Exploring the molecular characteristics of the malignant potential of gastric adenocarcinoma with enteroblastic differentiation.

AIMS: Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a rare subset of alpha-fetoprotein (AFP)-producing carcinomas with poor prognosis. However, the molecular features associated with the malignant potential of GEAD remain partially elucidated. METHODS AND RESULTS: In this study, the relationship between clinicopathological parameters and aggressive biological behaviour was analysed in 37 patients with GAED. The results showed that GAED tended to infiltrate the deep layer of the gastric wall and possessed more frequent vascular invasion than conventional gastric adenocarcinoma (CGA) (P < 0.001). All distant metastases were observed in the GAED group, not the CGA group (P < 0.001). High HER2 expression was found in nearly 24.32% of the informative cases, and none showed EBV-encoded RNA positivity or deficient mismatch repair. The most frequently mutated gene in GAED was p53. Programmed cell death-ligand 1 (PD-L1) immunostaining revealed 13 patients with a combined positive score (CPS) ≥ 5 (65%, 13 of 20). Thus, based on these molecular markers (immunostaining, in situ hybridisation and mutation analysis), GAED may be classified as a unique subgroup of the chromosomal instability subtype with HER2+ /EBV- /MSS/TP53+ /PD-L1+ . Next-generation sequencing analyses showed that mutations in the TOPI, ELOA and NOTCH3 genes were found only in GAED, and abnormally expressed genes in GAED were significantly enriched in hepatocellular carcinoma-, gland development-, and gastric cancer-related pathways. CONCLUSION: The HER2+ /EBV- /MSS/TP53+ /PD-L1+ profile and hepatocellular carcinoma-related pathways may be significant in the malignant potential of GAED. In addition to anti-HER2 therapy, immune check-point inhibitors may be an effective treatment option for patients with GAED.

RNAi-mediated silencing of Anxa2 inhibits breast cancer cell proliferation by downregulating cyclin D1 in STAT3-dependent pathway.

Although the upregulated expression of Anxa2 has been implicated in carcinogenesis, cancer progression, and poor prognosis of cancer patients, the detailed molecular mechanisms involved in these processes remain unclear. In this study, we investigated the effect of Anxa2 downregulation with small interference RNA on breast cancer proliferation. To explore molecular mechanisms underlying Anxa2-mediated cancer cell proliferation. We analyzed cell cycle distribution and signaling pathways using semi-quantitative real-time PCR and Western blotting. Anxa2 depletion in breast cancer cells significantly inhibited cell proliferation by decelerating cell cycle progression. The retarded G1-to-S phase transition in Anxa2-silenced cells was attributed to the decreased levels of cyclin D1, which is a crucial promoting factor for cell proliferation because it regulates G1-to-S phase transition during cell cycle progression. We provided evidence that Anxa2 regulates epidermal growth factor-induced phosphorylation of STAT3. The reduced expression of phosphorylated STAT3 is the main factor responsible for decreased cyclin D1 levels in Anxa2-silenced breast cancer cells. Our results revealed the direct relationship between Anxa2 and activation of STAT3, a key transcription factor that plays a pivotal role in regulating breast cancer proliferation and survival. This study provides novel insights into the functions of Anxa2 as a critical molecule in cellular signal transduction and significantly improves our understanding of the mechanism through which Anxa2 regulates cell cycle and cancer cell proliferation.

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer.

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.

[Relationship of c-FLIP(L) protein expression with molecular subtyping and clinical prognosis in invasive breast cancer].

