Reduction of HIF-1α/PD-L1 by Catalytic Topoisomerase Inhibitor Induces Cell Death Through Caspase Activation in Cancer Cells Under Hypoxia.

BACKGROUND/AIM: Under severe hypoxia, cellular apoptosis is induced through hypoxia-inducible factor 1, alpha subunit (HIF-1α)-dependent P53 accumulation and P53 phosphorylation via ataxia telangiectasia mutated and ataxia telangiectasia and RAD3-related (ATR) activation via replication stress-induced DNA damage response (DDR) activation. We previously demonstrated that the topoisomerase I catalytic inhibitor, 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), induced apoptosis in Jeko-1 and Panc-1 cells, both of which show cyclin D1 overexpression. After progression to the S phase facilitated by nuclear cyclin D1, an intra S phase checkpoint was induced in the presence of 3EZ,20Ac-ingenol, by ATR activation in response to replication stress-induced DDR. MATERIALS AND METHODS: In this study, we examined whether 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (3EE,20Ac-ingenol) might induce a higher degree of P53 phosphorylation and additional HIF-1α and P53 accumulation in response to replication stress-induced DDR activation under hypoxic conditions than under normoxic conditions, by controlling ATR activation. RESULTS: In the Panc-1 cells, 3EE,20Ac-ingenol induced P53 activation and HIF-1α-dependent P53 accumulation through cooperative ATR activation via hypoxia-induced DDR activation. Jeko-1 cells showed slight HIF-1α accumulation under hypoxia, but HIF-1α-dependent 53 accumulation was not observed in the presence of 3EE,20Ac-ingenol, so that the cells remained resistant to hypoxia. CONCLUSION: 3EE,20Ac-ingenol induces an intricate interplay between P53 and HIF-1α accumulation via ATR activation that results in a high P53 accumulation, which promoted transient expression and early disappearance of HIF-1α, accelerating cell death. Strong P53 accumulation and consequent phosphatase and tensin homolog deleted on chromosome 10 activation in Panc-1 cells also reduced HIF-1α accumulation and programmed death-ligand 1 expression, which resulted in intense apoptosis.

3EZ, 20Ac-ingenol-induced Apoptosis in Chemoresistant Cancers With Cyclin D1 Accumulation.

BACKGROUND/AIM: The topoisomerase 1 catalytic inhibitor 3EZ, 20Ac-ingenol specifically induces apoptosis through the activation of ATR and the up-regulation of PTEN by enhancing the DNA damage response (DDR) in human B lymphoma (BALL-1) cells. The accumulation of cyclin D1 in cancer is known to be related to chemoresistance to DNA damage agents and nuclei of BALL-1 cells exhibit high levels of cyclin D1. However, 3EZ, 20Ac-ingenol effectively induced apoptosis of BALL-1 cells. MATERIALS AND METHODS: Cell growth, protein levels, and apoptosis were determined by an MTT assay, immunoblotting and DNA fragmentation assay, respectively. RESULTS: 3EZ, 20Ac-ingenol strongly induced inhibition of cell proliferation and apoptosis in Jeko-1 and Panc-1 cell lines through the activation of tumor suppressor proteins and caspase 3. CONCLUSION: 3EZ, 20Ac-ingenol-induced apoptosis might occur in cells with cyclin D1 accumulation through enhancing DDR, regardless of the cancer cell type.

3EZ, 20Ac-ingenol induces cell-specific apoptosis in cyclin D1 over-expression through the activation of ATR and downregulation of p-Akt.

