Nrf3 alleviates oxidative stress and promotes the survival of colon cancer cells by activating AKT/BCL-2 signal pathway.

Oxidative stress is closely linked to tumor initiation and development, conferring a survival advantage to cancer cells. Therefore, understanding cancer cells' antioxidant molecular mechanisms is crucial to cancer therapy. In this study, we discovered that H2O2-induced oxidative stress increased Nrf3 expression in colon cancer cells. Overexpression of Nrf3 decreased H2O2-mediated cytotoxicity and apoptosis. Furthermore, Nrf3 reduced reactive oxygen species levels and malondialdehyde concentrations after H2O2 treatment. Mechanistically, H2O2-mediated cell apoptosis involves multiple signaling proteins, including Akt, bcl-2, JNK, and p38. An increase in Nrf3 expression in colon cancer cells treated with H2O2 partly reversed Akt/Bcl-2 inhibition, whereas it decreased activation of p38 and JNK. In addition, we found that increasing Nrf3 decreased stress-associated chemical-induced cell death, resulting in drug resistance. According to these results, Nrf3 is critical for drug resistance and oxidant adaptation.

A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124.

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 µM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

The Competition of Yin and Yang: Exploring the Role of Wild-Type and Mutant p53 in Tumor Progression.

The protein p53 is a well-known tumor suppressor that plays a crucial role in preventing cancer development [...].

Quantitative proteomics analysis revealed the potential role of lncRNA Ftx in cardiomyocytes.

OBJECTIVE: This study aims to decode the proteomic signature of cardiomyocytes in response to lncRNA Ftx knockdown and overexpression via proteomic analysis, and to study the biological role of lncRNA Ftx in cardiomyocytes.  METHODS: The expression level of the lncRNA Ftx in cardiomyocytes cultured in vitro was intervened, and the changes in protein levels in cardiomyocytes were quantitatively detected by liquid chromatography-mass spectrometry. The key molecules and pathways of the lncRNA-Ftx response were further examined by GO, KEGG, and protein interaction analysis. RESULTS: A total of 2828 proteins are quantified. With a 1.5-fold change threshold, 32 upregulated proteins and 49 downregulated proteins are identified in the lncRNA Ftx overexpression group, while 67 up-regulated proteins and 54 down-regulated proteins are identified in the lncRNA Ftx knockdown group. Functional clustering analysis of differential genes revealed that the lncRNA Ftx is involved in regulating cardiomyocyte apoptosis and ferroptosis and improving cellular energy metabolism. In addition, Hub genes such as ITGB1, HMGA2, STAT3, GSS, and LPCAT3 are regulated downstream by lncRNA Ftx. CONCLUSION: This study demonstrates that lncRNA Ftx plays a vital role in cardiomyocytes and may be involved in the occurrence and development of various myocardial diseases. It provides a potential target for clinical protection of the myocardium and reversal of myocardial fibrosis.

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes NOTCH1 and FAT1 to Repress HNSCC Cell Proliferation.

(1) Background: PTC124 (Ataluren) is an investigational drug for the treatment of nonsense mutation-mediated genetic diseases. With the exception of the TP53 tumor suppressor gene, there has been little research on cancers with nonsense mutation. By conducting a database search, we found that another two tumor suppressor genes, NOTCH1 and FAT1, have a high nonsense mutation rate in head and neck squamous cell carcinoma (HNSCC). PTC124 may re-express the functional NOTCH1 or FAT1 in nonsense mutation NOTCH1 or FAT1 in HSNCC (2) Methods: DOK (with NOTCH1 Y550X) or HO-1-u-1 (with FAT1 E378X) HNSCC cells were treated with PTC124, and the NOTCH1 or FAT1 expression, cell viability, and NOTCH1- or FAT1-related downstream gene profiles were assayed. (3) Results: PTC124 was able to induce NOTCH1 or FAT1 expression in DOK and HO-1-u-1 cells. PTC124 was able to upregulate NOTCH downstream genes HES5, AJUBA, and ADAM10 in DOK cells. PTC124 enhanced DDIT4, which is under the control of the FAT1-YAP1 pathway, in HO-1-u-1 cells. FLI-06 (a NOTCH signaling inhibitor) reversed PTC124-mediated cell growth inhibition in DOK cells. PTC124 could reverse TT-10 (a YAP signaling activator)-mediated HO-1-u-1 cell proliferation. (4) Conclusions: PTC124 can rescue nonsense mutation of NOTCH1 and FAT1 to repress HNSCC cell proliferation.