OBJECTIVE: To investigate the expression of apoptotic regulator c-FLIP(L) in invasive breast carcinoma tissues, and to evaluate its correlation with molecular subtyping and clinical prognosis. METHODS: Immunohistochemistry using EnVision staining for c-FLIP(L) was performed in 264 cases of invasive breast carcinomas and matched adjacent normal breast tissue samples from January 1996 to December 1999. ER, PR, HER2, Ki-67, CK5/6 and EGFR were evaluated by immunohistochemistry in order to classify the tumors into five molecular subtypes and the difference of c-FLIP(L) expression in these molecular subtypes was also analyzed. The influence of c-FLIP(L) expression on prognosis was evaluated by Kaplan-Meier curves and multi-factor Cox proportional risk model. RESULTS: High expression of c-FLIP(L) was observed in 84.5% (223/264) of cases of invasive breast carcinomas which were significantly higher than the 45.1% (119/264) of cases in adjacent normal epithelium of breast (χ² = 89.78, P = 0.000). The expression of c-FLIP(L) in luminal B (HER2 positive) and basal-like breast cancers was 78.1% (25/32) and 46.2% (18/39), respectively, with significant difference (P < 0.05). Moreover, the expression of c-FLIP(L) in luminal B (HER2 positive) was higher than in luminal A cancers (P < 0.05), and the expression of c-FLIP(L) in HER2 positive cancers was higher than in basal-like cancers (P < 0.01). C-FLIP(L) showed deep yellow staining in node positive breast cancer with a high-expression rate of 93.1% (134/144); whereas the expression was sporadic and light yellow in node negative breast cancer with a lower high-expressed rate of 72.5% (87/120, P < 0.01). C-FLIP(L) expression had significant influence on disease-free survival time, with c-FLIP(L)-positive patients showing poor prognosis (P < 0.01). Multi-factor Cox proportional risk model analysis showed that expression of c-FLIP(L), lymph nodes status and molecular subtypes were independent prognostic factors for invasive breast carcinomas (P < 0.05). CONCLUSIONS: C-FLIP(L) is highly expressed in invasive breast carcinomas, and its expression level is closely related to the molecular subtypes and clinical prognosis of breast cancer patients. Thus, c-FLIP(L) could be used as an important tumor marker for personalized cancer therapy and prognostic prediction.

Circulating endothelial cells and tumor blood volume as predictors in lung cancer.

The current criteria for evaluating antiangiogenic efficacy is insufficient as tumor shrinkage occurs after blood perfusion decreases. Tumor blood volume (BV) in computed tomography perfusion imaging and circulating endothelial cells (CEC) might predict the status of angiogenesis. The present study aimed to validate their representation as feasible predictors in non-small-cell lung carcinoma (NSCLC). A total of 74 patients was categorized randomly into two arms undergoing regimens of vinorelbine and cisplatin (Navelbine and platinum [NP]) with rh-endostatin or single NP. The response rate, perfusion imaging indexes and activated CEC (aCEC) during treatment were recorded. Progression-free survival (PFS) was determined through follow up. Correlations among the above indicators, response and PFS were analyzed: aCEC increased significantly in cases of progressive disease after single NP chemotherapy (P = 0.024). Tumor BV decreased significantly in cases with a clinical benefit in the combined arm (P = 0.026), whereas inverse correlations existed between ∆aCEC (post-therapeutic value minus the pre-therapeutic value) and PFS (P = 0.005) and between ∆BV and PFS (P = 0.044); a positive correlation existed between ∆aCEC and ∆BV. Therefore, both aCEC and tumor BV can serve as predictors, and detection of both indicators can help evaluate the chemo-antiangiogenic efficacy in NSCLC more accurately.

An anti-tumor nanoparticle, [Gd@C82(OH)22]n, induces macrophage activation.

[Gd@C82(OH)22]n, a fullerene-based nanoparticle, exhibits potent anti-tumor effects in mouse tumor-bearing models without detectable toxicity and the pathological studies revealed a massive infiltration of leukocytes in the residual tumors of [Gd@C82(OH)22]n-treated mice. We report here that [Gd@C82(OH)22]n promotes macrophages secreting pro-inflammatory cytokines IL-6 and TNF-alpha, enhances the expression of MHC-II and costimulatory molecules such as CD40 and CD54, increases endocytosis and cell adhesion. Furthermore, [Gd@C82(OH)22]n-treated macrophages became functionally activated as illustrated by their capacity to activate allogeneic T cells. Taken together, our results indicate that [Gd@C82(OH)22]n nanoparticle is a potent activator of macrophages, which may in part account for its potent anti-tumor effect.

[Comparison of the effect of rh-endostatin on intratumoral and myocardial micrangium in mice].