Acute lymphoblastic leukemia (ALL) samples exhibit an activated PI3K/Akt pathway, which suggests a general role of Akt in the development of leukemia. We have previously used western blot analysis to show that the catalytic topoisomerase (topo) inhibitor, 3EZ, 20Ac-ingenol, induced DNA damage response (DDR), which activated ATR, downregulated p-Akt through upregulation of PTEN level, and led to cell cycle arrest or apoptosis. In this study, we used ATR or PTEN siRNA and observed that the specific cell arrest and apoptosis of BALL-1 cells in DDR caused by 3EZ, 20Ac-ingenol was dependant on activation of ATR and downregulation of nuclear p-Akt through upregulation of PTEN. Moreover, some B cell lymphomas among ALLs overexpress cyclin D1. The DDR induced during the S-phase with 3EZ, 20Ac-ingenol treatment was increased by the intra S-phase checkpoint response that was triggered by the loss of nuclear cyclin D1 regulation in BALL-1 cells overexpressing cyclin D1. Although topo 1 catalytic inhibitors induce a decatenation checkpoint and subsequent G2/M phase arrest, the decatenation checkpoint caused by 3EZ, 20Ac-ingenol induced apoptosis only in the BALL-1 cells that accumulated cyclin D1.

Mechanism of the inhibition of leukemia cell growth and induction of apoptosis through the activation of ATR and PTEN by the topoisomerase inhibitor 3EZ, 20Ac-ingenol.

The PI3K/Akt signaling pathway is constitutively activated in various leukemias. In the present study, the topoisomerase inhibitor, 3EZ, 20Ac-ingenol, was more effective in inhibiting the growth of BALL-1 cells than that of normal lymphocyte cells. ATM/ATR protein levels were increased, PTEN protein was upregulated, and p-Akt protein was downregulated at early time points after treatment with 3EZ, 20Ac-ingenol. In further experiments, p53 protein expression was increased, and H2AX phosphorylation and p21 protein expression were induced after treatment with 3EZ, 20Ac-ingenol. Moreover, the activation of caspase 3 followed decrease in the Bcl-2/Bax ratio after treatment with 3EZ, 20Ac-ingenol, and accumulation of sub-G1 phase cells was observed in flow cytometry analyses. These data suggest that 3EZ, 20Ac-ingenol-induced DNA damage downregulates p-Akt and upregulates ATR leading to cell cycle arrest and increased apoptosis in BALL-1 cells.

3EZ,20Ac-ingenol, a catalytic inhibitor of topoisomerases, downregulates p-Akt and induces DSBs and apoptosis of DT40 cells.

We have previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase (topo) II inhibitory activity. Of these compounds, 3EZ,20Ac-ingenol inhibited topo I activity. Camptothecin, which inhibits the religation activity of topo I without interfering with the binding of topo I to DNA and induces topo I-mediated DNA cleavage, was used as a positive control. In this study, we found that 3EZ,20Ac-ingenol did not hamper the binding of topo I to DNA in the same manner as camptothecin but affected the inhibition of cleavage of one DNA strand. 3EZ,20Ac-ingenol inhibited cell proliferation by blocking cell cycle progression in the G2/M phase. To define the mechanism of inhibition of DT40 cell proliferation, the change in Akt activity was observed because Akt activity is regulated in response to DNA damage. Western blot analysis revealed that 3EZ,20Ac-ingenol downregulated the expression of p-Akt, and apoptosis was detected by the presence of DNA double-strand breaks and caspase 3 activation.

Characterization of cAMP response element-like sequence in gonadal specific aromatase promoter sequence of Xenopus embryos.

The p450 aromatase gene has a tissue-specific promoter that is regulated by specific transcriptional factors. In rats and humans, a cAMP response element-like sequence (CLS) and an NR5A1/NR5A2 binding sequence have been identified as cis elements in the aromatase promoter; these cis elements mediate cAMP-induced expression in the ovaries and testes. CLS is recognized by a cAMP-responsive element binding protein (CREB) as the principal component. In this study, we performed a gel shift assay to analyze the proteins that interact with the cis-element in Xenopus aromatase. An electrophpretic mobility gel shift assay (EMSA) and matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis of the proteins responsible for retarding the mobility of CLS revealed that ATF4 interacted in vitro with CLS in gonadal specific aromatase promoter sequence of Xenopus embryos. Although a significant difference was observed in aromatase mRNA expression between male and female gonads, no difference in the expression of ATF4 was observed between them at stage 50. With regard to aromatase expression in the gonad of Xenopus embryos, ATF4 might act in combination with multiple transcription factors as a trans-element of CLS in place of CREB.

Analysis of inhibition of topoisomerase IIalpha and cancer cell proliferation by ingenolEZ.

