Involvement of an aldo-keto reductase (AKR1C3) in redox cycling of 9,10-phenanthrenequinone leading to apoptosis in human endothelial cells

9,10-Phenanthrenequinone (9,10-PQ), a major quinone found in diesel exhaust particles, is considered to generate reactive oxygen species (ROS) through its redox cycling. Here, we show that 9,10-PQ evokes apoptosis in human aortic endothelial cells (HAECs) and its apoptotic signaling includes ROS generation and caspase activation. The 9,10-PQ-induced cytotoxicity was inhibited by ROS scavengers, indicating that intracellular ROS generation is responsible for the 9,10-PQ-induced apoptosis. Comparison of mRNA expression levels and kinetic constants in the 9,10-PQ reduction among 10 human reductases suggests that aldo-keto reductase 1C3 (AKR1C3) is a 9,10-PQ reductase in HAECs. In in vitro 9,10-PQ reduction by AKR1C3, the reduced product 9,10-dihydroxyphenanthrene and superoxide anions were formed, suggesting the enzymatic two-electron reduction of 9,10-PQ that thereby causes oxidative stress through its redox cycling. In addition, the participation of AKR1C3 in 9,10-PQ-redox cycling was confirmed by the data that AKR1C3 overexpression in endothelial cells augmented the ROS generation and cytotoxicity by 9,10-PQ, and the ROS scavengers inhibited the toxic effects. Pretreatment of the overexpressing cells with AKR1C3 inhibitors, flufenamic acid and indomethacin, suppressed the 9,10-PQ-induced GSH depletion. These results suggest that AKR1C3 is a key enzyme in the initial step of 9,10-PQ-induced cytotoxicity in HAECs.     

Involvement of carbonyl reductase in superoxide formation through redox cycling of adrenochrome and 9,10-phenanthrenequinone in pig heart

The effects of adrenochrome, a metabolite of epinephrine (adrenaline), and 9,10-phenanthrenequinone (PQ), a component of diesel exhaust particles, on the stereoselective reduction of 4-benzoylpyridine (4-BP) were examined in pig heart cytosol. PQ was a potent inhibitor for the 4-BP reduction, while adrenochrome was a poor inhibitor. A similar result was observed in the effects of adrenochrome and PQ on the reduction of all-trans retinal. Furthermore, although PQ mediated efficiently the formation of superoxide anion radical through its redox cycling in pig heart cytosol, adrenochrome had no ability to mediate the superoxide formation. These may be because the reactivity for adrenochrome, catalyzed by pig heart carbonyl reductase (PHCR), is much lower than that for PQ. The optimal pH for the reduction of PQ in pig heart cytosol was around 5.5. Dicumarol, a potent inhibitor of DT-diaphorase, had little effect on the time course of NADPH oxidation during the reduction of PQ. Therefore, it is concluded that PHCR plays a critical role in superoxide formation through redox cycling of PQ.     

Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.     

Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.     

Effect of Bcl—2 and caspase—3 on calcium distribution in apoptosis of HL—60 cells

Apoptosis manifests in two major execution programs downstream of the death signal:the caspase pathway and organelle dysfunction.An important antiapoptosis factor,Bcl-2 protein,contributes in caspase pathway of apoptosis.Calcium,an important intracellular signal element in cells,is also observed to have changes during apoptosis,which maybe affected by Bcl-2 protein.We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells,there‘s change of intracellular calcium distribution,oving from cytoplast especially Golgi‘s apparatus to nucleus and accumulating there with the highest concentration.We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells,which can be inhibited by overexpression of Bcl-2 protein.No sign of apoptosis or intracellular calcium movement from Golgi‘s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO,a specific inhibitor of caspase-3.The results indicate that activated caspase-2 can promote the movement of intracellular calcium from Golgi‘s apparatus to nucleus,and the process is inhibited by Ac-DEVD-CHO(inhibitor of caspase-3),and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3.Calcium relocalization in apoptosis seems to be irreversible,which is different from the intracellular calcium changes caused by growth factor.     

