Protein In Colon Cancer Cells Research Articles

Exploring the Molecular Targets and Therapeutic Potential of Coptisine in Colon Cancer: A Network Pharmacology Approach.

Colon cancer is a frequent malignancy, and surgery is still the primary therapy for people with colon cancer. Other treatments, including radiation, chemotherapy, and biologic therapy, may be utilized as a supplement. Chemotherapy, a prominent treatment for colon cancer, has failed to provide positive outcomes. This necessitates the development of more effective and less harmful treatment drugs. Coptisine was discovered to inhibit the development of colon cancer cell line HCT-116 in vivo, decrease the growth of HCT-116 cells, and cause apoptosis in vitro in colon cancer. Coptisine (COP) has shown antitumor activity in colon cancer, but its molecular mechanism and its molecular targets have not been fully understood. In this study, the biological behavior was verified in vitro. The targets of Huanglian alkaloids on colon cancer were predicted, and the protein-protein interaction (PPI) network was constructed. The core targets of safranine for colon cancer were extracted and analyzed by GO and KEGG enrichment to identify the possible molecular mechanisms of safranine treatment. Western blot was used to detect the changes of related pathway proteins in colon cancer cells. The differential expression of hub genes in colon cancer was analyzed using the GEPIA2 website. The binding ability of safranine to the target was verified by molecular docking. Finally, the targets were preliminarily verified by q-PCR analysis. Coptisine can inhibit the survival, migration, and proliferation of colon cancer cells DLD1 and HCT-116. Based on network pharmacology, ninety-one targets for colon cancer were screened. ESR1, ALB, AR, CDK2, PARP1, HSP90AB1, IGF1R, CCNE1, and CDC42 were found in the top 10. Enrichment analysis showed that these targets were mainly related to pathways in cancer, FC γ R-mediated phagocytosis, prostate cancer, progesterone-mediated oocyte maturation, the oestrogen signal pathway, proteoglycan in cancer and the PI3K-Akt signal pathway. WB results showed that after the treatment of colon cancer DLD1 cells with coptisine, the expression of P-AKT and AKT decreased, that of its downstream protein Bcl-2 decreased, and that of BAX increased. Differential expression analysis of hub genes showed that CCNE1, CDK2, HSP90AB1, and CHEK2 were upregulated in colon cancer samples, and molecular docking showed that these targets had a good ability to bind to coptisine. After the treatment of colon cancer DLD1 cells with coptisine, q-PCR results showed that CCNE1 and HSP90AB1 were significantly downregulated, while CDK2 and CHEK2 had no significant changes. Coptisine may be a candidate drug for the treatment of colon cancer, and its therapeutic effect may be related to the cancer pathway and PI3K-Akt signalling pathway. CCNE1 and HSP90AB1 may be potential targets of coptisine in the treatment of colon cancer.

NKD1 promotes glucose uptake in colon cancer cells by activating YWHAE transcription

Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation. HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1-/- cells), and SW620-nkd1-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells. In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P < 0.05). NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.

The effect of up-regulation of NLE1 gene expression on the invasion and migration of colon cancer cells and its mechanism

ObjectivesTo explore the regulatory effects of up-regulation of NLE1 gene expression in vitro and the mechanism of colon cancer cell invasion and migration. MethodsThe logarithmic growth phase human colonic fibroblasts CCD-18Co, colon cancer cells HT-29, and NLE1 interference group were transfected with NLE1 siRNA to interfere with expressing viral vectors and liposomes. The NLE1 negative control group was added with liposomes, and NLE1 was overexpressed. The expression changes of RNA interference on the target gene were analyzed. ResultsThe expression of NLE1 mRNA in colon cancer cells was significantly higher (P < 0.05). Likewise, the expression of NLE1 protein in colon cancer cells was significantly increased than in control colon cancer cell lines (p < 0.05). Compared with the NLE1 interference group and the NLE1 negative control group, the migration and invasion ability of colon cancer cells in the NLE1 overexpression group were significantly varied (P < 0.05)‘. ConclusionsUp-regulation of the NLE1 gene in colon cancer cells promotes cell migration and invasion of colon cancer cells and inhibits the apoptosis ability of colon cancer cells.

YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2.

Colon cancer is one of the most common causes of cancer-associated mortality. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β (YWHAB) has been reported to be aberrantly expressed in human colon cancer cells following alltrans retinoic acid treatment. PI3K regulatory subunit 2 (PIK3R2) has also been identified as a gene associated with colon cancer metastasis and tumor progression. The present study aimed to determine the role of YWHAB in colon cancer in addition to its detailed reaction mechanism. The expression levels of YWHAB and PIK3 'A0* R2 before or after transfection of YWHAB interference plasmids or PIK3R2 overexpression plasmids were examined by reverse transcription-quantitative PCR and western blotting. PI flow cytometry, Cell Counting Kit-8 and TUNEL assays were performed to measure the extent of cell cycle progression, proliferation and apoptosis. Additionally, the expression levels of G1-S cell-cycle transition regulator cyclin D1 and G1-checkpoint CDK inhibitor p21 and apoptosis marker proteins Bcl2 and Bax were assessed using western blotting. Subsequently, the Monarch Initiative database (https://monarchinitiative.org/) predicted the binding of YWHAB and PIK3R2, following which co-immunoprecipitation assay was utilized to assess their potential interaction. Furthermore, western blotting was performed to examine the expression levels of PI3K/AKT signaling pathway markers. It was revealed that YWHAB expression was upregulated in colon cancer cells compared with HIEC-6 human intestinal epithelial cells. Functionally, YWHAB depletion by transfection of YWHAB interference plasmids was demonstrated to suppress the proliferation of colon cancer cells whilst promoting cell cycle arrest at the G0/G1 phase and apoptosis, decreasing cyclin D1 and Bcl2 expression, and increasing p21 and Bax expression. Additionally, YWHAB was verified to bind to PIK3R2 and YWHAB knockdown decreased PIK3R2 expression. PIK3R2 overexpression by transfection of PIK3R2 overexpression plasmids reversed the effects of YWHAB knockdown on cell proliferation, cycle arrest, apoptosis, and apoptotic and cell cycle proteins in colon cancer cells. YWHAB knockdown reduced the levels of p-PI3K/PI3K and p-AKT/AKT and PIK3R2 overexpression also reversed the effects of YWHAB knockdown on the expression of PI3K/AKT signaling markers. In conclusion, these data suggest that YWHAB can activate the PI3K/AKT signaling pathway and participate in the malignant progression of colon cancer by targeting PIK3R2, which provides novel insights into the mechanism of colon cancer.

Proteomic identification of arginine-methylated proteins in colon cancer cells and comparison of messenger RNA expression between colorectal cancer and adjacent normal tissues.

PurposeIdentification of type I protein arginine methyltransferase (PRMT) substrates and their functional significance during tumorigenesis is becoming more important. The present study aimed to identify target substrates for type I PRMT using 2-dimensional (2D) gel electrophoresis (GE) and 2D Western blotting (WB).MethodsUsing immunoblot analysis, we compared the expression of type I PRMTs and endogenous levels of arginine methylation between the primary colorectal cancer (CRC) and adjacent noncancerous tissues paired from the same patient. To identify arginine-methylated proteins in HCT116 cells, we carried out 2D-GE and 2D-WB with a type I PRMT product-specific antibody (anti-dimethyl-arginine antibody, asymmetric [ASYM24]). Arginine-methylated protein spots were identified by mass spectrometry, and messenger RNA (mRNA) levels corresponding to the identified proteins were analyzed using National Center for Biotechnology Information (NCBI) microarray datasets between the primary CRC and noncancerous tissues.ResultsType I PRMTs and methylarginine-containing proteins were highly maintained in CRC tissues compared to noncancerous tissues. We matched 142 spots using spot analysis software between a Coomassie blue (CBB)-stained 2D gel and 2D-WB, and we successfully identified 7 proteins that reacted with the ASYM24 antibody: CACYBP, GLOD4, MAPRE1, CCT7, TKT, CK8, and HSPA8. Among these proteins, the levels of 4 mRNAs including MAPRE1, CCT7, TKT, and HSPA8 in CRC tissues showed a statistically significant increase compared to noncancerous tissues from patients using the NCBI microarray datasets.ConclusionOur results indicate that the method shown here is useful in identifying arginine-methylated proteins, and significance of arginine modification in the proteins identified here should be further identified during CRC development.

