The role of p62 in cell death and survival of 5-fluorouracil and oxaliplatin-resistant colorectal cancer cells.

The protein sequestosome 1 (p62/SQSTM1) is primarily known as a selective autophagy cargo receptor, but due to its multidomain structure, it also has roles in the ubiquitin-proteasome system, metabolism, cell death and survival signalling. The increase in p62 levels is detected in some types of cancers, including colorectal cancer (CRC). Chemoresistance is the main cause of high mortality rates of CRC patients. Since p62 can regulate both cell survival and death, it is a potential modulator of chemoresistance. The impact of p62 on molecular causes of chemoresistance in CRC cells is insufficiently analysed. Therefore, we aimed to determine the impact of p62 on apoptosis, RIPK1-pRIPK3 axis, and IL-8 levels in chemoresistant CRC cells. Our data revealed that p62 levels are higher in the 5-fluorouracil (5-FU)-resistant HCT116/FU subline compared to the parental cell line. 5-FU and oxaliplatin (OxaPt) treatment decreased p62 protein levels and it correlated with chemoresistance of HCT116 and DLD1 cell lines. The silencing of p62 increased CRC cell sensitivity to 5-FU and OxaPt, hence p62 is one of the factors supporting chemoresistance. The downregulation of p62 reduced the activation of caspase-3 and the levels of RIPK1 and pRIPK3. Furthermore, p62 silencing decreased the BAX/BCL2 ratio in the HCT116/FU subline and did not change the levels of apoptosis. Instead, p62 silencing reduced the amount of IL-8 protein. Our results show that p62 impacts chemoresistance by stimulating prosurvival signalling.

Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells.

NADPH:thioredoxin reductase (TrxR) is considered a potential target for anticancer agents. Several nitroheterocyclic sulfones, such as Stattic and Tri-1, irreversibly inhibit TrxR, which presumably accounts for their antitumor activity. However, it is necessary to distinguish the roles of enzymatic redox cycling, an inherent property of nitroaromatics (ArNO2), and the inhibition of TrxR in their cytotoxicity. In this study, we calculated the previously unavailable values of single-electron reduction potentials of known inhibitors of TrxR (Stattic, Tri-1, and 1-chloro-2,4-dinitrobenzene (CDNB)) and inhibitors identified (nitrofuran NSC697923 and nitrobenzene BTB06584). These calculations were according to the rates of their enzymatic single-electron reduction (PMID: 34098820). This enabled us to compare their cytotoxicity with that of model redox cycling ArNO2. In MH22a and HCT-116 cells, Tri-1, Stattic, CDNB, and NSC697023 possessed at least 10-fold greater cytotoxicity than can be expected from their redox cycling activity. This may be related to TrxR inhibition. The absence of enhanced cytotoxicity in BTB06548 may be attributed to its instability. Another known inhibitor of TrxR, tetryl, also did not possess enhanced cytotoxicity, probably because of its detoxification by DT-diaphorase (NQO1). Apart from the reactions with NQO1, the additional mechanisms influencing the cytotoxicity of the examined inhibitors of TrxR are their reactions with cytochromes P-450. Furthermore, some inhibitors, such as Stattic and NSC697923, may also inhibit glutathione reductase. We suggest that these data may be instrumental in the search for TrxR inhibitors with enhanced cytotoxic/anticancer activity.

Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.

Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce chemoresistance. Though the importance of autophagy in chemoresistance is established, the changes in autophagy in the case of acquired chemoresistance are poorly understood. In this study, we aimed to determine the changes of autophagy in the cellular model of acquired chemoresistance of colorectal cancer cell lines HCT116 and SW620, induced by 5-fluorouracil (5-FU) or oxaliplatin (OxaPt) treatment, and determine the susceptible factors for autophagy inhibition. Our results demonstrate that in the context of autophagy, 5-FU and OxaPt have different effects on HCT116 and SW620 cell lines and their chemoresistant sublines. 5-FU inhibits autophagic flux, while changes in the flux after OxaPt treatment are cell type- and dose-dependent, inducing autophagy reduction or increase. The chemoresistant subline of HCT116 cells derived by OxaPt differs from the subline derived by 5-FU treatment - it responds to OxaPt by upregulating ATG7 protein level and autophagic flux, in contrast to downregulation in cells derived by 5-FU. Moreover, 5-FU and OxaPt treatments significantly modulate protein levels of core-autophagy proteins ATG7 and ATG12. The potential effects of 5-FU and OxaPt on ATG protein levels should be taken into account to reduce chemoresistance by applying small interferingRNAs, targeting ATG proteins.

