Towards the Small and the Beautiful: A Small Dibromotyrosine Derivative from Pseudoceratina sp. Sponge Exhibits Potent Apoptotic Effect through Targeting IKK/NFκB Signaling Pathway

A dibromotyrosine derivative, (1′R,5′S,6′S)-2-(3′,5′-dibromo-1′,6′-dihydroxy-4′-oxocyclohex-2′-enyl) acetonitrile (DT), was isolated from the sponge Pseudoceratina sp., and was found to exhibit a significant cytotoxic activity against leukemia K562 cells. Despite the large number of the isolated bromotyrosine derivatives, studies focusing on their biological mechanism of action are scarce. In the current study we designed a set of experiments to reveal the underlying mechanism of DT cytotoxic activity against K562 cells. First, the results of MTT cytotoxic and the annexin V-FITC/PI apoptotic assays, indicated that the DT cytotoxic activity is mediated through induction of apoptosis. This effect was also supported by caspases-3 and -9 activation as well as PARP cleavage. DT induced generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) as indicated by flow cytometric assay. The involvement of ROS generation in the apoptotic activity of DT was further corroborated by the pretreatment of K562 cells with N-acetyl-cysteine (NAC), a ROS scavenger, which prevented apoptosis and the disruption of MMP induced by DT. Results of cell-free system assay suggested that DT can act as a topoisomerase II catalytic inhibitor, unlike the clinical anticancer drug, etoposide, which acts as a topoisomerase poison. Additionally, we found that DT treatment can block IKK/NFκB pathway and activate PI3K/Akt pathway. These findings suggest that the cytotoxic effect of DT is associated with mitochondrial dysfunction-dependent apoptosis which is mediated through oxidative stress. Therefore, DT represents an interesting reference point for the development of new cytotoxic agent targeting IKK/NFκB pathway.     

眼树莲提取物的体外抗肿瘤活性研究

为了探索眼树莲乙醇提取物和不同萃取部位的体外抗肿瘤作用,以人胃癌 SGC 细胞株、人肝癌 BEL 细胞株、 慢性髓原白血病 K562 细胞株、宫颈癌 Hela 细胞株、乳腺腺癌 MCF-7 细胞株、肺腺癌 A549 细胞株为供试细胞株,用 MTT 法对眼树莲不同提取部位进行体外抗肿瘤活性筛选,并观察细胞形态。结果表明：乙醇提取物（A）、石油醚部 位（B）、乙酸乙酯部位（C）均对 SGC 细胞具有明显的抑制性,IC50 值分别为 71.73、55.83、51.95 μg/mL,而对 Hela、 MCF-7、A549 3 种细胞有弱的抑制性。A、B 部位对 BEL 细胞有弱抑制性,C 部位对 BEL 细胞具有一定的抑制性,IC50 值为 72.62 μg/mL。B、C 部位对 K562 细胞具有明显的抑制性,IC50 值分别为 53.34、55.99 μg/mL。正丁醇部位（D） 对 SGC、BEL、K562、Hela、MCF-7、A549 这 6 种细胞均没有抗肿瘤抑制性。B、C 均可使相应的肿瘤细胞株的细胞 形态发生变化,引起细胞株的坏死,并呈现明显的剂量-时间-效应关系,为进一步研究眼树莲的抗肿瘤作用奠定基础。     

Induction of Apoptosis, G0/G1 Phase Arrest and Microtubule Disassembly in K562 Leukemia Cells by Mere15, a Novel Polypeptide from Meretrix meretrix Linnaeus

Mere15 is a novel polypeptide from Meretrix meretrix Linnaeus with cytotoxicity in solid cancer cells. In this study, we investigated its activity on human K562 chronic myelogenous leukemia cells. Mere15 inhibited the growth of K562 cells with IC₅₀ values of 38.2 μg/mL. Mere15 also caused concentration dependent induction of apoptosis, with overproduction of reactive oxygen species and loss of mitochondrial membrane potential. Moreover, Mere15 arrested cell cycle progression at G₀/G₁ phase of K562 cells in a concentration dependent manner. In addition, Mere15 caused the disassembly of the microtubule cytoskeleton in K562 cells and inhibited the polymerization of tubulin in a cell free system via interaction with tubulin. We concluded that Mere15 was cytotoxic to K562 leukemia cells and the cytotoxicity was related to the apoptosis induction, cell cycle arrest and microtubule disassembly. These results implied that Merer15 was a broad spectrum anticancer polypeptide, not only cytotoxic to various solid cancer cells but also to the chronic myelogenous leukemia cells. Mere15 may have therapeutic potential for the treatment of leukemia.     

