A morphometric tool applied to angiogenesis research based on vessel segmentation

To investigate the relationship between P-glycoprotein (Pgp), glutathione S-transferase π (GST-π) and topoisomerase II (Topo II) expression and human gastric cancer chemoresistance in vitro. Primary single-cell suspensions were prepared from fresh specimens of primary gastric cancer and exposed to hydroxycamptothecin (HCPT), cisplatin (CDDP), 5-fluorouracil (5-FU), adriamycin (ADM) and mitomycin (MMC) for 48 h. Cell metabolic activity and rate of inhibition were evaluated using tetrazolium (MTT) assay. Pgp, GST-π and Topo II expression was determined in gastric carcinoma tissue samples using immunohistochemistry. Chemosensitivity of the gastric cancer cells varied; the rates of inhibition of cells exposed to HCPT, CDDP and 5-FU were significantly higher than that of cells exposed to ADM and MMC (p?相似文献   

Upregulation of periostin prevents P53-mediated apoptosis in SGC-7901 gastric cancer cells

Periostin is frequently upregulated in human cancers including gastric cancer and implicated in cancer cell proliferation, invasion, and epithelial–mesenchymal transition. This study was undertaken to investigate the effects of periostin overexpression on the chemosensitivity of gastric cancer cells. We constructed a stable cell line overexpressing periostin in SGC-7901 human gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that periostin had no influence on the proliferation of SGC-7901 cells. Compared to empty vector-transfected cells, overexpression of periostin rendered SGC-7901 cells more resistant to cisplatin or 5-fluorouracil (5-FU)-induced apoptosis, accompanying with less release of cytochrome c from mitochondria and diminished cleavage of caspase-3 and poly (ADP-ribose) polymerase. Periostin-overexpressing cells treated with cisplatin or 5-FU showed significantly (p < 0.05) decreased expression of Bax and p53 proteins and increased expression of Bcl-2 protein, when compared to drug-treated mock counterparts. Restoration of p53 expression by delivering wild-type p53 gene resulted in a marked increase in drug-induced apoptosis in periostin-overexpressing SGC-7901 cells. Periostin overexpression elevated the phosphorylation of Akt. Pretreatment of periostin-overexpressing cells with an Akt inhibitor, MK-2206, partially rescued periostin-mediated inhibition of p53 expression and drug resistance. Taken together, our data indicate that periostin confers protection against cisplatin or 5-FU-induced apoptosis in SGC-7901 cells, likely through modulating the Akt/p53 pathway, and thus represents a potential therapeutic target in gastric cancer.     

Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer

Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.     

Dichloroacetate attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer through the regulation of glucose metabolism

In this study, we investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profiles of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20 mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues relative to the normal gastric tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20 mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism.     

Integration of DNA copy number alterations and transcriptional expression analysis in human gastric cancer

Background

Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level.

Principal Findings

We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis.

Conclusions

This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets.     

TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

Cisplatin is a cytotoxic platinum compound that triggers DNA crosslinking induced cell death, and is one of the reference drugs used in the treatment of several types of human cancers including gastric cancer. However, intrinsic or acquired drug resistance to cisplatin is very common, and leading to treatment failure. We have recently shown that reduced expression of base excision repair protein XRCC1 (X-ray repair cross complementing group1) in gastric cancerous tissues correlates with a significant survival benefit from adjuvant first-line platinum-based chemotherapy. In this study, we demonstrated the role of XRCC1 in repair of cisplatin-induced DNA lesions and acquired cisplatin resistance in gastric cancer by using cisplatin-sensitive gastric cancer cell lines BGC823 and the cisplatin-resistant gastric cancer cell lines BGC823/cis-diamminedichloridoplatinum(II) (DDP). Our results indicated that the protein expression of XRCC1 was significantly increased in cisplatin-resistant cells and independently contributed to cisplatin resistance. Irinotecan, another chemotherapeutic agent to induce DNA damaging used to treat patients with advanced gastric cancer that progressed on cisplatin, was found to inhibit the expression of XRCC1 effectively, and leading to an increase in the sensitivity of resistant cells to cisplatin. Our proteomic studies further identified a cofactor of 26S proteasome, the thioredoxin-like protein 1 (TXNL1) that downregulated XRCC1 in BGC823/DDP cells via the ubiquitin-proteasome pathway. In conclusion, the TXNL1-XRCC1 is a novel regulatory pathway that has an independent role in cisplatin resistance, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.     

Molecular Marker Identification for Relapse Prediction in 5-FU-Based Adjuvant Chemotherapy in Gastric and Colorectal Cancers

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.     

A Long Non-Coding RNA snaR Contributes to 5-Fluorouracil Resistance in Human Colon Cancer Cells

Several types of genetic and epigenetic regulation have been implicated in the development of drug resistance, one significant challenge for cancer therapy. Although changes in the expression of non-coding RNA are also responsible for drug resistance, the specific identities and roles of them remain to be elucidated. Long non-coding RNAs (lncRNAs) are a type of ncRNA (> 200 nt) that influence the regulation of gene expression in various ways. In this study, we aimed to identify differentially expressed lncRNAs in 5-fluorouracil-resistant colon cancer cells. Using two pairs of 5-FU-resistant cells derived from the human colon cancer cell lines SNU-C4 and SNU-C5, we analyzed the expression of 90 lncRNAs by qPCR-based profiling and found that 19 and 23 lncRNAs were differentially expressed in SNU-C4R and SNU-C5R cells, respectively. We confirmed that snaR and BACE1AS were downregulated in resistant cells. To further investigate the effects of snaR on cell growth, cell viability and cell cycle were analyzed after transfection of siRNAs targeting snaR. Down-regulation of snaR decreased cell death after 5-FU treatment, which indicates that snaR loss decreases in vitro sensitivity to 5-FU. Our results provide an important insight into the involvement of lncRNAs in 5-FU resistance in colon cancer cells.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression

Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.     

