Targeting the ZMIZ1-Notch1 signaling axis for the treatment of tongue squamous cell carcinoma.

Zinc finger MIZ-type containing 1 (ZMIZ1) is a transcriptional coactivator related to the protein inhibitors of activated STATs (PIAS) family. Mounting evidence suggests that ZMIZ1 plays a crucial role in the occurrence and development of cancers. The function of ZMIZ1 in tongue squamous cell carcinoma (TSCC) and the mechanisms underpinning its role in this disease have not been fully clarified. We performed qualitative ZMIZ1 protein expression analyses using immunohistochemistry in 20 patient-derived, paraffin-embedded TSCC tissue sections. We used RNAi to knock down ZMIZ1 expression in the CAL-27 TSCC cell line and quantified the impact of ZMIZ1 knock down on proliferation, migration and apoptosis via CCK-8, scratch assay and flow cytometry, respectively. We used qRT-PCR and western blotting to investigate the role of ZMIZ1 in this cell line. Finally, we established a model of lung metastasis in nude mice to replicate the in vitro results. ZMIZ1 protein was significantly more abundant in TSCC case tissue samples. ZMIZ1 knockdown reduced the invasion and metastases of TSCC tumor cells and promoted apoptosis. ZMIZ1 knockdown was associated with the down-regulation of Notch signaling pathway related factors Jagged1 and Notch1, and invasion and metastasis related factors MKP-1, SSBP2 and MMP7 in vitro and in vivo, at the mRNA level. In vitro and in vivo data suggest that knock down of ZMIZ1 may inhibit TSCC invasion and metastasis by modulating Notch signaling. ZMIZ1 inhibition may therefore represent a new therapeutic target for TSCC.

Targeting CCR2+ macrophages with BET inhibitor overcomes adaptive resistance to anti-VEGF therapy in ovarian cancer.

PURPOSE: Tumor-associated macrophages (TAMs) are known to contribute to adaptive resistance to anti-vascular endothelial growth factor (VEGF) antibody (AVA) therapy in ovarian cancer. BET (bromodomain and extra-terminal domain) inhibitors (BETi) may have unique roles in targeting TAMs. Our objective was to examine the effects of BETi on TAMs, especially in the context of enhancing the efficacy of AVA therapy. METHODS: We conducted a series of in vitro (MTT assay, apoptosis, flow cytometry, and RNA sequencing) and in vivo (xenograft ovarian cancer model) experiments to determine the biological effects of BETi combined with AVA in ovarian cancer. For statistical analysis, a two-tailed Student's t test (equal variance) or ANOVA was used for multiple groups' comparison, and p < 0.05 was considered significant. RESULTS: BETi resulted in a dose-dependent decrease in cell viability and induced apoptosis (p < 0.01) in ovarian cancer cells (SKOV3ip1, OVCAR5, and OVCAR8). Treatment with BETi significantly increased apoptosis in THP-1 monocytes and macrophages (PMA-differentiated THP-1; p < 0.01). Furthermore, BETi selectively induced greater apoptosis in M2-like macrophages (PMA and IL-4, IL-13-differentiated THP-1) (31.3%-36.1%) than in M1-like macrophages (PMA and LPS-differentiated THP-1) (12.4%-18.5%) (p < 0.01). Flow cytometry revealed that the percentage of M1-like macrophages (CD68+/CD80+) was significantly increased after treatment with low-dose BETi (ABBV-075 0.1 µM; p < 0.05), whereas the percentage of CD68+/CCR2+ macrophages was significantly decreased (p < 0.001); these findings suggest that BETi may selectively inhibit the survival of CCR2+ macrophages and re-polarize the macrophages into an M1-like phenotype. RNA-seq analysis revealed that BETi selectively targeted macrophage infiltration-related cytokines/chemokines in ovarian cancer (adjusted p < 0.05 and Log2 fold change ≥ 1.5). Finally, using in vivo ovarian cancer models, compared with control or monotherapy, the combination of BETi (ABBV-075) and bevacizumab resulted in greater inhibition of tumor growth and macrophage infiltration (p < 0.05) and longer survival of tumor-bearing mice (p < 0.001). CONCLUSIONS: Our findings indicate a previously unrecognized role for BETi in selectively targeting CCR2+ TAMs and enhancing the efficacy of AVA therapy in ovarian cancer.

Bi2O2CO3/red phosphorus S-scheme heterojunction for H2 evolution and Cr(VI) reduction.