Induction of apoptosis and non-apoptosis in human breast cancer cell line (MCF-7) by cisplatin and caffeine

Background: Molecular targeted therapy by different cell death inducers are recently considered in cancer therapy. The aim of this study was to compare the effect of cisplatin and inositol trisphosphate kinase inhibitor (caffeine) on human breast cancer cell line (MCF-7). The pattern of cell death in MCF-7 cells following the exposure to cisplatin and caffeine in individual and combination forms was characterized. Methods: MCF-7cells at late exponential phase were divided into two groups: control and experimental groups. Experimental group was exposed to cisplatin, caffeine and combination of them and control group was treated by vehicle. Forty-eight hours after incubation, floating and attached cells were collected separately. Flowcytometry analysis and electron microscopy were carried out on both attached and floating cells. Results: Two types of apoptotic and non-apoptotic cells were observed in the floating cells as well as in sub G1 cells of both experimental and control groups by electron microscopy. Both early and late stages of apoptosis were characterized and the attached cells remained unaffected. Conclusion: Although two different forms of cell death (apoptosis and non-apoptosis) were appeared in MCF-7 following exposure to cisplatin and caffeine, apoptosis was the major mechanism of cell death. The combination form of anti-cancer drugs with different mechanisms could decrease the dosage of employed anti-cancer drugs.Key Words: Cisplatin, Caffeine, Apoptosis     

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus,Containing Lactose-Binding Molecules,on Human Leukemic K562 Cells

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.     

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.     

Evaluation of growth inhibitory and apoptosis inducing activity of human calprotectin on the human gastric cell line (AGS)

BACKGROUND: Calprotectin is cytotoxic agent that its anti-tumor effects are governed through suppression of topoisomerase II; a key enzyme in apoptosis. In previous studies, cytotoxicity and apoptotic effects of calprotectin are shown on different cancer cell lines, but not human gastric cancer cell lines. In the present study, cytotoxicity and apoptotic effects of calprotectin on human gastric adenocarcinoma cancer cell line (AGS) were evaluated. METHODS: The AGS cells were exposed to the different concentrations of calprotectin for 24, 48 and 72 hours. Cell proliferation was assessed using dimethylthiazol diphenyl tetrazolium bromide assay and dye exclusion tests. For evaluation of cytotoxic mechanism in calprotectin on AGS cells, flow cytometric analysis was performed. RESULTS: Our results revealed that calprotectin induces growth inhibition of AGS in a dose- and time-dependent manner. Results of this investigation showed that sensitivity of AGS cells to cytotoxic effect of human calprotectin was highly remarkable. In addition, growth inhibitory effect of this cytotoxic agent mostly was governed through induction of apoptosis in the AGS cells. CONCLUSION: These findings indicated that calprotectin induces growth inhibition and apoptosis in the AGS cells.     

Comparative Analysis of the Cytotoxic Effects of Okadaic Acid-Group Toxins on Human Intestinal Cell Lines

The phycotoxin, okadaic acid (OA) and dinophysistoxin 1 and 2 (DTX-1 and -2) are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP). Data on the toxicity of the OA-group toxins show some differences with respect to the in vivo acute toxicity between the toxin members. In order to investigate whether OA and congeners DTX-1 and -2 may induce different mechanisms of action during acute toxicity on the human intestine, we compared their toxicological effects in two in vitro intestinal cell models: the colorectal adenocarcinoma cell line, Caco-2, and the intestinal muco-secreting cell line, HT29-MTX. Using a high content analysis approach, we evaluated various cytotoxicity parameters, including apoptosis (caspase-3 activation), DNA damage (phosphorylation of histone H2AX), inflammation (translocation of NF-κB) and cell proliferation (Ki-67 production). Investigation of the kinetics of the cellular responses demonstrated that the three toxins induced a pro-inflammatory response followed by cell cycle disruption in both cell lines, leading to apoptosis. Our results demonstrate that the three toxins induce similar effects, as no major differences in the cytotoxic responses could be detected. However DTX-1 induced cytotoxic effects at five-fold lower concentrations than for OA and DTX-2.     

