Search

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Visceral Leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani, is a potentially fatal disease. The only orally bioavailable drug miltefosine is toxic and the effective liposomal Amphotericin B (AmBisome) is limited by its prohibitive cost and requirement for parenteral administration. Therefore, finding a new potential drug candidate and an alternative delivery system is imperative. We report that Betulinic acid (BA), a pentacyclic triterpenoid from Betula alba bark, was loaded onto uniformly spherical PLGA nanoparticles (BANPs; diameter 187.5 ± 5.60 nm) coated with Lactoferrin (Lf-BANPs). The amastigotes count in macrophages was more effectively reduced by Lf-BANP than BA and BANP. Lf-BANPs reduced the pro-parasitic, anti-inflammatory cytokine IL-10, but increased nitric oxide (NO), production in L. donovani-infected macrophages indicating that Lf-BANP possesses a significant anti-leishmanial activity., (Copyright © 2018. Published by Elsevier Ltd.)

Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un-responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down-modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug-resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56 + cells during VL, but was down-regulated after treatment. In addition, MFI of CD58 on CD56 + cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine- or Ambisome-resistant Ld. The ratio of CD56 + CD58 + IFN-γ + /CD56 + CD58 + IL-10 + cells was reduced by 6.98-fold after stimulation with Ld. Further, an antagonist to CD58 or its counter-receptor CD2 down-regulated CD56 + NK cell recruitment across a polycarbonate trans-membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56 + cells and their migration to the infection site in association with CD2., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A new small library of 2-aminobenzoyl amino acid hydrazide derivatives and quinazolinones derivatives was synthesized and fully characterized by IR, NMR, and elemental analysis. The activity of the prepared compounds on the growth of Leishmania aethiopica promastigotes was evaluated. 2-Benzoyl amino acid hydrazide showed higher inhibitory effect than the quinazoline counterpart. The in vitro antipromastigote activity demonstrated that compounds 2a, 2b, 2f and 4a had IC 50 better than standard drug miltefosine and comparable activity to amphotericin B deoxycholate, which indicates their high antileishmanial activity against Leishmania. aethiopica. Among the prepared compounds; 2-amino-N-(6-hydrazinyl-6-oxohexyl)benzamide 2f (IC 50 =0.051μM) has the best activity, 154 folds more active than reference standard drug miltefosine (IC 50 =7.832μM), and half fold the activity of amphotericin B (IC 50 =0.035μM). In addition, this compound was safe and well tolerated by experimental animals orally up to 250mg/kg and parenterally up to 100mg/kg., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid which regulates many cancer-related processes, including cellular proliferation. The Hippo signaling pathway consists of a cascade of tumor suppressive kinases Mst1/2 and Lats1/2 and their downstream targets YAP and TAZ which are generally pro-proliferative transcriptional regulators. Direct phosphorylation by Lats1/2 causes inhibition or degradation of YAP/TAZ and down-regulation of their target genes. We found SPC treatment of MDA-MB-435S breast cancer cells to strongly inhibit their proliferation and to induce a sustained Lats2 protein expression (6-24h). Therefore, we hypothesized that Hippo signaling might mediate the anti-proliferative SPC response. We also saw a cell density-dependent increase in S127-phosphorylated YAP (pS127-YAP) and a decrease in mRNA levels of YAP target genes (CTGF, Cyr61) in response to long (9h) SPC treatment. Knockdown of S1P receptor 2 (S1P 2 ) prevented the SPC-induced up-regulation of Lats2 and attenuated the anti-proliferative effect of SPC. However, while knockdown of Lats2 alone or in combination with Lats1 expectedly increased basal proliferation it did not attenuate the SPC-induced inhibition of proliferation. Exogenous expression of wild-type or kinase-dead Lats2 and knockdown of YAP/TAZ also had no effect on the anti-proliferative SPC response. It has been previously shown that activation of S1P 2 -G 12/13 by sphingosine-1-phosphate (S1P) leads to rapid de-phosphorylation and up-regulation of YAP. Similarly, we saw a decrease in pS127-YAP and an increase in total YAP levels with short (1h) SPC treatment as well as a subsequent transient increase in YAP target gene expression. Inhibition of S1P 2 prevented the SPC-induced YAP de-phosphorylation. The rapid YAP activation and subsequent up-regulation of Lats2 mRNA does not constitute a negative feedback loop as knockdown of YAP/TAZ did not inhibit SPC-induced Lats2 expression. In conclusion, in this study we show that SPC is able to regulate Hippo signaling in a dual and opposite manner, causing an initial activation of YAP followed by an inhibition. However, even the strong SPC-induced effects seen in Lats2 and YAP did not mediate the anti-proliferative SPC response., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Some novel heteroretinoid-bisbenzylidine ketone hybrids have been synthesized and evaluated for their in vitro antileishmanial activity against intramacrophagic amastigotes of Leishmania donovani. Among all the nine synthetic compounds, five compounds (7c, 7d and 7f-h) have shown significant (less than 7μM) activity against intramacrophagic amastigotes. The IC50 values of these compounds were found better than the reference drugs sodium stibogluconate (SSG) and miltefosine. This study helped us in identifying the new class of compounds that could be exploited as potent antileishmanial agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A new series of aryl substituted ketene dithioacetals 6a-h was synthesized and evaluated for their in vitro and in vivo antileishmanial activity against Leishmania donovani. Two compounds exhibited significant in vitro activity against intracellular amastigotes of L. donovani with IC(50) values 3.56 and 5.12 μM and were found promising as compared with reference drug, miltefosine. On the basis of good Selectivity Indices (S.I.), they were further tested for their in vivo response against L. donovani/hamster model and showed significant inhibition of parasite multiplication 78% and 83%, respectively. These compounds were better than the existing antileishmanials in respect to IC(50) and SI values, but were less active than miltefosine in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

