In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells

(+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dose-dependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase-3 activation, which indicate apoptosis induction.     

Isolation and characterization of a mannoglucan from edible Cordyceps sinensis mycelium

Cordyceps sinensis is a well known tonic food or invigorant with broad-spectrum medicinal properties that is widely used in the People's Republic of China. A neutral mannoglucan 1 with a molar mass of approximately 7.7x10(3) Da was obtained from the 0.05 M acetate buffer extract of C. sinensis mycelium. It had [alpha](D)(20)+126 (c 0.2, H(2)O) and consisted of Man and Glc units in the molar ratio of 1:9. A combination of chemical analysis and NMR and IR spectroscopy together with digestion with alpha-D-amylase showed 1 to have a alpha-D-glucan backbone with (1-->4)- and (1-->3)-linkages, and the side chains of alpha-D-(1-->6)-Manp were attached to the backbone via O-6 of alpha-(1-->3)-Glcp residues. Compound 1 showed weak cytotoxicity activity against SPC-I (IC(50)=63 microg/mL) cancer line, and no obvious cytotoxicity activities against BCAP37 (IC(50)>100 microg/mL) and SW480 (IC(50)>100 microg/mL) cancer lines.     

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.     

Inhibitory effects of ethyl acetate extract of Cordyceps sinensis mycelium on various cancer cells in culture and B16 melanoma in C57BL/6 mice

The cultivated mycelium of a Cordyceps sinensis (Cs) fungus was sequentially extracted by petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH) and hot water. All solvent extracts except hot water extract showed a significant and dose-dependent inhibitory effect on the proliferation of four cancer cell lines, MCF-7 breast cancer, B16 mouse melanoma, HL-60 human premyelocytic leukemia and HepG2 human hepatocellular carcinoma, with IC(50) values below 132 microg/ml. The EtOAc extract, in particular, had the most potent effect against all four cancer cell lines, with IC(50) between 12 microg/ml (on B16) and 45 microg/ml (on MCF-7). In contrast, it had much lower cytotoxicity against normal mouse bone marrow cells. The EtOAc extract contained carbohydrates, adenosine, ergosterol and trace amount of cordycepin, of which ergosterol and related compounds were identified as a major class of active constituents contributing to the in vitro cytotoxicity. In an animal test, the EtOAc extract showed significant inhibiting effect on B16-induced melanoma in C57BL/6 mice, causing about 60% decrease of tumor size over 27 days. Our results suggest that the EtOAc extract of Cs fungal mycelium has strong anti-tumor activity and is a potential source of natural anti-tumor products.     

In vivo stimulatory effect of Cordyceps sinensis mycelium and its fractions on reproductive functions in male mouse

Cordyceps sinensis (CS), an Ascomycetes fungus parasitic to Lepidoptera larvae, has been traditionally used as nutritious food for the enhancement on sexual performance and the restitution of impairment in sexual function in Chinese society. We have previously demonstrated the stimulatory effect of CS and its fractions on steroidogenesis both on primary mouse Leydig cells and MA-10 mouse Leydig tumor cells. In the present studies, we determined the in vivo effects of CS and its fractions on steroidogenesis in mouse. Different concentrations of CS and CS fractions (0.02 and 0.2 mg/g body weight) were fed to immature or mature mice from 1 to 7 days. The plasma levels of testosterone were evaluated by radioimmunoassay. The weights of reproductive organs were also determined. Results illustrated that CS significantly induced plasma testosterone levels both in immature and mature mice in 3 and/or 7 days treatment (p < 0.05). F2 and F3 at 0.02 and/or 0.2 mg/g body weight for different feeding duration could also significantly stimulated plasma testosterone levels both in immature and mature mice (p < 0.05). In general, CS, F2 and F3 didn't have considerable effect on the weights of reproductive organs. Taken together, these studies illustrate that CS and its fractions significantly stimulated in vivo mouse testosterone production.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate     

Induction of differentiation rescues HL-60 cells from Rana catesbeiana ribonuclease-induced cell death

Rana catesbeiana ribonuclease (RC-RNase) exerted strong anti-tumor activity and its cytotoxicity was shown to correlate with differentiation stages of three different hepatoma cell lines. In this study, we demonstrate different RC-RNase cytotoxicity in undifferentiated HL-60 cells and in those that had been induced to differentiate by retinoic acid or dimethylsulfoxide. RC-RNase showed cytotoxicity in undifferentiated HL-60 cells, but not in HL-60 cells undergoing terminal differentiation. Furthermore, the caspase-9/caspase-3 pathway was activated when RC-RNase induced death in undifferentiated HL-60 cells and induction of differentiation led to a reversal of the caspase activation pathway.     

