P388 Leukemia Cells Research Articles

The conformations of discodermolide in DMSO.

(+)-Discodermolide (1), a polyhydroxylated lactone isolated from the marine sponge Discodermia dissoluta,1 is currently a high-profile substance for its promise as an immunosuppressive agent1,2 and an anti-cancer drug. The compound induces apoptosis in human breast cancer cells,3 inhibits the in vitro proliferation of murine P388 leukemia cells,1 and combines synergistically with Taxol to suppress the proliferation of human carcinoma cells.4 Biomechanistically, discodermolide resembles Taxol and epothilone in its ability to bind to microtubules, effect tubulin polymerization, and promote mitotic arrest.5 Not surprisingly, these optimistic findings have stimulated a number of laboratories to pursue the total synthesis of the compound.6,7

Cytotoxic activity of Typhonium flagelliforme (Araceae)

The plant Typhonium flagelliforme (Araceae), commonly known as the 'rodent tuber', is often included as an essential ingredient in various herbal remedies recommended for cancer therapies in Malaysia. Various extracts prepared from either the roots, tubers, stems or leaves were tested for cytotoxic activity on murine P388 leukaemia cells using the MTT assay method. Both the chloroform (IC50 = 6.0 microg/mL) and hexane (IC50 = 15.0 microg/mL) extract from the 'roots and tubers' exhibited weak cytotoxic activity. The hexane extract (IC50 = 65.0 microg/mL) from the 'stems and leaves' exhibited weaker cytotoxic activity than the chloroform extract (IC50 = 8.0 microg/mL). Although the juice extract from the 'roots and tubers' is frequently consumed for cancer treatment, it exhibited poor cytotoxic activity. Further analysis using an amino acid analyser revealed that the juice extract contained a high concentration of arginine (0.874%). A high tryptophan content (0.800%) was confirmed by NMR and HPLC analysis.

A conformational and structure-activity relationship study of cytotoxic 3,5-bis(arylidene)-4-piperidones and related N-acryloyl analogues.

A series of 3,5-bis(arylidene)-4-piperidones 1 and related N-acryloyl analogues 2 were prepared as candidate cytotoxic agents with a view to discerning those structural features which contributed to bioactivity. A number of the compounds were markedly cytotoxic toward murine P388 and L1210 leukemic cells and also to human Molt 4/C8 and CEM neoplasms. Approximately 40% of the IC50 values generated were lower than the figures obtained for melphalan. In virtually all cases, the N-acyl compounds were significantly more bioactive than the analogues 1. In general, structure-activity relationships revealed that the cytotoxicity of series 1 was correlated positively with the size of the aryl substituents, while in series 2, a -sigma relationship was established. In particular, various angles and interatomic distances were obtained by molecular modeling, and the presence of an acryloyl group on the piperidyl nitrogen atom in series 2 affected the relative locations of the two aryl rings. This observation, along with some differences in distances between various atoms in series 1 and 2, may have contributed to the disparity in cytotoxicity between 1 and 2. The results obtained by X-ray crystallography of representative compounds were mainly in accordance with the observations noted by molecular modeling. Selected compounds interfered with the biosynthesis of DNA, RNA, and protein in murine L1210 cells, while others were shown to cause apoptosis in the human Jurkat leukemic cell line. This study has revealed the potential of these molecules for development as cytotoxic and anticancer agents.

Acetylated analogues of the microtubule-stabilizing agent discodermolide: preparation and biological activity.

A series of eight discodermolide acetates have been prepared using natural (+)-discodermolide and evaluated for in vitro cytotoxicity against the cultured murine P-388 leukemia cells. The acetylated analogues showed a significant variation of cytotoxicity and suggested the importance of C-11 and C-17 hydroxyl groups for potency. The preparation and structure elucidation of the new analogues are described.

Reversal of anticancer drug resistance by macrolide antibiotics in vitro and in vivo.