Semi-Synthesis,Cytotoxic Evaluation,and Structure—Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC₅₀ value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.     

Smenospongine,a Sesquiterpene Aminoquinone from a Marine Sponge,Induces G1 Arrest or Apoptosis in Different Leukemia Cells

Smenospongine, a sesquiterpene aminoquinone isolated from the marine sponge Dactylospongia elegans, was previously reported by us to induce erythroid differentiation and G1 phase arrest of K562 chronic myelogenous leukemia cells. In this study, we investigated the effect of smenospongine on the cell cycles of other leukemia cells, including HL60 human acute promyelocytic leukemia cells and U937 human histiocytic lymphoma cells by flow cytometric analysis. Smenospongine induced apoptosis dose-dependently in HL60 and U937 cells. The smenospongine treatment increased expression of p21 and inhibited phosphorylation of Rb in K562 cells, suggesting the p21-Rb pathway play an important role in G1 arrest in K562 cells. However, the p21 promoter was not activated by the smenospongine treatment based on a luciferase assay using the transfected K562 cells. Smenospongine might induce p21 expression via another mechanism than transactivation of p21 promoter.     

A Non-digestible Fraction of the Common Bean (Phaseolus vulgaris L.) Induces Cell Cycle Arrest and Apoptosis During Early Carcinogenesis

We have previously demonstrated that the non-digestible fraction (NDF) from common cooked beans (P. vulgaris L., cv Negro 8025) inhibits azoxymethane (AOM)-induced colon cancer and influences the expression of genes involved in the induction of apoptosis and cell cycle arrest through the action of butyrate. The objective of this study was to identify cell cycle alterations and morphological changes induced by treatment with AOM and to examine the formation of colonic aberrant crypt foci (ACF) in male Sprague Dawley rats fed with these beans. Rats were fed control diets upon arrival and were randomly placed into four groups after one week of acclimatization: control, NDF (intragastric administration), NDF?+?AOM and AOM. Rats treated with NDF?+?AOM exhibited a significantly lower number of total colonic ACF with a notable increase in the number of cells present in the G₁ phase (83.14 %); a decreased proliferation index was observed in the NDF?+?AOM group when compared to AOM group. NDF?+?AOM also displayed a higher number of apoptotic cells compared to AOM group. NDF of cooked common beans inhibited colon carcinogenesis at an early stage by inducing cell cycle arrest of colon cells and morphological changes linked to apoptosis, thus confirming previous results obtained with gene expression studies.     

The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line

BACKGROUND: The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. METHODS: The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. RESULTS: While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. CONCLUSION: Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment.     

Fucoidan Independently Enhances Activity in Human Immune Cells and Has a Cytostatic Effect on Prostate Cancer Cells in the Presence of Nivolumab

Fucoidan compounds may increase immune activity and are known to have cancer inhibitory effects in vitro and in vivo. In this study, we aimed to investigate the effect of fucoidan compounds on ex vivo human peripheral blood mononuclear cells (PBMCs), and to determine their cancer cell killing activity both solely, and in combination with an immune-checkpoint inhibitor drug, Nivolumab. Proliferation of PBMCs and interferon gamma (IFNγ) release were assessed in the presence of fucoidan compounds extracted from Fucus vesiculosus, Undaria pinnatifida and Macrocystis pyrifera. Total cell numbers and cell killing activity were assessed using a hormone resistant prostate cancer cell line, PC3. All fucoidan compounds activated PBMCs, and increased the effects of Nivolumab. All fucoidan compounds had significant direct cytostatic effects on PC3 cells, reducing cancer cell numbers, and PBMCs exhibited cell killing activity as measured by apoptosis. However, there was no fucoidan mediated increase in the cell killing activity. In conclusion, fucoidan compounds promoted proliferation and activity of PBMCs and added to the effects of Nivolumab. Fucoidan compounds all had a direct cytostatic effect on PC3 cells, as shown through their proliferation reduction, while their killing was not increased.     