Arsenic Trioxide Promotes Tumor Progression by Inducing the Formation of PGCCs and Embryonic Hemoglobin in Colon Cancer Cells.

Arsenic trioxide (ATO) has been used to treat acute promyelocytic leukemia. However, it is not effective in treating solid tumors such as colorectal cancer. We have previously reported that polyploid giant cancer cells (PGCCs) exhibiting the characteristics of cancer stem cells can be generated by various inducers. In this study, ATO was used to induce the formation of PGCCs in LoVo and Hct116 colon cancer cell lines. The migration, invasion, and proliferation abilities of colon cancer cells with and without ATO treatment were assessed by wound-healing, transwell, and plate colony formation assays. The expression of epithelial to mesenchymal transition-related proteins and erythroid differentiation-related proteins in colon cancer cells was further evaluated by western blot and immunocytochemical assays. LoVo and Hct116 cells were transfected with a eukaryotic expression vector for green fluorescent protein (GFP), red fluorescent protein (RFP), H2B-GFP, and H2B-mCherry to study PGCCs formation via cell fusion. WB and ICC assays were performed to assess the expression of cell fusion-related proteins. MG132, small interfering RNA-glial cell missing 1 (GCM1), and chromatin immunoprecipitation-polymerase chain reaction assays were performed to study the role of GCM1/syncytin-1-mediated cell fusion. Clinically, the significance of cell fusion-related proteins and erythroid differentiation-related proteins expression in human colorectal cancer tissues was evaluated. Results of our study showed that ATO induced the formation of PGCCs, and the daughter cells derived from PGCCs gained a mesenchymal phenotype and exhibited strong migration, invasion, and proliferation abilities. PGCCs also produced embryonic hemoglobin-delta and -zeta with strong oxygen-binding ability and erythroid differentiation-related proteins after ATO treatment. In addition, cell fusion was observed during the formation of PGCCs, indicated by the presence of yellow fluorescence via the GCM1/syncytin-1 signaling pathway. Clinically, the expression of cell fusion-related and erythroid differentiation-related proteins gradually increased with the progression of human colorectal cancer tissues. In conclusion, ATO can promote tumor progression by inducing the formation of PGCCs via GCM1/syncytin-1-mediated cell fusion. PGCCs can produce daughter cells with high invasion and migration abilities and embryonic hemoglobin with strong oxygen binding ability, promoting survival of tumor cells in a hypoxic microenvironment.

CHK Methylation Is Elevated in Colon Cancer Cells and Contributes to the Oncogenic Properties.

Src is an important oncogene that plays key roles in multiple signal transduction pathways. Csk-homologous kinase (CHK) is a kinase whose molecular roles are largely uncharacterized. We previously reported expression of CHK in normal human colon cells, and decreased levels of CHK protein in colon cancer cells leads to the activation of Src (Zhu et al., 2008). However, how CHK protein expression is downregulated in colon cancer cells has been unknown. We report herein that CHK mRNA was decreased in colon cancer cells as compared to normal colon cells, and similarly in human tissues of normal colon and colon cancer. Increased levels of DNA methylation at promotor CpG islands of CHK gene were observed in colon cancer cells and human colon cancer tissues as compared to their normal healthy counterparts. Increased levels of DNA methyltransferases (DNMTs) were also observed in colon cancer cells and tissues. DNA methylation and decreased expression of CHK mRNA were inhibited by DNMT inhibitor 5-Aza-CdR. Cell proliferation, colony growth, wound healing, and Matrigel invasion were all decreased in the presence of 5-Aza-CdR. These results suggest that increased levels of DNA methylation, possibly induced by enhanced levels of DNMT, leads to decreased expression of CHK mRNA and CHK protein, promoting increased oncogenic properties in colon cancer cells.

