leupeptins and Colorectal-Neoplasms

To elucidate the underlying oncogenic mechanism of zinc finger protein 746 (ZNF746), current study was conducted in colorectal cancers (CRCs). Herein, ZNF746 was overexpressed in HCT116, SW620, and SW480 cells, which was supported by CRC tissue microarray and TCGA analysis. Also, DNA microarray revealed the differentially expressed gene profile particularly related to cell cycle genes and c-Myc in ZNF746 depleted HCT116 cells. Furthermore, ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization by immunoprecipitation and immunofluorescence, while ZNF746 and c-Myc exist mainly in nucleoplasm. Conversely, ZNF746 depletion attenuated phosphorylation of c-Myc (S62) and glycogen synthase kinase 3β (GSK3β) (S9) and also activated p-c-Myc (T58), which was reversed by GSK3 inhibitors such as SB-216763 and Enza. Also, c-Myc degradation by ZNF746 depletion was blocked by knockdown of F-box/WD repeat-containing protein 7 (FBW7) ubiquitin ligase or proteosomal inhibitor MG132. Additionally, the growth of ZNF746 depleted HCT116 cancer cells was retarded with decreased expression of ZNF746 and c-Myc. Overall, these findings suggest that ZNF746 promotes CRC progression via c-Myc stability mediated by GSK3 and FBW7.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Disease Progression; F-Box-WD Repeat-Containing Protein 7; G1 Phase Cell Cycle Checkpoints; Gene Expression Profiling; Glycogen Synthase Kinase 3 beta; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; Indoles; Leupeptins; Male; Maleimides; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Phosphorylation; Proto-Oncogene Proteins c-myc; Repressor Proteins; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous

Dichloroacetate (DCA) is a metabolic reprogramming agent that reverses the Warburg effect, causing cancer cells to couple glycolysis to oxidative phosphorylation. This has been shown to induce apoptosis and reduce the growth of various types of cancer but not normal cells. Colorectal cancer cells HCT116, HCT116 p53(-/-), and HCT116 Bax(-/-), were treated with DCA in vitro. Response to treatment was determined by measuring PDH phosphorylation, apoptosis, proliferation, and cell cycle. Molecular changes associated with these responses were determined using western immunoblotting and quantitative PCR. Treatment with 20 mM DCA did not increase apoptosis, despite decreasing levels of anti-apoptotic protein Mcl-1 after 6 h, in any of the cell lines observed. Mcl-1 expression was stabilized with MG-132, an inhibitor of proteasomal degradation. A decrease in Mcl-1 correlated with a decrease in proliferation, both of which showed dose-dependence in DCA treated cells. Cells showed nuclear localization of Mcl-1, however cell cycle was unaffected by DCA treatment. These data suggest that a reduction in the prosurvival Bcl-2 family member Mcl-1 due to increased proteasomal degradation is correlated with the ability of DCA to reduce proliferation of HCT116 human colorectal cancer cells without causing apoptosis.

Topics: Antimetabolites; Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Dichloroacetic Acid; Gene Knockout Techniques; HCT116 Cells; Humans; Leupeptins; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Tumor Suppressor Protein p53

Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

Topics: Antineoplastic Agents; Cell Cycle; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; HCT116 Cells; Humans; Leupeptins; NF-kappa B; Nitriles; Phosphorylation; Point Mutation; Proteasome Endopeptidase Complex; Proteolysis; Silybum marianum; Silymarin; Sulfones; Threonine

It was previously reported that the histone deacetylase inhibitor (HDACI) trichostatin A (TSA) induced B cell lymphoma 2 (Bcl-2)-associated X protein (Bax)-dependent apoptosis in colorectal cancer (CRC) cells. In addition, Ku70 has been identified as a regulator of apoptosis, the mechanism of which proceeds via interacting with Bax. The aim of the present study was to investigate the role of Ku70 in TSA-induced apoptosis in the CRC cell lines HCT116 and HT29. The results showed that TSA induced the acetylation of Ku70, which was found to be associated with increased apoptosis. In addition, TSA treatment promoted the release of Bax from its complex with Ku70. Bax was then detected to have translocated from the cytoplasm into the mitochondria, while cytochrome c was detected to have translocated from the mitochondria into the cytoplasm. Furthermore, knockdown of Ku70 using small interfering RNA decreased TSA-induced apoptosis as well as downregulated the expression of Bax. These effects were rescued through pre-treatment of cells with the proteasome inhibitor MG132. In conclusion, the results of the present study suggested that Ku70 acetylation mediated TSA-induced apoptosis in CRC cells. In addition, Ku70 was found to be indispensable in TSA-induced apoptosis due to its role in protecting Bax from proteosomal degradation.