β-carbolines from various natural and synthetic sources have been known to show diverse biological activities. As a part of our current ongoing project to search for potent natural product-derived anti-leishmanial compounds, we have synthesized a series of substituted 1-aryl-β-carboline derivatives. A total of 22 compounds were synthesized and tested in vitro against Leishmania donovani, out of which 6 compounds (4, 5, 10, 11, 19 and 22) showed notably more activity than the standard miltefosine (IC(50) 12.07±0.82 μM), with compound 4 being the most potent (IC(50) 2.16±0.26 μM)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, has recently been reported to modulate actin cytoskeleton rearrangement. We have previously demonstrated Fyn tyrosine kinase is involved in SPC-induced actin stress fiber formation in fibroblasts. However, Fyn-dependent signaling pathway remains to be elucidated. The present study demonstrates that RhoA-ROCK signaling downstream of Fyn controls stress fiber formation in SPC-treated fibroblasts. Here, we found that SPC-induced stress fiber formation was inhibited by C3 transferase, dominant negative RhoA or ROCK. SPC activated RhoA, which was blocked by pharmacological inhibition of Fyn activity or dominant negative Fyn. Constitutively active Fyn (ca-Fyn) stimulated stress fiber formation and localized with F-actin at the both ends of stress fibers, both of which were prevented by Fyn translocation inhibitor eicosapentaenoic acid (EPA). In contrast, inhibition of ROCK abolished only the formation of stress fibers, without affecting the localization of ca-Fyn. These results allow the identification of the molecular events downstream SPC in stress fiber formation for a better understanding of stress fiber formation involving Fyn., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A series of new 2-[3-(2-alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids was synthesized and their antileishmanial activity was evaluated against Leishmania infantum amastigotes. The majority of the new analogues were significantly less cytotoxic than miltefosine while, antiparasitic activity depended on the length of the 2-alkyloxy substituent. The most potent compounds were {2-[[[3-(2-hexyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5b) and {2-[[[3-(2-octyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5c)., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved.