Ropivacaine inhibits the proliferation and migration of colorectal cancer cells through ITGB1

ABSTRACT To study whether ropivacaine inhibits the proliferation and migration of colon cancer cells through ITGB1 (Integrin beta-1). First, the effect of ropivacaine on cell proliferation and migration was detected by MTT and Transwell. DAPI staining, annexin V staining and Western blot were used to detect the expression of apoptosis-related proteins to investigate the effect of ropivacaine on cell apoptosis. Using bioinformatics software to predict the potential drug targets of ropivacaine. RT-PCR, Western blot and immunofluorescence verify the distribution and expression of the drug target ITGB1, and detect its downstream-related proteins to further prove that ropivacaine affects colon cancer cells by acting on ITGB1 protein. 1. Ropivacaine significantly inhibited the proliferation of colon cancer cells and promoted their apoptosis 2. Ropivacaine could interact with ITGB1 protein, and inhibited the expression of ITGB1 protein in colon cancer cells, thereby affecting its downstream signaling pathway. Ropivacaine regulates the function of colon cancer cells by targeting the expression of ITGB1 protein and affecting the activation of its downstream signaling pathways. Abbreviation: Integrin beta-1 (ITGB1); 3-(45)-dimethylthiahiazo (-z-y1)-35-di- phenytetrazoliumromide (MTT); 4. 6-diamimo-2-phenyl indole (DAPI); Reverse transcrption PCR (RT-PCR); Colorectal cancer (CRC); Local anesthetics (LA); voltage-gated sodium channel (VGSC); dulbecco s modifed eade medium (DMEM); propidium iodide (PI); dodecyl sulf ate, sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE); Polyvinylidene Fluoride (PVDF); BCL2 associated X (Bax); Focal Adhesion Kinase (FAK); extracellular signal-regulated kmase (ERK); alpha serme threcnime-proteim kinase (AKT); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Tris-buffered salme with 0.1% Tween 20 (TBST); Similarty ensemble approach (SEA)

Targeting LncRNA EPIC1 to inhibit human colon cancer cell progression.

Long non-coding RNA EPIC1 (Lnc-EPIC1) binds MYC protein, which is essential for MYC function and expression of MYC target genes. The current study tested its expression and potential functions in human colon cancer cells. We show that Lnc-EPIC1 expression is elevated in human colon cancer tissues and primary human colon cancer cells. Whereas its expression is relatively low in normal colon tissues and colon epithelial cells. In the primary human colon cancer cells, Lnc-EPIC1 siRNA largely inhibited cancer cell growth, proliferation, migration and invasion. Further, Lnc-EPIC1 silencing induced significant apoptosis activation in colon cancer cells. Conversely, ectopic overexpression of Lnc-EPIC1 augmented colon cancer cell growth, proliferation, migration and invasion. RNA-immunoprecipitation and RNA pull-down results confirmed that Lnc-EPIC1 directly binds MYC protein in colon cancer cells. MYC target proteins, including cyclin A, cyclin D and CDK9, were downregulated with Lnc-EPIC1 silencing, but upregulated after Lnc-EPIC1 overexpression in colon cancer cells. Further Lnc-EPIC1 silencing or overexpression failed to alter functions of MYC-knockout colon cancer cells. Collectively, overexpressed Lnc-EPIC1 is important for the progression of human colon cancer cells.