Components of NOTCH Signaling for Uterine Cancer Patients' Prognosis.

New molecular biomarkers that could have an independent prognostic value in endometrial cancer are currently under investigation. Recently, it was suggested that genetic changes in the Notch signaling pathway could be associated with the development of endometrial carcinoma. This study aimed to determine the expression of the Notch signaling pathway components in tumour and adjacent normal uterine tissue and to evaluate their importance for the survival of uterine cancer patients. The present study was performed on uterine body samples collected from 109 patients and paired adjacent noncancerous endometrial tissue samples. Kaplan-Meier curves and Cox regression were used for survival analyses. Expression alterations of NOTCH2, NOTCH3, NOTCH4, JAG2, and HES1 were evaluated as independent and significant prognostic factors for uterine cancer patients.

Notch and Endometrial Cancer.

The human endometrium is a unique, highly dynamic tissue that undergoes cyclic changes of cell proliferation, differentiation, and death. Endometrial cancer is the most common malignancy among women in developed countries. Importantly, the incidence of endometrial cancer is rising in high-income countries. Currently histological classification is used for subtyping of endometrial cancer, while ongoing research is evaluating markers for more accurate molecular classification. Evolutionary conserved Notch signaling pathway regulates diverse cellular processes such as proliferation, differentiation, and cell invasion. Accumulating evidence links aberrant Notch signaling with diseases such as hyperplasia and endometrial cancer. This chapter summarizes the current state of Notch signaling investigations in the endometrium, endometriosis, and endometrial cancer.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

Exogenous interleukin-1α signaling negatively impacts acquired chemoresistance and alters cell adhesion molecule expression pattern in colorectal carcinoma cells HCT116.

Proinflammatory cytokine and chemokine signaling from the tumor microenvironment is thought to be crucial for developing and sustaining colorectal cancer by regulating a multitude of pathways associated with a variety of cellular mechanisms. Among these pathways there is acquired chemoresistance, which is usually a major obstacle in the way towards successful chemotherapeutic treatment of advanced colorectal cancer cases. Despite of an emerging body of data published on the role of cytokine signaling network in cancer, little is known about the effects of the upstream cytokine interleukin-1α (IL-1α) signaling to the cancer cells. In this study we have shown that the increase in exogenous IL-1α signaling increases chemosensitivity of both chemosensitive and chemoresistant colorectal cancer cell lines, treated with a widely used cytotoxic antimetabolite 5-fluorouracil (5-FU). This was a result of increased cell death but not of the changes in 5-FU-induced cell cycle arrest. Noticeably, combined exogenous IL-1α and 5-FU treatment had significant effects on the expression of cell adhesion molecules, suggesting a decrease in adhesion-dependent chemoresistance and, on the other hand, an increase in metastatic potential of the cells. These results lead to a conclusion that modulation of IL-1 receptor activity could have applications as a part of combination therapy for advanced and highly metastatic colorectal cancers.

Changes in the expression of Notch and Wnt signalling molecules in human endometrial cancer.

BACKGROUND: Endometrial cancer is the  sixth most frequent type of cancer among women worldwide. Type  I adenocarcinomas account for 80-85% of endometrial cancer cases and sometimes require more aggressive treatment than the remaining part of this group. Therefore, molecular markers to stratify adenocarcinomas are needed. MATERIALS AND METHODS: In this study, we analysed Notch and Wnt signalling in human endometrial cancer cases to evaluate these pathway elements as potential biomarkers for type  I endometrial cancer. Endometrial samples were obtained from 47 women undergoing surgery for stage I-IV endometrial cancer in the National Cancer Institute (Vilnius, Lithuania) in 2015-2016. The expression at the mRNA level of signalling molecules genes (NOTCH1, NOTCH2, NOTCH3, NOTCH4, JAG1, JAG2, DLL1, HES1, AXIN2 and CTNNB1) was analysed by the quantitative real-time polymerase chain reaction. Relative expression of NOTCH1, NOTCH4, HES1 and ß-catenin proteins in endometrioid adenocarcinoma was evaluated by the Western blot method. RESULTS: The  expression level of Notch receptors, ligands, and the target gene, as well as CTNNB1 and AXIN2, was reduced in stage I endometrioid adenocarcinoma if compared to the adjacent non-tumour tissue. The expression of all receptors, ligands, and target molecules was reduced in adenocarcinomas of later stages. The statistically significant correlations between transcript amounts of Notch receptors and ligands were found. There was a statistically significant difference in the  gene expression of Notch signalling pathway components between different tumour differentiation grade samples. A positive correlation between mRNA and protein the expression level of NOTCH1, NOTCH4, HES1 was determined in stage I samples. CONCLUSIONS: Analysis of 47 human endometrial cancer samples revealeda reduction in the transcript levels of Notch and Wnt signalling molecule compared to the adjacent non-tumour tissue. These results suggest tumour suppressor function of Notch and Wnt signalling in human endometrial cancer. More detailed research on these signalling pathways should reveal their importance as potential biomarkers.