Red phosphorus (RP) has a suitable energy band structure and excellent photocatalytic properties. However, there are some problems, such as low quantum efficiency and serious photogenerated electron-hole recombination. The S-scheme heterostructure shows great potential in facilitating the separation and transfer of photogenerated carriers and obtaining strong photo-redox ability. Herein, hydrothermally treated red phosphorus (HRP) was combined with Bi2O2CO3 to construct Bi2O2CO3/HRP S-scheme heterojunction composite. The Bi2O2CO3 content was optimized, and the 5 %Bi2O2CO3/HRP composite obtained at 5 %Bi2O2CO3 mass fraction exhibited the strongest photoreduction ability. The Cr(VI) photoreduction and photolytic hydrogen production rates were as high as 0.22 min-1 and 157.2 µmol •h-1, which were 7.3 and 3.0 times higher than those of HRP, respectively. The promoted photocatalytic activity could be attributed to the formation of S-scheme heterojunctions, which accelerated the separation and transfer of useful photogenerated electron-hole pairs, while enhancing the recombination of relatively useless photogenerated electron-hole pairs, thereby resulting in the highest photocurrent density (17.3 µA/cm2) of the 5 %Bi2O2CO3/HRP composite, which was 1.6 and 4.3 times higher than pure Bi2O2CO3 (10.5 µA/cm2) and pure HRP (4.0 µA/cm2), respectively. This work would provide an advanced approach to enhance the photocatalytic activity of RP.

A fluorescent paper biosensor for the rapid and ultrasensitive detection of zearalenone in corn and wheat.

Zearalenone (ZEN) is a kind of estrogen-like mycotoxin which contaminates primary crops and their products under natural conditions and becomes a serious hazard to human health. In this study, we prepared a sensitive and specific anti-ZEN monoclonal antibody (mAb) belonging to the IgG2b subclass, with a 50%-inhibitory concentration of 0.034 ng mL-1. A lateral flow fluorescence microsphere immunochromatographic test strip (FM-ICTS) for the rapid and ultrasensitive detection of zearalenone in corn and wheat samples was developed based on this mAb. After optimizing experimental parameters, the visual limit of detection (LOD) of the strip assay in both corn and wheat samples was 2.5 ng mL-1, and the cut-off value was 25 ng mL-1. The LOD was calculated to be 0.68 ng mL-1 in corn samples and 0.48 ng mL-1 in wheat samples. Recovery experiments showed that the test results of the strip were consistent with those of ic-ELISA. As a result, this FM-ICTS assay is reliable, simple and sensitive, and can be used for rapid detection of ZEN in corn and wheat.

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer.

BACKGROUND: Our pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. METHODS: We evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. RESULTS: EphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25-0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. CONCLUSIONS: These findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

Sustained Adrenergic Activation of YAP1 Induces Anoikis Resistance in Cervical Cancer Cells.

Chronic stress-related hormones modulate tumor pathogenesis at multiple levels; however, the molecular pathways involved in stress and cervical cancer progression are not well understood. We established a preclinical orthotopic mouse model of cervical cancer and used the model to show that daily restraint stress increased tumor growth and metastatic tumor burden. Exposure to norepinephrine significantly protected cervical cancer cells from anoikis. We demonstrated that YAP1 was dephosphorylated and translocated from the cytoplasm to the nucleus by norepinephrine, a process initiated by ADRB2/cAMP/protein kinase A activation. Furthermore, anoikis resistance and YAP1 activation induced by norepinephrine could be rescued by a broad ß-adrenergic receptor antagonist, propranolol. Collectively, our results provide a pivotal molecular pathway for disrupting pro-tumor neuroendocrine signaling in cervical cancer.

Prospective pilot trial with combination of propranolol with chemotherapy in patients with epithelial ovarian cancer and evaluation on circulating immune cell gene expression.

OBJECTIVE: To determine the feasibility of pharmacologic beta-adrenergic blockade in women with newly diagnosed stage II-IV epithelial ovarian cancer (EOC) throughout primary treatment. METHODS: Patients initiated propranolol prior to beginning chemotherapy or surgery. Feasibility was assessed as proportion able to complete 6 chemotherapy cycles while on adrenergic suppression. Descriptive statistics summarized surveys, and paired changes were analyzed using signed rank tests. Random-intercept Tobit models examined immune response. RESULTS: Median age was 59.9; 88.5% were stage IIIC/IV; and 38.5% underwent primary debulking. Thirty-two patients were enrolled; 3 excluded because they never took propranolol; an additional 3 didn't meet inclusion criteria, leaving 26 evaluable. Eighteen of 26 (69%), 90% credible interval (CI) of 53-81%, completed 6 chemotherapy cycles plus propranolol (an 82% posterior probability that the true proportion of success is ≥60%). Among the 23 patients with baseline and six month follow up data, overall QOL, anxiety, and depression improved (P < 0.05) and leukocyte expression of pro-inflammatory genes declined (P = 0.03) after completion of therapy. Decrease from baseline of serum IL-6 and IL-8 preceded response to chemotherapy (P < 0.0014). Change from baseline IL-10 preceded complete response. CONCLUSION: Use of propranolol during primary treatment of EOC is feasible and treatment resulted in decrease in markers of adrenergic stress response. In combination with chemotherapy, propranolol potentially results in improved QOL over baseline.