Sphingosylphosphorylcholine (SPC) is the major component of high-density lipoproteins (HDL) in blood plasma. The bioactive lipid acts mainly via G protein coupled receptors (GPCRs). Similar to ligands of other GPCRs, SPC has multiple biological roles including the regulation of proliferation, migration, angiogenesis, wound healing and heart rate. Lysophospholipids and their receptors have also been implicated in cell differentiation. A potential role of SPC in stem cell or tumour cell differentiation has been elusive so far. Here we examined the effect of SPC on the differentiation of mouse embryonic stem (ES) cells and of human NB4 promyelocytic leukemia cells, a well established tumour differentiation model. Our data show that mouse embryonic stem cells and NB4 cells express the relevant GPCRs for SPC. We demonstrate both at the level of morphology and of gene expression that SPC induces neuronal and cardiac differentiation of mouse ES cells. Furthermore, SPC induces differentiation of NB4 cells by a mechanism which is critically dependent on the activity of the MEK-ERK cascade. Thus, the bioactive lipid SPC is a novel differentiation inducing agent both for mouse ES cells, but also of certain human tumour cells.

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.

The alkylphosphocholine miltefosine (n-hexadecylphosphocholine, MT) has been introduced recently as a very effective drug for the oral treatment of human leishmaniasis. However, the parasiticidal mechanism of MT at a molecular level is far from being understood. Here we report the synthesis and biological characterization of 16-mercaptohexadecylphosphocholine, a thiol analog of MT which was designed to facilitate the search of MT interacting targets within the parasite by a variety of analytical methods. This analog presents the same leishmanicidal effect as the parent drug against Leishmania donovani promastigotes and Leishmania pifanoi axenic amastigotes, and has been used to develop an affinity chromatography method to attempt the isolation of putative Leishmania proteins that bind to the phosphocholine part of the molecule.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.

We studied the mechanism of sphingosylphosphorylcholine (SPC)-induced contraction in feline ileal smooth muscle cells. Western blotting revealed that G protein subtypes of Galpha(i1), Galpha(i3) and Galpha(o) existed in feline ileum. Galpha(i3) antibody penetration into permeabilized cells decreased SPC-induced contraction. In addition, incubation of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) with membrane fraction increased its binding to Galpha(i3) subtype after SPC treatment, suggesting that the signalling pathways invoked by SPC were mediated by Galpha(i3) protein. MAPK kinase (MEK) inhibitor PD98059 blocked the contraction significantly, but p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 did not. Chelerythrine and neomycin also inhibited the contraction. However, cotreatment of PD98059 and chelerythrine showed no significant difference. Phosphorylation of p44/42 MAPK was increased by SPC treatment, which was reversed by pretreatment of inhibitors of signalling molecules that decreased SPC-induced contraction previously. The same result was obtained in the assay of MAPK activity., (Copyright 2002 Elsevier Science Inc.)

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

The aim of this study was to determine the antitumour effects of D-21266 in a rodent tumour model. Hexadecylphosphocholine (INN: Miltefosine) represents the first anticancer agent which was specifically formulated for topical use in cancer patients. The development as an oral drug was hampered by the gastrointestinal toxicity. Hexadecylphosphocholine derivatives were sought with a better therapeutic index. Octadecyl-(1,1-dimethyl-4-piperidylio) phosphate (D-21266) was identified as a suitable candidate. This compound is highly active in vitro inhibiting the growth of a number of human cancer cell lines. Mammary carcinomas were induced in Sprague-Dawley rats using DMBA, and oral doses of D-21266, in various schedules, were given to the animals. A high antineoplastic potency was observed without inducing loss of body weight at highly effective doses. The antitumour effect could be enhanced by introducing a dose schedule consisting of a high loading dose followed by a low maintenance dose, both of which are only marginally active when given alone. Therefore, D-21266 with its favourable pharmacological and toxicological profile, warrants evaluation in the clinic. However, the concept of clinical trials requires new approaches to dose finding and response evaluation, because the dose-response relationship of this compound is distinctly different from that of classical cytostatic agents.