Topics: Acetylation; Antigens, Nuclear; Apoptosis; bcl-2-Associated X Protein; Colorectal Neoplasms; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Ku Autoantigen; Leupeptins

Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

Topics: Animals; Benzimidazoles; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Colorectal Neoplasms; Dasatinib; Enzyme Activation; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Heterografts; Humans; Kaplan-Meier Estimate; Leupeptins; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Paxillin; Phosphorylation; Proportional Hazards Models; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Sulfonamides

Despite the effectiveness of histone deacetylase inhibitors, proteasome inhibitors and cytotoxic drugs on human cancers, none of these types of treatments by themselves has been sufficient to eradicate the disease. The combination of different modalities may hold enormous potential for eliciting therapeutic results. In the current study, we examined the effects of treatment with the histone deacetylase inhibitor (HDACI) apicidin (APC) in combination with proteasome inhibitors on human colorectal cancer cells. The molecular mechanisms of the combined treatments and their potential to sensitize colorectal cancer cells to chemotherapies were also investigated. Cancer cells were exposed to the agents alone and in combination, and cell growth inhibition was determined by MTT and colony formation assays. HDAC, proteasome and NF-κB activities as well as reactive oxygen species (ROS) were monitored. Cell cycle perturbation and induction of apoptosis were assessed by flow cytometry. The expression of cell cycle/apoptosis- and cytoprotective/stress-related genes was determined by quantitative PCR and EIA, respectively. The potentiation of cancer cell sensitivity to chemotherapies upon APC/PI combination treatment was also studied. The combination of APC and MG132, PI-1 or epoxomicin potently inhibited cancer cell growth, disrupted the cell cycle, induced apoptosis, decreased NF-κB activity and increased ROS production. These events were accompanied by the altered expression of genes associated with the cell cycle, apoptosis and cytoprotection/stress regulation. The combination treatment markedly enhanced the chemosensitivity of colorectal cancer cells (50-3.7 x 10(4)-fold) in a drug-, APC/PI combination- and colorectal cancer subtype-dependent manner. The results of this study have implications for the development of com-binatorial treatments that include HDACIs, PIs and conventional chemotherapeutic drugs, suggesting a potential therapeutic synergy with general applicability to various types of cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Drug Synergism; Humans; Leupeptins; NF-kappa B; Oligopeptides; Peptides, Cyclic; Proteasome Endopeptidase Complex; Reactive Oxygen Species

Cyclins are essential for cell proliferation, the cell cycle and tumorigenesis in all eukaryotes. UbcH10 regulates the degradation of cyclins in a ubiquitin-dependent manner. Here, we report that UbcH10 is likely involved in tumorigenesis. We found that cancer cells exposed to n-acetyl-leu-leu-norleucinal (ALLN) treatment and UbcH10 depletion exhibit a synergistic therapeutic effect. Abundant expression of UbcH10 drives resistance to ALLN-induced cell death, while cells deficient in UbcH10 were susceptible to ALLN-induced cell death. The depletion of UbcH10 hindered tumorigenesis both in vitro and in vivo, as assessed by colony formation, growth curve, soft agar and xenograft assays. These phenotypes were efficiently rescued through the introduction of recombinant UbcH10. In the UbcH10-deficient cells, alterations in the expression of cyclins led to cell cycle changes and subsequently decreases in tumorigenesis. The tumorigenesis of xenograft tumors from UbcH10-deficient cells treated with ALLN was decreased relative to wild-type cells treated with ALLN in nude mice. On the molecular level, we observed that UbcH10 deficiency enhances the activation of caspase 8 and caspase 3 but not caspase 9 to impair cell viability upon ALLN treatment. Collectively, our results suggest that, as an oncogene, UbcH10 is a potential drug target for the treatment of colorectal cancer.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Caspases; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclins; Dependovirus; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Leupeptins; Mice; Mice, Knockout; Mice, Nude; Signal Transduction; Ubiquitin; Ubiquitin-Conjugating Enzymes; Xenograft Model Antitumor Assays