DPEP1 promotes the proliferation of colon cancer cells via the DPEP1/MYC feedback loop regulation

DPEP1 is highly expressed in the colorectal carcinoma tissues and colon cancer cells. However, the function and underlying mechanism of DPEP1 in the colon cancer cells are still poorly understood. Here, we found that transcription factor MYC could occupy on the DPEP1 promoter and activate its activities, and DPEP1 was up-regulated by MYC proteins in mRNA and protein levels in a dose-dependent manner in colon cancer cells. The expression levels of DPEP1 were positively correlated with that of MYC in colorectal tumor tissues. Moreover, Laser confocal images and Co-immunoprecipitation (Co-IP) revealed that DPEP1 and MYC proteins could bind to each other in the colon cancer cells. In turn, DPEP1 could enhance the stability of MYC proteins by extending the half-life of MYC proteins in colon cancer cells. Thus, DPEP1 and MYC proteins might form a positive feedback loop to maintain their high expression levels in colon cancer cells. In function, the MTT, EdU, Clone Formation assays and xenograft tumors assays demonstrated that DPEP1 could boost the proliferation of colon cancer cells through the DPEP1/MYC positive feedback loop in vitro and in vivo. Theoretically, DPEP1 may serve as a colon cancer biomarker and a novel target of colorectal carcinogenesis therapy.

Significant expression of tafazzin (TAZ) protein in colon cancer cells and its downregulation by radiation

Aim: To demonstrate the radiation responses of tafazzin (TAZ) protein in colon cancer.Methods: TAZ expression was examined in colon cancer cell lines SW480, KM12C, SW620 and KM12L4a. KM12C and KM12L4a cell lines were used for this experiment with exposure to X- and UV rays (mW/cm2). HCT15 cell line was used to test the expression of TAZ by using an anti-TAZ drug, namely 9-fluorenone, which is a Hippo-YAP/TAZ signaling inhibitor. The experimentation also involved exposing HCT15 cell line, to UV radiation. Cell proliferation and apoptosis studies were carried out. TAZ interactions with oncoproteins were screened and the oncoproteins Livin, MAC30 and FXYD-3 were considered for in silico protein–protein interaction studies.Results: TAZ protein was significantly downregulated after 2 Gy radiations. 9-Fluorenone inhibited the expression of TAZ. Action of 9-fluorenone along with radiation, decreased the percentage of proliferation and increased apoptosis. Computational studies predicted that TAZ interacts with the oncoproteins Livin, MAC30 and FXYD-3.Conclusions: Our results suggest that TAZ plays a significant role in non-metastatic KM12C cells and is predominantly seen in the colon cancer cells isolated from primary stages of cancer. Thus, use of TAZ protein as a biomarker will be an efficient way to detect tumors in the early stages and treatment may be modulated with radiation before surgery/therapy.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation.

PurposeThe serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression.Materials and MethodsCEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis.ResultsCEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner.ConclusionCEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

CAMP‐Phosphodiesterase PDE4D as a Target for Colon Cancer Therapy

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) are part of a superfamily of isoenzymes that hydrolyze and inactivate the second messenger cAMP. Recent studies have shown that treatment with non‐selective PDE4 inhibitors impairs cancer cell proliferation and tumor growth, cell migration, epithelial to mesenchymal transition and tumor angiogenesis in various types of cancers; suggesting that PDE4s represent a potential target for the development of novel cancer therapeutics. However, non‐selective PDE4 inhibitors are known to induce a number of side effects, including nausea and emesis, diarrhea and weight loss that limit their clinical utility. The PDE4 family comprises four subtypes/genes, PDE4A to D. As each plays unique and non‐overlapping physiological and pathophysiological roles in the body, targeting individual PDE4 subtypes may serve to dissect the therapeutic benefits from the side effects of non‐selective PDE4 inhibitors. To this end, we have explored the role of PDE4 and individual PDE4 subtypes in five human colon cancer cell lines. Treatment with the archetypal non‐selective PDE4 inhibitor Rolipram ablated the chief portion of total cAMP hydrolytic activity in cell extracts prepared from all five colon cancer cell lines, suggesting that PDE4s are the main cAMP‐PDE expressed in these cells. Treatment with non‐selective PDE4 inhibitors such as Rolipram significantly reduced the viability of colon cancer cells in the presence of low doses of the adenylyl cyclase activator forskolin; confirming the potential of PDE4 as a therapeutic target. In addition, treatment with Rolipram attenuated cancer cell migration assessed in wound scratch assays. Western blotting and immunoprecipitation with PDE4 subtype‐selective antibodies revealed that PDE4 activity and protein in colon cancer cells is largely due to PDE4D, with little to none of the other PDE4 subtypes (PDE4A‐C) being expressed. If colon cancer cells exclusively express PDE4D, then the effect of non‐selective PDE4 inhibitors on cell viability/motility must necessarily have been mediated via inhibition of PDE4D, thus identifying PDE4D as the preferred target for development of a therapeutic. We will be testing this hypothesis by assessing the effect of selective PDE4D inactivation on various cancer cell functions. The ultimate goal is to determine whether selective inhibition of PDE4D retains the cancer therapeutic benefits, but exhibits an improved safety profile compared to the non‐selective PDE4 inhibitors available to date.

