RV-59 suppresses cytoplasmic Nrf2-mediated 5-fluorouracil resistance and tumor growth in colorectal cancer.
ABSTRACT: Our previous studies indicated that tumor invasion and 5-flurouracil (5-FU) resistance in colorectal cancer (CRC) was more affected by cytoplasmic localization of expressed Nrf2 (cNrf2) than by nuclear localization (nNrf2), indicating a need for novel antitumor agents to overcome 5-FU resistance and improve outcomes in patients with CRC. In the present study, 20 nitrogen-substituted anthra[1,2-c][1,2,5] thiadiazole-6,11-dione derivatives were collected to verify the compound most able to suppress cell growth in nuclear location sequence (NLS)-mutated Nrf2-transfected shNrf2-HCT116 stable clones that have high cNrf2 expression. The MTT assay indicated that these high-cNrf2-expressing shNrf2-HCT116 stable clones exhibited the lowest percentage survival when treated with RV-59 than with the other 19 compounds. As expected, the high-cNrf2-expressing cells also showed a higher value for the inhibitory concentration of 50% cell survival (IC50) for 5-FU when compared with Nrf2-knockdown HCT116 stable clones (17.74 ?M vs. 5.34 ?M). Interestingly, a lower RV-59 IC50 value was seen in the high-cNrf2-expressing stable clones than in the Nrf2-knockdown stable clones (3.55 ?M vs. 16.81 ?M). A similar low RV-59 IC50 value was observed in high-cNrf2-expressing NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones and p53 null (-/-) HCT116 cells (4.2 ?M vs. 4.4 ?M), whereas the IC50 value was 17.6 ?M in normal colon FHC epithelial cells. Colony-forming assays confirmed that RV-59 treatment inhibited colony formation in NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones and in p53-/- HCT116 cells. Annexin-V/PI staining showed an involvement of apoptosis in the inhibitory effect of RV-59 on cell viability. A nude mouse xenograft tumor model showed that RV-59 efficiently suppressed tumor growth induced by transplanted NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones without affecting the body weight of the nude mice over the 37 day experimental period. These results strongly suggest that RV-59 may be a novel antitumor agent for suppression of 5-FU resistance and may have therapeutic potential for improving outcomes in patients with cNrf2-expressing tumors.
Project description:Nuclear Nrf2 (nNrf2) binding to the antioxidant response element may promote chemoresistance in colorectal cancer. However, the shuttling of Nrf2 between cytoplasm and nucleus in colon cancer cells has revealed the possibility that cytoplasmic location of Nrf2 (cNrf2) may play a specific role in chemoresistance. Transfection of a nuclear location sequence (NLS)-wild-type or NLS-mutated Nrf2 expression vector into a stable shNrf2 HCT116 clone using the MTT assay to examine whether chemoresistance induced by cNrf2 may be greater than nNrf2. Different specific inhibitors and small hairpin (sh)RNAs of targeting genes were used to verify the mechanistic action of cNrf2 in chemoresistance and further confirmed by an animal model. The association of cNrf2 with chemotherapeutic response in patients with colorectal cancer was statistically analyzed. The MTT assay indicated that cNrf2 may play a more important role than nNrf2 in conferring 5-fluorouracil (5-FU) and oxaliplatin resistance in HCT116 cells. Mechanistically, cNrf2-induced PSMD4 expression was responsible for chemoresistance in the NLS-mutated Nrf2-tranfected shNrf2HCT116 clone via the NF-?B/AKT/?-catenin/ZEB1 cascades. The tumor burden induced by the NLS-mutated Nrf2-transfected shNrf2HCT116 clone was completely suppressed by treatment with 5-FU in combination with carfilzomib. A higher prevalence of unfavorable chemotherapeutic response in colorectal cancer patients with cNrf2, PSMD4-positive, p-p65-positive, and nuclear ?-catenin tumors was observed when compared to their counterparts. cNrf2 may play a more important role than nNrf2 in the chemoresistance of colorectal cancer. Activation of the NF-?B/AKT/?-catenin/ZEB1 cascade by PSMD4 may be responsible for cNrf2-mediated chemoresistance.CNrf2 may play a more important role than nNrf2 in conferring 5-FU and oxaliplatin resistance. This observation in patients seemed to support the findings of the cell and animal models and suggested that PSMD4 may be responsible cNrf2-mediated chemoresistance via the NF-?B/AKT/?-catenin /ZEB1 cascades.
Project description:Two common single nucleotide polymorphisms (SNPs) of the human TLR4 gene, namely Asp299Gly (D299G) and Thr399Ile (T399I), have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type (WT) and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC50-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells.
Project description:BACKGROUND: Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication. RESULTS: The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2. CONCLUSIONS: Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.
Project description:Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.
Project description:HCT116 cells transfected with lentiviral vectors expressing two different N-BLR shRNAs (clones #3-1 and #4-7) and empty vector control Overall design: Two HCT116 stable clones expressing N-BLR shRNAs (named clones #3-1 and #4-7) compared to empty vector control. Biological replicate: two stable clones vs empty vector controls
Project description:Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.
