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Inhibition of lactate dehydrogenase A by diclofenac sodium induces apoptosis in HeLa cells through activation of AMPK.

Cancer cells exhibit a unique metabolic preference for the glycolytic pathway over oxidative phosphorylation for maintaining the tumor microenvironment. Lactate dehydrogenase A (LDHA) is a key enzyme that facilitates glycolysis by converting pyruvate to lactate and has been shown to be upregulated in multiple cancers due to the hypoxic tumor microenvironment. Diclofenac (DCF), a nonsteroidal anti-inflammatory drug, has been shown to exhibit anticancer effects by interfering with the glucose metabolism pathway. However, the specific targets of this drug remain unknown. Using in silico, biochemical, and biophysical studies, we show that DCF binds to LDHA adjacent to the substrate binding site and inhibits its activity in a dose-dependent and allosteric manner in HeLa cells. Thus, DCF inhibits the hypoxic microenvironment and induces apoptosis-mediated cell death. DCF failed to induce cytotoxicity in HeLa cells when LDHA was knocked down, confirming that DCF exerts its antimitotic effects via LDHA inhibition. DCF-induced LDHA inhibition alters pyruvate, lactate, NAD+, and ATP production in cells, and this could be a possible mechanism through which DCF inhibits glucose uptake in cancer cells. DCF-induced ATP deprivation leads to mitochondria-mediated oxidative stress, which results in DNA damage, lipid peroxidation, and apoptosis-mediated cell death. Reduction in intracellular ATP levels additionally activates the sensor kinase, adenosine monophosphate-activated protein kinase (AMPK), which further downregulates phosphorylated ribosomal S6 kinase (p-S6K), leading to apoptosis-mediated cell death. We find that in LDHA knocked down cells, intracellular ATP levels were depleted, resulting in the inhibition of p-S6K, suggesting the involvement of DCF-induced LDHA inhibition in the activation of the AMPK/S6K signaling pathway.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Molecular mechanism of the unusual biphasic effects of the natural compound hinokitiol on iron-induced cellular DNA damage.

Hinokitiol is a natural monoterpene compound found in the heartwood of cupressaceous plants that have anticancer and anti-inflammatory properties. However, few studies have focused on its effect on iron-mediated cellular DNA damage. Here we show that hinokitiol exhibited unusual biphasic effects on iron-induced DNA damage in a molar ratio (hinokitiol/iron) dependent manner in HeLa cells. Under low ratios (<3:1), hinokitiol markedly enhanced DNA damage induced by Fe(II) or Fe(II)-H2O2; However, when the ratios increased over 3:1, the DNA damage was progressively inhibited. We found that the total cytoplasmic and nuclear iron concentration increased as the ratios of hinokitiol/iron increased. However, the cellular level of labile iron pool (LIP) only increased at ratios lower than 3, and the ROS generation is consistent with LIP change. Hinokitiol was found to interact with iron to form lipophilic hinokitiol-iron complexes with different stoichiometry and redox-activity by complementary applications of various analytical methods. Taken together, we propose that the enhancement of iron-induced cellular DNA damage by hinokitiol at low ratios (<3:1) was due to formation of lipophilic and redox-active iron complexes which facilitated cellular iron uptake and •OH production, while the inhibition at ratios higher than 3 was due to formation of redox-inactive iron complexes. These new findings will help us to design more effective drugs for the prevention and treatment of a series of iron-related diseases via regulating the two critical physicochemical factors (lipophilicity and redox activity of iron complexes) by simple natural compounds with iron-chelating properties.

Baicalein-rich fraction of Oroxylum indicum triggers mitochondrialmediated apoptosis pathway through MAPK transduction in cervical cancer cells

Baicalein was proved to have an apoptotic effect on a wide range of cancer cells in numerous studies. In this study, the apoptotic effect of baicalein-rich fraction from Oroxylum indicum leaves on human cervical cancer cells (SiHa and HeLa) were investigated. In this case, BRF-induced cell death was regulated by the activation of JNK/p38 and deactivation of ERK. Apart from that, BRF was able to trigger mitochondrial-mediated apoptosis pathway by downregulating Bcl-2 as well as upregulating Bax, caspase-9 and caspase-3. Indeed, MAPK transduction was found to be involved in BRF action in modulating mitochondrial Bcl-2 family, Bcl-2 and Bax. In SiHa cells, ERK and p38 pathway regulated BRF stimulation on mitochondrial-mediated pathway. Yet, BRF-induced this apoptosis pathway in ERK/JNK/p38-dependent manner in HeLa cells. Therefore, BRF induced and augmented the cell death of human cervical cancer cells through MAPK transduction.

