Enhanced anti-tumour effects of Vinca alkaloids given separately from cytostatic therapies.

BACKGROUND AND PURPOSE: In polychemotherapy protocols, that is for treatment of neuroblastoma and Ewing sarcoma, Vinca alkaloids and cell cycle-arresting drugs are usually administered on the same day. Here we studied whether this combination enables the optimal antitumour effects of Vinca alkaloids to be manifested. EXPERIMENTAL APPROACH: Vinca alkaloids were tested in a preclinical mouse model in vivo and in vitro in combination with cell cycle-arresting drugs. Signalling pathways were characterized using RNA interference. KEY RESULTS: In vitro, knockdown of cyclins significantly inhibited vincristine-induced cell death indicating, in accordance with previous findings, Vinca alkaloids require active cell cycling and M-phase transition for induction of cell death. In contrast, anthracyclines, irradiation and dexamethasone arrested the cell cycle and acted like cytostatic drugs. The combination of Vinca alkaloids with cytostatic therapeutics resulted in diminished cell death in 31 of 36 (86%) tumour cell lines. In a preclinical tumour model, anthracyclines significantly inhibited the antitumour effect of Vinca alkaloids in vivo. Antitumour effects of Vinca alkaloids in the presence of cytostatic drugs were restored by caffeine, which maintained active cell cycling, or by knockdown of p53, which prevented drug-induced cell cycle arrest. Therapeutically most important, optimal antitumour effects were obtained in vivo upon separating the application of Vinca alkaloids from cytostatic therapeutics. CONCLUSION AND IMPLICATIONS: Clinical trials are required to prove whether Vinca alkaloids act more efficiently in cancer patients if they are applied uncoupled from cytostatic therapies. On a conceptual level, our data suggest the implementation of polychemotherapy protocols based on molecular mechanisms of drug-drug interactions. LINKED ARTICLE: This article is commented on by Solary, pp 1555-1557 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12101.

Neutral sphingomyelinase inhibitor C11AG prevents lipopolysaccharide-induced macrophage activation.

Stimulation of macrophages by lipopolysaccharide (LPS) leads to the synthesis of proinflammatory cytokines and nitric oxide (NO) and, as a consequence, to endotoxic shock. We provide evidence that LPS stimulates the activity of a membrane-associated neutral sphingomyelinase (nSMase) and that this activity is mandatory for the liberation of nuclear factor-kappa B (NF kappa B) and the induction of inducible NO-synthase (iNOS). With the aid of a newly developed, selective inhibitor of nSMase, C11AG, we could distinguish between nSMase-dependent and -independent LPS-induced signals. C11AG blocked LPS-stimulated sphingomyelin degradation and NF kappa B activation without interfering with p42 tyrosine phosphorylation. Concomitantly, the expression of iNOS was found to be reduced both in mononuclear cells and in murine endotoxemia. Therefore, specific inhibitors of nSMase may define a new class of antiinflammatory substances.

Antitumoral activity of a sulphur-containing platinum complex with an acidic pH optimum.

UNLABELLED: Platinum complexes are essential tools for cancer treatment despite their toxic side effects. Here we describe a new platinum complex with sulphurs as complexing atoms (thioplatin). PURPOSE: To demonstrate that the antitumoral activity of a new sulphur-containing platinum compound (thioplatin) depends on a slightly acidic pH. METHODS: Platinum uptake by tumour cells and interaction with DNA was determined at slightly acidic or alkaline pH. To demonstrate low in vivo toxicity the effects of thioplatin on body weight, blood urea nitrogen, white blood cell count and the histopathological appearance of small intestines and kidneys were evaluated at doses that displayed antitumoral effects against human small-cell lung cancer and human colorectal cancer xenotransplants in nude mice. RESULTS: The slightly acidic pH optimum of thioplatin was proven by the altered electrophoretic mobility of plasmid DNA, quantitation of the platinum content in the DNA of tumour cells and cytotoxicity studies. Thioplatin displayed antitumoral activity without severe side effects such as weight loss, renal ischaemia, destruction of villi in the small intestine or leukopenia as observed at comparable doses of cisplatin. Furthermore, probably due to its lipophilic nature, thioplatin was taken up readily even by cisplatin-resistant cells. In vivo studies with human tumour xenografts in nude mice showed a therapeutic index of thioplatin five to ten times higher than that of cisplatin.

Neutral sphingomyelinase-inhibiting guanidines prevent herpes simplex virus-1 replication.