The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis

Besides its preventive action on bone resorption the third generation bisphosphonate zoledronic acid (ZOL) has been shown to display potent inhibitory action on the formation of bone metastases of various human cancers. Recent research also indicates an antitumoral effect on primary tumors and visceral metastases. Here we investigate for the first time the effect of ZOL on the human colon carcinoma cell line HCT-116. ZOL strongly inhibited the proliferation and soft agar colony formation of HCT-116 cells and caused a G1 cell cycle arrest in a population of ZOL treated cells. This cell cycle arrest was accompanied by an induction of apoptosis via a caspase dependent mechanism. Activation of Caspases 3, 7, 8 and 9, cleavage of PARP as well as the release of cytochrome C into the cytosol were detected in HCT-116 cells treated with low micromolar concentrations of ZOL. The induction of the mitochondrial pathway of apoptosis was accompanied by a translocation of Bax into the mitochondria, Bid activation and a decrease of overall Bcl-2 expression. We also detected a cytosolic increase of apoptosis inducing factor (AIF), a trigger of caspase-independent apoptosis. Taken together, our data indicate a potent antitumoral and apoptosis inducing effect of ZOL on HCT-116 colon carcinoma cells. Lilian Sewing and Florian Steinberg contributed equally to this work.     

Hypertonicity induced apoptosis in HL-60 cells in the presence of intracellular potassium

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K⁺. We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.     

Effects of recombinant human PLD2 on proliferation and apoptosis of HL-60 cells

In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.     

An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells

An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.     

Effect of dimethyl sulfides on the induction of apoptosis in human leukemia Jurkat cells and HL-60 cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me(2)S), dimethyl disulfide (Me(2)S(2)), dimethyl trisulfide (Me(2)S(3)) and dimethyl tetrasulfide (Me(2)S(4)) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 muM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me(2)S(4) asymptotically equal to Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for Jurkat cells and Me(2)S(4) > Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for HL-60 cells. Me(2)S(3) and Me(2)S(4) significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect on human myeloid leukemia cells in this study. The water extract of P. urinaria induced the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria and this inhibitory effect of fumonisin B1 could be eliminated by the addition of ceramide. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. The P. urinaria-induced apoptosis in HL-60 cells is mediated through a ceramide-related pathway.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

A novel cleavage product formed by autoxidation of lycopene induces apoptosis in HL-60 cells

Dietary carotenoids have been thought to have beneficial effects on human health through their antioxidant activity, provitamin A activity, and effects on cancer cell propagation. Recent studies suggest that oxidation products or metabolites are involved in biological activities of carotenoids. We previously reported that an autoxidation mixture of lycopene induced apoptosis in HL-60 human promyelocytic leukemia cells, but lycopene alone did not. In the present study, bioassay-directed fractionations of autoxidized lycopene led to isolation of a novel cleavage product of lycopene. Spectral analyses elucidated its structure as (E,E,E)-4-methyl-8-oxo-2,4,6-nonatrienal (MON), suggesting the formation through the oxidative cleavages at the 5, 6- and 13, 14-double bonds of lycopene. MON was proved to cause a dose-dependent reduction of viability in HL-60 cells with morphological changes such as chromatin condensation and nuclear fragmentation. Treatment of HL-60 cells with MON could induce DNA fragmentation and increase apoptotic cells in a time- and dose-dependent manner. The MON treatment could enhance both caspase 8 and caspase 9 activities. Moreover, it reduced the expression of Bcl-2 and Bcl-X_L proteins, whereas it had no effect on the level of Bax protein. These results clearly indicated that MON induced apoptosis in HL-60 cells, associated with the down regulation of Bcl-2 and Bcl-X_L and the activation of caspase cascades. The concentration of MON attained by treatment of the autoxidized lycopene preparation was far less than the IC₅₀ (10 μM) value of MON alone in reducing the viability of HL-60 cells. The fractionation of the oxidized lycopene indicated the presence of other active oxidation products. Thus, unidentified products as well as MON would be responsible for the apoptosis-inducing activity of the autoxidized lycopene.     

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.     

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53^+/+ and HCT116p53^−/− colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53^+/+ and HCT116p53^−/− cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax. This work was supported by the U.S. Air Force Office of Scientific Research/DOD MURI grant on Subcellular Responses to Narrow Band and Wide Band Radio Frequency Radiation, administered by Old Dominion University, and the American Cancer Society.     