Zinc finger protein 91 (ZFP91) activates HIF-1α via NF-κB/p65 to promote proliferation and tumorigenesis of colon cancer.

Zinc finger protein 91 (ZFP91) has been reported to be involved in various biological processes. However, the clinical significance and biological role of ZFP91 in colon cancer remains unknown. Here, we show that ZFP91 expression is upregulated in patients with colon cancer. We found that ZFP91 upregulated HIF-1α at the levels of promoter and protein in colon cancer cells. Using chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter gene assay, we found that NF-κB/p65 is required for the binding of ZFP91 to the HIF-1α promoter at −197/−188 base pairs and for the transcriptional activation of HIF-1α gene mediated by ZFP91. Flow cytometry, 5-ethynyl-2′-deoxyuridine (EdU) incorporation and tumor xenograft assay demonstrated that ZFP91 enhanced cell proliferation of colon cancer through upregulating HIF-1α in vitro and in vivo. Furthermore, ZFP91 is positively associated with HIF-1α in human colon cancer. Thus, we concluded that ZFP91 activates transcriptional coregulatory protein HIF-1α through transcription factor NF-κB/p65 in the promotion of proliferation and tumorigenesis in colon cancer cell. ZFP91 may serve as a driver gene to activate HIF-1α transcription in the development of cancer.

Periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway

In the present study, we aimed to explore the effects of periostin, a cell adhesion protein, on chemoresistance in colon cancer cells. Reverse-transcription polymerase chain reaction and Western blot analyses were employed to detect periostin expression in SW480 and HT-29 colon cancer cells treated with oxaliplatin or fluorouracil (5-FU). Small interfering RNA was used to downregulate endogenous periostin. Annexin-V/propidium iodide staining was performed to analyze the effects of periostin on drug-induced apoptosis. The results showed that treatment with oxaliplatin or 5-FU elevated both the mRNA and protein levels of periostin in SW480 and HT-29 cells. Silencing of periostin significantly (P < 0.01) augmented drug-induced apoptosis in colon cancer cells, coupled with enhanced cleavage of caspase-3 and poly(ADP-ribose) polymerase. Mechanistic studies revealed that periostin silencing significantly (P < 0.01) suppressed the expression of survivin, an antiapoptotic protein in colon cancer cells. Enforced expression of survivin repressed drug-induced apoptosis in periostin-depleted SW480 and HT-29 cells. Additionally, periostin overexpression increased the expression of survivin and the phosphorylation of Akt, which was reversed by pretreatment with the phosphatidylinositol 3-kinase (PI3K)-specific inhibitor LY294002. Taken together, our data demonstrate that periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway.

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells.

Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis

Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain (CBD) of Ku70 (amino acids 471–478). Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A), limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.