Trisomy for chromosome 7 is frequently observed as an initiating event in sporadic colorectal cancer. Although unstable chromosome numbers and recurrent aneuploidies drive a large fraction of human cancers, targeted therapies selective to pre-neoplastic trisomic cells are non-existent. We have previously characterized a trisomy 7 cell line (1CT+7) spontaneously derived from normal diploid human colonic epithelial cells that aberrantly expresses the epidermal growth factor receptor (EGFR, chromosome 7p11). Recent studies identified AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) as a pharmacological inhibitor of aneuploid murine fibroblast proliferation. Here, we report that AICAR induces profound cytostatic and metabolic effects on 1CT+7 cells, but not on their isogenic diploid counterpart. Dose-response experiments indicate that 1CT+7 cells are fourfold preferentially sensitive to AICAR compared to diploid cells. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction (P=0.0025) in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cells in vitro and in xenograft tumors in vivo. Our data collectively support the pharmacological compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential anticancer agent for aneuploidy-driven colorectal cancer.

Topics: Aminoimidazole Carboxamide; Aneuploidy; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; Epithelial Cells; ErbB Receptors; Humans; Hypoglycemic Agents; Intestinal Mucosa; Leupeptins; Mice; Neoplasm Transplantation; Proteasome Endopeptidase Complex; Ribonucleotides; Transplantation, Heterologous; Trisomy; Ubiquitination

Abnormalities in the STAT3 pathway are involved in the oncogenesis of several cancers. However, the mechanism by which dysregulated STAT3 signaling contributes to the progression of human colorectal cancer (CRC) has not been elucidated, nor has the role of JAK, the physiological activator of STAT3, been evaluated. To investigate the role of both JAK and STAT3 in CRC progression, we inhibited JAK with AG490 and depleted STAT3 with a SiRNA. Our results demonstrate that STAT3 and both JAK1 and 2 are involved in CRC cell growth, survival, invasion, and migration through regulation of gene expression, such as Bcl-2, p1(6ink4a), p21(waf1/cip1), p27(kip1), E-cadherin, VEGF, and MMPs. Importantly, the FAK is not required for STAT3-mediated regulation, but does function downstream of JAK. In addition, our data show that proteasome-mediated proteolysis promotes dephosphorylation of the JAK2, and consequently, negatively regulates STAT3 signaling in CRC. Moreover, immunohistochemical staining reveals that nuclear staining of phospho-STAT3 mostly presents in adenomas and adenocarcinomas, and a positive correlation is found between phospho-JAK2 immunoreactivity and the differentiation of colorectal adenocarcinomas. Therefore, our findings illustrate the biologic significance of JAK1, 2/STAT3 signaling in CRC progression and provide novel evidence that the JAK/STAT3 pathway may be a new potential target for therapy of CRC.

Topics: Apoptosis; Cadherins; Cell Cycle; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Down-Regulation; Focal Adhesion Kinase 1; Humans; Janus Kinase 1; Janus Kinase 2; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Protein Kinase Inhibitors; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Vascular Endothelial Growth Factor A

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.

Topics: Acetylcysteine; Apoptosis; Butyrates; Caspases; Cell Division; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Therapy, Combination; G2 Phase; Humans; Leupeptins; Mitosis; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Ubiquitins