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2'E,4'Z-decadienoyl) ingenol and 3-O-(2'E,4'Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIalpha activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX.

Characterization of an activating transcription factor 4 gene containing a consensus phosphorylation site for PKA in the gonads of Xenopus embryos.

Activating transcription factor / cyclic-AMP response element-binding protein (ATF/CREB) has been implicated as a key regulator in the transcriptional control of many genes. In this study, we isolated and characterized a full-length cDNA that encodes a CRE-binding protein 2 (CREB2) called ATF4 in Xenopus embryos. Like other CREB 2 transcription factors, the 342-amino acid ATF4 protein contains a carboxyl terminal leucine-zipper motif, an adjacent basic domain, and an amino terminal leucine-zipper motif. Unlike other CREB2 (ATF4) proteins, the ATF4 isolated from the gonads of Xenopus embryos contains a consensus phosphorylation site for protein kinase A (PKA). In a gel shift analysis, ATF4 bound to a CLS sequence in the promoter of Xenopus aromatase.

Isolation of novel isoforms of estrogen receptor genes from Xenopus gonad and brain.

We isolated four variants of ER alpha mRNA, two each from the brain and gonad of the Xenopus embryo (brain, bER alpha 1 and bER alpha 2; gonad, gER alpha 1 and gER alpha 2). The N-terminal-domain of ER alpha differed between the brain and gonad. While the C-terminal region downstream of the DNA-binding domain of ER alpha differed between ER alpha 1 and ER alpha 2 in both the brain and gonad, each of ER alpha 1 and ER alpha 2 contained the same sequence sets (bER alpha 1 and gER alpha 1; bER alpha 2 and gER alpha 2) between the brain and gonad. Both bER alpha 1 and gER alpha 1 contained the same full-length, C-terminal estrogen receptor, whereas the C terminus of bER alpha 2 and gER alpha 2 lacked half of the DNA-binding, ligand-binding, and transcriptional activation domain. Although gER alpha was mainly expressed in the gonad, both bER alpha and gER alpha were expressed in the brain of the Xenopus embryo at stage 50. These isoforms might be transcribed by alterative splicing of a single gene. Two gonad ER beta s differing in the length of the N-terminal sequence were isolated from the Xenopus gonad. While ER beta was expressed in the gonad, it was not expressed in the brain of the Xenopus embryo at stage 50.

Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad.

Analysis of 5'-RACE clones revealed two Cyp19 transcript variants (gonad p450 aroms 1 and 2) in the gonads and one Cyp transcript (brain p450 arom) in the brain that differed in their 5'-untranslated region (UTR). Since all cDNAs contained an identical open reading frame, it is considered that while Xenopus aromatase may be encoded by a single Cyp19 gene, it is transcribed by tissue-specific promoters, each of which may be regulated by a distinct set of transcriptional factors. While gonad p450 aroms 1 and 2 and brain p450 arom were expressed in the gonads, brain p450 arom was the predominantly expressed aromatase gene in the brain, with a low expression level of gonad p450 arom 1.

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1.

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.

Inhibition of cellular proliferation by diterpenes, topoisomerase II inhibitor.

We examined the effects of 12 terpene compounds derived from the roots of Euphorbia kansui on the proliferative activity of Xenopus embryo cells. Eight of these compounds showed significant inhibition of cellular proliferation even at low concentrations, while four of them needed to be present at higher concentrations to inhibit cellular proliferation. In order to define the mechanism of inhibition of cellular proliferation by these compounds, the effects of diterpene compounds on the activity of topoisomerase II were measured. Most of the diterpene compounds that inhibited cellular proliferation also inhibited topoisomerase II activity.

Expression of the gonadal p450 aromatase gene of Xenopus and characterization of the 5'-flanking region of the aromatase gene.

Investigation by RQ-RT-PCR revealed that transcription of the p450 aromatase gene is activated at stage 50, when sex determination of the female begins, and that aromatase gene expression is also activated by exogenously administrated estradiol. In order to determine the molecular basis underlying the specific activation of aromatase gene expression during sex differentiation and in response to exogenous estradiol, we isolated the 5'-flanking fragment of the gene and characterized the promoter sequence. We demonstrated binding sequences to a specific trans-activating factor upstream of the p450 aromatase promoter II, the cAMP response element binding protein/activating transcription factor family, and steroidogenic factor-1. An estrogen response element half-site sequence that recognize an estrogen receptors, was also found.