P63 and P73 Activation in Cancers with p53 Mutation.

The members of the p53 family comprise p53, p63, and p73, and full-length isoforms of the p53 family have a tumor suppressor function. However, p53, but not p63 or p73, has a high mutation rate in cancers causing it to lose its tumor suppressor function. The top and second-most prevalent p53 mutations are missense and nonsense mutations, respectively. In this review, we discuss possible drug therapies for nonsense mutation and a missense mutation in p53. p63 and p73 activators may be able to replace mutant p53 and act as anti-cancer drugs. Herein, these p63 and p73 activators are summarized and how to improve these activator responses, particularly focusing on p53 gain-of-function mutants, is discussed.

NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner.

The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation.

Homocysteine Suppresses Autophagy Through AMPK-mTOR-TFEB Signaling in Human THP-1 Macrophages.

ABSTRACT: Hyperhomocysteinemia is an independent risk factor for atherosclerosis. It is known that macrophage autophagy plays a protective role in atherosclerosis and that hyperhomocysteinemia is strongly linked to autophagy. Therefore, it is of great significance to study the molecular mechanisms underlying the effect of homocysteine (Hcy) on macrophage autophagy. This study aimed to investigate the effects of Hcy on autophagy in a human acute monocytic leukemia cell line (THP-1). The Hcy-treated THP-1 cells exhibited increased levels of the autophagy substrate SQSTM1 (p62) and decreased levels of the autophagy markers LC3 II/I and Beclin-1, indicating a decrease in autophagy in vitro. Furthermore, Western blotting showed that Hcy significantly increased the levels of p-mTOR and nuclear TFEB and decreased the levels of p-AMPK and cytoplasmic TFEB. These data suggest that Hcy inhibits autophagosome formation in human THP-1 macrophages through the AMPK-mTOR-TFEB signaling pathway. Our findings provide new insights into the mechanisms of atherosclerotic diseases caused by Hcy.

Nrf3 Promotes 5-FU Resistance in Colorectal Cancer Cells via the NF-κB/BCL-2 Signaling Pathway In Vitro and In Vivo.

Increasing evidence indicates that nuclear factor, erythroid 2-like 3 (Nrf3) is connected with tumorigenesis. However, the relationship between Nrf3 and tumor drug resistance remains elusive. In this study, we investigated the effect and mechanism of action by which Nrf3 regulated the sensitivity of colon cancer cells to 5-fluorouracil (5-FU). We found Nrf3 was significantly increased in colon cancer tissues. Furthermore, we observed that Nrf3 knockdown and overexpression can significantly affect the sensitivity of colon cancer cells to 5-FU in vitro and in vivo. Moreover, Nrf3 promoted the expression of RELA, P-RELA, and BCL-2. Inhibition of NF-κB partly reversed the effects of Nrf3 overexpression, resulting in the resistance of colon cancer cells to 5-FU. Overall, the study revealed that Nrf3 was connected to the sensitivity of colon cancer cells to 5-FU, and its possible mechanism was related to the NF-κB signaling pathway, which provided a new target for overcoming the resistance of colon cancer cells to 5-FU.

RNA-seq and In Vitro Experiments Reveal the Protective Effect of Curcumin against 5-Fluorouracil-Induced Intestinal Mucositis via IL-6/STAT3 Signaling Pathway.