Abstract 2084: Claudin-1 induces resistance to anoikis in Src dependent manner in colon cancer cells

Abstract Anoikis, a complete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), is often dysfunctional in metastatic cancer cells. We along with other groups have demonstrated that expression of Claudin-1, a tight junction protein, is markedly increased and mislocalized in colon cancer and helps promote the tumor progression especially metastasis. However the underlying mechanism remains poorly understood. To explore, we examined effect of claudin-1 expression upon anoikis. Colon cancer cells genetically manipulated (stable overexpression in SW480 cells and RNA-interference based gene silencing in SW620 cells) for claudin-1 expression were cultured on polyHEMA-coated culture dishes to induce anoikis. Results demonstrated a positive correlation between claudin-1 expression and resistance to anoikis. We also observed a positive correlation between claudin-1 expression, Src (L4A1) and Akt phosphorylation (Ser 473) and Bcl-2 expression in the cells subjected to anoikis. Notably, use of PP2, a Src-kinase inhibitor, inhibited claudin-1-dependent resistance to anoikis (2.5fold) as well as the increases in Akt-phosphorylation and Bcl-2 expression, and thus suggested a causal role of Src-activation in claudin-1-dependent resistance to anoikis. Our further studies revealed that claudin-1 associates with Src in a multi-protein complex that also included ZO-1, a PDZ-domain containing tight junction protein, known to associate with claudin-1and Src. Importantly, claudin-1 contains a PDZ-binding domain in its c-terminal. Deletion of the c-terminal domain of claudin-1 resulted in the loss of the physical association between claudin-1 and Src and a significant loss in claudin-1-dependent resistance to anoikis. Thus, our findings supported a role of claudin-1 in the regulation of colon cancer progression through its association with Src and dependent cell signaling. Our further analysis using a large database of colon cancer patient samples (microarray analysis; 260 colon cancer and 10 adjacent normal colon samples) showed a significant and positive correlation between claudin-1 and Src expression. Taken together, we here report a novel partnering between claudin-1 and Src proteins in colon cancer cells which has a function implication in the regulation of anoikis in a claudin-1/Src/PI3-kinase/Bcl-2-dependent manner. Our findings have clinical significance and have the potential to open potential new therapeutic opportunity in colon cancer treatment and/or management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2084. doi:1538-7445.AM2012-2084

The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer

PHLPP is a family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt. Our recent studies have demonstrated that the ubiquitin proteasome pathway plays an important role in the downregulation of PHLPP in colorectal cancer. In this study, we show that the deubiquitinase USP46 stabilizes the expression of both PHLPP isoforms by reducing the rate of PHLPP degradation. USP46 binds to PHLPP and directly removes the polyubiquitin chains from PHLPP in vitro and in cells. Increased USP46 expression correlates with decreased ubiquitination and upregulation of PHLPP proteins in colon cancer cells, whereas knockdown of USP46 has the opposite effect. Functionally, USP46-mediated stabilization of PHLPP and the subsequent inhibition of Akt result in a decrease in cell proliferation and tumorigenesis of colon cancer cells in vivo. Moreover, reduced USP46 protein level is found associated with poor PHLPP expression in colorectal cancer patient specimens. Taken together, these results indentify a tumor suppressor role of USP46 in promoting PHLPP expression and inhibiting Akt signaling in colon cancer.

Guggulsterone-Mediated Enhancement of Radiosensitivity in Human Tumor Cell Lines

Purpose: To observe the effect of guggulsterone (GS) on the radiation response in human cancer cell lines. Materials and methods: The radiation response of cancer cells treated with GS was observed by cell survival studies, cell growth assay, NF-κB activity assay, western blotting of some key growth promoting receptors, the DNA repair protein γH2AX, and flow cytometry for DNA analyses. Results: GS inhibited radiation induced NF-κB activation and enhanced radiosensitivity in the pancreatic cell line, PC-Sw. It reduced both cell cycle movement and cell growth. GS reduced ERα protein in MCF7 cells and IGF1-Rβ protein in colon cancer cells and pancreatic cancer cells and inhibited DNA double strand break (DSB) repair following radiation. Conclusion: GS induced radiation sensitization may be due to several different mechanisms including the inhibition of NF-κB activation and reductions in IGF1-Rβ. In addition, GS induced γH2AX formation, primarily in the S-phase, indicates that DNA DSB's in the S-phase may be another reason for GS induced radiosensitivity. ERα down-regulation in response to GS suggests that it can be of potential use in the treatment of estrogen positive tumors that are resistant to tamoxifen.