1. The combined effects of the macrolide antibiotics erythromycin, josamycin, clarithromycin and YM17K (3,4'-dideoxy mycaminosyl tylonolide hydrochloride) on in vitro intracellular accumulation of vinblastine or cyclosporine (Cs)A and on the in vivo antitumour activity of vinblastine were investigated using mouse leukaemia P388 cells (P388/S) and anticancer drug-resistant (P388/ADR) cells. These effects were compared with those of a calcium antagonist (verapamil) or immunosuppressants (FK506 and CsA). 2. All tested macrolide antibiotics increased the accumulation of both vinblastine and CsA in P388/ADR cells in a dose-dependent manner, but their potency was lower than that of verapamil, CsA or FK506. 3. When vinblastine (200 microg/kg) was administered intraperitoneally with each of the macrolide antibiotics (10 or 100 mg/kg) or with verapamil (25 mg/kg) once a day for 10 days in P388/ADR-bearing mice, combined effects of vinblastine with the macrolide antibiotics (erythromycin, clarithromycin and YM17K) or verapamil were observed. 4. The present study suggests that macrolide antibiotics may overcome anticancer drug resistance by inhibiting the binding of vinblastine or CsA to P-glycoprotein in P388/ADR cells. 5. We believe that these results are encouraging for combination chemotherapy to overcome P-glycoprotein-dependent anticancer drug-resistant tumours in clinical practice.

Comparative QSAR studies on substituted bis-(acridines) and bis-(phenazines)-carboxamides: a new class of anticancer agents

Quantitative structure–activity relationships have been formulated for two sets of DNA binding topoisomerase agents (bis-acridines and bis-phenazines) acting on murine P388 leukemia cells, murine Lewis lung carcinoma (LLC) cells and human Jurkat leukemia wild-type (JLC) cells. For the acridines, all three QSARs (1–3) show only a (small negative) hydrophobic effect. In sharp contrast, the phenazines in all three studies (4–6) show a strong hydrophobic effect, with the optimum ClogP being near 7.3 for all examples. This suggests that, despite the structural similarity of the compounds, different modes of enzyme and/or DNA binding may be involved.

Inhibition of topoisomerase II by the marine alkaloid ascididemin and induction of apoptosis in leukemia cells

Ascididemin (ASC) is a pentacyclic DNA-intercalating agent isolated from the Mediterranean ascidian Cystodytes dellechiajei. This marine alkaloid exhibits marked cytotoxic activities against a range of tumor cells, but its mechanism of action remains poorly understood. We investigated the effects of ASC on DNA cleavage by human topoisomerases I and II. Relaxation assays using supercoiled DNA showed that ASC stimulated double-stranded cleavage of DNA by topoisomerase II, but exerted only a very weak effect on topoisomerase I. ASC is a conventional topoisomerase II poison that significantly promoted DNA cleavage, essentially at sites having a C on the 3′ side of the cleaved bond (−1 position), as observed with etoposide. The stimulation of DNA cleavage by topoisomerase I in the presence of ASC was considerably weaker than that observed with camptothecin. Cytotoxicity measurements showed that ASC was even less toxic to P388 leukemia cells than to P388CPT5 cells resistant to camptothecin. In addition, the marine alkaloid was found to be equally toxic to HL-60 leukemia cells sensitive or resistant to mitoxantrone. It is therefore unlikely that topoisomerases are the main cellular targets for ASC. This alkaloid was found to strongly induce apoptosis in HL-60 and P388 leukemia cells. Cell cycle analysis showed that ASC treatment was associated with a loss of cells in the G1 phase accompanied with a large increase in the sub-G1 region. Cleavage experiments with poly(ADP-ribose) polymerase (PARP) revealed that caspase-3 was a mediator of the apoptotic pathway induced by ASC. The DNA of ASC-treated cells was severely fragmented. Collectively, these findings indicate that ASC is a potent inducer of apoptosis in leukemia cells.

Effects of 1-methyl-3-propyl-7-butylxanthine (MPBX) on idarubicin-induced antitumor activity and bone marrow suppression.