Gene regulation analysis of the effects of evodiamine on tongue squamous cell carcinoma.

OBJECTIVE: To use gene chip technology to study the effects of evodiamine (EVO) on the gene expression profile of tongue squamous cell carcinoma (TSCC) CAL-27 cell line, for the purpose of analyzing the mechanisms underlying the effects of EVO on gene expression and functional regulation of TSCC cells at the gene level. METHOD: Differentially expressed genes in CAL-27 cells treated with EVO were detected using gene chip technology and analyzed using ingenuity pathway analysis. RESULTS: Microarray results showed that there were 1243 differentially expressed genes following treatment with CAL-27 cells; 684 genes were upregulated and 559 were downregulated. Classical pathway analysis revealed a total of 89 signal transduction pathways with upregulated gene set enrichment, including lipopolysaccharide/interleukin (IL)-1-mediated inhibition of retinoid X receptor (RXR) function, agrin interactions at neuromuscular junctions, cholecystokinin/gastrin-mediated signaling, toll-like receptor signaling, and IL-6 signaling. A total of 39 signal transduction pathways were enriched for the downregulated genes, including interferon signals, liver X receptor/RXR activation signals, and RhoGDI signals. In the disease and function analysis, the upregulated genes were enriched in viral infection, RNA virus replication, viral replication, cancer cell invasion, cell invasion, and other related functions, while downregulated genes were enriched in neuromuscular diseases, and leukocyte differentiation, antiviral response, connective tissue cell death and other functions. CONCLUSIONS: Gene chip analysis offers an effective means of screening differential gene expression between EVO-treated TSCCs and controls, thus providing a sound basis for further research.

Inhibiting Nuclear Phospho-Progesterone Receptor Enhances Antitumor Activity of Onapristone in Uterine Cancer.

Although progesterone receptor (PR)-targeted therapies are modestly active in patients with uterine cancer, their underlying molecular mechanisms are not well understood. The clinical use of such therapies is limited because of the lack of biomarkers that predict response to PR agonists (progestins) or PR antagonists (onapristone). Thus, understanding the underlying molecular mechanisms of action will provide an advance in developing novel combination therapies for cancer patients. Nuclear translocation of PR has been reported to be ligand-dependent or -independent. Here, we identified that onapristone, a PR antagonist, inhibited nuclear translocation of ligand-dependent or -independent (EGF) phospho-PR (S294), whereas trametinib inhibited nuclear translocation of EGF-induced phospho-PR (S294). Using orthotopic mouse models of uterine cancer, we demonstrated that the combination of onapristone and trametinib results in superior antitumor effects in uterine cancer models compared with either monotherapy. These synergistic effects are, in part, mediated through inhibiting the nuclear translocation of EGF-induced PR phosphorylation in uterine cancer cells. Targeting MAPK-dependent PR activation with onapristone and trametinib significantly inhibited tumor growth in preclinical uterine cancer models and is worthy of further clinical investigation. Mol Cancer Ther; 17(2); 464-73. ©2017 AACR.

Phase II trial of bevacizumab with dose-dense paclitaxel as first-line treatment in patients with advanced ovarian cancer.

