Prolyl hydroxylase 3 interacts with Bcl-2 to regulate doxorubicin-induced apoptosis in H9c2 cells

Prolyl hydroxylases (PHDs) are dioxygenases that use oxygen as a co-substrate to hydroxylate proline residues. Three PHD isoforms (PHD1, PHD2 and PHD3) have been identified in mammalian cells. PHD3 expression is upregulated in some cardiac diseases such as cardiomyopathy, myocardial ischemia-reperfusion injury and congestive heart failure, all of which are associated with apoptosis. However, the role of PHDs in cardiomyocyte apoptosis remains unknown. Here, we have found that exposure of embryonic rat heart-derived H9c2 cells to doxorubicin (DOX) induced cell apoptosis as evaluated by caspase-3/7 activity, mitochondrial membrane potential (Δψm) and cell viability, and that this apoptosis was linked to PHD3 upregulation. PHD inhibition or PHD3 silencing substantially ameliorated DOX-induced apoptosis, but PHD1 or PHD2 knockdown did not significantly influence apoptosis. Furthermore, immunoprecipitation experiments showed that PHD3 upregulation reduced the formation of the Bax-Bcl-2 complex, inhibiting the anti-apoptotic effect of Bcl-2. Thus, PHD3 upregulation may be partially responsible for DOX-induced cardiomyocyte apoptosis via its interaction with Bcl-2. Inhibition of PHD3 is likely to be cardioprotective against apoptosis in some heart disorders.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Relationship between reactive oxygen species and heme metabolism during the differentiation of Neuro2a cells

Although neuronal cells are highly vulnerable to oxidative stress, recent studies suggest that production of reactive oxygen species (ROS) increases during and is essential for neuronal differentiation. In addition, we have previously found that heme biosynthesis is up-regulated during retinoic acid-induced differentiation of Neuro2a cells. In the current study, we showed that this up-regulation of heme biosynthesis during differentiation is ROS-dependent. Furthermore, we found that ROS-dependent induction of heme oxygenase, which degrades heme and acts as an anti-oxidant, and catalase, another anti-oxidant enzyme that contains heme as a prosthetic group, occurs during differentiation. These results suggest that heme biosynthesis following the degradation of heme protects Neuro2a cells from oxidative stress caused by ROS during differentiation.     

Urolithin A induces cell cycle arrest and apoptosis by inhibiting Bcl-2, increasing p53-p21 proteins and reactive oxygen species production in colorectal cancer cells

Colorectal cancer (CRC) is the second most common gastrointestinal cancer globally. Prevention of tumor cell proliferation and metastasis is vital for prolonging patient survival. Polyphenols provide a wide range of health benefits and prevention from cancer. In the gut, urolithins are the major metabolites of polyphenols. The objective of our study was to elucidate the molecular mechanism of the anticancer effect of urolithin A (UA) on colorectal cancer cells. UA was found to inhibit the cell proliferation of CRC cell lines in a dose-dependent and time-dependent manner in HT29, SW480, and SW620 cells. Exposure to UA resulted in cell cycle arrest in a dose-dependent manner along with alteration in the expression of cell cycle–related protein. Treatment of CRC cell lines with UA resulted in the induction of apoptosis. Treatment of HT29, SW480, and SW620 with UA resulted in increased expression of the pro-apoptotic proteins, p53 and p21. Similarly, UA treatment inhibited the anti-apoptotic protein expression of Bcl-2. Moreover, exposure of UA induced cytochrome c release and caspase activation. Furthermore, UA was found to generate reactive oxygen species (ROS) production in CRC cells. These findings indicate that UA possesses anticancer potential and may be used therapeutically for the treatment of CRC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01189-8.     

