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As the frequent emergency of resistant tumor cells during treatment, the development of new agents with new modes of action attracts a great deal of interest. Polybia-MPI was a short cationic alpha-helical amphiphilic peptide that has selective toxicity toward cancer cells but no hemolytic activity. Its target selectivity is based on the binding preference to membranes containing anionic phospholipids by electrostatic driving. Its ability to make PI and trypan blue permeate into tumor cells at the same rate (within minutes), suggests a killing mechanism that involves plasma membrane perturbation. SEM and confocal microscopy experiments verified that the cell died as a result of acute injury and bursting, suggesting necrosis. As compared to the conventional chemotherapy, polybia-MPI targets at the cell membrane rather than enters into the cell to exert its action. So it is difficult for tumor cells to develop resistance to polybia-MPI during treatment and its action is not affected by the common multi-drug resistant mechanism. Although this is an initial study that looked at its in vitro activity rather than the in vivo activity, with the increasing resistance of conventional chemotherapy, polybia-MPI may offer a novel therapeutic strategy in the treatment of multi-drug resistant cancer.

The fungal metabolite apicidin (cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)) inhibited the growth of HL-60 cells in a concentration-dependent manner (100-1000 nM). At higher concentrations (>300 nM), cell death was induced. At 100 nM, it induced hyperacetylation of histone H4 time-dependently, while trichostatin A induced transient hyperacetylation. Apicidin (10-100 nM) increased the cells having nitroblue tetrazolium-reducing activity and expressing CD11b but not CD14 and CD15. The expression of CD11b by apicidin was long lasting, while that by trichostatin A was transient. In K562 cells, apicidin at 10-100 nM did not inhibit cell growth nor express CD11b, CD14 and CD15. Our findings indicate that apicidin inhibits proliferation and induces the early stage of differentiation of HL-60 cells.

In contrast to their inactive parent compound triptycene (code name TT0), new synthetic analogs (TT code number) mimic the antitumor effects of the anthracycline quinone antibiotic daunorubicin (DAU) in the nM range in vitro but have the additional advantage of also blocking nucleoside transport and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones may induce DNA fragmentation at 24 h by an active mechanism that requires RNA and protein syntheses and protease activities, the most cytotoxic of them, TT24, was tested for its ability to induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early marker of apoptosis. PARP-1 cleavage starts at 2-3 h and is maximally induced at 6 h by 1.6 microM concentrations of TT24 and DAU in wild-type drug-sensitive HL-60-S cells. However, in MDR HL-60-RV cells, PARP-1 cleavage is still induced by 4 microM TT24 but not by 4-10 microM DAU. The magnitude of PARP-1 cleavage may increase with the number of quinoid rings in the triptych structure and, in contrast to TT0, all lead antitumor TT bisquinones share the ability to fully induce PARP-1 cleavage in HL-60-S cells. A 1 h pulse treatment is sufficient for TT24 and DAU to induce PARP-1 cleavage at 6 h. Since the abilities of TT24 and DAU to induce PARP-1 cleavage are inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone but not by N-tosyl-L-phenylalanine chloromethyl ketone, caspase-mediated apoptosis may be involved in the mechanism by which these quinone antitumor drugs induce the proteolytic cleavage of PARP-1 at 6 h and the internucleosomal fragmentation of DNA at 24 h in the HL-60 tumor cell system.

Etoposide (VP-16) is an anticancer agent that induces apoptosis in human leukemic cell lines such as U937 and HL60. We performed RNase protection assays, with two distinct cRNA panels covering most of caspase and BCL-2-related genes, using total RNA from cell lines exposed to various concentrations of the drug. Our results show that VP-16 down-regulates expression of most surveyed genes with the noticeable exception of casp-2S mRNA that is up regulated whereas casp-2L mRNA is decreased. Since these mRNAs are produced by the alternative splicing of exon 9, we devised a reverse transcriptase-polymerase chain reaction method using primers from exons 8 and 10 to demonstrate that VP-16 stimulates the production of exon 9-containing sequences, irrespective of active transcription. However, this effect is specific of the 3'-end of the CASP-2 gene since no difference in the relative amounts of the 5'-end of the mRNA species is detected. Nevertheless, the level of full-length casp-2L mRNA together with that of procaspase-2L protein, which is pro-apoptotic, are decreased under VP-16 treatment, suggesting that an early cell response to treatment by cytotoxic agents is to down-regulate expression of selected pro-apoptotic proteins.

The present study shows that stress signaling plays a role in differentiation of K562, PANC1, HT29 and HL60 tumor cells: (1) Butyrate induced differentiation in K562, PANC1, and HT29 cells can be inhibited by SB203580, a specific inhibitor of p38 stress activated protein kinase. (2) Heat shock and hyperosmolarity increase expression of differentiation markers in K562, HT29, HL60 and in K562, PANC1, and HT29 cells, respectively. (3) Conversely, environmental stress induced differentiation in K562, HT29, and PANC1 cells can be inhibited by SB203580 and quercetin, a compound with heat shock pathway inhibiting activity. (4) Butyrate and environmental stress enhance either additively or synergistically differentiation of K562, HT29, PANC1 or HL60 cells, respectively. Stress signaling pathways might be an interesting pharmacologic target for differentiation therapy of malignant disease.