Gentiopicroside Inhibits Cell Growth and Migration on Cervical Cancer via the Reciprocal MAPK/Akt Signaling Pathways

Cervical cancer (CC) is a common gynecological malignancy and represents a major global health challenge. Chemotherapeutic agents are commonly applied in treatment of CC, while along various adverse effects and chemotherapy resistance. As an iridoid glycoside compound, gentiopicroside (GPS) possesses the characteristic of the better availability and lower toxicity effect on cancer treatment. In the present study, we investigated that GPS exhibited the anticancer effect on HeLa cells through the inhibition of cell growth, induced apoptosis, cycle arrest, and suppressed migration. Furthermore, the possible mechanism or the targets of GPS was also clarified. The results revealed that GPS exerted an anti-proliferation effect in a dose- and time-dependent manner in HeLa cells, in contrast, with the less inhibiting proliferation effects on normal cell line (HUVEC). Moreover, GPS arrested cells at G2/M phase and induced apoptosis through mitochondrial apoptotic pathway. More significantly, GPS dramatically inhibited the migration of HeLa cells and regulated the matrix metalloproteinase expression through the MAPK and Akt signaling pathways, of which MAPK1 was an underlying target in GPS against HeLa cells.

In Vitro Cytotoxic Effect Of Various Fruits On Human Cervical Carcinoma Cells

Objective: The aim of this study was to evaluate the antiproliferative activities of some the fruits, traditionally used in folk medicine. Therefore, the fruits of Fructus cynosbati (Rosehip), Rubus fruticosus (Blackberry), Sorbus torminalis (Wild service tree), Vaccinium myrtillus (Blueberry) and Vitis labrusca (Isabella grape) were used in the present study.Material and Methods: Methanol extracts of the fruits were obtained and added to ARPE-19 (no-trasformed) and HeLa (transformed) culture media at various concentrations. Taxol and DMSO were used as positive and negative controls, respectively. After 48 h of cell incubation, the cytotoxic effect of the extracts was assessed by MTT. Cell viability values ​​were calculated for each group. The statistical analysis of the data was performed using unpaired t-test. p values ​​less than 0.05 were considered significant.Findings: V. labrusca showed dose dependent cytotoxic activity against both cell lines. In additions, V. myrtillus and R. fruticosus extracts exhibited moderate cytotoxic effect on both cell lines at higher concentrations. Extracts of F. cynosbati and S. torminalis were found to decrease the cell viability in a dose-dependent manner in HeLa cells and the results were statistically significant (p &amp;lt;0.05). Additionally, S. torminalis extract was found to be the most effective extract against the HeLa cells. Conclusion: These results demonsrated that S. torminalis methanol extract has a potent in vitro antiproliferative effect toward the human cervical cancer cell line HeLa. Further work needs to be done on isolation and purification of specific bioactive compounds in S. torminalis methanol extract.

New e:b-Friedo-Hopane Type Triterpenoids from Euphorbia peplus with Simiarendiol Possessing Significant Cytostatic Activity against HeLa Cells by Induction of Apoptosis and S/G2 Cell Cycle Arrest.

Seven rare e:b-friedo-hopane-type triterpenoids including four new (1–4) and three known (5–7) ones with 5 being first reported as a natural product, together with five other known triterpenoids (8–12), were isolated from the nonpolar fractions of the ethanolic extract of Euphorbia peplus. Structural assignments for these compounds were based on spectroscopic analyses and quantum chemical computation method. The structural variations for the C-21 isopropyl group, including dehydrogenation (1 and 3) and hydroxylation at C-22 (simiarendiol, 2), were the first cases among e:b-friedo-hopane-type triterpenoids. Simiarendiol (2) bearing a 22-OH showed significant cytostatic activity against HeLa and A549 human tumor cell lines with IC50 values of 3.93 ± 0.10 and 7.90 ± 0.31 μM, respectively. The DAPI staining and flow cytometric analysis revealed that simiarendiol (2) effectively induced cell apoptosis and arrested cell cycle at the S/G2 phases in a dose-dependent manner in HeLa cells.

Zac1 regulates IL-11 expression in osteoarthritis.