Hexadecylphosphocholine (HePC, Miltefosine) is a representative of the group of alkyl-lysophosphocholines showing remarkable antitumoral activity in in vitro experiments and in experimental animal tumour models. The epidermoid tumour cell line KB, which is highly sensitive to HePC (half-maximal growth inhibiting concentration, IC50: 1.2 microM; half lethal concentration, LC50: 2.8 microM), was slowly adapted to increasing concentrations of HePC. After 14 months, the adaptation process was stopped at a concentration of 10 micrograms/ml (23.5 microM). At this point, the KB cells tolerated high doses of HePC (IC50: 41.2 microM; LC50: 87.1 microM). The resistant cells (KBr) also showed crossresistance to the other well studied ether-lysophospholipids, Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, OMG-3PC; ET18OCH3) and Ilmofosine (1 S-hexadecyl-2-methoxymethyl-rac-(1-thio-3-hydroxy)propyl-3-phosphocho lin e, BM 41.440). Comparison of the KB and KBr cells showed that total lipid phosphate, ether-lipid content, vinyl-ether-lipid content, protein content as well as cholesterol content were unchanged. Furthermore, no changes were observed in the lipid composition between KB and KBr cells. Uptake of choline was also unchanged in both cells, but the uptake of D-myo-inositol was lower by a factor of two in the KBr cells. However, in KB cells, the addition of HePC induced a 50% reduction of D-myo-inositol-uptake, whereas in KBr cells inositol uptake was unchanged. Differences in HePC uptake and HePC metabolism were apparent between the KB and KBr cell lines. KBr cells showed a 3-fold lower uptake for HePC and a 3- to 4-fold faster metabolism of HePC than KB cells. However, the amount of non-metabolised HePC after 2 days of incubation with 1 microgram/ml HePC (LC50: 1.2 microgram/ml) in KB cells was 3- to 4-fold lower than the amount of HePC in KBR cells at 10 micrograms/ml (LC50: 37 micrograms/ml), indicating that KBr cells can incorporate higher amounts of HePC than KB cells without adverse effects for cell growth and viability. This seems to indicate that mechanisms other than slower uptake and faster metabolism are involved in the induction of resistance to HePC in KBr cells.

Hexadecylphosphocholine (HePC) is an analogue of the antiproliferative alkyllysophospholipids (ALP). As these lipid-like compounds interfere with membrane lipid metabolism at several sites, we studied the effects of HePC on uptake and metabolism of inositol and choline, two important phospholipid precursor molecules in two sensitive cell lines, Raji and KB, and in a resistant variant of KB cells, KBr. HePC substantially inhibited the membrane uptake of inositol and of choline in KB and Raji. Inositol uptake of KBr cells was constitutively low and was not further decreased by HePC. In all three cell lines, uptake inhibition of choline was less pronounced. Uptake inhibition showed characteristics of a non-specific effect, probably due to the physicochemical properties of HePC as a "lyso" structure. Decreased uptake of inositol did not affect phosphoinositide synthesis. Cellular phosphatidylcholine (PC) metabolism seemed to be affected through inhibition of choline incorporation and enhancement of PC degradation in the two sensitive cells. In KBr cells, these effects were not observed.

The effect of pretreatment with miltefosine (MIL) on the antineoplastic activity of cyclophosphamide (CPA) was evaluated in subcutaneous benzo(a)pyrene-induced sarcomas (BPS) of the rat. MIL alone had no antineoplastic effect on this autochthonous tumour, but enhanced the chemotherapeutic effect of CPA. Conversely, MIL counteracted the myelotoxicity of CPA in normal adult rats. Although the nadir of the leucocyte count remained unchanged, the recovery phase was considerably shortened, an effect which resembled the pharmacological action of GM-CSF.