Structural conservation of residues in BH1 and BH2 domains of Bcl-2 family proteins

The sequence of Bcl-2 homology domains, BH1 and BH2, is known to be conserved among anti- and pro-apoptotic members of Bcl-2 family proteins. But structural conservation of these domains with respect to functionally active residues playing role in heterodimerization-mediated regulation of apoptosis has never been elucidated. Here, we have suggested the formation of an active site by structurally conserved residues in BH1 (glycine, arginine) and BH2 (tryptophan) domains of Bcl-2 family members, which also accounts for the functional effect of known mutations in BH1 (G145A, G145E) and BH2 (W188A) domains of Bcl-2.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Induction of apoptosis by Meretrix lusoria through reactive oxygen species production, glutathione depletion, and caspase activation in human leukemia cells

Apoptosis-induced directed fractionation and purification was used to identify the bioactive components of hard clams (HC), Meretrix lusoria. Two stereoisomers of epidioxysterol were previously identified as the active compounds in the ethyl acetate fraction (HC-EA). The molecular mechanism of HC-EA-induced apoptosis was also investigated in this study. Dissipation of mitochondrial membrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of pro-caspase-9 and -3 processing preceded apoptosis in HL-60 cells, confirmed by DNA fragmentation, chromatin condensation, changes in the cell membrane and the appearance of a sub-G1 DNA peak. Furthermore, treatment with HC-EA caused a rapid loss of intracellular glutathione content and stimulation of reactive oxygen species (ROS). Antioxidants such as catalase, N-acetylcysteine, pyrrolidine dithiocarbamate, and superoxide dismutase, but not allopurinol and diphenylene iodonium, significantly inhibited HC-EA-induced cell death. Apoptosis was completely prevented by a pan-caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK). The induction of apoptosis by M. lusoria may prove to be a pivotal mechanism for its cancer chemopreventive action.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.     

Distinct patterns of mitochondrial changes precede induction of apoptosis by all-trans-retinoic acid and N-(4-hydroxyphenyl)retinamide in MCF7 breast cancer cells

The biochemical mechanisms of apoptosis-induction by all-trans-retinoic acid (atRA) and N-(4-hydroxyphenyl)retinamide (4HPR) in cultured MCF7 cancer cells were studied by multiparameter flow cytometry. Retinoid treatment induced formation of two biochemically distinct cell subpopulations, which preceded the appearance of cells with fragmented nuclei. Exposure to atRA led to a transient increase in NADH level and mitochondrial oxidative turnover and a slow decline in reduced thiol level and mitochondrial membrane potential, suggesting that atRA treatment induces a transient defense mechanism. The synthetic retinoid 4HPR, in contrast, caused a gradual decrease in mitochondrial oxidative turnover and cardiolipin level together with a small decline in mitochondrial membrane potential, suggesting that 4HPR induces oxidation of cardiolipin and subsequent leakage of the mitochondria. Co-incubation with cyclosporin A, an inhibitor of the mitochondrial permeability transition, did not prevent formation of fragmented nuclei or induction of changes in mitochondrial parameters by retinoids. Thus, the mitochondrial permeability transition does not appear to be involved in retinoid induction of apoptosis in MCF7 cells. Retinoid exposure of diploid human mammary epithelial cells induced mild oxidative stress but did not lead to formation of two cell subpopulations. We conclude that atRA and 4HPR induce apoptosis in MCF7 cells by two distinct and novel biochemical mechanisms.     

Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells

The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax^−/− cells to roscovitine or purvalanol for 24 h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax^−/− cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect.     

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.     

Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr⁴⁰⁹, Val⁴¹¹, and Gly⁴¹⁴ showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe³⁸⁸, Thr³⁸⁹, and Val³⁹⁶ in cytoplasmic loop i were essential for efficient H⁺ translocation. Ala⁴³⁶ and Pro⁵⁶⁰ in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H⁺-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme.     

Drug screening assay based on the interaction of intact Keap1 and Nrf2 proteins in cancer cells

Background

The Nrf2–Keap1 interaction is the major regulatory pathway for cytoprotective responses against oxidative and electrophilic stresses. Keap1, a substrate protein of a Cul3-dependent E3 ubiquitin ligase complex, is a negative regulator of Nrf2. The use of chemicals to regulate the interaction between Keap1 and Nrf2 has been proposed as a strategy for the chemoprevention of degenerative diseases and cancers.

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.