中国绿茶的抗肿瘤作用 Ⅰ.龙雾茶提取物抗癌体外实验

一定浓度的绿茶提取物,对人胃腺癌细胞具有明显的细胞毒作用,细胞抑制程度与提取物的剂量和接触时间成正相关;能够抑制肿瘤细胞DNA合成,阻止癌细胞由G_1期向S期移行,其阻断效应是发生在细胞分化的早期阶段;对致癌物质亚硝基吗啉的合成具有显著的阻断作用,阻断率为92.4％;对辐射损伤所产生的有害自由基具有明显的清除能力,因而具有防癌和抗癌作用。     

Antiproliferation and Apoptosis on RKO Colon Cancer by <Emphasis Type="Italic">Millingtonia hortensis</Emphasis>

Millingtonia hortensis is a medicinal plant widely used in many Asian countries. An aqueous crude extract of this plant has been shown the apoptosis induction on RKO colon cancer cells. However, its mechanism remains unknown. To learn more about this plant extract, we partially purified the crude extract using Sephadex LH-20 and three aqueous fractions were collected. Each fraction was investigated for cytotoxicity using MTT assay. Fraction 1 showed antiproliferative effect on RKO cells with dose-dependent manner, while fraction 2 and 3 had no effect. Induction of apoptosis was determined using flow cytometry and DNA fragmentation method. Apoptotic cell numbers and the appearance of fragmented DNA increased with dose-dependent manner after treatment with fraction 1 for 48 h. We further investigated the expression of apoptotic protein by western blot analysis. Fraction 1 decreased the expression of anti-apoptotic protein, Bcl-x_L and p-Bad, while pro-apoptotic protein Bad, was not changed. Fraction 1 also decreased the expression of p-Akt and slightly increased the level of total Akt. These results indicated that fraction 1 is able to inhibit cell proliferation and induce apoptosis on RKO cells by decreasing the expression of Bcl-x_L, p-Bad and p-Akt which are involving in survival of cancer cells.     

Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat)

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 – 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.     

Separacenes A–D,Novel Polyene Polyols from the Marine Actinomycete,Streptomyces sp

Separacenes A–D (1–4), novel polyene polyols, were isolated from Streptomyces sp. collected from the southern area of Jeju Island, Korea. The chemical structures of 1–4 were established by NMR, mass, UV, and IR spectroscopy as well as the modified Mosher’s method. Separacenes A–B (1–2), which share an identical planar structure but possess different relative configurations, bear tetraene units flanked by two diol moieties, whereas the stereoisomeric separacenes C–D (3–4) possess a triene moiety between two diol substructures. Separacenes A–D each contain a terminal olefinic methylene. Separacene A displayed inhibitory activity against Candida albicans isocitrate lyase and weak cytotoxicity against both the colon carcinoma cell line HCT-116 and the lung cancer cell line A549.     

A new hydroxylated nonaprenylhydroquinone from the Mediterranean marine sponge Sarcotragus spinosulus

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated.     

Study of Structure–Activity Relationships of the Marine Alkaloid Fascaplysin and Its Derivatives as Potent Anticancer Agents

Marine alkaloid fascaplysin and its derivatives are known to exhibit promising anticancer properties in vitro and in vivo. However, toxicity of these molecules to non-cancer cells was identified as a main limitation for their clinical use. Here, for the very first time, we synthesized a library of fascaplysin derivatives covering all possible substituent introduction sites, i.e., cycles A, C and E of the 12H-pyrido[1-2-a:3,4-b’]diindole system. Their selectivity towards human prostate cancer versus non-cancer cells, as well as the effects on cellular metabolism, membrane integrity, cell cycle progression, apoptosis induction and their ability to intercalate into DNA were investigated. A pronounced selectivity for cancer cells was observed for the family of di- and trisubstituted halogen derivatives (modification of cycles A and E), while a modification of cycle C resulted in a stronger activity in therapy-resistant PC-3 cells. Among others, 3,10-dibromofascaplysin exhibited the highest selectivity, presumably due to the cytostatic effects executed via the targeting of cellular metabolism. Moreover, an introduction of radical substituents at C-9, C-10 or C-10 plus C-3 resulted in a notable reduction in DNA intercalating activity and improved selectivity. Taken together, our research contributes to understanding the structure–activity relationships of fascaplysin alkaloids and defines further directions of the structural optimization.