The effects of 1‐methyl‐3‐propyl‐7‐butylxanthine (MPBX), a xanthine derivative, on idarubicin (IDA)‐induced antitumor activity against P388 leukemia cells (P388) and bone marrow suppression were examined. In P388 tumor‐bearing mice, the combination of MPBX with IDA increased the antitumor activity of IDA. The IDA concentration in the tumors in the MPBX combination group increased by 2.0‐fold compared to the level in the IDA‐alone group. On the other hand, as regards IDA‐induced bone marrow suppression, the combination of MPBX with IDA reduced the decrease in the bone marrow cell number by 30% compared to that in the IDA‐alone group. In addition, the IDA concentration in the bone marrow cells was decreased by the combination of MPBX with IDA. An in vitro experiment showed that MPBX facilitated IDA influx and suppressed IDA efflux in P388 cells. In conclusion, the combination of MPBX with IDA increased the antitumor activity and decreased the bone marrow suppression. Therefore, we expect that the combination of MPBX with IDA will be useful for leukemia chemotherapy.

Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance

Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (−)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.

Potentiation of Doxorubicin-Induced Apoptosis of Resistant Mouse Leukaemia Cells by Ivermectin

The apoptosis and cell cycle effect of doxorubicin were evaluated in the presence and absence of ivermectin in mouse doxorubicin-resistant P388 leukaemia cells. Ivermectin (2 μM) increased the sensitivity to doxorubicin of multidrug resistant (MDR) mouse leukemic P388 cells and significantly enhanced the apoptosis and intracellular accumulation of doxorubicin in resistant cells, but had no effect on parent cells. Using the fluorescent potential probe, 3,3′-dihexyl-oxacarbocyanine, we found that ivermectin induced a plasma membrane potential increase in resistant cells. Ivermectin also enhanced doxorubicin-induced G2/M blockade of the cell cycle in resistant cells. It is possible that ivermectin could reverse resistance by direct interaction with the P-glycoprotein or other components of the altered MDR cell membrane.

4-Aza-2,3-dehydro-4-deoxypodophyllotoxins: simple aza-podophyllotoxin analogues possessing potent cytotoxicity

4-Aza-2,3-dehydro-4-deoxypodophyllotoxin analogues 3a–n were synthesized through quinolines 2a–n. Comparison of their cytotoxicity against P-388 leukemia cells revealed that the steric effects of the ring B substituents on the activity are greater than the electronic effects, while the presence of a methoxy group on the ring E is not essential to exhibit potent cytotoxicity. Analogues 3a and 3b proved to be more than twice as cytotoxic as natural podophyllotoxin (1).

Synthesis and biological activity of platinum(II) and palladium(II) thiosalicylate complexes with mixed ancillary donor ligands

A series of mixed-ligand thiosalicylate complexes of the type [M(SC6H4CO2)(PPh3)L] (M = Pt, L = pyridine (py), 4-methylpyridine, imidazole, 4-picolinic acid hydrazide {C5H4N[C(O)NHNH2]-4}; M = Pd, L = pyridine) have been prepared by the one-pot reaction of [PtCl2(cod)] or [PdCl2(cod)] (cod = cycloocta-1,5-diene) with one equivalent of PPh3 and thiosalicylic acid (HSC6H4CO2H) in the presence of the nitrogen base. Single-crystal X-ray diffraction studies on [Pt(SC6H4CO2)(PPh3)(py)] and the previously reported complex [Pt(SC6H4CO2)(PPh3)(XyNC)] (Xy = 2,6-xylyl) indicates that the complexes have different geometries, though in each case the two ligands of highest trans-influence are mutually cis. The reaction of [PtCl2(cod)] with PPh3, thiosalicylic acid and ammonia led to the isolation of crystals of the dinuclear thiosalicylate-bridged complex [Pt(SC6H4CO2)(PPh3)(NH3)Pt(SC6H4CO2)(PPh3)], which was characterised by X-ray crystallography and electrospray mass spectrometry. The conformation of the thiosalicylate ligand can vary widely with values for the dihedral angle between the ligand plane and the platinum(II) coordination plane varying from 12.4° to 70.9° in the seven complexes so far determined. The biological activities of selected platinum and palladium thiosalicylate and related complexes against P388 leukemia cells, bacteria and fungi are also reported. The complex [Pt(SCH2CO2)(PPh3)2] shows the highest activity against P388 cells (IC50 671 ng mL−1).