OBJECTIVE: To compare the effect of rh-endostatin on micrangium in tumor and myocardial tissue in nude mice. METHODS: Nude mice were randomized into 4 groups (10 mice in each group), blank control group (without tumor burden, received NS 100 µl×d(-1) injection), drug control group (without tumor burden, received rh-endostatin 400 µg×d(-1) injection), model group (with tumor burden, received NS 100 µl×d(-1) injection) and treatment group (with tumor burden, received rh-endostatin 400 µg×d(-1) injection) for 28 days. The tumor volume and body weight of the mice were measured before and after administration. The expression of CD34, MMP-2, MMP-9, HIF-1α and VEGF in the myocardium and tumor were detected by immunohistochemistry. The vascular structure was observed by immunoenzymatic CD34 and Masson double staining. RESULTS: The increase of tumor volume of the treatment group [(48.18 ± 37.31) mm(3)] was significantly lower than that in the model group [(113.80 ± 73.27) mm(3)). The changes of body weight was not significant different among the four groups. After treated with rh-endostatin, the expressions of MMP-9 and VEGF in tumors were significantly down-regulated, but the expressions of MMP-2 and HIF-1α in the tumor were not. The microvessel density (MVD) in the tumors of treatment group was significantly decreased compared with that of model group. The proportion of tumor vessels covered by collagen in the treatment group was increased compared with that of the model group. However, MVD and micrangium in myocardium were not changed significantly. CONCLUSION: Rh-endostatin can decrease the expression of MMP-9, VEGF and MVD, inhibit the tumor growth and normalize tumor micrangium in tumor but not weaken the MMPs and MVD of mature micrangium in myocadium.

[Observation of the antitumor effect of endostar combined with docetaxel under different administration sequences].

OBJECTIVE: To observe and analyze the antitumor effect of endostar combined with docetaxel under different administration sequences. METHODS: Nude mice with xenograft tumor (A549 cell line) were randomized into 3 groups, 8 mice/group: (1) Concurrent administration group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d1-d19); (2) Endo-first group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d16-d34); (3) Model group (positive control, tumor-bearing mice without treatment, each mouse: physiological saline, 100 µl/d, d1-d35, water for injection, 200 µl/d, d1-d35, every 3 days), and blank control group (negative control, normal mice without treatment, 8 mice), the administration method was the same to the model group. The volume of tumor and the weight of mouse were measured during treatment. Circulating endothelial cells (CECs) were detected by flowcytometry, and the expression of matrix metalloproteinase (MMP-2, MMP-9), the tissue inhibitor of MMP (TIMP-1, TIMP-2), the extracellular MMP inducer (EMMPRIN), CD34, α-smooth muscle actin (α-SMA) were determined by immunohistochemistry. RESULTS: The tumor growth of concurrent administration group (39.94 mm(3)) was lower than that of the endo-first group [(99.57 ± 74.48) mm(3)] during treatment, both of them were smaller than that of the model group [(217.67 ± 95.44) mm(3), P < 0.05]. The amount of CECs in the endo-first group [(77.25 ± 24.02) cells/10(4) cells] was more than that of the concurrent administration group [(25.86 ± 11.77) cells/10(4) cells], the model group [(14.71 ± 11.07) cells/10(4) cells], and the blank control group [(12.90 ± 11.20) cells/10(4) cells, P < 0.01]. The expression of MMPs in the treatment groups was obviously downregulated. The expressions of TIMP-1 in the endo-first group and TIMP-2 in the concurrent administration group were upregulated (P < 0.05). The expression of EMMPRIN was significantly down-regulated in the concurrent administration group (P < 0.05). The MVD and α-SMA expressions of the treatment groups were less than that of the model group (P < 0.05). CONCLUSION: In comparison with the endo-first group, the anti-tumor effect and survival quality of the concurrent administration group are better. Both of the administration groups may have "vascular normalization effect" by down-regulating MMPs expression through different points, and inhibit the cancer-induced stromal reaction, restraining the cancer progress to a certain extent. The changes of CECs should be a dynamic process with an initial rise in the early-stage suggesting the decrease of vascular bed and subsequent decline ascribed to apoptosis of CECs and the tumor-regression after combined therapy. Investigation of its dynamic changes may be helpful to know the change of tumor burden and vascular bed and predict the antitumor effect.

Anxa2 plays a critical role in enhanced invasiveness of the multidrug resistant human breast cancer cells.