OBJECTIVES: To assess the tolerability and efficacy of bevacizumab with carboplatin and weekly paclitaxel as first-line adjuvant therapy for advanced stage ovarian cancer. METHODS: After IRB approval, this single-institution, phase II study enrolled patients with stage III or IV epithelial ovarian cancer after primary cytoreductive surgery to treatment with carboplatin (AUC 5), weekly paclitaxel (80mg/m2), and bevacizumab (15mg/kg) every 3weeks for at least 6cycles. The primary endpoint was tolerability of at least 4cycles of therapy, with a target treatment success rate of >60%. Secondary endpoints included progression-free survival (PFS) and response rate. Plasma biomarkers were analyzed by the multiplex ELISA assays. RESULTS: Thirty-three patients were enrolled with 30 evaluable patients receiving at least one cycle of combination treatment. Twenty-three patients (77%) were able to complete at least 4cycles of therapy per protocol, and the posterior probability that the treatment success rate is >60% is 0.77. Twenty-one patients (70%) were able to complete ≥6cycles of therapy. Median PFS was 22.4months for patients with optimal (R0) compared to 16.9months for optimal≤1cm (HR 1.71, 95% CI 0.58-4.98, p=0.33), and 16.9months for suboptimal>1cm (HR 3.75, 95% CI 1.05-13.34, p=0.04) disease. Increases in mean Flt-3L was significantly higher in responders versus non-responders (83.4 vs. 28pg/mL, p=0.05). CONCLUSIONS: Adjuvant bevacizumab with dose-dense chemotherapy is associated with acceptable toxicity and a high likelihood of completing 4cycles of therapy. Dynamic changes in Flt-3L may represent a predictive marker to treatment response.

Dll4 Inhibition plus Aflibercept Markedly Reduces Ovarian Tumor Growth.

Delta-like ligand 4 (Dll4), one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti-VEGF therapy. Here, we examined the biologic effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biologic effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibit tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse-phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior antitumor effects in ovarian cancer models compared with either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel (P < 0.05 for the combination vs. aflibercept). The superior antitumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared with the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach. Mol Cancer Ther; 15(6); 1344-52. ©2016 AACR.

Notch3 pathway alterations in ovarian cancer.

The Notch pathway plays an important role in the growth of high-grade serous ovarian (HGS-OvCa) and other cancers, but its clinical and biologic mechanisms are not well understood. Here, we found that the Notch pathway alterations are prevalent and significantly related to poor clinical outcome in patients with ovarian cancer. Particularly, Notch3 alterations, including amplification and upregulation, were highly associated with poor patient survival. Targeting Notch3 inhibited ovarian cancer growth and induced apoptosis. Importantly, we found that dynamin-mediated endocytosis was required for selectively activating Jagged-1-mediated Notch3 signaling. Cleaved Notch3 expression was the critical determinant of response to Notch-targeted therapy. Collectively, these data identify previously unknown mechanisms underlying Notch3 signaling and identify new, biomarker-driven approaches for therapy.

A galectin-3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells.

BACKGROUND: A common polymorphism, rs4644, coding for Pro64 or His64 of the carbohydrate-binding protein galectin-3, influences the susceptibility of galectin-3 to cleavage by matrix metalloproteinases and is associated with breast cancer incidence. Because forced expression of galectin-3 in a galectin-3 null breast cancer cell line confers sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the authors sought to determine whether the His64/Pro64 polymorphism of galectin-3 affects the sensitivity to TRAIL. METHODS: Genomic DNA of breast cell lines was analyzed for the single nucleotide polymorphism rs4644, and cytotoxicity was determined with the MTT assay. RESULTS: When a collection of 9 breast cancer cell lines that express galectin-3 was examined for lectin, galactoside-binding, soluble, 3 (LGALS3) genotype and sensitivity to doxorubicin and TRAIL, doxorubicin sensitivity was not found to be related to LGALS3 genotype. In contrast, none of the 5 cell lines that were homozygous for Pro64 galectin-3 were found to be sensitive to TRAIL, but 2 of 2 homozygous His64 cell lines and 1 of 2 heterozygous His64 cell lines were sensitive to TRAIL. Forced expression of galectin-3 of defined genotype in galectin-3 null cells was used to more directly test the effect of the Pro64His mutation on TRAIL sensitivity. High levels of expression of His64 galectin-3 rendered BT549 cells sensitive to TRAIL and resistant to doxorubicin, but cells expressing Pro64 galectin-3 remained resistant to TRAIL and sensitive to doxorubicin. CONCLUSIONS: The results of the current study indicate that the naturally occurring Pro64His mutation in galectin-3 increases sensitivity to death receptor-mediated apoptosis. This finding could be relevant to disparities in breast cancer outcomes across population groups, and could guide the design of future clinical trials of TRAIL-based therapies.

Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity.