Interleukin (IL)-11, a member of the IL-6 family of cytokines, exerts pleiotropic effects under normal and various disease conditions. We assessed IL-11 expression regulation and the IL-11/IL-6 ratio in osteoarthritis (OA) to better guide clinical therapeutic decision-making. Our findings suggest that Zac1, a zinc finger protein that regulates apoptosis and cell cycle arrest, is a transcription factor regulating IL-11 expression. Zac1 overexpression or knockdown respectively induced or suppressed IL-11 expression in HeLa cells. Zac1 acted synergistically with AP-1, human papillomavirus E2, and hypoxia inducible factor 1 alpha (HIF1α). IL-11 expression under various conditions, including hypoxia or treatment with phorbol 12-myristate 13-acetate or copper sulfate. Recombinant IL-11-induced phosphorylation of signal transducer and activator of transcription 3 at tyrosine 705 was reduced in a dose-dependent manner in HeLa cells. Cross-talk between Zac1, IL-11, p53, and suppressor of cytokine signaling 3 was differentially affected by copper sulfate, digoxin, and caffeine. Finally, aggressive vs. conventional treatment of OA patients was primarily determined by IL-6 levels. However, we suggest that OA patients with higher IL-11 levels may respond well to conventional treatments, even in the presence of high IL-6.

Abstract 1475: Transcription of BUB1 is regulated by the CDE and CHR elements through the cell cycle

Abstract To ensure the accuracy of chromosome segregation during cell division, eukaryotic cells have developed a surveillance system, the mitotic checkpoint, which delays anaphase onset until all kinetochores are properly attached to microtubules emanating from opposite spindle poles. One of the core components of this checkpoint is Bub1 protein. Bub1 protein is encoded in the BUB1 gene, which is known to have a low basal levels of transcripts during interphase and increases its transcriptional activity in G2 and mitosis. However, the mechanisms involved in the regulation of BUB1 are not fully understood. Some of the possible components that participate in the transcriptional regulation of BUB1 are the cell cycle-dependent element (CDE) and the cell cycle homology region (CHR) element. The CDE/CHR are DNA sequences of 6 nucleotides, rich in CGs, located at proximal promoter regions of genes, and recruit repressive and activation factors during the initial and late phases of cell cycle, respectively. For instance, the promoters of CDC25C, CCNA2 and CDK1 genes contain the CDE/CHR elements and have high expression in G2 and mitosis. Thus, the aim of this study was to identify the mechanisms involved in the transcriptional regulation of BUB1 in a cell cycle dependent manner, using RT-qPCR, immunoblotting, flow cytometry, luciferase reporter gene assay, Methyl Sensitive-PCR. In this study, we unravel the mechanisms involved in the transcriptional regulation of BUB1 through the cell cycle. We found that it's expressed in a cell cycle-dependent manner in HeLa cells, being highly expressed during G2/M, followed by a rapid decline of both the mRNA and protein levels at the G1 phase. Also, we identified that the CDE/CHR elements are located at the BUB1 promoter and participate in its repression during the early phases of cell cycle, and his activation in G2 and mitosis. Finally, we found that the BUB1 promoter is located inside a CpG island which is mainly unmethylated in cancer cell lines regardless the cell type, indicating that DNA methylation might not be a determinant factor involved in its transcription. However, it's still unclear whether DNA methylation can affect BUB1 transcriptional activity through the cell cycle, particularly the methylation at specific CpG sites, such as those contained in the CDE and CHR elements. In summary, BUB1 transcription is regulated in a cell cycle dependent manner by the CDE/CHR elements located at its promoter. Also, DNA methylation is not a determinant factor implicated in its transcriptional activity. Nevertheless, whether BUB1 transcriptional deregulation can contribute towards the development of a cancer phenotype is still unclear. Thus, this study contributes to the understanding of the mechanisms implicated on the transcriptional deregulation of cell cycle genes, such as BUB1. Citation Format: Fernando Luna, Marco A. Andonegui, Fernanda Cisneros, Alfredo Cantu, Luis A. Herrera. Transcription of BUB1 is regulated by the CDE and CHR elements through the cell cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1475.