Multidrug resistance (MDR) is the major cause of failure in cancer chemotherapy. Recent reports even suggest that MDR is associated with elevated invasion and metastasis of tumor cells. In the current study, we used a proteomic approach to identify genes that play an important role in MDR induced cell migration. 2D-PAGE and MALDI-TOF/MS-based proteomics approach were used to separate and identify differentially expressed proteins between MCF-7 and MCF-7/ADR, a p-glycoprotein-overexpressing adriamycin-resistance breast cancer cell line. Annexin a2 (Anxa2) was identified as highly expressed in MCF-7/ADR cells, but not in MCF-7 cells. Small interference RNA-mediated gene suppression demonstrated that Anxa2 was required for enhanced cell proliferation and invasion of the MCF-7/ADR cells. Down-regulation of Anxa2 alone was not sufficient to revert the cell sensitivity to adriamycin, suggesting that Anxa2 was not required for MDR phenotype. Taken together, our results showed that expression of Anxa2 is enhanced when cancer cells, MCF-7, acquired drug resistance and it plays an essential role in MDR-induced tumor invasion.

Expression level of beta protein 1 mRNA in Chinese breast cancer patients: a potential molecular marker for poor prognosis.

Recent studies revealed high ectopic beta protein 1 (BP1) expression in breast cancer. Remarkably, up to 100% (18/18) of estrogen receptor (ER)-negative tumors and 89% (25/28) of tumors from African American women were BP1-positive. However, the role of BP1 in breast cancer development and its clinical significance still has not been well defined. In the present study, we analyzed the quantitative level of BP1 mRNA in breast carcinomas using real-time polymerase chain reaction and aimed to elucidate its association with tumor characteristics and patient prognosis. Our data showed that BP1 mRNA was expressed at significantly higher levels in tumors with lymph node metastasis, with a high histological grade, and in those that were of ER-negative status. Furthermore, overexpression of BP1 was significantly associated with poor outcome of patients harboring tumors with a high histological grade and negative ER. Using both in vitro and in vivo systems, we also showed that the transcript level of BP1 was positively correlated to the growth rate of breast tumor cells. Taken together, our results support the notion that BP1 might contribute to breast neoplastic transformation or tumor progression and suggest for the first time that BP1 mRNA level has potential as a prognostic predictor for breast cancer.

Sequence variations of mitochondrial DNA D-loop region are highly frequent events in familial breast cancer.

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations. The non-coding displacement (D)-loop, especially a mononucleotide repeat (poly-C) between 303 and 315 nucleotides (D310), has been recently identified as a frequent hotspot of mutations in human neoplasia, including breast cancer. To further explore the sequence variations of mitochondrial D-loop region in familial breast cancer and their possible associations with breast cancer risk, PCR-SSCP and direct DNA sequencing methods were used to detect the variants of the mtDNA D-Loop in 23 familial breast cancer patients as well as three high-risk cancer families. Compared to that in sporadic breast tumors (53.3%, 16/30) and healthy blood donors (6.7%, 2/30), we identified a total of 126 sequence alterations in 23/23 (100%) of familial breast cancer patients, including eight novel nucleotide variants. Among these changes, A to G at nt.263, T to C at nt.489, T to C at nt.310, TC insertion at nt.311, CA deletion at nt.522, and C to G at nt.527 were highly frequent ones. In addition, among three high-risk cancer families, we found that individuals affected with breast cancer harbored more mtDNA sequence variants in mtDNA D310 area than other affected family members. Together, our data indicate that sequence variants within the mtDNA D-Loop region are frequent events in Chinese familial breast cancer patients. Some of these nucleotide abnormalities, particularly those in D310 segment, might be involved in the breast carcinogenesis and could be included in a panel of molecular biomarkers for cancer susceptibility early-detection strategy.

[Biological effect of endostatin on transplanted human lung adenocarcinoma Calu-6 tumor in nude mice].