Gene expression signature analysis identifies vorinostat as a candidate therapy for gastric cancer

Background

Gastric cancer continues to be one of the deadliest cancers in the world and therefore identification of new drugs targeting this type of cancer is thus of significant importance. The purpose of this study was to identify and validate a therapeutic agent which might improve the outcomes for gastric cancer patients in the future.

Methodology/Principal Findings

Using microarray technology, we generated a gene expression profile of human gastric cancer–specific genes from human gastric cancer tissue samples. We used this profile in the Broad Institute''s Connectivity Map analysis to identify candidate therapeutic compounds for gastric cancer. We found the histone deacetylase inhibitor vorinostat as the lead compound and thus a potential therapeutic drug for gastric cancer. Vorinostat induced both apoptosis and autophagy in gastric cancer cell lines. Pharmacological and genetic inhibition of autophagy however, increased the therapeutic efficacy of vorinostat, indicating that a combination of vorinostat with autophagy inhibitors may therapeutically be more beneficial. Moreover, gene expression analysis of gastric cancer identified a collection of genes (ITGB5, TYMS, MYB, APOC1, CBX5, PLA2G2A, and KIF20A) whose expression was elevated in gastric tumor tissue and downregulated more than 2-fold by vorinostat treatment in gastric cancer cell lines. In contrast, SCGB2A1, TCN1, CFD, APLP1, and NQO1 manifested a reversed pattern.

Conclusions/Significance

We showed that analysis of gene expression signature may represent an emerging approach to discover therapeutic agents for gastric cancer, such as vorinostat. The observation of altered gene expression after vorinostat treatment may provide the clue to identify the molecular mechanism of vorinostat and those patients likely to benefit from vorinostat treatment.     

Silencing of XB130 Is Associated with Both the Prognosis and Chemosensitivity of Gastric Cancer

XB130 is a newly characterized adaptor protein that was reported to promote thyroid tumor growth, but its role in the progression of other kinds of cancer such as gastric cancer (GC) remains unknown. Accordingly, we investigated the association between XB130 expression and the prognosis of GC patients. The subjects were 411 patients with GC in stages I to IV. XB130 expression was examined in surgical specimens of GC. Kaplan-Meier analysis and the Cox proportional hazards model were used to assess the prognostic significance of XB130 for survival and recurrence. Moreover, GC cells stably transfected with XB130 short hairpin RNA were established to analyze the effect of XB130 on sensitivity of chemotherapy. The results show that both XB130 mRNA and protein expression were detectable in normal gastric tissues. The overall survival time of stage IV patients and the disease-free period after radical resection of GC in stage I-III patients were significantly shorter when immunohistochemical staining for XB130 was low than when staining was high (both p<0.05). XB130 expression also predicted tumor sensitivity to several chemotherapy agents. Viability of both XB130-silenced SGC7901 cells and wild-type cells was suppressed by 5-fluorouracil (5-FU), cisplatin, and irinotecan in a dose-dependent way, but cisplatin and irinotecan were more sensitive against sXB130-silenced GC cells and 5-FU showed higher sensitivity to wild-type cells. When treated by 5-FU, patients with high expression of XB130 tumors had a higher survival rate than those with low expression tumors. These findings indicate that reduced XB130 protein expression is a prognostic biomarker for shorter survival and a higher recurrence rate in patients with GC, as well as for the response to chemotherapy.     

中药组方黄芩汤抑制胃癌细胞增殖的实验研究

目的探讨黄芩汤对胃癌细胞生长增殖及干细胞标志物表达的影响。 方法采用体外优化培养的胃癌细胞株SGC-7901模型,分别加入生理盐水、5-FU、黄岑汤和5-FU+黄芪汤干预,MTT法检测胃癌细胞增殖情况,Real-time PCR检测胃癌干细胞相关表面标志物CD44、EpCAM、CD90的表达情况;并进一步利用胃癌细胞裸鼠荷瘤模型,观察黄岑汤协同5-FU对体内肿瘤生长的抑制效果。细胞增殖曲线多组间数据比较采用重复测量数据方差分析,单时间点多组数据采用单因素方差分析,组间比较采用Tukey法。 结果优化培养的胃癌细胞SGC- 7901呈球形生长,细胞活性染色显示良好的活性状态;MTT结果显示,黄岑汤组（0.44±0.04）能够抑制胃癌细胞的增殖,从第4天起与对照组（0.59±0.02）相比,差异具有统计学意义（F = 39.550,P < 0.01）;黄岑汤联合5-FU组（0.36±0.04）加强5-FU对肿瘤细胞的抑制作用,从第3天起与对照组（0.50±0.01）相比,差异具有统计学意义（F = 10.670,P < 0.01）,与5-FU组（0.42±0.03）对比,差异具有统计学意义（F = 10.670,P < 0.05）;Real-time PCR结果显示黄岑汤联合5-FU可抑制胃癌细胞中肿瘤干细胞相关表面标志物CD44、EpCAM、CD90表达（P < 0.01）;移植瘤生长抑制实验发现黄芪汤组对荷瘤鼠移植瘤的生长具有抑制作用,与对照组相比移植瘤体积减小,差异具有统计学意义（P < 0.01）,黄岑汤联合5-FU组与5-FU组相比对移植瘤抑制效果更明显,差异具有统计学意义（P < 0.01）。 结论黄芪汤能抑制胃癌细胞的生长和增殖,并能协同5-FU产生抗肿瘤作用。     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.