Although first-line chemotherapy drugs, including 5-fluorouracil (5-FU), remain one of the major choice for cancer treatment, the clinical use is also accompanied with dose-depending toxicities, such as intestinal mucositis (IM), in cancer patients undergoing treatment. IM-induced gastrointestinal adverse reactions become frequent reason to postpone chemotherapy and have negative impacts on therapeutic outcomes and prognosis. Various studies have evidenced the anticancer role of curcumin in many cancers; except for this effect, studies also indicated a protective role of curcumin in intestinal diseases. Therefore, in this study, we investigated the effect of curcumin on inflammation, intestinal epithelial cell damage in an IM model. 5-FU was used to induce the model of IM in intestinal epithelial cells, and curcumin at different concentrations was administrated. The results showed that curcumin efficiently attenuated 5-FU-induced damage to IEC-6 cells, inhibited the levels of inflammatory cytokines, attenuated the 5-FU-induced inhibition on cell viability, and displayed antiapoptosis effect on IEC-6 cells. Further RNA-sequencing analysis and experiment validation found that curcumin displays its protective effect against 5-FU-induced IM in intestinal epithelial cells by the inhibition of IL-6/STAT3 signaling pathway. Taken together, these findings suggested that curcumin may be provided as a therapeutic agent in prevention and treatment of chemotherapy-induced IM.

12-Epi-Napelline Inhibits Leukemia Cell Proliferation via the PI3K/AKT Signaling Pathway In Vitro and In Vivo.

This study aimed to investigate the inhibitory effect of 12-epi-napelline on leukemia cells and its possible mechanisms. The inhibitory effects of 12-epi-napelline on K-562 and HL-60 cells were evaluated using the CCK-8 assay, cell cycle arrest and apoptosis were detected by flow cytometry, and the expression of related proteins was measured by western blot. A K-562 tumor model was established to evaluate the antitumor effect of 12-epi-napelline in vivo. A reduction in leukemia cell viability was observed after treatment with 12-epi-napelline. It was determined that the cell cycle was arrested in the G0/G1 phase, and the cell apoptosis rate was increased. Moreover, caspase-3 and Bcl-2 were downregulated, whereas cleaved caspase-3 and caspase-9 were upregulated. Further study revealed that 12-epi-napelline could suppress the expression of PI3K, AKT, p-AKT, and mTOR. Insulin-like growth factor 1 (IGF-1) attenuated 12-epi-napelline-induced apoptosis and ameliorated the repression of PI3K, AKT, p-AKT, and mTOR by 12-epi-napelline. Animal experiments clearly showed that 12-epi-napelline inhibited tumor growth. In conclusion, 12-epi-napelline restrained leukemia cell proliferation by suppressing the PI3K/AKT/mTOR pathway in vitro and in vivo.

BGN/TLR4/NF-B Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells.

Human Toll-like receptor (TLR) signaling plays a vital role in intestinal inflammation by activating the NF-B pathway. By querying GENT2 datasets, we identified the gene expression level of TLR2 and TLR4 as being substantially increased in colorectal cancer. Introduction of shRNAs for TLR4 but not TLR2 dramatically recovered disialyl Lewisa and sialyl 6-sulfo Lewisx glycans, which are preferentially expressed in non-malignant colonic epithelial cells and could serve as ligands for the immunosuppressive molecule Siglec-7. We screened several TLR4 ligands and found that among them BGN is highly expressed in cancers and is involved in the epigenetic silencing of Siglec-7 ligands. Suppression of BGN expression substantially downregulated NF-B activity and the marker H3K27me3 in the promoter regions of the SLC26A2 and ST6GalNAc6 genes, which are involved in the synthesis of those glycans, and restored expression of normal glycans as well as Siglec-7 binding activities. We show that in the presence of TLR4, inflammatory stimuli initiate a positive loop involving NF-B that activates BGN and further enhances TLR4 activity. Present findings indicate a putative mechanism for the promotion of carcinogenesis by loss of immunosuppressive ligands by the BGN/TLR4/ NF-B pathway.

SSEA3 and Sialyl Lewis a Glycan Expression Is Controlled by B3GALT5 LTR through Lamin A-NFYA and SIRT1-STAT3 Signaling in Human ES Cells.