Wnt/beta-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a beta-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/beta-catenin pathway in colon cancer cells, as well as the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, short hairpin RNA, or full-length galectin-3 cDNA, and effects on beta-catenin levels, subcellular distribution, and Wnt signaling were determined. Galectin-3 levels correlated with beta-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased beta-catenin protein levels but no change in beta-catenin mRNA levels, suggesting that galectin-3 modulates beta-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear beta-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased beta-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase-3beta (GSK-3beta) dephosphorylation and increased GSK activity, increasing beta-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT, suppressed TCF4 transcriptional activity induced by galectin-3 whereas LiCl, a GSK-3beta inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3beta phosphorylation and activity via the phosphatidylinositol 3-kinase/AKT pathway, and, thus, the degradation of beta-catenin in colon cancer cells.

Anti-EGFR and ErbB-2 antibodies attenuate cyclooxygenase-2 expression and cooperatively inhibit survival of human colon cancer cells.

Cyclooxygenase-2 (COX-2) is a transcriptional target and downstream effector of the ErbB-1 (EGFR) and ErbB-2 signaling pathways. We found that anti-EGFR and anti-ErbB-2 antibodies inhibited ERK phosphorylation and downregulated COX-2 protein expression in HCA-7 human colon carcinoma cells. Both antibodies also augmented the cytotoxic effects of the selective COX-2 inhibitor, NS-398. Inhibition of EGFR and ErbB-2 attenuated cell growth by increasing cell death, and the antibody combination suppressed cell growth to a greater extent than did either antibody alone. In conclusion, EGFR and ErbB-2 regulate ERK-mediated COX-2 expression and their selective inhibition enhanced NS-398-induced cell death. Cooperative inhibition of cell growth by EGFR and ErbB-2 blockade suggests the therapeutic potential of targeting multiple ErbB receptors.

Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac prevents experimental colon cancer and can regress precancerous polyps in humans. Sulindac sulfide inhibits cyclooxygenase (COX)-mediated prostaglandin synthesis and retards the growth of cultured colon cell lines primarily by inducing apoptosis. Given the known role of mitogen-activated protein kinase (MAPK) in signal transduction and the regulation of cell survival and death, we determined the effect of sulindac sulfide on MAPK activation, COX-2 expression, and apoptosis induction in HCA-7 human colon cancer cells. Sulindac sulfide treatment was associated with activation of ERKp44/42 and p38 MAPK in a dosage- and time-dependent manner, and also activated upstream MEK. Similar results were seen in HCT-15 cells and also with the selective COX-2 inhibitor NS398. ERKp44/42 and p38 activation were accompanied by an induction of COX-2 protein expression. Selective inhibitors of sulindac sulfide-induced ERKp44/42 (PD98059) and p38 MAPK (SB203580) activation also suppressed the induction of COX-2 by this NSAID. Furthermore, both MAPK inhibitors significantly augmented sulindac sulfide-induced apoptosis, as did suppression of constitutive COX-2 using antisense oligonucleotides. In conclusion, MEK/ERK and p38 MAPK activation mediate COX-2 induction by sulindac sulfide. Selective inhibitors of these MAPKs potentiate apoptosis induction by this NSAID, suggesting a novel strategy for the prevention or treatment of colorectal cancer.

Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells.

The cyclooxygenase-2 (COX-2) gene encodes an inducible enzyme that converts arachidonic acid to prostaglandins and is up-regulated in colorectal neoplasms. Evidence indicates that COX-2 may regulate apoptosis and can influence the malignant phenotype. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzymes and induce apoptosis in colorectal cancer cell lines, which may contribute to their antitumor effects. To determine whether forced COX-2 expression modulates susceptibility to drug-induced apoptosis, HCT-15 colon carcinoma cells were stably transfected with the COX-2 cDNA, and two clones overexpressing COX-2 were isolated. Selective COX-2 (NS398) and nonselective (sulindac sulfide) COX inhibitors, as well as 5-fluorouracil (5-FU), induced apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling in a dosage-dependent manner. Forced COX-2 expression significantly attenuated induction of apoptosis by all three of the drugs compared with parental HCT-15 cells. NSAIDs and 5-FU induced the mitochondrial release of cytochrome c as well as caspase-3 and -9 activation, and to a much lesser extent, caspase-8. COX-2-overexpressing cells showed reduced cytochrome c and caspase activation, relative to parental cells. A specific inhibitor of caspase-3 restored cell survival after drug treatment. COX-2 transfectants were found to overexpress the antiapoptotic Bcl-2 mRNA and protein relative to parental cells. In conclusion, forced COX-2 expression significantly attenuates apoptosis induction by NSAIDs and 5-FU through predominant inhibition of the cytochrome c-dependent apoptotic pathway. COX-2-mediated up-regulation of Bcl-2 suggests a potential mechanism for reduced apoptotic susceptibility.