Vosaroxin induces mitochondrial dysfunction and apoptosis in cervical cancer HeLa cells: Involvement of AMPK/Sirt3/HIF-1 pathway

Vosaroxin is a quinolone-derivative anticancer agent with inhibitory activity on type II DNA topoisomerases (TOP2). The aim of the present study was to investigate its cytotoxic effect and potential molecular mechanisms in human cervical cancer HeLa cells. Vosaroxin decreased cell viability and increased lactate dehydrogenase (LDH) release in a dose- and time-dependent manner in HeLa cells, but not in normal cervical epithelial cells. Vosaroxin also induced apoptosis and increased caspase-3 activity in HeLa cells. These effects were accompanied by increased mitochondrial reactive oxygen species (ROS) generation, lipid peroxidation, mitochondrial swelling and reduced ATP production. Western blot analysis showed that vosaroxin significantly reduced hypoxia-inducible factor 1α (HIF-1α) protein levels. However, it had no effect on HIF-1α protein degradation and HIF-1α mRNA levels. The results showed that vosaroxin inhibited the synthesis of HIF-1α protein and interfered with the dimerization of HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, vosaroxin stimulated mitochondrial enzyme activities and superoxide dismutase 2 (SOD2) deacetylation via activating (Sir2 like protein 3) Sirt3. More importantly, vosaroxin-induced inhibition on HIF-1α and its cytotoxic effects, as measured by cell viability, LDH release and apoptosis, were partially prevented by Sirt3 knockdown or the AMP-activated protein kinase (AMPK) inhibitor compound C. Overall, vosaroxin is demonstrated to be a chemotherapeutic agent targeting the Sirt3/HIF-1 pathway and could be beneficial for inducing cytotoxicity in human cervical cancer cells.

ATF6 ubiquitylation is required for its transcriptional activity and degradation

IntroductionATF6α is an endoplasmic reticulum (ER) transmembrane protein that senses the accumulation of misfolded proteins in the ER of cells during ER stress. Upon ER stress, ATF6α is proteolytically cleaved. The resulting N‐terminal fragment becomes an active transcription factor, i.e. N‐ATF6α, which induces genes that adaptively reprogram ER protein folding. N‐ATF6α exerts potent but transient transcriptional activation of its target genes; the transient nature of its activity is due in part to its rapid degradation upon engagement in transcription. The transcriptional activation domain (TAD) and degradation domain (degron) of N‐ATF6α map to the same N‐terminal region of N‐ATF6α, implying that transcriptional activity and degradation are linked. Moreover, mutations introduced into the TAD of N‐ATF6α that decrease its activity also decrease its degradation, indicating that N‐ATF6α degradation is coupled with its transcriptional activity. To elucidate how and why N‐ATF6α degradation and activation are coupled, we examined the mechanism of N‐ATF6α degradation, positing that ubiquitylation may target it for proteasome‐mediated degradation. Accordingly, this study focused primarily on delineating whether N‐ATF6α is ubiquitylated, and if so, what domain(s) of N‐ATF6α are ubiquitylated, as well as investigating the functional consequences of such ubiquitylation.MethodsHeLa cells were co‐transfected with DNA constructs encoding FLAG‐tagged N‐ATF6α and with constructs encoding HA‐ubiquitin. To examine ubiquitylation, FLAG‐N‐ATF6α was FLAG immunoprecipitated (IP'd), then subjected to SDS‐PAGE followed by immunoblotting (IB) with anti‐HA or anti‐FLAG antibody. Specific ubiquitylation, i.e. the level of ubiquitylation per quantity of ATF6, was defined as the ratio of HA/FLAG. A series of mutations was introduced into N‐ATF6α to map the ubiquitylation domain(s) on N‐ATF6α and to assess the functional effects of the mutations that exhibited altered ubiquitylation. Transfected N‐ATF6α function was determined by assessing its ability to induce canonical ATF6 target genes by IB.ResultsIn the absence of ER stress, wild type (WT) N‐ATF6α was ubiquitylated in a proteasome‐dependent manner in HeLa cells. A series of mutations in the N‐terminus of N‐ATF6α that progressively decreased its ability to induce ATF6 target genes coordinately decreased its specific ubiquitylation, as well as its degradation. Using this mutation approach, the ubiquitylation domain of N‐ATF6α was mapped to the N‐terminal 39 amino acids. To determine whether engagement in transcription affected ATF6 ubiquitylation, the effect of mutating the DNA binding domain (DBD) of WT N‐ATF6α was examined. Interestingly, the DBD mutation disengaged ATF6 from DNA, which led to a loss of its transcriptional activation, as expected, and to a dramatic increased its specific ubiquitylation.ConclusionN‐ATF6α is ubiquitylated in a domain within the first 39 amino acids of the N‐terminus. This ubiquitylation is required for full ATF6 activity as a transcription factor, as well as its degradation. Moreover, deubiquitylation, degradation and transcriptional engagement were shown to be coordinate processes.Support or Funding InformationNational Institutes of Health (NIH) grants R01 HL75573, R01 HL104535, R01 HL127439, and P01 HL085577This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitofusin-2 Triggers Cervical Carcinoma Cell Hela Apoptosis via Mitochondrial Pathway in Mouse Model