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol3P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 microM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC zeta is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC zeta during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC zeta activation. The activity of PI 3-K is essential for PKC zeta activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC zeta during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC zeta in the process of PMA-induced monocytic differentiation.

Eugenia jambos L. (Myrtaceae) is an antipyretic and anti-inflammatory herb of Asian folk medicine. A 70% acetone extract exerted the strongest cytotoxic effects on human leukemia cells (HL-60) from a preliminary screening of 15 plants. The cytotoxic principles were separated by bio-assay-guided fractionation to HL-60 cells; two hydrolyzable tannins (1-O-galloyl castalagin and casuarinin) were isolated from the 70% acetone extract. All significantly inhibited human promyelocytic leukemia cell line HL-60 and showed less cytotoxicity to human adenocarcinoma cell line SK-HEP-1 and normal cell lines of human lymphocytes and Chang liver cells. Thus, these compounds were exhibited the dose-dependent manner in HL-60 cells and the IC(50) were 10.8 and 12.5 microM, respectively. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G(2)/M phase, and a concomitant increase of cell population at G(1) phase. The apoptosis induced by these two compounds was also demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that the cytotoxic mechanism of both antitumor principle constituents might be the induction of apoptosis in HL-60 cells.

A total of 23 ferulic acid (FA) derivatives were synthesized, and investigated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) activation and superoxide (O(2)(-)) generation. Most of the derivatives showed significant EBV activation suppression or cytotoxicity at a concentration of 100 microM, with FA15 as the most potent suppressor. In both assays, FA6-FA17, bearing straight- or branched-alkyl side chains, exhibited marked suppression of O(2)(-) generation, with both FA16 and FA17 being highly active, while FA itself was virtually inactive. The activity differences seen between FA16/FA17 and FA are attributable, at least in part, to their cellular incorporating efficiencies. Further, both FA15 and FA21 attenuated the expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins, while FA did not. Our results suggest that these novel FA derivatives are effective chemopreventive agents.

Cuphiin D1 (CD1), a new macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert antitumor activity both in vitro and in vivo. In this study, we explored the mechanism of the CD1-induced antitumor effect on human promyelocytic leukemia (HL-60) cells. The results showed that CD1 induced cytotoxicity in HL-60 cells and the IC50 was 16 microM after 36 h treatment. HL-60 cells treated with CD1 for 36 h decreased the uptake of [3H]-labeled thymidine, uridine and leucine in a dose dependent manner. Electron micrographs demonstrated that HL-60 cells treated with 16 microM CD1 for 36 h exhibited chromatin condensation, indicating the apoptosis occurrence. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G2/M phase, and a concomitant increase of cell population at G1 phase. CD1 also caused DNA fragmentation and inhibited Bcl-2 expression in the HL-60 cells. These results suggest that the inhibition of Bcl-2 expression in HL-60 cell might account for the mechanism of CD1-induced apoptosis.

A clonogenic assay was tested in order to determine the effects of low intensity ultrasound on HL-60 cells in the presence of cytosine arabinoside (Ara-C). HL-60 cells were exposed to ultrasound at an intensity of 0.3 W/cm2 (48 kHz). Cells were then cultured for 8 days and the number of colonies was statistically analyzed (ANOVA). Ultrasound exposure alone for 120 s resulted in no significant decrease of colonies compared to non-treated cells (P0 = 0.1426). Significant differences (P0 < 0.005) were obtained between ultrasound treated and untreated cells in the presence of various concentrations of Ara-C (2 x 10(-9), 1 x 10(-8), 2 x 10(-8), 5 x 10(-8), 1 x 10(-7) M). Morphological evaluation of ultrasound irradiated cells with scanning electron microscopy showed minor disruption of cell surface and disappearance of microvilli. These observations suggests that low intensity ultrasound altered the cell membrane thus resulting in change in Ara-C uptake into HL-60 cells.

Induction of hematopoietic differentiation in human promyelocytic leukemia cells (HL-60) by new synthetic drugs or natural products has recently been recognized as a new strategy in the identification and testing of potential cancer chemopreventive and/or chemotherapeutic agents. 2-(Allythio) pyrazine (2-AP) is a pyrazine derivative of allysulfide, which has been suggested to be a potential cancer chemopreventive agent in previous in vivo and in vitro experiments. In the present study, we have investigated the inducing effect of granulocytic differentiation in HL-60 cells by 2-AP. Treatment of HL-60 cells with various concentrations of 2-AP (1-100 microM) for 7 days showed the induction of granulocytic differentiation following both morphological examination and NBT (nitroblue tetrazolium) testing (up to 40 and 52%, respectively). The expressions of bcl-2 and c-myc were down-regulated during granulocytic differentiation of HL-60 cells (up to 40%). The immunoblots for G1 cyclins in the G1-S phase transition (cyclin D1 and E) showed a progressive decrease of their expressions in both concentration- and time-dependent manners (up to 30 and 50%, respectively). These results suggest that 2-AP could induce the differentiation of HL-60 cells and might have potent cancer chemoprevention and/or chemotherapy roles in human leukemias.

