 48 h to observe the change of the cell proliferation rate. 3-BrPA of different concentrations was co-cultivated with LOVO cells for 24 h, and then enzyme labeling instrument was used to measure the activity of caspase-3, a cell apoptosis signal. 3-BrPA and CaCl2 were added into the culture fluid of LOVO cells and then ultra-violet spectrum was used to detect the mitochondrial permeability transition pore (PTP) opening degree. Flow cytometry (FCM), with addition of 10 microg/ml Rh123, was used to measure the mitochondrial membrane potential (DeltaPsim) of LOVO cells. RESULTS: Both HK-II mRNA and HK-II protein were expressed in the HCT-116, LOVO, HT-19, and SW480 cells. Treated by 3-BrPA combined with ADM of the concentrations of 0.05 and 0.1 microg/ml for 48 h, the cell proliferation inhibiting rates in the 4 lines were increased from 5.1% +/- 1.3% and 10.5% +/- 2.0% to 46.5% +/- 3.2% and 57.9% +/- 3.3% respectively (all P < 0.01). Treated by 3-BrPA combined with L-OHP of the concentration of 0.5 and 1.0 microg/ml for 48 h, the cell proliferation inhibiting rates were increased from 19.2% +/- 6.1% and 32.2% +/- 2.2% to 48.4% +/- 3.2% and 60.5% +/- 4.6% respectively (all P < 0.01). 24 h after the co-cultivation of 3-BrPA of different concentrations, the caspase-3 activity of the LOVO cells was increased along with the increase of the concentration of 3-BrPA (P = 0.000). The PTP opening degrees induced by 3-BrPA and CaCl(2) of the LOVO cells were 40.0% +/- 3.5% and 37.4% +/- 2.3% respectively (P = 0.348). After treated with 100 microml/L 3-BrPA for 1, 3, and 5 h, the DeltaPsim decrease rates of the LOVO cells were 12.7%, 15.4%, and 26.8% respectively. CONCLUSION: HK-II gene may be an effective therapeutic target for gene may be an effective therapeutic target for gene may be an effective therapeutic target for colon cancer.

Clinical benefit of gemcitabine plus cisplatin 3-week regimen for patients with advanced non-small cell lung cancer: a prospective observational study.

BACKGROUND: Platinum-based chemotherapy has been proved effective in patients with advanced non-small cell lung cancer (NSCLC). This study evaluated the effectiveness and safety of first-line chemotherapy with gemcitabine plus cisplatin (GEM-Cis) 3-week regimen in routine care of Chinese patients with advanced NSCLC. METHODS: Two hundred and twenty-one patients with NSCLC stage IIIb or IV were enrolled and 209 were eligible for effectiveness and safety analysis. The median age was 58 (range 29 to 79) years. The percents of cases in stage IV and stage IIIb were 52.2% and 47.8%; of Karnofsky performance score (KPS) less than 80 and 80 - 100 were 37.3% and 62.7% and of adeno-cancer and non-adeno-cancer were 59.8% and 40.2%. The average number of completed chemotherapy cycles was three. Measures of effectiveness included clinical benefit, significant clinical response (SCR) and adverse effects of GEM-Cis in the treatment of NSCLC at stages IIIb/IV. RESULTS: KPS increased from 79 +/- 9 at baseline to 86 +/- 10 after chemotherapy (P < 0.01). Lung cancer symptom scale (LCSS) score of pain, dyspnea and cough increased from 77 +/- 24, 74 +/- 22 and 63 +/- 19 to 92 +/- 15, 90 +/- 14 and 86 +/- 15, respectively (P < 0.01). The clinical benefit rate was 85.2% [95% confidence interval (CI) 80.3% - 90.0%]. The SCR was 89.5% (95% CI 85.3% - 93.7%). Median survival time was 7.8 months (95% CI 7.1 months-9.1 months). Sixty-four patients (30.6%) experienced an adverse effect that was deemed clinically significant. Only one patient (0.5%) was hospitalized due to chemotherapy related adverse effects. Life-threatening toxicity was observed in two patients (1.0%). CONCLUSION: First-line chemotherapy with GEM-Cis in the routine care of Chinese patients with advanced NSCLC is effective and safe.

[Identification bcr-abl fusion gene in leukemia cells with oligonucleotide microarray].

To explore the application value of bcr-abl fusion gene deterction microarray in diagnosis, typing, choosing of treatment variant and prognosis judgment, probe for fusion gene detection was designed, oligonucleotide microarray was prepared; total RNA was extracted, reverse-transcripted and labeled by fluorescence, then cDNA was hybridized with microarray in order to detect bcr-abl fusion gene in leukemia cells. The results showed that better reaction conditions were gained by exploration of hybridizotion temperature and elution conditions, bcr-abl fusion gene in leukemia cells was detected by prepared miccroarray. In conclusion, oligonucleotide microarray is effective in detecting the fusion gene and has some unique advantages and certain clinical application value, but has some deficiency too. If microarray can be improved further, it could be used widely in the field of hematology.