thiostrepton and Colorectal-Neoplasms

Tripartite motif-containing proteins (TRIM) play a crucial role in carcinogenesis. Little attention has been focused on the possible functions of TRIM6 on carcinogenesis.. The expression levels of TRIM6 were assessed in colorectal cancer (CRC) samples. TRIM6 expression was knocked down in CRC cell lines, and subjected to Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation and cell cycle assays. Immunoprecipitation and proteomics analysis was performed to identify potential associated proteins of TRIM6.. TRIM6 expression was up-regulated in CRC samples and TRIM6 expression may be an independent prognostic marker for CRC. Knocking down TRIM6 expression suppressed CRC cell proliferation, induced cell cycle arrested at G2/M phase and increased sensitivity to 5-fluorouracil and oxaliplatin. TIS21, an anti-proliferative protein involved in the regulation of G2/M arrest, was identified as an interaction partner of TRIM6. Moreover, CRC cells with TRIM6 overexpression showed decreased TIS21 protein stability. TIS21 ubiquitination was increased in CRC cells overexpressing TRIM6, but not in those overexpressing TRIM6 E3 catalytic mutant (C15A). Further, Lys5 was essential for TRIM6 mediated TIS21 ubiquitination. TIS21 overexpression reversed the induced effects of TRIM6 overexpression on CRC cell proliferation, and the levels of forkhead box M1 (FoxM1), phosphorylated FoxM1, Cyclin B1 and c-Myc. Thiostrepton, a specific inhibitor for FoxM1, was less effective in anti-proliferative activity against CRC cells with lower level of TRIM6 in vitro and in vivo.. Our study suggests that TRIM6 promotes the progression of CRC via TIS21/FoxM1.

Topics: Aged; Animals; Anti-Bacterial Agents; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Forkhead Box Protein M1; G2 Phase Cell Cycle Checkpoints; HCT116 Cells; HEK293 Cells; Humans; Immediate-Early Proteins; M Phase Cell Cycle Checkpoints; Male; Mice; Mice, Nude; Middle Aged; Random Allocation; Thiostrepton; Tripartite Motif Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic agent for colorectal cancer (CRC). However, frequently occurred 5-FU resistance poses a great challenge in the clinic. Elucidating the underlying mechanisms and developing effective strategies against 5-FU resistance are highly desired. Here we identified the upregulation of FOXM1 in 5-FU nonresponsive CRC patients by gene expression profile analysis and 5-FU-resistant CRC cells by qRT-PCR assay. Silencing of FOXM1 promoted the sensitivity of CRC cells to 5-FU by enhancing cell apoptosis, while overexpression of FOXM1 conferred CRC cells with 5-FU resistance both in vitro and in vivo. Furthermore, we showed that genetic and pharmacological inhibition of FOXM1 resensitized resistant CRC cells to 5-FU treatment. Mechanistically, FOXM1 promoted the transcription of ABCC10 by directly binding to its promoter region. Notably, treatment with ABCC10 inhibitor reversed FOXM1-induced resistance to 5-FU in vivo. Clinical investigation revealed that the levels of FOXM1 and ABCC10 were positively correlated in CRC tissues. Therefore, FOXM1 promotes 5-FU resistance by upregulating ABCC10, suggesting that FOXM1/ABCC10 axis may serve as a potential therapeutic target for 5-FU resistance in CRC patients.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Binding Sites; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Fluorouracil; Forkhead Box Protein M1; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; Multidrug Resistance-Associated Proteins; Promoter Regions, Genetic; RNA Interference; Signal Transduction; Thiostrepton; Time Factors; Transcription, Genetic; Transfection; Xenograft Model Antitumor Assays

Cross-talk between deregulated signaling pathways in cancer cells causes uncontrolled growth and proliferation. These cancers cells become more aggressive and quickly develop resistance to therapy. Therefore targeting of these deregulated pathways simultaneously can result in efficient cell death of cancer cells. In this study we investigated co-expression of Cox-2 and FoxM1 in a cohort of colorectal carcinoma (CRC) samples and also examined whether inhibition of Cox-2 and FoxM1 simultaneously can lead to inhibition of cell viability and induction of apoptosis in colorectal cancer cell lines and in vivo xenografts.. Protein expression of Cox-2 and FoxM1 was determined in a large cohort of 770 clinical CRC samples in a tissue micro-array format by immunohistochemistry. Cell death was measured using live dead assay. Apoptosis was measured by annexin V/PI dual staining. Immunoblotting was performed to examine the expression of proteins. Calcusyn software was utilized to estimate the synergistic doses using chou and Talalay method.. Co-expression of Cox-2 and FoxM1 was detected in 33.3 % (232/697) of CRC's and associated with an aggressive phenotype characterized by younger age (p = 0.0191), high proliferative index marker; Ki-67 (p = 0.004) and MMP-9 (p = 0.0116) as well as activation of AKT (p = 0.0214). In vitro, inhibition of FoxM1 and Cox-2 with pharmacological inhibitors; Thiostrepton and NS398 resulted in efficient down-regulation of FoxM1 and Cox-2 expression along with in-activation of AKT and inhibition of colony formation, invasion and migratory capability of CRC cells. In addition, there was also inhibition of cell viability and induction of apoptosis via the mitochondrial apoptotic pathway in CRC cell lines. Finally, treatment of CRC xenograft tumors in nude mice with combination of Cox-2 and FoxM1 inhibitors inhibited tumor growth significantly via down-regulation of Cox-2 and FoxM1 expression.. These findings demonstrate that co-expression of Cox-2 and FoxM1 might play a critical role in the pathogenesis of CRC. Therefore, targeting of these pathways simultaneously with sub toxic doses of pharmacological inhibitors can be a potential therapeutic approach for the treatment of this subset of CRC.

Topics: Adult; Aged; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression; Humans; Male; Mice; Middle Aged; Mitochondria; Neoplasm Grading; Neoplasm Staging; Nitrobenzenes; Signal Transduction; Sulfonamides; Thiostrepton; Tumor Burden; Xenograft Model Antitumor Assays

To identify genes potentially playing an important role in the progression of colorectal carcinoma (CRC), we screened global gene expression using cDNA expression array on 41 CRC tissue samples and 25 noncancerous colorectal tissue samples. Among the up-regulated genes, forkhead box M1 (FOXM1) has been shown to play a critical role in pathogenesis of various malignancies. Using immunohistochemistry on 448 Saudi CRC samples in tissue microarray format, FoxM1 protein overexpression was seen in 66% of CRC tissues and was significantly associated with poorly differentiated and highly proliferative tumors (P = 0.0200 and 0.0018, respectively). FoxM1 expression was also significantly associated with MMP-9 protein expression (P = 0.0002). In vitro data using CRC cell lines showed that inhibition of FoxM1 by thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. Overexpression of FoxM1 potentiated cell proliferation, cell transformation, and migration/invasion of CRC cells via up-regulation of FoxM1 target genes MMP2 and MMP9 and protected these cells from thiostrepton-mediated antiproliferative effects. Finally, in vivo, overexpression of FoxM1 promoted growth of CRC-cell line xenograft tumors in nude mice. Altogether, our data indicate that FoxM1 signaling contributes to aggressiveness in a subset of CRC and that the FOXM1 gene may serve as a useful molecular biomarker and potential therapeutic target.

Topics: Adult; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cytoprotection; DNA, Complementary; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Middle Aged; Middle East; Molecular Targeted Therapy; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Thiostrepton; Transfection