B3GALT5 is involved in the synthesis of embryonic stem (ES) cell marker glycan, stage-specific embryonic antigen-3 (SSEA3). This gene has three native promoters and an integrated retroviral long terminal repeat (LTR) promoter. We found that B3GALT5-LTR is expressed at high levels in human ES cells. B3GALT5-LTR is also involved in the synthesis of the cancer-associated glycan, sialyl Lewis a. Sialyl Lewis a is expressed in ES cells and its expression decreases upon differentiation. Retinoic acid induced differentiation of ES cells, decreased the short form of NFYA (NFYAs), increased phosphorylation of STAT3, and decreased B3GALT5-LTR expression. NFYAs activated, and constitutively-active STAT3 (STAT3C) repressed B3GALT5-LTR promoter. The NFYAs and STAT3C effects were eliminated when their binding sites were deleted. Retinoic acid decreased the binding of NFYA to B3GALT5-LTR promoter and increased phospho-STAT3 binding. Lamin A repressed NFYAs and SSEA3 expression. SSEA3 repression mediated by a SIRT1 inhibitor was reversed by a STAT3 inhibitor. Repression of SSEA3 and sialyl Lewis a synthesis mediated by retinoic acid was partially reversed by lamin A short interfering RNA (siRNA) and a STAT3 inhibitor. In conclusion, B3GALT5-LTR is regulated by lamin A-NFYA and SIRT1-STAT3 signaling that regulates SSEA3 and sialyl Lewis a synthesis in ES cells, and sialyl Lewis a is also a ES cell marker.

Roles of p53 Family Structure and Function in Non-Canonical Response Element Binding and Activation.

The p53 canonical consensus sequence is a 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases. C(A/T)(A/T)G is the core sequence and purine (Pu) and pyrimidine (Py) bases comprise the flanking sequence. However, in the p53 noncanonical sequences, there are many variations, such as length of consensus sequence, variance of core sequence or flanking sequence, and variance in number of bases making up the spacer or AT gap composition. In comparison to p53, the p53 family members p63 and p73 have been found to have more tolerance to bind and activate several of these noncanonical sequences. The p53 protein forms monomers, dimers, and tetramers, and its nonspecific binding domain is well-defined; however, those for p63 or p73 are still not fully understood. Study of p63 and p73 structure to determine the monomers, dimers or tetramers to bind and regulate noncanonical sequence is a new challenge which is crucial to obtaining a complete picture of structure and function in order to understand how p63 and p73 regulate genes differently from p53. In this review, we will summarize the rules of p53 family non-canonical sequences, especially focusing on the structure of p53 family members in the regulation of specific target genes. In addition, we will compare different software programs for prediction of p53 family responsive elements containing parameters with canonical or non-canonical sequences.

Synergistic activation of the NEU4 promoter by p73 and AP2 in colon cancer cells.

More than 50% of colon cancers bear mutations in p53, one of the most important tumor suppressors, and its family members p63 or p73 are expected to contribute to inhibiting the progression of colon cancers. The AP2 family also acts as a tumor suppressor. Here we found that p73 and AP2 are able to activate NEU4, a neuraminidase gene, which removes the terminal sialic acid residues from cancer-associated glycans. Under serum starvation, NEU4 was up-regulated and one of the NEU4 target glycans, sialyl Lewis X, was decreased, whereas p73 and AP2 were up-regulated. Sialyl Lewis X levels were not, however, decreased under starvation conditions in p73- or AP2-knockdown cells. p53 and AP2 underwent protein-protein interactions, exerting synergistic effects to activate p21, and interaction of p53 with AP2 was lost in cells expressing the L350P mutation of p53. The homologous residues in p63 and p73 are L423 and L377, respectively. The synergistic effect of p53/p63 with AP2 to activate genes was lost with the L350P/L423P mutation in p53/p63, but p73 bearing the L377P mutation was able to interact with AP2 and exerted its normal synergistic effects. We propose that p73 and AP2 synergistically activate the NEU4 promoter in colon cancer cells.

Epigenetic silencing of the synthesis of immunosuppressive Siglec ligand glycans by NF-κB/EZH2/YY1 axis in early-stage colon cancers.