Significance of Notch and Wnt signaling for chemoresistance of colorectal cancer cells HCT116.

5-fluorouracil (5-FU) and oxaliplatin (OxaPt) are the main chemotherapeutics for colorectal cancer (CRC). Chemotherapy response rates for advanced CRC remain low, primarily due to intrinsic or acquired chemoresistance. The importance of Notch and Wnt signaling for carcinogenesis of CRC as well as crosstalk of Notch and Wnt signaling with many oncogenic signaling pathways suggest that Notch and Wnt pathways could be responsible for chemoresistance. In this study, we compared changes in Notch and Wnt signaling after 5-FU and OxaPt treatment in CRC cells HCT116 and its chemoresistant sublines HCT116/FU and HCT116/OXA. The levels of Notch1 receptor intracellular domain NICD1 and non-phosphorylated ß-catenin, the reporters of Notch and Wnt signaling, were upregulated in untreated chemoresistant HCT116/FU and HCT116/OXA cells. Our data suggest that Notch inhibitor RO4929097 (RO) and Wnt inhibitor XAV939 (XAV) enhance the survival potential of OxaPt-treated cells. The protein level of Notch target gene HES1 was significantly upregulated in chemoresistant HCT116/FU and HCT116/OXA cells, compared to HCT116. HES1 silencing increased viability of HCT116 and its chemoresistant sublines after 5-FU or OxaPt treatment. The results of HES1 downregulation coincide with RO and XAV effects on cell viability of OxaPt-treated cells.

The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116.

Colorectal cancer is one of the most common malignant diseases and is a leading cause of cancer mortality in the Western world. Primary or acquired resistance to chemotherapeutic drugs is a common phenomenon which causes a failure in cancer treatment. A diverse range of molecular mechanisms has been implicated in drug resistance: DNA damage repair, alterations in drug metabolism, mutation of drug targets, increased rates of drug efflux, and activation of survival signaling pathways. The aim of this study was to investigate the expression of CXCL8-CXCR1/2 pathway, its impact on cell proliferation and cytokine expression in human colorectal carcinoma HCT116 cells, and their chemotherapy-resistant subline. We found that IL-1 alpha stimulates the production of CXCL8 through IL-1 receptor signaling. Our data indicate that CXCL8 is upregulated in chemoresistant subline of colorectal cancer cells HCT116, and modulation of CXCR2 pathway can be a target for proliferation inhibition of chemoresistant colorectal cancer cells.

NOTCH1, NOTCH3, NOTCH4, and JAG2 protein levels in human endometrial cancer.

BACKGROUND AND OBJECTIVE: Notch signaling is a conserved developmental pathway, which plays an important role in the regulation of cell proliferation, differentiation and death. Deregulation of Notch pathway has been connected with the carcinogenesis in a variety of cancers. The aim of this study was to investigate the level of the Notch signaling pathway proteins (NOTCH1, 3, 4 and JAG2) in the samples from human endometrial cancer. MATERIALS AND METHODS: The amount of the Notch receptors NOTCH1, 3, 4 and ligand JAG2 protein was determined by Western blot analysis in the samples from stage I endometrial cancer and adjacent nontumor endometrial tissue of 22 patients. RESULTS: The level of NOTCH4 receptor was 1.7 times lower in stage I endometrial cancer as compared with the healthy tissue of the same patients (P=0.04). The protein level of ligand JAG2 was significantly reduced by 2.5 times in stage IB endometrial adenocarcinoma samples (P=0.01). It was reduced in the majority of stage IB adenocarcinomas. There were no significant changes in the protein amount of NOTCH1 and NOTCH3 receptors comparing stage I endometrial adenocarcinoma and healthy tissues. CONCLUSIONS: The reduced amount of NOTCH4 and JAG2 proteins and the decreased level of mRNA coding Notch proteins, as reported in our previous studies, supports the notion that Notch pathway has rather tumor-suppressive than oncogenic role in human endometrial cancer cells. It suggests that Notch pathway activation is a potential therapeutic target.

Down-regulated expression of Notch signaling molecules in human endometrial cancer.