We synthesized a series of new guanidinium derivatives and studied the inhibitory activity on both neutral sphingomyelinase and herpes simplex virus-1 (HSV-1) replication. The lipophilic quality of the molecules was found to be correlated with the inhibitory potential of the compounds. Undecylidene-aminoguanidine was superior to derivatives with 10, 8 or 6 carbon atoms whereas propylidene-aminoguanidine was completely inactive. Decylidene-aminoguanidine was the most active derivative, with 10 carbon atoms. Various cyclic saturated isomers were inferior to the linear molecule. Aromatic cyclic residues were superior to saturated cyclic residues. The most active compound was a derivative containing 11 carbon atoms, undecylidene-aminoguanidine (C11AG), which inhibited the replication of HSV-1 by 50% at a concentration of 2.6 microM while cytotoxic adverse effects were only observed at a concentration of 31 microM. Expression of immediate early gene ICP-4 and concomitantly of HSV-1 specific DNA replication was found to be a target of C11AG. This result suggests that C11AG interferes with cellular signal transduction mechanisms that regulate expression of HSV-1 immediate early genes. C11AG was shown to inhibit neutral sphingomyelinase without affecting phospholipase A2, phosphatidylcholine-specific phospholipase C and phospholipase D.

The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C.

The effect of the antiviral, antitumoural xanthate D609 on the activity of phospholipase A2, C (PC- and Pi-specific) and D was investigated. D609 is the first model substance of a new concept of antiviral therapy that interferes with cellular regulation mechanisms, rather than with virus coded enzymes. Exclusively phosphatidylcholine (PC) specific phospholipase C (PC-PLC) was found to be inhibited in a dose-dependent manner. Enzyme activity was determined either as the rate of acid release from PC or as the rate of phosphorylcholine production form 3H labelled PC. Lineweaver-Burk plots revealed D609 as a competitive inhibitor of PC-PLC with a Ki of 6.4 microM. In addition, D609 competitively inhibited PC-PLC mediated cleavage of P-nitrophenylphosphorylcholine (p-NPP), a pseudo-substrate of PC-PLC with a Ki of 8.8 microM. These data suggest that D609 competes with the phosphorylcholine residue of PC for binding to PC-PLC.

Experimental treatment of equine sarcoid using a xanthate compound and recombinant human tumour necrosis factor alpha.

A xanthate compound with antiviral and antitumoural activities, tricyclodecan-9-yl-xanthogenate (D609) in combination with the potassium salt of the lauric acid (KC12) and, in a further investigation, the above-mentioned substances together with recombinant human TNF alpha (rh-TNF alpha), were tested on equine sarcoid tumours for therapeutic efficacy. A pilot investigation on 5 healthy horses showed that the compounds were well-tolerated; apart from a local, temporary oedema at the injection site, no other clinical symptoms were observed after subcutaneous administration of volumes from 0.1 to 10 ml per injection. The tested concentrations of D609 and KC12 (5 mg/ml solution) and of rh-TNF alpha (50 micrograms/ml) were used for the treatment experiments. The repeated injections of the compounds to 11 sarcoid affected horses were also well-tolerated, except by one horse. In this case the treatment had to be interrupted after two injections because of severe reaction, i.e. fever and lameness due to oedemas. Five horses (n = 6 sarcoids) were treated by local, subcutaneous injection of D609 and KC12 under the tumour at intervals of 3 weeks. On one periocular sarcoid the compounds were applied as an ointment. After a follow-up period of 18 months, 5 tumours did completely regress and one remained unchanged. The periocular tumour showed a reduction in size. Five horses (n = 9 sarcoids) were then treated with a combination of D609, KC12 and rh-TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

TNF accelerates the S-phase of the cell cycle in tumor cells.

The reduction of glucose supply induced the killing of tumor cells by tumor necrosis factor (TNF) in vivo and in vitro. In contrast, normal cell lines were resistant to TNF regardless of the presence or absence of glucose. Epidermal growth factor (EGF) did not exert a cytotoxic effect on tumor cells in the absence of glucose. Therefore, the killing mechanism of TNF under conditions of reduced glucose supply was investigated. Flow cytometry experiments and studies of kinetics revealed that the S-phase of the cell cycle was prolonged in the absence of glucose. After TNF treatment, the S-phase was found to be shortened and the rate of 3H-thymidine incorporation into DNA was increased, whereas EGF failed to exert such an effect. DNA synthesis and entry into mitosis are known to be regulated by cyclin A. In serum-starved tumor cells (HeLa) we have observed increased cyclin A synthesis within 10 hr, in parallel with enhancement of DNA synthesis and shortening of the S-phase after TNF treatment. We conclude that, under conditions of low glucose supply, TNF can assume the role of a growth factor in transformed cells.

Lauric acid inhibits the maturation of vesicular stomatitis virus.