Normal colonic epithelial cells express sialyl 6-sulfo Lewisx and disialyl Lewisa on their cell surface, which are ligands for the immunosuppressive molecule Siglec-7. Expression of these normal glycans is frequently lost upon malignant transformation by silencing DTDST and ST6GalNAc6 at the early stage of colorectal carcinogenesis, and leads to production of inflammatory mediators that facilitate carcinogenesis. Indeed, by querying The Cancer Genome Atlas datasets, we confirmed that the level of DTDST or ST6GalNAc6 mRNA is substantially decreased at the early stage of colorectal carcinogenesis. Cultured colon cancer cell lines were used in this study including DLD-1, HT-29, LS174T and SW620. Their promoter regions were strongly marked by repressive mark H3K27me3, catalyzed by EZH2 that was markedly upregulated in early stage of colorectal carcinogenesis. Suppression of EZH2 substantially downregulated H3K27me3 mark and upregulated DTDST and ST6GalNAc6 as well as expression of normal glycans and Siglec-binding activities. Transcription factor YY1 was vital for the recruitment of PRC2-containing EZH2 to both promoters. Inhibition of NF-κB substantially reduced EZH2 transcription and restored their mRNAs as well as the production of normal Siglec ligand glycans in the results obtained from in vitro studies on cultured colon cancer cell lines. These findings provide a putative mechanism for promotion of carcinogenesis by loss of immunosuppressive molecules by epigenetic silencing through NF-κB-mediated EZH2/YY1 axis.

Gangliosides and Tumors.

Tumor-associated gangliosides play important roles in regulation of signal transduction induced by growth-factor receptors including EGFR, FGFR, HGF and PDGFR in a specific microdomain called glycosynapse in the cancer cell membranes, and in interaction with glycan recognition molecules involved in cell adhesion and immune regulation including selectins and siglecs. As the genes involved in the synthesis and degradation of tumor-associated gangliosides were identified, biological functions became clearer from the experimental results employing forced overexpression and/or knockdown/knockout of the genes. Studies on the regulatory mechanisms for their expression also achieved great advancements. Epigenetic silencing of glycan-related genes is a dominant mechanism in glycan alteration at early stages of carcinogenesis. Development of hypoxia resistance involving activation of a transcription factor HIF, and acquisition of cancer stem cell-like characteristics through epithelial-mesenchymal transition are important mechanisms for glycan modulations in the later stages of cancer progression. In the initial stages of studies, the gangliosides which specifically appear in cancers attracted attention under the name of tumor-associated gangliosides. However, it became apparent that not only the cancer-associated gangliosides but also the normal gangliosides present in nonmalignant cells and tissues perform important biological functions, and some of them tend to disappear in cancer cells resulting in the loss of the physiological functions, and this sometimes facilitates progression of cancers.

Downregulation of miR-199a/b-5p is associated with GCNT2 induction upon epithelial-mesenchymal transition in colon cancer.

ß-1,6-N-acetylglucosaminyltransferase 2 (GCNT2), which encodes a key glycosyltransferase for blood group I antigen synthesis, is induced upon epithelial-mesenchymal transition (EMT). Our results indicate that GCNT2 is upregulated upon EMT induced with epidermal growth factor and basic FGF in cultured human colon cancer cells. GCNT2 knockdown or overexpression decreases or increases, respectively, malignancy-related characteristics of colon cancer cells and I antigen levels. MiR-199a/b-5p is markedly downregulated upon EMT in colon cancer cells. Here, we find that miR-199a/b-5p consistently regulates GCNT2 expression in reporter assays and that it binds directly to the GCNT2 3' untranslated region intracellularly in RNA-induced silencing complex-trap assays. Overexpression of miR-199a/b-5p decreases GCNT2 expression and suppresses I antigen production. Based on these findings, we propose that miR-199a/b-5p regulates GCNT2 and I antigen expression in colon cancer cells undergoing EMT.

A/T gap tolerance in the core sequence and flanking sequence requirements of non-canonical p53 response elements.

The canonical core sequence of the p53 response element, CATG, has a two-base A/T gap. Previously, we found that p53 can also activate a non-canonical four-base A/T gap CATATG core sequence. In this study, we investigated the possible number of A/T bases used by p53 and showed that a six-base A/T gap CATATATG core sequence was the maximum A/T gap in the p53 response element that could be upregulated by p53 and p63. Canonical and non-canonical p53 response elements also have three-base flanking sequences. A/T bases could be substituted by G/C bases, including CACACG and CGTGTG, but not CGCGCG. We found that the SV40 promoter with functional six- and two-base A/T gap core sequences could be activated by TAp63γ and that TAp63γ could upregulate SV40 small and large T antigens expression in COS7 cells. We also found that the distal region of PUMA promoter with functional two six-base A/T gap core sequences could be activated by TAp63γ in 293T cells. These new findings could provide novel rules for the non-canonical p53 family response element and could extend the entire p53 family regulation network.