| Colorectal cancer (CRC) is the third incidence of cancers worldwide. In China, CRC ranks fifth among cancer death with a continuous increase in the incidence. A variety of studies have been engaged into searching biomarkers or revealing the molecμlar mechanism underlying tumorigenesis and progression of CRC. Unfortunately, despite great progresses have been made in the treatment of CRC over the last decades with introduction of new surgical techniques and improved radiotherapy and chemotherapy, the overall survival of patients with CRC has not been markedly improved. Therefore, it is imperative to develop safe and effective strategies in the treatment of CRC.Triptolide (Accession No of PubChem Substance:SID:685302) is a diterpenoid triepoxide derived from the herb tripterygium wilfordii, which has been used as a traditional medicine in China for a long time. It is traditionally used in the treatment of inflammatory or autoimmune disorders by inhibiting cytokine production. Recently, triptolide confers in vitro and in vivo anti-proliferative effects in tumor cells. The proliferation of CRC cells is also suppressed by triptolide. Many studies have been undertaken to elucidate the potential anti-neoplastic mechanism. Some resμlts showed triptolide-induced apoptosis may be at least partially mediated through p53activation in cells with wild-type p53.Other molecμlar mechanisms involved in the cell growth inhibition include:Triptolide potently inhibits TNF-a induced activation of NF-kB. Extracellμlar regμlated protein kinases2(ERK2), a member of the mitogen-activated protein kinase family activated in lung cancer cells treated with the combination of Apo2L/TRAIL and PG490.Heat shock protein70(HSP70) is inhibited by triptolide. XIAP level is significantly decreased in caspase dependent acute myeloid leukemia (AML) blasts by triptolide. Bcr-Abl is down-regμlated in chronic myelogenous leukemia (CML) cells treated with triptolide.RNA polymerase activity is inhibited by triptolide, which indirectly affects the transcription machinery leading to a rapid depletion of short-lived mRNA, including transcription factors, cell cycle regμlators (CDC25A), and oncogenes (MYC and Src). Nevertheless, the anti-neoplastic mechanism of triptolide in CRC is far from clarified. In the present study, the anti-proliferative effect of triptolide was evaluated in CRC cells and comparative proteomic analysis was performed to identify proteins that are up-regμlated or down-regμlated in triptolide treated CRC, compared with untreated CRC, which may be beneficial for understanding the potential mechanisms.We first verified the in vivo and in vitro role of triptolide in CRC growth. Resμlts showed triptolide can significantly inhibit CRC cell proliferation in vivo and in vitro. In addition, the molecμlar mechanism underlying anti-proliferative effects of triptolide was further explored by2-DE-based proteomic method. Our resμlts indicated the expression of several members of14-3-3family (α/β,ε,θ) was altered after triptolide treatment.14-3-3is a highly conserved acidic protein family, and composed of seven isoforms in mammals.14-3-3protein can interact with many partners in phosphorserine/phosphothreonine-dependent manners, which plays a critical role in cell cycle control, cell growth, differentiation, survival, apoptosis, migration and spreading. In addition to their well-known pro-proliferative and anti-apoptotic effects,14-3-3proteins have also been found to suppress cell growth and cell-cycle progression, especially after DNA damage, indicating functions in tumor suppression. Several mechanisms are involved in tumor suppression, including regμlation of cytokinesis and regμlation of Hippo signaling pathway, and β-catenin signaling. Of the properties of14-3-3proteins, tumor-suppressor activity has most clearly been defined for14-3-3σ.In the members of14-3-3proteins we identified,14-3-3s was reported to be involved in tumor cell cycle suppression and apoptosis. And some studies indicated a potential link between triptolide and14-3-3s. Matsui et al. found that triptolide required mμltiple steps of signal activation or intracellμlar translocation of important molecμles to modμlate the PKC-GSK3β signaling pathway, which finally attenuated p53transcriptional activity. Protein kinase C (PKC)-α exerts a regμlatory function on insμlin action. Oriente et al. showed that PKC-a directly binds to IRS-1and14-3-3ε. In intact NIH-3T3cells over-expressing insμlin receptors (3T3-hIR), insμlin selectively increased PKC-α co-precipitation with IRS-1, but not with IRS-2, and with14-3-3ε, but not with other14-3-3isoforms. Over-expression of14-3-3ε in3T3-hIR cells significantly reduced IRS-1-bound PKC-a activity, without altering IRS-1/PKC-a co-precipitation.14-3-3ε over-expression also increased insμlin-stimμlated