Background/Aims: Cervical carcinoma continues to be one of the most dangerous cancer types, and more effective therapies are urgently needed for cervical carcinoma treatment. Mitochondria-associated Mitofusin 2 has influence on the progression of many cancers. In the current study, we aimed to focus on the cell apoptotic effects of Mfn2 on cervical carcinoma HeLa cells in vitro and to try to explore its underlying mechanisms. Moreover, we investigated the anticancer potential of Mfn2 in a cervical carcinoma mouse model. Methods: Adenovirus-Mfn2 (Adv-Mfn2) was used to deliver mfn2 into HeLa cells and tumour tissues in a nude mouse model. CCK-8, TUNEL assay, Western blot and immunohistochemical staining were performed to detect the effects of Mfn2. The mRNA level of Mfn2 was determined by quantitative realtime PCR (qRT-PCR) analysis. The effect of Mfn2 on cell apoptosis was investigated by flow cytometry. Flow cytometry was used to assess the change of the mitochondrial membrane potential of the cells treated with JC-1 assay. Mfn2, Bax, Bcl-2, cytochrome c, cleaved caspase-3, and cleaved caspase-9 protein levels were analysed by Western blot. Results: Data from CCK-8 and flow cytometry showed that Mfn2 could inhibit proliferation and induce apoptosis in a dose- and time-dependent manner in HeLa cells. JC-1 test results revealed that the membrane potential of the mitochondrial decreased in a dose-dependent manner in HeLa cells after Adv-Mfn2 treatment. The data from Western blot confirmed that higher cytosolic amounts of cytochrome c with increasing doses of Adv-Mfn2 signified the onset of the intrinsic apoptotic pathway. Levels of cleaved caspase-3 and cleaved caspase-9 increased in HeLa cells with Adv-Mfn2 treatment. We also found significant increases in the Bax level and a decreased Bcl-2 level with Adv-Mfn2 treatment. We further confirmed that Mfn2 could significantly inhibit the growth of the cervical tumour in the xenografted cervical carcinoma mouse model. After a 9-day-treatment, the tumours of the Adv-mfn2 group were inhibited and induced into apoptosis. The results demonstrated that the overexpression of Mfn2 could not only increase the levels of Bax and Bid in cervical tumour cells but also decrease the phosphorylation of Bad and the expression of Bcl-2. Conclusion: These studies suggested that the overexpression of Mfn2 could trigger cervical tumour apoptosis in vitro and in vivo, which was related to the mitochondrial pathway, and may provide a new treatment target for cervical carcinoma.

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2 +

Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

In vitro anticancer activity of gold(III) complexes with some esters of (S,S)-ethylenediamine-N,N'-di-2-propanoic acid.

Five novel gold(III) complexes of general formulas [AuCl2{(S,S)-R2eddip}]PF6, ((S,S)-eddip = (S,S)-ethylenediamine-N,N'-di-2-propanoate, R = n-Bu, n-Pe, i-Bu, i-Am, cPe; 1-5, respectively) were synthesized and characterized by UV/Vis, IR and NMR spectroscopy and mass spectrometry. DFT calculations indicated that (R,R)-N,N'-configuration diastereoisomers were the most stable for 1-5. 3 is stable in DMSO for at least 24 h, but immediate hydrolysis in PBS occurs. 3 is readily reduced with ascorbic acid and forms adducts with bovine serum albumin (BSA). In vitro anticancer activity of the gold(III) complexes against human cervix adenocarcinoma HeLa, human myelogenous leukemia K562, human melanoma Fem-x tumor cell lines, as well as against non-cancerous human embryonic lung fibroblast cell line MRC-5 was determined using MTT assay. Complex 4 showed highest activity and selectivity (IC50(Fem-x) = 1.3 ± 0.2; IC50(MRC-5)/IC50(Fem-x) = 72.5 ± 12.4), 4 times more active and 28 times more selective than cisplatin. Complexes induced apoptotic mode of death in a time-dependent manner in HeLa cells.

Baohuoside I suppresses invasion of cervical and breast cancer cells through the downregulation of CXCR4 chemokine receptor expression.