OBJECTIVE: To assess the effect of endostatin on growth and neoplastic angiogenesis in transplanted human lung adenocarcinoma Calu-6 tumor in nude mice. METHODS: To treat Calu-6 tumor-bearing mice with endostatin at different doses, and to record the changes of the tumor size. The expressions of survivin, VEGF, COX-2 and MVD in tumor tissue were examined by immunohistochemistry staining, circulating endothelial cells (CECs) by flow cytometry and mRNA of CD146 and CD105 by RT-PCR and real-time PCR. RESULTS: After endostatin treatment, the tumor size was conspicuously shrunk, and the expressions of survivin, COX-2 and VEGF protein and MVD in tumor tissue decreased concomitantly with the significant difference between each of trial groups and control group (all P < 0.05). Both CECs and mRNA of CD146 and CD105 diminished remarkably. A positive correlation between both exhibition and change of amount of activated CECs and survivin, VEGF expression and MVD count in tumor tissue was found. CONCLUSION: Endostatin can decrease the expression of survivin, COX-2, VEGF and MVD, and to inhibit the growth of transplanted tumor. Activated CECs may probably serve as an ideal marker to predict the efficacy and prognosis of anti-angiogenesis therapy.

Depletion of mitochondrial DNA by ethidium bromide treatment inhibits the proliferation and tumorigenesis of T47D human breast cancer cells.

In order to investigate the role of mitochondrial DNA (mtDNA) in human breast cancer cell proliferation and apoptosis, a mtDNA-deficient cell line, T47D rho(0), was generated following a long-term exposure to ethidium bromide (EtBr). T47D rho(0) cells showed a marked decrease in mitochondrial membrane potential (DeltaPsi(m)). However, the apoptosis rate of T47D rho(0) cells was the same as that of their parental cells, suggesting that the change in DeltaPsi(m) was insufficient to induce cell death. Electromicroscopy revealed a profound alteration of mitochondrial morphology, which was consistent with the loss of mtDNA and the decrease in DeltaPsi(m). Disruption of mtDNA resulted in a slower proliferation rate in tissue culture and a reduction in anchorage-independent growth. An in vivo assay revealed a severe impairment of tumorigenicity in T47D rho(0) cells, indicating the biological relevance of in vitro studies. Taken together, our results suggest that the integrity of mtDNA plays a critical role in human breast cancer cell proliferation and tumorigenesis.

FGF1 and IGF1-conditioned 3D culture system promoted the amplification and cancer stemness of lung cancer cells.

Lung cancer stem cells (LCSCs) are considered as the cellular origins of metastasis and relapse of lung cancer. However, routine two-dimensional culture system (2D-culture) hardly mimics the growth and functions of LCSCs in vivo and therefore significantly decreases the stemness activity of LCSCs. In this study, we constructed a special BME-based three-dimensional culture system (3D-culture) to amplify LCSCs in human lung adenocarcinoma cell line A549 cells and found 3D-culture promoted the enrichment and amplification of LCSCs in A549 cells displaying higher proliferation potential and invasion activity, but lower apoptosis. The expression and secretion levels of FGF1 and IGF1 were dramatically elevated in 3D-culture compared to 2D-culture. After growing in FGF1 and IGF1-conditioned 3D-culture, the proportion of LCSCs with specific stemness phenotypes in A549 cells significantly increased compared to that in conventional 3D suspension culture system. Further results indicated that FGF1 and IGF1 promoted the amplification and cancer stemness of LCSCs dependent on MAPK signaling pathway. Our data firstly established a growth factors-conditioned 3D-culture for LCSCs and demonstrated the effects of FGF1 and IGF1 in promoting the enrichment and amplification of LCSCs which might provide a feasible cell model in vitro for both mechanism study and translational research on lung cancer.

Predictors for the efficacy of Endostar combined with neoadjuvant chemotherapy for stage IIIA (N2) NSCLC.

BACKGROUND: Endostar (rh-endostatin) is a new recombinant human endostatin, which could inhibit cell proliferation, angiogenesis, and tumor growth. OBJECTIVE: To explore anti-angiogenesis short-term efficacy combined with neoadjuvant chemotherapy for stage IIIA (N2) non-small cell lung cancer (NSCLC), and identify the potential predictive factors. METHODS: We pathologically examined 26 patients diagnosed with stage IIIA (N2) NSCLC who received NP chemotherapy alone or combined with Endostar, respectively. RESULTS: Our results indicated that total clinical benefit rate (CBR) 87.5% and 64% (p= 0.76), respectively. The clinical benefit (CB) patients in the treatment group showed significant changes in endothelial progenitor cells (EPC), vascular endothelial growth factor (VEGF), blood flow (BF), permeability surface (PMS), and microvascular density (MVD) before and after treatment. Compared with CB patients in the control group, changes in EPC and MVD (only) before and after treatment were significant. The variation of EPC, PMS, and MVD before and after treatment in the treatment group showed positive correlation with tumor regression rate (TRR) and the variation of MVD, whereas those of EPC and PMS demonstrated positive correlations with variation of MVD before and after treatment. CONCLUSION: Our findings suggested that PMS and EPC may be used as a predictive factor for the short-term efficacy of the combined therapy in NSCLC.