Humic acid induces apoptosis in human premyelocytic leukemia HL-60 cells

It has been shown that humic acid (HA), a phenolic polymer, exhibits pro-oxidant and cytotoxic effects. In this study, HA induction of apoptosis was studied using cultured human premyelocytic leukemia HL-60 cells. Treatment at a range of HA concentrations (50-400 microg/ml) resulted in dose-and time-dependent sequences of events marked by apoptosis, as demonstrated through by apoptotic features such as loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. This HA-induced apoptosis in the HL-60 cells was mainly associated with the release of cytochrome c from the mitochondria. Furthermore, apoptosis in the HL-60 cells was accompanied by the activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a major component in the apoptotic cell death mechanism. Although the HA-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data reported herein reveals that HA exerts antiproliferative action and growth inhibition on HL-60 cells through induction of apoptosis, which may have anticancer properties potentially useful for the development of new drug products.     

Curcumin abolishes apoptosis resistance of calcitriol-differentiated HL-60 cells

Exposure of HL-60 cells to 1,25-dihydroxyvitamin D(3) (calcitriol) induces their differentiation into monocytes. This terminal differentiation is associated with acquired resistance to many proapoptotic stimuli. Here we show that differentiated HL-60 cells undergo apoptosis upon curcumin treatment although they retain resistance to apoptosis induced by a topoisomerase poison - etoposide. Curcumin induced changes of nuclear morphology, DNA fragmentation, release of cytochrome c as well as caspase activation in both differentiated and undifferentiated cells. Experiments performed in other laboratories suggested that curcumin initiates apoptosis by DNA damage that results from topoisomerase II poisoning. We measured gammaH2AX expression, a marker of DNA double strand breaks, in both undifferentiated and differentiated HL-60 cells treated with curcumin or etoposide. In etoposide-treated undifferentiated cells early gammaH2AX expression correlated with initiation phase of apoptosis. In contrast, in curcumin-treated cells gammaH2AX expression correlated with apoptotic DNA fragmentation, which is characteristic for the execution phase of apoptosis. Our experiments show that curcumin overcomes the resistance of calcitriol-differentiated HL-60 cells to DNA-damage-induced apoptosis by activating other cell signaling pathways leading to cell death of HL-60.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Acetone extract of Angelica sinensis inhibits proliferation of human cancer cells via inducing cell cycle arrest and apoptosis

Angelica sinensis (Oliv.) Diels, a traditional Chinese medicine, has been widely prescribed in treatment of gynecological diseases. Bio-based assays for extracts of Angelica sinensis showed that the acetone extract (AE-AS) had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells. The IC₅₀ values of AE-AS on mentioned cancer cells ranged from 35 to 50 μg/ml after 24 h of treatment. After 72 h of exposure, AE-AS (40 μg/ml) significantly reduced A549 cell proliferation to 24 ± 3.2% of control. In A549 cells, the cell cycle analysis showed that AE-AS induced a significant increase in the number of cells in G0/G1, with a concomitant decrease in the number of cells in S phase. AE-AS-induced chromatin changes and apoptosis of A549 cells were confirmed by Hoechst 33342 DNA staining and annexin V staining. A549 cells treated with AE-AS caused activation of caspase-9 and -3, and AE-AS-induced apoptosis could be inhibited by the broad-spectrum caspase inhibitor, z-VAD-fmk. The Western blot indicated the AE-AS-triggered apoptosis is mediated via suppression of Bcl-2 oncoprotein expression rather than p53 or Bax. Besides, AE-AS decreased the levels of cdk4 protein was observed. These results indicate that the AE-AS could induce G1/S arrest and activate the mechanism of apoptosis in human cancer cells. Extracts obtained from different methods of fractionation might possess distinct bioactivity. These results prompted us to further evaluate the in vivo anticancer effects and elucidate the chemical composition profile of AE-AS.     

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrix-metalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p<0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.     

Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.     

Induction of Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells by a Tunisian Gerboui Olive Leaf Extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.     

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.     

Apoptosis of human leukemic HL—60 cells induced to differentiate by treatment with RA or DMSO

In present study,we studied the effect of all-trans retinoic acid(ATRA)and dimethylsulfoxide(DMSO)on the induction of apoptosis in HL-60 cell line.Based on morphological changes by Hochest 33342 staining and identification of internuclesomal NDA celeavage by gel electrophoresis,we observed aberrant nuclear chromatin condensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method.We found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6d after the initiation of the treatment However,Such an obvious apoptotic peak was not identified in DMSO-differentiated cells.Combining the research accomplished before.our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.     

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.