More than 90 percent of cancer-mediated deaths are due to metastasis, but the mechanisms that control metastasis remain poorly understood. Thus, the therapy targeting this process has been challenged constantly, but no therapy has yet been approved. CXC chemokine receptor 4 (CXCR4), a Gi protein-coupled receptor for the CXC chemokine ligand (CXCL) 12/stromal cell derived factor (SDF) 1α, is known to be expressed in various tumors. Recently, the CXCL12/CXCR4 axis has emerged as a key mediator of tumor metastasis; therefore, the possibility that identification of CXCR4 inhibitors can be a promising strategy for abrogating metastasis has been considered. In this report, we investigate baohuoside I, a component of Epimedium koreanum, as a regulator of CXCR4 expression as well as function in cervical cancer and breast cancer cells. We observed that baohuoside I downregulated CXCR4 expression in a dose- and time-dependent manner in HeLa cells. Treatment with a pharmacological proteasome and lysosomal inhibitors did not have a substantial effect on baohuoside I's ability to suppress CXCR4 expression. When we investigated the molecular mechanism of action, it was observed that the suppression of CXCR4 expression occurred at the level of mRNA. The decrease in the level of CXCR4 expression caused by baohuoside I was correlated with inhibition of the CXCL12-induced invasion of both cervical and breast cancer cells. Overall, our results show that baohuoside I exerts its antimetastatic effect through the downregulation of CXCR4 expression and, thus, has the potential to play a role in the suppression of cancer metastasis.

Non-coding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding.

Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5′ to 3′) eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction.

Roscovitine-treated HeLa cells finalize autophagy later than apoptosis by downregulating Bcl‑2.

The cell cycle is tightly regulated by the family of cyclin-dependent kinases (CDKs). CDKs act as regulatory factors on serine and threonine residues by phosphorylating their substrates and cyclins. CDK‑targeting drugs have previously demonstrated promising effects as cancer therapeutics both in vitro and in vivo. Roscovitine, a purine‑derivative and specific CDK inhibitor, has been demonstrated to arrest the cell cycle and induce apoptosis in a number of different cancer cell lines, including HeLa cervical cancer cells. In the present study, roscovitine was able to decrease both the cell viability and cell survival as well as induce apoptosis in a dose‑dependent manner in HeLa cells by modulating the mitochondrial membrane potential. The decrease of anti‑apoptotic B-cell lymphoma 2 (Bcl‑2) and Bcl-2 extra large protein expression was accompanied by the increase in pro‑apoptotic Bcl-2-associated X protein and P53-upregulated modulator of apoptosis expression. The marked decrease in Bcl‑2 following exposure to roscovitine (20 µM) for 48 h prompted us to determine the autophagic regulation. The outcome revealed that roscovitine triggered Beclin‑1 downregulation and microtubule-associated light chain 3 cleavage starting from 12 h of incubation. Another biomarker of autophagy, p62, a crucial protein for autophagic vacuole formation, was diminished following 48 h. In addition, monodansyl cadaverin staining of autophagosomes also confirmed the autophagic regulation by roscovitine treatment. The expression levels of different Bcl‑2 family members determined whether apoptosis or autophagy were induced following incubation with roscovitine for different time periods. Downregulation of pro‑apoptotic Bcl‑2 family members indicated induction of apoptosis, while the downregulation of anti‑apoptotic Bcl‑2 family members rapidly induced autophagosome formation in HeLa cells.

KCTD1 suppresses canonical Wnt signaling pathway by enhancing β-catenin degradation.

The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both in vivo by co-immunoprecipitation as well as in vitro through GST pull-down assays. We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.

Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin

Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration.

Reducing TDP-43 aggregation does not prevent its cytotoxicity

BackgroundTAR DNA-binding protein 43 (TDP-43) is a protein that is involved in the pathology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In patients with these neurodegenerative diseases, TDP-43 does not remain in its normal nuclear location, but instead forms insoluble aggregates in both the nucleus and cytoplasm of affected neurons.ResultsWe used high density peptide array analysis to identify regions in TDP-43 that are bound by TDP-43 itself and designed candidate peptides that might be able to reduce TDP-43 aggregation. We found that two of the synthetic peptides identified with this approach could effectively inhibit the formation of TDP-43 protein aggregates in a concentration-dependent manner in HeLa cells in which a mutated human TDP-43 gene was overexpressed. However, despite reducing aggregation, these peptides did not reduce or prevent cell death. Similar results were observed in HeLa cells treated with arsenite. Again we found reduced aggregation, in this case of wild type TDP-43, but no difference in cell death.ConclusionsOur results suggest that TDP-43 aggregation is associated with the cell death process rather than being a direct cause.