We have demonstrated previously that a phosphorothioate antisense oligonucleotide to the p65 subunit of the inducible transcription factor NF-kappaB produced rapid changes in the expression of leukocyte integrin CD11b (Mo 1) and in the adhesion of dimethylsulfoxide (DMSO)-differentiated HL-60 cells stimulated by 12-O-tetradecanoylphorbol 13-acetate. We have also shown that a variety of agents which inhibit NF-kappaB, including vitamin E and related antioxidants, curcumin and several non-steroidal anti-inflammatory agents, significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of 1,25-dihydroxyvitamin D3 (vitamin D3). To provide further evidence that interference with the activation of NF-kappaB affects the maturation of HL-60 leukemia cells by creating an environment conducive to terminal differentiation, we measured the effects of phosphorothioate antisense oligonucleotides to the various subunits of NF-kappaB on the differentiation of HL-60 cells produced by low levels of vitamin D3. When used alone these oligonucleotides had no significant effect on the differentiation of HL-60 cells. However, the antisense oligomer to the Rel A subunit of NF-kappaB markedly increased the extent of differentiation produced by low levels of vitamin D3. An enhancement of the differentiation of HL-60 cells induced by vitamin D3 was also obtained by several transcription factor decoys designed to mimic the consensus sequences of genes activated by Rel A. The findings provide additional support for the concept that inhibition of the activation of NF-kappaB may be involved in regulating the entry of promyelocytic leukemia cells into a differentiation pathway.

The ability of caffeine to modulate hyperthermia induced apoptosis was investigated in the human promyelocytic cell line HL60. Mild hyperthermia has been shown to be a strong inducer of apoptosis in many cell lines of lymphoblastoid lineage. In this investigation HL60 cells were simultaneously exposed to caffeine (concentrations ranging from 0 to 20 mM), and a brief hyperthermic treatment (43 degrees C) for 1 h and then allowed a recovery time of 12 h. Approximately 50% of a cell population receiving the hyperthermia treatment died by apoptosis within 12 h, as determined by the comet assay, whereas cells that received concomitant treatments of caffeine with heat shock displayed an apparent suppression of apoptotic induction and enhanced cell survival.

We evaluated the effect of sulindac sulfide (SS), which reduces cell number and induces apoptosis in cultured colon cancer cells (CCCs), on expression of the proliferation markers PCNA and Ki-67 in HT-29 and HCT-15 CCCs; only the former express cyclooxygenases. DNA content and PCNA/Ki-67 expression were analyzed by bivariate flow cytometry. SS inhibited cell proliferation, determined by the reduced expression of PCNA and Ki-67, roughly by half at 72 h, and induced apoptosis (accounting for about two-thirds and one-third of the reduction in cell number, respectively). A similar effect of SS occurred in HT-29 and HCT-15 CCCs, and also in non-colonic cells, indicating that this rather general effect of SS on cultured cells is not dependent on inhibition of prostaglandin synthesis.

The crude saponins from the shoots (edible part of asparagus) of asparagus (asparagus crude saponins; ACS) were found to have antitumor activity. The ACS inhibited the growth of human leukemia HL-60 cells in culture and macromolecular synthesis in a dose and time dependent manner. The ACS at 75-100 micrograms/ml range was cytostatic. ACS concentrations greater than 200 micrograms/ml were cytocidal to HL-60 cells. The ACS at 6 and 50 micrograms/ml inhibited the synthesis of DNA, RNA and protein in HL-60 cells by 41, 5, and 4, respectively, or by 84, 68 and 59%, respectively. The inhibitory effect of ACS on DNA synthesis was irreversible.

This is the first observation that active an oxygen-producing system showed cross-resistance to vincristine (VCR) resistant cells or other anticancer agent-resistant cells. The extent of cross-resistance against oxygen radicals and anticancer agents in wild type and VCR resistant human promyelocytic leukemia cell line, HL-60 and HL-60/VCR, is compared. Superoxide was generated by reaction with hypoxanthine(HX)-xanthine oxidase(XO). HL-60/VCR was 81-fold resistant to VCR, 11.8-fold resistant to adriamycin, and 8.5-fold resistant to the XO concentration required for 50% growth inhibition compared with HL-60. Because oxygen radicals injure the cell membrane, the results indicate an increased resistance to membrane damage by oxygen radicals in drug resistant cells.