[A quantitative immuno-fluorescence analysis of alpha-tubulin and gamma-tubulin proteins in precancerous lesion and carcinoma of the breast and their clinical significance].

OBJECTIVE: In order to explore the correlation between the centrosome aberration and oncogenesis of the breast carcinoma, the expression of alpha-tubulin and gamma-tubulin proteins in breast precancerous lesions, ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC) was investigated. METHODS: Quantitative immunofluorescence analysis was performed for measuring centrosome proteins by FITC-labeled monoclonal anti-alpha and anti-gamma-tubulin antibodies in 90 cases with precancerous lesions, DCIS and IDC of the breast, respectively. Normal breast tissue from 30 cases were taken as control group. RESULTS: The average of positive (FITC-labeled) cells were 3.2, 11.6, 14.8, 23.1 (alpha-tubulin) and 3.3, 10.7, 14.5, 24.5 (gamma-tubulin) in four groups, respectively. There were significant differences of alpha-tubulin or gamma-tubulin expression among those groups (P = 0.000), respectively. The highest expression quantity was in IDC group and the lowest was in normal breast tissue. Their expression was significantly associated with cellular proliferation and differentiation. CONCLUSION: There is over-expression of the centrosome tubulin protein in the precancerous stage of the breast. The centrosome aberration may play an important role during the crucial early step of oncogenesis and it may promote the cellular cancerization or transformation into malignancy. Quantitative immuno-fluorescence analysis and immunohistochemistry can be complementary each other.

[Circulating endothelial cells in the peripheral blood of advanced NSCLC patients].

OBJECTIVE: To investigate the changes and clinical value of circulating endothelial cells (CEC) in the peripheral blood of advanced NSCLC patient. METHODS: Sixty-seven advanced NSCLC patients were randomly divided into either the treatment group with NP plus endostatin or control group with NP alone. Level of CEC and cytokeratin (CK) in the peripheral blood were measured by flow cytometry. RESULTS: The response rate and benefit rate was 44.4%, 80.0% in the treatment group, and 27.3%, 50.0% in the control group, respectively (P = 0.176 and P = 0.012). Time to tumor progression (TTP) was 146.7 days in the treatment group and 91.1 days in the control group (P = 0.061). However, when the cut-off of TTP was defined as > 170 days, there was a significant difference between two groups (cut-off = 170, P = 0.034; cut-off = 180, P = 0.009). The number of CEC decreased by 0.29 +/- 0.47 in the treatment group and by 0.01 +/- 0.43 in the control group (P = 0.033). The correlation between CEC and CK was found to be positive either before (r = 0.381, P = 0.013) or after the treatment (r = 0.450, P = 0.004). CONCLUSION: Chemotherapy combined with endostatin is superior to chemotherapy alone in the treatment of NSCLC. CEC, as a biomarker, may be useful in predicting the efficacy of the combined treatment.

Expression of centrosomal tubulins associated with DNA ploidy in breast premalignant lesions and carcinoma.

The centrosome plays an essential role in chromosomal segregation during cell division. Centrosome dysfunction might lead to aneuploidy and chromosomal instability. Invasive breast tumors with centrosome amplification often show aneuploidy. Flow cytometry (FCM) was used to examine the aneuploidy rate in 30 cases of each of the following seven tissue types: normal breast tissue, usual ductal hyperplasia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, high-grade ductal carcinoma in situ, low-grade invasive ductal carcinoma, and high-grade invasive ductal carcinoma. Centrosomal α, γ-tubulin expression was examined by FCM immunofluorescence and compared between diploid and aneuploid cells. The aneuploidy rate was 0, 6.7%, 26.7%, 30.0%, 46.7%, 56.7%, and 86.7%, respectively, in the seven tissue types. The percentage of cells expressing α- and γ-tubulins was significantly different between the seven groups, and the positive rate of α- and γ-tubulin expression in ADH, DCIS and IDC was higher than that in NBT and UDH. The percentage of cells expressing α- and γ-tubulins in the diploid state was significantly lower than that in the aneuploid state. Expression of centrosomal α- and γ-tubulins seems to be associated with DNA ploidy in breast premalignant lesions and carcinoma during the progression of breast cancer.