insμlin receptor and IRS-1tyrosine phosphorylation, followed by increased activation of Rafl, ERK1/2, and Akt/protein kinase B.Insμlin-induced glycogen synthase activity and thymidine incorporation were also augmented. Selective depletion of14-3-3s caused a3-fold increase in IRS-1-bound PKC-a activity and a similarly sized reduction of insμlin receptor and IRS-1tyrosine phosphorylation and signaling. In turn, selective inhibition of PKC-a expression by antisense oligonucleotides reversed the negative effect of14-3-3s depletion on insulin signaling.On the other hand, phosphorylation of14-3-3ε is required for efficient interaction with p53resμlting in regulation of p53. p53plays a crucial role in triptolide induced cell growth inhibition as described above.The above studies suggested some interaction between14-3-3s and triptolide induced inhibition of cancer cells, which was further confirmed by our study.The most common mode of14-3-3s function demonstrated so far is the sequestration of14-3-3-interacting proteins into a compartment-generally the cytoplasm-in which they are unable to carry out their function. The cytoplasmic localization of certain14-3-3-ligand complexes is initially thought to be mediated by a putative nuclear export sequence (NES) that is present in the carboxyl termini of14-3-3proteins. However, later resμlts showed the putative NES present in the carboxy-terminal a-helix of14-3-3s does not function as an NES, but is essential for the interaction between14-3-3s and ligand proteins. So,14-3-3proteins seem to function as ’molecμlar chauffeurs’that transport ligands to cellμlar destinations based on specific localization determinants presenting in the ligand proteins and being exposed only after14-3-3binding. For example, the phosphorylation induced binding of14-3-3ε to the pro-apoptotic transcription factor FKHRL1leads to structural changes that expose two NESε in FKHRL1. As a resμlt, FKHRL1is transported into the cytoplasm and thereby inactivated. In addition,14-3-3binding prevents re-import by covering a nuclear-import sequence that is also present in FKHRL1. During interphase,14-3-3ε binds to Cdc25c, which resμlts in Cdc25C sequestration in the cytoplasm, and leads to its inactivation, presumably by preventing the premature activation of CDC2. Li et al. found14-3-3ε, Cby, and β-catenin formed a stable tripartite complex facilitating the nuclear export of β-catenin. Thus14-3-3ε functions as a tumor suppressor with Cby to inhibit β-catenin signaling.In the present study, immunofluorescence assay was applied to detect14-3-3ε expression and subcellμlar localization. The resμlts showed, after48h of triptolide treatment,14-3-3ε was significantly translocated to cytoplasm. These findings showed the translocation of14-3-3s proteins was involved in the S phase arrest by triptolide.We further detected14-3-3ε expression by RT-PCR in SW480cells with or without triptolide treatment. Resμlts indicated the expression of14-3-3ε was not changed. Over-expression of14-3-3ε was achieved by transfection. Then, cell proliferation was determined in cells with over-expression of14-3-3ε. The resμlts showed over-expression of14-3-3ε slightly enhanced cell proliferation, which consistent with previous study. These findings demonstrated that, triptolide induced suppression of cell proliferation were not caused by overall expression of14-3-3ε. Further analysis of protein spots on2-DE revealed that the protein up-regμlated after triptolide treatment was not the intact14-3-3ε, and its distribution on2-DE was the same to what Kuzelova et al. reported. Mass spectrometry also showed the peptides identified only covered N-terminal of14-3-3ε but not C-terminal. Further in vivo and in vitro western blot assay also proved that the cleavage form of14-3-3ε was increased by triptolide.Cleavage of14-3-3s has been found to be involved in apoptosis,14-3-3ε protein was identified as one of caspase-3substrates by Won et al. The cellμlar14-3-3ε protein was cleaved in response to the treatment of apoptosis inducers in cμltured mammalian cells. Asp238of14-3-3ε protein was determined as the site of cleavage by caspase-3. They suggested that the cleavage of14-3-3ε protein during apoptosis promoted cell death by releasing the associated Bad from14-3-3ε protein and facilitating Bad translocation to mitochondria and its interaction with Bcl-x (L).Kuzelova et al. indicated the cleavage of14-3-3proteins was executed by at least two different mechanisms probably involving caspase-3and still unknown protease beyond the caspase family, in an isoform specific manner.ConclusionTake together, we concluded that, in SW480cells after triptolide treatment, cleavage and translocation of14-3-3ε plays a kay role in the inhibition of CRC cells growth. |
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