Project description:Colorectal cancer (CRC) has become a predominant cancer and accounts for approximately 10% of cancer-related mortality. Drug resistance still remains a priority mortality factor for patients due to no available therapeutic alternatives. The purpose of the present study was to investigate the underlying molecular mechanisms how eukaryotic translation initiation factor 3 subunit G (EIF3G) resensitized 5-Fu-resistant human CRC cells (HCT116/5-Fu) to 5-fluorouracil (5-Fu).Multiple cellular and molecular biology experiments were performed in the present study, such as CCK-8, western blotting and flow cytometry.We found that EIF3G is highly expressed at RNA and protein levels in HCT116/5-Fu cells compared with HCT116 cells using quantitative real-time polymerase chain reaction and Western blot analysis. In addition, silencing EIF3G enhanced 5-Fu-induced apoptosis in HCT116/5-Fu cells. Moreover, EIF3G silencing decreased the activity of the drug-related proteins MDR1 and MRP levels in HCT116/5-Fu cells. Finally, the xenograft tumor model further confirmed that EIF3G resensitized HCT116/5-Fu tumors to 5-Fu. We observed that EIF3G silencing followed by 5-Fu administration had a synergistic interaction effect on HCT116/5-Fu in vitro and in vivo.These findings demonstrate that EIF3G is a targetable regulator of chemoresistance in CRC, and inhibiting EIF3G in combination with 5-Fu might be a potential therapeutic strategy for colon cancer.
Project description:5-fluorouracil (5-FU), one of the first-line chemotherapeutic agents for the treatment of gastrointestinal malignancies, has shown limited efficacy. The expression of thymidylate synthase (TYMS) has been reported to be associated with the resistance to 5-FU. Here, we demonstrate that the enhanced HSP90 function and subsequent activation of Src induce expression of TYMS and acquired resistance to 5-FU in colon cancer. We show that the persistent 5-FU treatment granted 5-FU-sensitive HCT116 colon cancer cells morphologic, molecular, and behavioral characteristic of the epithelial-mesenchymal transition (EMT), contributing to emergence of acquired resistance to 5-FU. HCT116/R, a HCT116 colon cancer cell subline carrying acquired resistance to 5-FU, showed increased expression and activation of HSP90's client proteins and transcriptional up-regulation of TYMS. Forced overexpression of HSP90 or constitutive active Src in HCT116 cells increased TYMS expression. Conversely, pharmacological blockade of HSP90 or Src in HCT116/R cells effectively suppressed the changes involved in 5-FU resistance in vitro and xenograft tumor growth, hematogenous spread, and metastatic tumor development in vivo. This study suggests a novel function of HSP90-Src pathway in regulation of TYMS expression and acquisition of 5-FU resistance. Thus, therapeutics targeting this pathway may be an effective clinical strategy to overcome 5-FU resistance in colon cancer.
Project description:Most patients with advanced colorectal cancer (CRC) eventually develop resistance to systemic combination therapy. miR-195-5p and miR-497-5p are downregulated in CRC tissues and associated with drug resistance. Sensitization to 5-FU, oxaliplatin, and irinotecan by transfection with miR-195-5p and miR-497-5p mimics was studied using cell viability and clonogenic assays in cell lines HCT116, RKO, DLD-1, and SW480. In addition, proteomic analysis of transfected cells was implemented to identify potential targets. Significantly altered proteins were subjected to STRING (protein-protein interaction networks) database analysis to study the potential mechanisms of drug resistance. Cell viability analysis of transfected cells revealed increased sensitivity to oxaliplatin in microsatellite instable (MSI)/P53 wild-type HCT116 and RKO cells. HCT116 transfected cells formed significantly fewer colonies when treated with oxaliplatin. In sensitized cells, proteomic analysis showed 158 and 202 proteins with significantly altered expression after transfection with miR-195-5p and miR-497-5p mimics respectively, of which CHUK and LUZP1 proved to be coinciding downregulated proteins. Resistance mechanisms of these proteins may be associated with nuclear factor kappa-B signaling and G1 cell-cycle arrest. In conclusion, miR-195-5p and miR-497-5p replacement enhanced sensitivity to oxaliplatin in treatment naïve MSI/P53 wild-type CRC cells. Proteomic analysis revealed potential miRNA targets associated with the cell-cycle which possibly bare a relation with chemotherapy sensitivity.
Project description:Background:ACY1215 is a selective histone deacetylase 6 (HDAC6) inhibitor, and can suppress tumor growth for many human cancers. However, its role in colon cancer and its impact on the chemotherapeutic effect of 5-fluorouracil (5-Fu) are largely unknown. The aim of the present study is to explore the effect of ACY1215 on cell growth, migration, invasion and apoptosis, along with its impact on the chemotherapeutic effect of 5-Fu in HCT116 cells. Methods:HCT116 cells were treated with ACY1215 with or without 5-Fu, and cell viability, proliferation, migration, invasion and apoptosis were explored. Results:The cell viability, colony formation number, wound closure rate, and migrated cell numbers of HCT116 cells significantly decreased, while the apoptotic cells significantly increased with the increased concentration of ACY1215 (P<0.05). The combination of ACY1215 and 5-Fu was more potent than either drug alone, as indicated by an increase of apoptotic cells, and by a decrease of cell viability, colony formation number, wound closure rate and migrated cell numbers. The expression of phosphorylated mitogen-activated protein kinases (pMEK) and phosphorylated extracellular-signal regulated protein kinase (pERK) were decreased when HCT116 cells were cultured with ACY1215. Conclusions:Selective HDAC6 inhibitor, ACY1215, could inhibit the cell proliferation, migration and invasion, and induce apoptosis of HCT116 colon cancer cells. Furthermore, ACY1215 may enhance the chemotherapeutic effect of 5-Fu in HCT116 cells.