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Cell Death & Disease May 2019Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a...
Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a deubiquitinase that is overexpressed in TNBC and correlates with poor prognosis. UCHL3 deubiquitinates RAD51 thereby promoting the recruitment of RAD51 to DNA damage sites and augmenting DNA repair. Therefore, UCHL3 overexpression can render cancer cells resistant to DNA damage inducing chemo and radiotherapy, and targeting UCHL3 can sensitize TNBC to radiation and chemotherapy. However, small molecule inhibitors of UCHL3 are yet to be identified. Here we report that perifosine, a previously reported Akt inhibitor, can inhibit UCHL3 in vitro and in vivo. We found low dose (50 nM) perifosine inhibited UCHL3 deubiquitination activity without affecting Akt activity. Furthermore, perifosine enhanced Olaparib-induced growth inhibition in TNBC cells. Mechanistically, perifosine induced RAD51 ubiquitination and blocked the RAD51-BRCA2 interaction, which in turn decreased ionizing radiation-induced foci (IRIF) of Rad51 and, thereby, homologous recombination (HR)-mediated DNA double strand break repair. In addition, combination of perifosine and Olaparib showed synergistic antitumor activity in vivo in TNBC xenograft model. Thus, our present study provides a novel therapeutic approach to optimize PARP inhibitor treatment efficiency.
Topics: Animals; Antineoplastic Agents; Apoptosis; BRCA2 Protein; Cell Line, Tumor; Female; Humans; Mice; Mice, Nude; Phosphorylcholine; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Rad51 Recombinase; Radiation, Ionizing; Recombinational DNA Repair; Transplantation, Heterologous; Triple Negative Breast Neoplasms; Ubiquitin Thiolesterase; Ubiquitination; RNA, Guide, CRISPR-Cas Systems
PubMed: 31113933
DOI: 10.1038/s41419-019-1628-8 -
Molecular & Cellular Proteomics : MCP Jun 2019Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide...
Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.
Topics: Antioxidants; Collagen; Dermis; Dithiothreitol; Down-Regulation; Endoplasmic Reticulum Stress; Extracellular Matrix Proteins; Fibroblasts; Homeostasis; Humans; Mitogen-Activated Protein Kinases; Oxidation-Reduction; Phosphorylation; Phosphorylcholine; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 30890563
DOI: 10.1074/mcp.RA118.001140 -
Translational Cancer Research Dec 2018Perifosine, is a third generation alkylphospholipid analog which has promising anti-tumor efficacy in clinical trials of refractory/recurrent neuroblastoma (NB)....
BACKGROUND
Perifosine, is a third generation alkylphospholipid analog which has promising anti-tumor efficacy in clinical trials of refractory/recurrent neuroblastoma (NB). However, perifosine's mechanism of action remains unclear. Previously, we have shown that perifosine changes global proteome and acetylome profiles in NB.
METHODS
To obtain a more comprehensive understanding of the perifosine mechanism, we performed a quantitative assessment of the lysine ubiquitylome in SK-N-AS NB cells using SILAC labeling, affinity enrichment and high-resolution liquid chromatography combined with mass spectrometry analysis. To analyse the data of ubiquitylome, we performed enrichment analysis with gene ontology (GO), the Encyclopedia of Genes and Genomes (KEGG) pathway, ubiquitylated lysine motif, protein complex and protein domain. Protein-protein interaction was conducted to explore the crosstalk between ubiquitylome and previous global proteome/acetylome. Co-immunoprecipitation and western blotting were used to validate the results of the ubiquitylome analysis.
RESULTS
Altogether, 3,935 sites and 1,658 proteins were quantified. These quantified ubiquitylated proteins participated in various cellular processes such as binding, catalytic activity, biological regulation, metabolic process and signaling pathways involving non-homologous end-joining, steroid biosynthesis and Ras signaling pathway. Ubiquitylome and proteome presented negative connection. We identified 607 sites which were modified with both ubiquitination and acetylation. We selected 14 proteins carrying differentially quantified lysine ubiquitination and acetylation sites at the threshold of 1.5 folds as potential targets. These proteins were enriched in activities associated with ribosome, cell cycle and metabolism.
CONCLUSIONS
Our study extends our understanding of the spectrum of novel targets that are differentially ubiquitinated after perifosine treatment of NB tumor cells.
PubMed: 30761266
DOI: 10.21037/tcr.2018.11.30 -
Frontiers in Aging Neuroscience 2018Neuroinflammation and autophagy dysfunction are closely related to the development of neurodegeneration such as Parkinson's disease (PD). However, the role of autophagy...