Dysfunctional activation of neurotensin/IL-8 pathway in hepatocellular carcinoma is associated with increased inflammatory response in microenvironment, more epithelial mesenchymal transition in cancer and worse prognosis in patients.

AIM: To investigate the role of neurotensin (NTS) in hepatocellular carcinoma (HCC) sub- grouping and the clinical and pathological significance of activation of NTS/IL-8 pathway in HCC. METHODS: The genome-wide gene expression profiling were conducted in 10 pairs of cancer tissues and corresponding normal adjacent tissues samples using Affymetrix GeneChip® Human Genome U133 Plus 2.0 microarray to screen differentially expressing genes and enrich dysfunctional activated pathways among different HCC subgroups. The levels of NTS protein and multiple inflammation and epithelial mesenchymal transition (EMT) related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin, ß-Catenin and Vimentin were examined in 64 cases of paraffin-embedded HCC samples using immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) were compared. RESULTS: A subgroup of HCC characterized by up-regulated NTS expression was accompanied by up-regulated inflammatory responses and EMT. The direct interaction between NTS and IL-8 was identified by pathway enrichment analysis. Significantly increased IL-8 protein was confirmed in 90.91% of NTS(+) HCC samples and significantly positively correlated to the levels of NTS protein in cancer tissues (P = 0.036), which implied activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 correlated with co-expression of NTS and IL-8. Increased infiltration of CD68(+) macrophages and more cancer cells displaying EMT features were found in NTS(+)IL-8(+) samples. The co-expression of NTS and IL-8 in cancer significantly correlated with the clinical outcomes, as the mortality rate of NTS(+)IL-8(+) HCC patients is 2.5-fold higher than the others after the surgery (P = 0.022). Accordingly, the OS of NTS(+)IL-8(+) HCC patients significantly decreased who are under a higher hazard of death at an expected hazard ratio (HR) of 3.457. CONCLUSION: Dysfunctional activation of the NTS/IL-8 pathway was detected in HCC which is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.

Effects of recombinant human endostatin and its synergy with cisplatin on circulating endothelial cells and tumor vascular normalization in A549 xenograft murine model.

PURPOSE: Endostatin can normalize the tumor vasculature to some extent. However, exact length of its time window and corresponding markers for tumor vascular normalization are needed to be explored. METHODS: The A549 lung adenocarcinoma xenograft murine model was treated with recombinant human endostatin (rh-endostatin) for 14 days. Cisplatin was combined in different schedules. The effects of rh-endostatin on circulating endothelial cells (CECs) by flow cytometry, tumor vasculature and angiogenesis-related factors by confocal microscope and immunohistochemistry, and anti-tumor efficacy of cytotoxic drugs were observed. RESULTS: The activated CECs (aCECs) were increased on day 7 and decreased on day 10, and apoptotic CECs were increased on day 10. Tumor vasculature was transiently normalized with increased collagen coverage, decreased vessel permeability, intratumoral hypoxia, and microvascular density from day 7 to 10 after rh-endostatin administration. Extracellular matrix metalloproteinase inducer, vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 were transiently decreased by rh-endostatin from day 4 to 10, whereas the opposite effects were observed with tissue inhibitors of matrix metalloproteinase (TIMP)-1 and TIMP-2. The maximal anti-tumor effects of cisplatin were observed on administration from day 5 to 9 after rh-endostatin initial administration. CONCLUSIONS: Rh-endostatin could transiently normalize tumor vasculature, probably via regulation of both pro- and anti-angiogenesis factors. The synergistic efficacy of anti-angiogenesis and chemotherapy was found during "the normalization window". CEC could be a feasible blood biomarker for defining "vascular normalization window" and providing the evidence to make an optimizing combination therapeutic schedule in human tumor.