High In-Vitro Antitumour Activity of Triphenyltin Coumarin 3‐Carboxylate and its Coordination Complexes With Monodentate Oxygen Donor Ligands Against the Epstein Barr Virus (EBV)‐DNA Positive Raji and the P‐388 Murine Leukaemia Cell Lines, and Evidence for the Suppression by Organotin of the Early Antigen Complex in the EBV Lytic Cycle

Triphenyltin coumarin-3-carboxylate and its coordination complexes with ethanol, triphenylphosphine oxide, triphenylarsine oxide, diphenylcyclopropenone and quinoline N-oxide exhibited high in vitro cytotoxicity (LC50 values in the range 0.25-3.4 μg/mL) when tested against EBV-DNA positive Raji cells and P-388 leukaemia cells, compared to the standard drug 5-Fluorouracil, which showed LC50 values of 11 and >50 μg/mL, respectively, against these cells. Additional tests performed on the Raji cells incubated with the quinoline N-oxide complex in the presence of the tumour promoters, TPA and sodium butyrate, revealed that the diffused and restricted protein components of the early antigen complex were suppressed relative to the control containing only the promoters, indicating impaired function of the genes involved as transactivators in the early lytic cycle of the EBV. The failure of the restriction enzymes Eco R1 and Hind III to cleave the extracted DNA from such treated cells in contrast to the control, coupled with the amplification of the BMLF-1 gene by the PCR technique which was realised only with the DNA of the control and not of the treated sample, point to a punitive interaction of the organotin with the nuclear DNA of the Raji cells.

Synthesis of (−)-4-aza-4-deoxypodophyllotoxin from (−)-podophyllotoxin

(−)-4-Aza-4-deoxypodophyllotoxin ( 4) was synthesized from (−)-podophyllotoxin ( 1) through C-ring cleavage, Curtius rearrangement and intramolecular N-alkylation. Analogue 4 showed potent cytotoxicity against P388 leukemia cells.

Effect of a 7-tesla homogeneous magnetic field on mammalian cells

When two types of mammalian cells, mouse leukemia cells, P388, and Chinese hamster fibroblast cells, V79, were grown under a 7-tesla (T) homogeneous magnetic field which was produced by a newly constructed superconducting magnet biosystem (SBS), the growth pattern of cells under 7 T magnetic field and the geomagnetic field control showed no differences. The DNA distribution of the two cells was compared by flow cytometry after exposure to 7 T for 3 and 24 h, but there was no significant differences between magnet-exposed cells and unexposed cells. When the two types of cells were exposed to different concentrations of the antitumor agent, bleomycin (BLM), for 3 h under 7 T, their viable cell numbers were almost the same as that of the control although sensitivity to BLM was different between the two cells. These results suggest that the 7 T homogeneous magnetic field exerts no adverse effects on mammalian cells.

Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin

We reported previously that pironetin and its derivatives were potent inhibitors of cell cycle progression at the M-phase and showed antitumour activity against a murine tumour cell line, P388 leukaemia, transplanted in mice. In this paper, we investigated the mechanism of action of pironetins in antitumour activity and cell cycle arrest at the M-phase. As reported previously for murine leukaemia P388 cells, pironetin showed antitumour activity in a dose-dependent manner in the human leukaemia cell line HL-60. Since DNA fragmentation was observed in both P388 and HL-60 cells, the antitumour activity of pironetin is thought to be due to the induction of apoptosis. Pironetin also induced the rapid phosphorylation of Bcl-2 before formation of the DNA ladder in HL-60 cells, as seen with several tubulin binders. These results suggest that the antitumour activity of pironetin is due to apoptosis caused by the phosphorylation of Bcl-2, and that pironetin targets the microtubules. Pironetin and demethylpironetin exhibited reversible disruption of the cellular microtubule network in normal rat fibroblast 3Y1 cells. However, epoxypironetin, which contains epoxide instead of the double bond of pironetin, showed only weak activity. Since the concentrations that inhibit cell cycle progression at the M-phase were the same as those for disruption of the microtubule network, it was suggested that the mitotic arrest induced by pironetin was the result of the loss of the mitotic spindle. These compounds also inhibited the microtubule-associated protein-induced and glutamate-induced tubulin assembly in vitro. Pironetin inhibited the binding of [3H]vinblastine, but not that of [3H]colchicine, to tubulin, and the Kd values revealed that the affinity of pironetin for tubulin is stronger than that of vinblastine. These results suggest that pironetins are novel antitumour agents which inhibit microtubule assembly.

Synthesis, mode of action, and biological activities of rebeccamycin bromo derivatives.

Bromo analogues of the natural metabolite rebeccamycin with and without a methyl substituent on the imide nitrogen were synthesized. The effects of the drugs on protein kinase C, the binding to DNA, and the effect on topoisomerase I were determined. The drugs' uptake and their antiproliferative activities against P388 leukemia cells sensitive and resistant to camptothecin, their antimicrobial activity against a Gram-positive bacterium (B. cereus), and their anti-HIV-1 activity were measured and compared to those of the chlorinated and dechlorinated analogues. Dibrominated imide 5 shows a remarkable activity against topoisomerase I, affecting both the kinase and DNA cleavage activity of the enzyme. The marked cytotoxic potency of this compound depends essentially on its capacity to inhibit topoisomerase I.

Cisplatin combined with tiopronin or sodium thiosulfate: cytotoxicity in vitro and antitumor activity in vivo.

We have previously reported that the thiol compound tiopronin protects rat kidneys in vitro against the toxic activity of cisplatin. The influence of tiopronin and sodium thiosulfate (STS) on the cytotoxicity of cisplatin has been investigated on P388 leukemic cells in vitro after 3 days. The combination has also been investigated in vivo in BDF1 mice bearing a P388 s.c. tumor. In contrast to STS, tiopronin did not significantly reduce the cytotoxic activity of cisplatin in vitro and nor did it affect the uptake of platinum (cisplatin-derived), binding to DNA or the percentage of interstrand cross-links (%ISCL) formation. The co-administration of cisplatin (4 mg/kg) and tiopronin (150 and 300 mg/kg) to BDF1 female mice bearing a s.c. P388 tumor produced a significant reduction in tumor growth similar to that of a single 6 mg/kg dose of cisplatin. Interestingly, pre-incubation in vitro of either tiopronin or STS for 2 h with the species formed from cisplatin by hydrolysis demonstrated their ability in inhibiting the cytotoxicity of these reactive platinum products. These results indicate that tiopronin does not reduce the cytotoxicity of cisplatin in vitro, as STS does. This may be, at least partly, because of a different effect of the two thiol compounds on the cellular uptake and binding of platinum to DNA. Notably, tiopronin substantially reduced tumor growth in mice treated with a non-toxic dose of cisplatin (p < or = 0.0277), suggesting some positive influence of this thiol compound on the antitumor properties of cisplatin. The ability of tiopronin to protect in vitro against the cytotoxicity of the aquation products of cisplatin may be related to its nephroprotective effect.

New semisynthetic vinca alkaloids: chemical, biochemical and cellular studies.

A new semisynthetic anti-tumour bis-indol compound, KAR-2 [3′-(β-chloroethyl)-2′,4′-dioxo-3,5′-spiro-oxazolidino-4-deacetoxy-vinblastine] with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti-calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes. © 1999 Cancer Research Campaign

Enhanced binding to DNA and topoisomerase I inhibition by an analog of the antitumor antibiotic rebeccamycin containing an amino sugar residue.

Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments. The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH-->NH2 substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole-based antitumor agents.