Neuroinflammation and autophagy dysfunction are closely related to the development of neurodegeneration such as Parkinson's disease (PD). However, the role of autophagy in microglia polarization and neuroinflammation is poorly understood. TNF-α, which is highly toxic to dopaminergic neurons, is implicated as a major mediator of neuroinflammation in PD. In this study, we found that TNF-α resulted in an impairment of autophagic flux in microglia. Concomitantly, an increase of M1 marker (iNOS/NO, IL-1β, and IL-6) expression and reduction of M2 marker (Arginase1, Ym1/2, and IL-10) were observed in TNF-α challenged microglia. Upregulation of autophagy via serum deprivation or pharmacologic activators (rapamycin and resveratrol) promoted microglia polarization toward M2 phenotype, as evidenced by suppressed M1 and elevated M2 gene expression, while inhibition of autophagy with 3-MA or Atg5 siRNA consistently aggravated the M1 polarization induced by TNF-α. Moreover, Atg5 knockdown alone was sufficient to trigger microglia activation toward M1 status. More important, TNF-α stimulated microglia conditioned medium caused neurotoxicity when added to neuronal cells. The neurotoxicity was further aggravated when Atg5 knockdown in BV2 cells but alleviated when microglia pretreatment with rapamycin. Activation of AKT/mTOR signaling may contribute to the changes of autophagy and inflammation as the AKT specific inhibitor perifosine prevented the increase of LC3II (an autophagic marker) in TNF-α stimulated microglia. Taking together, our results demonstrate that TNF-α inhibits autophagy in microglia through AKT/mTOR signaling pathway, and autophagy enhancement can promote microglia polarization toward M2 phenotype and inflammation resolution.
PubMed: 30515090
DOI: 10.3389/fnagi.2018.00378 -
Molecular Cancer Therapeutics Feb 2019Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA...
Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA production and phosphorylates many histone-modulating enzymes, resulting in their activation or inhibition. Therefore, understanding the therapeutic impact of AKT inhibition on chromatin-related events is essential. Here, we report that AKT inhibition in prostate-specific PTEN knockout mice significantly induces di- and trimethylation of H3K4 with concomitant reduction in H3K9 acetylation. Mechanistically, we observed that AKT inhibition reduces expression of the H3K4 methylation-specific histone demethylases KDM5 family, especially KDM5B expression at transcriptional levels. Furthermore, we observed that AKT negatively regulates miR-137 levels, which transcriptionally represses KDM5B expression. Overexpression of miR-137 significantly reduced KDM5B and increased H3K4 methylation levels but failed to change AKT phosphorylation. Overall, we observed that AKT transcriptionally regulates KDM5B mainly via repression of miR-137. Our data identify a mechanism by which AKT kinase modulates the prostate cancer epigenome through regulating H3K4 methylation. Additional studies on AKT inhibition-mediated induction of H3K4 methylation will help in designing strategies to enhance the therapeutic efficacy of PI3K/AKT inhibitors.
Topics: Acetylation; Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Humans; Jumonji Domain-Containing Histone Demethylases; Male; Methylation; Mice; MicroRNAs; Nuclear Proteins; PTEN Phosphohydrolase; Phosphorylcholine; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Repressor Proteins; Xenograft Model Antitumor Assays
PubMed: 30446585
DOI: 10.1158/1535-7163.MCT-18-0141 -
Evidence-based Complementary and... 2018We recently reported that ETAS 50, a standardized extract from the stem, exerted anti-inflammatory effects on ultraviolet-B- (UV-B-) irradiated normal human dermal...
We recently reported that ETAS 50, a standardized extract from the stem, exerted anti-inflammatory effects on ultraviolet-B- (UV-B-) irradiated normal human dermal fibroblasts (NHDFs) by inhibiting nuclear factor-B p65 nuclear import and the resulting interleukin-1 (IL-1) expression. To further elucidate the antiphotoaging potency of ETAS 50, we examined the anti-inflammatory effects on UV-B-irradiated NHDFs by focusing on the stress-activated mitogen-activated protein kinase (MAPK) and Akt signaling pathways. NHDFs were treated with 1 mg/mL of ETAS 50 or dextrin (vehicle control) after UV-B irradiation (20 mJ/cm) for different time periods. Phosphorylation levels of c-Jun N-terminal kinase (JNK), p38 MAPK, and Akt were analyzed by western blotting. IL-6 mRNA levels were analyzed by real-time polymerase chain reaction. UV-B-irradiated NHDFs showed increased phosphorylation levels of JNK, p38 MAPK, and Akt, as well as increased mRNA levels of IL-6. ETAS 50 treatment after UV-B irradiation suppressed the increased phosphorylation levels of Akt without affecting those of JNK and p38 MAPK. ETAS 50 as well as Akt inhibitor Perifosine repressed UV-B irradiation-induced IL-6 mRNA expression. These results suggest that ETAS 50 treatment represses UV-B irradiation-induced IL-6 expression by suppressing Akt phosphorylation. The present findings demonstrate the potential of ETAS 50 to prevent photoaging by attenuating UV-B irradiation-induced proinflammatory responses in skin fibroblasts.