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.     

Effect of enzyme-assisted extract of Sargassum coreanum on induction of apoptosis in HL-60 tumor cells

In order to effectively prepare useful components from Sargassum coreanum, enzyme-assisted extraction was adapted to extraction processing of the seaweed. Our previous studies have shown that Neutrase extract of S. coreanum exhibited the strongest antioxidant activities among ten enzymatic extracts. It is reported that many antioxidant substances have anticancer or anticarcinogenic properties. Thus, Neutrase extract, which possessed a strong antioxidant activity in the previous study, was separated into four different molecular weight fractions (<5, 5–10, 10–30, and >30?kDa) which were screened for inhibitory activities against cancer cell growth. The >30-kDa fraction inhibited cell growth more than the other fractions. We separated crude polysaccharide (CPS) from the >30-kDa fraction to show that CPS increased DNA fragmentation, apoptotic body, and apoptotic cells with hypodiploid DNA contents in HL-60 cells. This indicates that CPS suppressed the growth of cells through apoptosis. The CPS gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. The CPS was 40.85% fucose, 27.24% galactose, and 12.99% glucose, as analyzed by bio-liquid chromatography. These findings suggest that apoptosis effect can be mediated by CPS in HL-60 cells.     

The influence of Trisenox on actin organization in HL-60 cells

The aim of this study was to show the influence of Trisenox (arsenic trioxide, ATO) on cytoplasmic and nuclear F-actin organization in HL-60 human leukemia cell line. Changes in localization were determined with the use of fluorescence microscopy and flow cytometry. Alterations, in both cytoplasmic and nuclear actin, were observed in cells exposed to ATO. F-actin network underwent accumulation and formed aggregates, that were very often placed under the cell membrane in whole cells and at the periphery of isolated nuclei. Addition of ATO also induced apoptosis and a decrease in G2 phase cells. These results suggest the influence of actin on the formation of apoptotic bodies and also participation of this protein in apoptotic alterations within nuclei, i.e. chromatin reorganization.     

Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines

We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product.     

Fruits and barks extracts of Zanthozyllum heitzii a spice from Cameroon induce mitochondrial dependent apoptosis and Go/G1 phase arrest in human leukemia HL-60 cells

Background

Zanthoxylum heitzii is a spice used to prepare several dishes and to treat tumors, syphilis, malaria, cardiac palpitations, urogenital infections in the west region of Cameroon, but the antitumor mechanisms and chemical composition are not yet investigated.This study was aimed to determine the antiproliferative effects of four extracts from the fruits and barks of Zanthoxyllum heitzii (Rutaceae) on apoptosis in human promyelocytic cells, their mechanisms and the chemical composition. The 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the fifty percent inhibition (IC₅₀) concentration of the cell lines after treatment. The effect on morphology was observed using a light or fluorescence microscopy. The rate of apoptosis and the cell cycle were measured using flow cytometry (FCM). The phytochemical analysis of the extract was carried with HPLC/MS methods.

Results

The phytochemical analysis of the extracts indicated the presence of four known polyphenols (Syringic acid, Juglon, Luteolin and Myricetin) in both fruits and barks of Z. heitzii but in different quantities. Syringic acid and Myricetin concentrations were between 17-21 fold higher in the fruits than the stem bark. Rhamnetin (393.35 μg/mL) and Oleuropein (63.10 μg/mL) were identified only in the stem barks of Z. heitzii. Among the four extracts tested for cytotoxicity properties, only the methanol extract of fruits and barks significantly inhibited cell proliferation of HL-60 cells with IC₅₀ value of 20 μg/mL and 12 μg/mL respectively. HL-60 cells treated with Z. heitzii extracts significantly produced reactive oxygen species (ROS) with concurrent loss of mitochondrial membrane potential (MMP). Modifications in the DNA distribution and enhanced of G1/G0 phase cell cycle arrest were observed in a concentration dependent manner.

Conclusions

Polyphenols from Z. heitzii plant exert inhibitory effect on HL-60 cells through the reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and cell cycle destabilization.