We investigated the hypothesis that the antiproliferative effect of hexadecylphosphocholine (HePC) and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is mediated through the inhibition of cellular protein kinase C (PKC). In the sensitive HL60 cell line, ID50 and LD50 values of 5.6 and 5.3 microM, respectively (HePC), and of 3.8 and 4.2 microM, respectively (ET-18-OCH3) were obtained. In the more resistant K562 cell line, these values were 69.1 and > 97 microM, respectively (HePC) and 7.8 and 76.8 microM, respectively (ET-18-OCH3). Treatment of both cell lines with HePC and ET-18-OCH3 (25 microM) for 2 h did not lead to PKC translocation. However, a 30% reduction of PKC activity, mainly due to a decrease in the cytosolic compartment, was found. Half maximal stimulation of PKC translocation by phorbolester (TPA) in HL60 and K562 cells, which were pretreated for 2 h with 25 microM of the lipids, resulted in a 20-30% decrease of membrane-bound PKC, whereas the cytosolic form was found to be unchanged. In the same experimental setting, dioctanoylglycerol (DIC8)-stimulated PKC translocation was not affected by HePC or ET-18-OCH3. However, a 10-20% reduction of PKC enzyme activity in the membrane and in the cytosolic fraction was obtained. These findings indicate that HePC and ET-18-OCH3 do not interfere with PKC translocation but rather mediate a general decrease of the enzyme activity in the membrane and cytosol of the cells. Since the extent of PKC inhibition was somewhat similar in the sensitive HL60 and the resistant K562 cell line, inhibition of PKC is probably not a prerequisite for the antiproliferative action of HePC and ET-18-OCH3.

Suramin has shown antitumour activity in vitro and in vivo. At plasma levels higher than 200 microM there is, however, excessive toxicity. We have, therefore, attempted to improve the antitumour effects of suramin in vitro by combining it with several other antitumour agents. The MCF-7 mammary carcinoma and PC3 prostate cancer cell lines were exposed continuously to suramin and the other agents for 6 days. The sulphorhodamine B (SRB) assay was used for the assessment of growth inhibition. The dose-response interactions were evaluated using the median-effect analysis with the Chou and Talalay computer programme. In the MCF-7 cell line, the combination of suramin plus doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) or tumour necrosis factor (TNF) resulted in synergistic growth inhibition, whilst its combination with miltefosine (HPC) was antagonistic. In the PC-3 cell line, suramin plus CDDP or TNF was synergistic, whilst its combination with DXR, 5-FU and HPC was antagonistic. All tested combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and with the combination of both IFN-alpha+IFN-gamma were not synergistic. The synergistic effect of suramin with DXR was schedule dependent. Pretreatment (addition of DXR on day 1 and suramin on days 2-5) was additive at the IC50 level, in both cell lines. Addition of DXR at day 5 was more effective than simultaneous exposure. We found a synergistic effect for the combination of suramin with CDDP and TNF in both cell lines. In addition the combination with DXR and 5-FU was synergistic in MCF-7. Sequential administration of DXR-suramin or suramin-DXR increased the growth inhibition.

Hexadecylphosphocholine (HePC), an experimental and clinical antitumour agent of the alkyllysophospholipid group, was tested for its radiosensitising effect on a panel of nine human mammary cancer cell lines in vitro. Growth inhibition by ionising radiation and recovery from it were not influenced by pretreatment with HePC in most cases, except for two cell lines expressing an activated ras oncogene. In the latter we found an enhanced radioresistance that was abolished by pretreatment with HePC. Our results suggest that HePC may act as a radiosensitiser for cells carrying an activated ras oncogene.

The antineoplastic agent, hexadecylphosphocholine, a phospholipid analogue, inhibited phosphatidylserine-activated protein kinase C in vitro at concentrations higher than 40 mumol/l. The half-inhibitory concentration (IC50) was 62 mumol/l. Another alkylphosphocholine, dodecylphosphocholine, did not have an inhibitory effect on protein kinase C. At the same concentrations, hexadecylphosphocholine antagonised the phorbol ester-stimulated proliferation of Madin-Darby canine kidney cells whereas dodecylphosphocholine had no effect. In addition, phorbol ester-induced morphological changes of these epithelial cells were antagonised by hexadecylphosphocholine. Both effects of hexadecylphosphocholine, the inhibition of protein kinase C and the antagonisation of the altered cell morphology induced by phorbol ester, were comparable to those observed after treatment with sphingosine, a known protein kinase C inhibitor. We conclude that one possible mechanism of the antineoplastic action of hexadecylphosphocholine is mediated by inhibition of protein kinase C.