Notch signaling pathway is a highly conserved developmental pathway, which plays an important role in the regulation of cellular proliferation, differentiation and apoptosis. Deregulation of Notch pathway has been connected with the carcinogenesis in a variety of cancers. In this study, we investigated the expression of Notch receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4), ligands (JAG1, JAG2 and DLL1) and target gene HES1. Fifty paired samples of endometrial cancer and adjacent nontumor endometrial tissue from endometrial cancer patients were analyzed by quantitative PCR. The mRNA levels of all investigated molecules were lower in endometrial cancer compared to adjacent nontumor tissue. The expression of NOTCH1, NOTCH4 and DLL1 in IB stage adenocarcinoma was significantly lower (P < 0.05) than the expression in IA stage adenocarcinoma. Significant correlations were found between mRNA expression levels of Notch target gene HES1 and several Notch signaling molecules: NOTCH1, NOTCH3, DLL1 (P < 0.001) and NOTCH2, JAG2 (P < 0.05). This supports the notion that Notch pathway can function as tumor suppressor in human endometrial cancer.

Epigenetic changes by zebularine leading to enhanced differentiation of human promyelocytic leukemia NB4 and KG1 cells.

Aberrant DNA methylation is a critical epigenetic process involved in gene expression of tumor cells. Diverse DNA methyltransferase inhibitors are being studied as potential anticancer drugs, and there is interest in developing novel and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth inhibition, did not induce myeloid differentiation but triggered concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1 cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at pharmacological dose (1 µM for NB4 cells) and higher (3 µM for KG1 cells) increased granulocytic differentiation in both cell lines. Pretreatment for 24 or 48 h with zebularine before the treatment with different doses of RA alone or RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted in significant acceleration and enhancement of differentiation and cell cycle arrest at G0/1. Zebularine alone or in sequential combination with RA decreased expression of DNMT1, caused fast and time-dependent expression of pan-cadherin and partial demethylation of E-cadherin but not tumor suppressor p15. When used in combination with RA, zebularine increased expression of both genes transcript and protein. Zebularine induced regional chromatin remodeling by local histone H4 acetylation and histone H3-K4 methylation in promoter sites of methylated E-cadherin and also in the promoter of unmethylated p21 as evidenced by chromatin immunoprecipitation assay. Our results extend the spectrum of zebularine effects and the evaluation its utility in acute myeloid leukemia therapy based on epigenetics.

Damage targeted to the mitochondrial interior induces autophagy, cell cycle arrest and, only at high doses, apoptosis.

It is generally accepted that permeabilization of the outer mitochondrial membrane is a crucial event in the induction of apoptosis. In order to know how the cell would respond to the damage targeted not to the outer mitochondrial membrane, but to the mitochondrial interior, we followed the changes in cell morphology and some biochemical parameters triggered by ROS in the mitochondrial inner space. The experiments were carried out in epidermoid carcinoma A431 cells. For stimulated production of singlet oxygen in the inner space of mitochondria, we employed photodynamic treatment (PDT) mediated by a cationic photosensitizer 7-diamino-2,8-dimethyl-5-phenylphenazinium chloride (Safranin O, Safr), accumulating in the inner space of mitochondria. At low to intermediate cytotoxic doses (up to CD50 reducing cellular viability by 50%), Safr-PDT did not reveal hallmarks of dead cells, and the decrease of cellular viability could be attributed to cell cycle inhibition, and enhanced autophagy. High Safr-PDT doses (beyond CD70) did induce cell death by apoptosis involving release of cytochrome c and caspase-3 activation, in addition to enhanced autophagy.

Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior.

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.

The cloning and characterization of Tetrahymena pyriformis translation elongation factor 1b alpha and gamma subunits.

The multi-subunit eukaryotic translation elongation factor 1 (eEF1) consists of two functionally distinct parts: G-protein eEF1A and guanine nucleotide exchange factor eEF1B. Here, we report on the cloning of cDNAs of both the alpha and gamma subunits of the eEF1B from the ciliated protozoan Tetrahymena pyriformis. The open reading frame of the eEF1Bgamma cDNA encodes a 399-amino acid long polypeptide with a calculated molecular mass of 45.2 kDa. The eEF1Balpha cDNA contains an open reading frame encoding a polypeptide of 228 amino acids. The calculated molecular mass of this protein is 25.2 kDa. The overall deduced amino acid sequences of eEF1Balpha and eEF1Bgamma show a considerable homology with the families of alpha and gamma proteins from other eukaryotic organisms. We demonstrated that eEF1Bgamma is an RNA-binding protein which is able to bind to different RNAs.