Mechanism of proliferation arrest of embryonic cells of Xenopus by diterpene compounds.

Three diterpene compounds isolated from the anti-cancer herbal medicine kansui, namely, kansuinin B, 20-OD-ingenol Z, and 20-OD-ingenol E, specifically inhibited the proliferation of isolated embryonic cells from Xenopus embryos. We conducted a cytologic study to determine the mechanism underlying the arrest of the cellular proliferation by these compounds. While kansuinin B and 20-OD-ingenol Z treatment decreased the cell numbers in the S phase and the M phase substages of the cell cycle, 20-OD-ingenol E inhibited mitosis.

Analysis of genes related to expression of aromatase and estradiol-regulated genes during sex differentiation in Xenopus embryos.

We analyzed the genes that exhibit transcriptional changes during sex differentiation in Xenopus, using fluorescent differential display (FDD). Search was then undertaken for sequences that were homologous to the differentially displayed DNA. In this report, trans-acting factors of activating transcription factor 4 (ATF 4) and heat shock proteins were selected, on the basis of homology, from candidate genes thought to be involved in the expression cascade of aromatase and estrogen receptor genes. The stage and tissue specificities and the effect of estradiol treatment on the expression of these genes were then examined using real-time quantitative polymerase chain reaction (RQ-RT-PCR). The expression of ATF 4, a member of the ATF/cAMP-responsive element-binding protein (CREB) family of genes, peaked in the gonads at stage 50 of development. Interestingly, expression of the genes encoding the heat shock cognate protein70. II (Hsc70. II) and the heat shock protein 70 (Hsp70) binding protein was strongly activated at stages 50 and 48 of development, respectively. The three genes revealed a higher transcription activity in the gonads than in other tissues. Although the expression of all of the genes encoding ATF 4, aromatase, Hsc70. II, and Hsp70 binding protein was activated in vitro by estrogen treatment, that of Hsc70. II and Hsp70 binding protein was found to be transient.

Selective inhibition of the growth of cancer cells by diterpenes selected with embryonic cells of Xenopus.

We used Xenopus embryo cells with a cell cycle of 20-30 min to detect inhibitory effects on cell proliferation. Inhibition of proliferation was observed when isolated embryonic cells were incubated for 16 h in a simple salt solution containing the well-known anticancer drugs 5-fluorouracil and adriamycin. In addition, three diterpene compounds isolated from the anticancer herbal medicine kansui: kansuinin B, 20-OD-ingenol Z, and 20-OD-ingenol E specifically inhibited the proliferation of isolated embryonic cells. The inhibitory compounds selected using the embryonic cells also inhibited proliferation of certain mammalian cell types.

Effects of proteasome inhibitor (lactacystin) and cysteine protease inhibitor (E-64-d) on processes of mitosis in Xenopus embryonic cells.

At least two different protease pathways have been implicated in the degradation that is required to control the eukaryotic cell cycle; these two pathways center on the activities of ubiquitin/proteasome and cysteine protease. The proteasome inhibitors, lactacystin and AcLLnL and the cysteine protease inhibitor E-64-d were tested for their ability to inhibit the cell cycles of Xenopus embryos. Lactacystin, AcLLnL and E-64-d all caused the complete arrest of the cell cycle. To define the specific cell cycle processes that were affected by the two inhibitors, we performed a cytological analysis. Inhibition of the cell cycle by lactacystin and E-64-d occurred during prophase and metaphase. The number of cells that arrested in prophase was 1.4-times higher in the E-64-d-treated group than in the control group and the number of arrested cells in the lactacystin-treated group was 1.4-times higher than in the E-64-d-treated group. The number of cells that arrested in metaphase was 3-to-4-times higher in the E-64-d and lactacystin groups than in the control group. These results indicate that both cysteine protease(s) and proteasomes are involved in the prophase and metaphase stages of cell division.