PubMed: 30108645
DOI: 10.1155/2018/1547120 -
Cell Death & Disease Jul 2018Chemotherapy represents an important treatment option for colorectal cancer (CRC), but only half of the patients benefit from these regimens. We explored the potential...
Chemotherapy represents an important treatment option for colorectal cancer (CRC), but only half of the patients benefit from these regimens. We explored the potential predicting value and mechanism of PIK3CA mutation in CRC chemotherapy. CRC specimens from 440 patients were retrospectively collected and examined with a fluorescence PCR-based method. The correlation of first-line chemotherapy response and PIK3CA mutation was evaluated according to follow-up and medical records. The underlying mechanism of PIK3CA mutation in chemotherapy resistance was assessed with CRC tumors and primary cells. The mutation frequency of the PIK3CA gene in CRC patients was 9.55%, which was correlated with late TNM staging and lower histological grade. The CRC patients with PIK3A mutation showed worse response to first-line chemotherapy than those without PIK3CA mutation. PIK3A mutation tumor cells showed poor sensitivity to first-line chemotherapy in vitro and in vivo. PIK3CA mutation induced PI3K/Akt signaling activation to increase LGR5 CRC stem cells survival and proliferation, from which lead to chemotherapy resistance. Furthermore, PIK3CA /LGR5 expression was an independent detrimental factor for CRC patients. Our findings indicated that PIK3CA mutation induced PI3K/Akt activation contributed to CRC stem cells survival and proliferation, from which cells further resistance to chemotherapy. PIK3CA /LGR5 expression was a potential biomarker for monitoring chemotherapy resistance in CRC.
Topics: Adult; Aged; Aged, 80 and over; Cell Survival; Chromones; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Female; Fluorouracil; HCT116 Cells; Humans; Male; Middle Aged; Morpholines; Multivariate Analysis; Mutation; Phosphorylcholine; Receptors, G-Protein-Coupled; Retrospective Studies
PubMed: 29970892
DOI: 10.1038/s41419-018-0776-6 -
Hiwi overexpression does not affect proliferation, migration or apoptosis of liver cancer cells or .Oncology Letters Jun 2018Piwi like RNA-mediated gene silencing 1 (Hiwi) is a human homolog of the Piwi gene family that has been reported to be upregulated in hepatocellular carcinoma (HCC). The...
Piwi like RNA-mediated gene silencing 1 (Hiwi) is a human homolog of the Piwi gene family that has been reported to be upregulated in hepatocellular carcinoma (HCC). The present study aimed to investigate the role of Hiwi in the initiation and development of HCC and . Adenovirus-mediated Hiwi overexpression was established in primary murine hepatocytes and SMMC7721 HCC cells. Cell viability and proliferation were assessed using MTT and EdU assays, respectively. Cell migration was measured using a scratch migration assay. The cell cycle was assessed using flow cytometry, and the expression of genes associated with the epithelial mesenchymal transition (EMT) was assessed using reverse transcription-quantitative polymerase chain reaction. SMMC7721 cells that stably express Hiwi were also generated and injected subcutaneously into the nude mice, and tumor growth was examined. Recombinant adenovirus encoding green fluorescent protein or Hiwi was delivered by injection into the tail vein, and its effect on murine hepatocyte gene expression was studied. The present study revealed that the overexpression of Hiwi did not affect the proliferation or migration of liver cancer cells and failed to suppress perifosine- or doxorubicin-induced apoptosis . The tumors of mice that were injected with Hiwi-expressing SMMC7721 cells were not significantly larger compared with mice that were injected with control SMMC7721 cells. Hiwi overexpression did not noticeably alter the expression of genes involved in EMT, either or . The results of the present study indicate that although expression of Hiwi is associated with HCC development and progression in the clinic, it does not act as an oncogene in liver cancer cells.