Upregulation of nuclear factor (NF)-kappaB is found in many forms of cancer. Activation of NF-kappaB in cancer cells by chemotherapy or radiation can blunt the ability of this therapy to induce cell death. Proteasome inhibitors stimulate apoptosis in part via prevention of NF-kappaB activation. We sought to determine whether constitutive NF-kappaB activity is present in human colon cancer. In addition we studied whether alterations of NF-kappaB activity with a proteasome inhibitor would prevent colon cancer cell growth and induce apoptosis. We demonstrated constitutive transcriptional activation of NF-kappaB in SW48 and SW116 colon cancer cells by luciferase and electromobility shift assays. This was confirmed by p65 immunostaining. This activity was further induced in the presence of chemotherapy. In colon cancer specimens constitutive activation of NF-kappaB was observed in the majority of tumors. Treatment with the proteasome inhibitor (MG-132) inhibited growth and also stimulated apoptosis of colon cancer cells. We conclude that inhibition of NF-kappaB activation may be a logical therapy for certain cancers. This can be done via specific approaches on molecules necessary for keeping NF-kappaB inactivated in the cytoplasm. Other potentially useful ways to promote apoptosis in cancer cells include the utilization of proteasome inhibitors. Such inhibitors are currently being evaluated in clinical trials.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Colorectal Neoplasms; Cysteine Endopeptidases; Deoxycytidine; DNA, Neoplasm; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Enzyme Precursors; Gemcitabine; Gene Transfer Techniques; Humans; Leupeptins; Luciferases; Multienzyme Complexes; NF-kappa B; Proteasome Endopeptidase Complex; Rats; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation

Chemoresistance may involve the anti-apoptotic transcriptional regulator, nuclear factor-kappa B (NF-kappa B). The purpose of this study was to determine whether chemotherapy induces NF-kappa B activation in a human colon cancer cell line (SW48) and whether NF-kappa B is constitutively activated in colorectal cancer.. SW48 cells were incubated with gemcitabine hydrochloride (Gemzar) in the presence and absence of the 26s proteasome inhibitor, MG132, and NF-kappa B binding (electrophoretic mobility shift assay), DNA synthesis (tritiated thymidine uptake), cell viability (3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide assay), and apoptosis (caspase-3 activity) were measured at 24 hours. NF-kappa B binding (electrophoretic mobility shift assay) was also assayed in 10 colorectal cancer tumors.. SW48 cells demonstrated constitutive NF-kappa B binding that was enhanced by gemcitabine hydrochloride in a dose-dependent manner. MG132 inhibited NF-kappa B binding and enhanced gemcitabine hydrochloride's inhibition of DNA synthesis (gemcitabine hydrochloride = 73% +/- 1.4% vs gemcitabine hydrochloride + MG132 = 6% +/- 0.4%, P <.05), cell killing (gemcitabine hydrochloride = 87% +/- 2.0 vs gemcitabine hydrochloride + MG132 = 25% +/- 1.3%, P <.05), and caspase-3 activity (gemcitabine hydrochloride = 870 +/- 17.4 vs gemcitabine hydrochloride + MG132 = 1075 +/- 20.4, P <.05). NF-kappa B binding was increased in 8 of 10 colorectal cancer tumors compared with adjacent normal mucosa.. Gemcitabine hydrochloride enhances NF-kappa B binding in a colorectal cancer cell line, whereas inhibition of NF-kappa B enhances gemcitabine hydrochloride's antitumor activity. NF-kappa B is also activated in human colorectal cancer. NF-kappa B may identify chemoresistant tumors, whereas inhibition of NF-kappa B may be a novel, biologically based therapy. (Surgery 2001;130:363-9).

Topics: Antimetabolites, Antineoplastic; Caspase 3; Caspases; Colon; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Deoxycytidine; DNA; Gemcitabine; Humans; Intestinal Mucosa; Leupeptins; NF-kappa B; Protein Binding; Rectum; Tumor Cells, Cultured

beta-catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals. Deregulated beta-catenin protein expression, due to mutations in the beta-catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein. Increased expression of beta-catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor. To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess beta-catenin on p53. We report here that overexpression of beta-catenin results in accumulation of p53, apparently through interference with its proteolytic degradation. This effect involves both Mdm2-dependent and -independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells. Increased p53 activity may provide a safeguard against oncogenic deregulation of beta-catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression.

Topics: Adaptor Proteins, Signal Transducing; Animals; beta Catenin; Cell Line; Cell Nucleus; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Desmoplakins; Dishevelled Proteins; Gene Expression; Humans; Leupeptins; Mice; Mutation; Nuclear Proteins; Phosphoproteins; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Signal Transduction; Solubility; Trans-Activators; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Up-Regulation