PubMed: 29928347
DOI: 10.3892/ol.2018.8585 -
Cancer Management and Research 2018Chemotherapy is still the primary adjuvant strategy of cancer therapy; however, the emergence of multi-drug resistance has been a cause for concern. Autophagy has been...
BACKGROUND
Chemotherapy is still the primary adjuvant strategy of cancer therapy; however, the emergence of multi-drug resistance has been a cause for concern. Autophagy has been demonstrated to have a protective role against chemotherapeutic drugs in cancer cells, and autophagy inhibition is generally considered to be a promising therapeutic strategy. However, the paucity of effective and specific autophagy inhibitors limits its application.
PURPOSE
The objective of this study was to explore the effect of DCA, small molecular anti-tumor agent, on the autophagy regulation and chemosensitization in NSCLC cells.
METHODS
We investigated the autophagy regulation of dichloroacetate (DCA) by laser confocal microscopy and western blotting in A549 and H1975 cell lines. The MTT assay and flow cytometry was performed for explore the chemosensitization effectiveness of DCA. The results were verified with subcutaneous tumor model in nude mice and the immunohistochemistry was applied for assessing the level of cell apoptosis and autophagy in vivo post treatment.
RESULTS
We found that DCA, which exhibited antitumor properties in various carcinoma models, induced apoptosis of non-small cell lung cancer cells (NSCLC) by inhibiting cancer cell autophagy. Furthermore, Perifosine, an AKT inhibitor, can greatly weaken the capacity of inducing apoptosis by DCA. The results indicate that the AKT-mTOR pathway, a main negative regulator of autophagy, is involved in the DCA-induced inhibition of autophagy. Then, we detected the effectiveness of autophagy inhibition by DCA. When used in co-treatment with the chemotherapeutic drug paclitaxel (PTX), DCA markedly decreased cell autophagy, enhanced apoptosis and inhibited proliferation in A549 and H1975 cells. The results of the xenograft experiment demonstrate that co-treatment of PTX and DCA can significantly decrease cell proliferation in vivo and prolong the survival of mice.
CONCLUSION
Our results suggest that DCA can inhibit cell autophagy induced by chemotherapeutics, providing a new avenue for cancer chemotherapy sensitization.
PubMed: 29844702
DOI: 10.2147/CMAR.S156530 -
European Review For Medical and... Apr 2018It is to study the stimulation and possible active mechanism of miRNA-21 on AGS proliferation of gastric cancer.
OBJECTIVE
It is to study the stimulation and possible active mechanism of miRNA-21 on AGS proliferation of gastric cancer.
MATERIALS AND METHODS
AGS gastric cancer cells were cultivated in vitro and then divided into the blank control group, the PGE2 (prostaglandin E2) group, the anti-miRNA-21 group and the PGE2 + anti-miRNA-21 group and the MTT and the flow cytometry methods were adopted to test the effect of PGE2 or/and anti-miRNA-21 intervention on AGS cell proliferation and apoptosis and the differences to miRNA-21 expression. In addition, the cells were also divided into the blank control group, the PGE2 group, the PGE2 + Perifosine group, the PGE2 + anti-miRNA-21 group and the PGE2 + anti-miRNA-21 + Perifosine group and the MTT and flow cytometry methods were adopted to test the effect of Perifosine intervention on AGS cell proliferation and apoptosis and on PTEN and p-AktmRNA and protein expressions.
RESULTS
Compared with the control group, AGS cell proliferation activity increased significantly, the apoptosis rate decreased and the miRNA-21tmRNA and protein expression increased in the PGE2 group (p < 0.05); compared with the PGE2 group, the AGS cell proliferation rate decreased, the apoptosis rate increased and the miRNA-21mRNA and protein expressions decreased (p < 0.05) in the anti-miRNA-21 group and the PGE2 + anti-miRNA-21 group. In addition, after intervention of Perifosine, the AGS cell proliferation rate decreased, the apoptosis rate increased, the PTEN mRNA and protein expressions increased and the pAktmRNA and protein expressions decreased (p < 0.05).
CONCLUSIONS
miRNA-21 may promote the growth of gastric cancer cells by adjusting and controlling PTEN/Akt signal passage mediated PEG2.
Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Dinoprostone; Humans; MicroRNAs; PTEN Phosphohydrolase; Stomach Neoplasms
PubMed: 29687845
DOI: 10.26355/eurrev_201804_14717