In the presence of lauric acid (C12), the production of infectious vesicular stomatitis virus (VSV) was inhibited in a dose-dependent manner. The inhibitory effect was reversible; after removal of C12 the antiviral effect disappeared. In addition, the chain length of the monocarboxylic acids proved to be crucial, as those with shorter or longer chains were less effective or had no antiviral activity. Concomitant with the C12-induced inhibition was the stimulation of triacylglycerol synthesis, increasing the amount up to ninefold. Analysis of the antiviral mechanism of C12 revealed that the correct assembly of the viral components was disturbed, but viral RNA and protein synthesis remained unimpaired. By cell fractionation and Western blot analysis the amount of viral M protein located in the plasma membrane was found to be markedly reduced after treatment with C12, whereas in the cytoplasm the quantity of M protein was similar to that in untreated cells. C12 did not influence M protein synthesis, but prevented the binding of M protein to the host cell membrane, where the protein plays an essential role in virus assembly. Thus, treatment of VSV-infected cells with C12 resulted in inhibition of virus release. It is suggested that the newly synthesized triacylglycerols might interact with the host cell plasma membrane and interfere with virus maturation.

Hexadecylphosphocholine differs from conventional cytostatic agents.

Alkylphosphocholines, and especially their main representative hexadecylphosphocholine (HPC), show high anticancer activity in methylnitrosourea (MNU)-induced autochthonous rat mammary carcinoma. The regression of MNU-induced rat mammary carcinoma during HPC treatment can be evaluated by computed tomography and sonography. This allows a noninvasive monitoring of therapy in vivo (tumor size, morphology, and blood supply). Both diagnostic modalities can show a rapid concentric decrease in tumor volume as well as the appearance of cystic, scarry, and necrotic areas in the tumor tissue as a result of HPC treatment. In addition, prior to, during and after therapy tumor perfusion can be assessed by color Doppler sonography in vivo. A more than 4-fold difference in HPC efficacy was observed when the colony growth of explanted MNU-induced mammary carcinoma cells was measured in the methylcellulose colony assay (IC50 = 180 mumol HPC/l) and the Hamburger Salmon colony assay (IC50 = 740 mumol HPC/l). In the latter assay, growth of concomitantly seeded untransformed cells, especially of fibroblasts, is much lower than in the methyl-cellulose colony assay. We therefore assume that the antitumor efficacy of HPC against MNU-induced mammary carcinoma is enhanced by neighboring cells such as fibroblasts. Cell culture experiments with the three MNU-induced rat mammary carcinoma cell clones 1-C-2, 1-C-30, and 1-C-32 revealed IC50 values in the range of 50-70 mumol HPC/l. The volume of 1-C-2 cells increased up to 4-fold after 72 h of permanent exposure to 100 mumol HPC/l, a concentration that completely inhibited proliferation of tumor cell numbers without being cytotoxic. Nucleotide triphosphate levels dropped significantly after 24 h and were slowly restored in spite of continued exposure. After 72 h, they nearly reached those levels observed in plateau-phase cells. This suggests that HPC-induced growth inhibition has similarities with physiologically occurring growth arrest. Finally, replication of RNA viruses and DNA viruses was suppressed 30-fold and 7-fold, respectively, at low concentrations of HPC (12 mumol/l), which caused no or negligible growth inhibition in the virus-harboring cells, thus demonstrating specific antiviral activity of HPC. From these observations we conclude that HPC differs in many important aspects from conventional cytostatic agents and is certainly worth following-up in further investigations.

Glucose depletion enhances the anti-tumor effect of TNF.

Treatment of human carcinoma xenotransplants in athymic mice with recombinant human tumor necrosis factor (rh TNF) causes necrosis mainly in the central parts of the tumors, while peripheral sections remain mitotically active. As tumors are known to be supplied with adequate glucose exclusively in their periphery, the influence of the lack of glucose on the cytotoxic activity of rh TNF was studied. The absence of glucose enhanced the killing of tumor cell lines by rh TNF in tissue culture. Meth-A, a cell line known to be resistant to TNF in vitro but highly sensitive to it in vivo, was readily killed in tissue-culture medium lacking glucose. All non-transformed cell lines tested were found to be resistant to rh TNF, regardless of the presence or absence of glucose. In tumor-bearing mice a reduction of the blood glucose content augmented by insulin led to increased anti-tumor efficiency of rh TNF. The enhanced anti-tumor activity was reflected both in histological sections of the tumor xenotransplants, by extensive central necroses, and by reduction of the tumor volumes.

Binding of NF-kB to the HIV-1 LTR is not sufficient to induce HIV-1 LTR activity.

Human immunodeficiency virus type 1 (HIV-1) spends a significant part of its life cycle as latent provirus in nonactivated cells. It induction requires mitogen stimulation. TPA treatment induces HIV-1 transcription by protein kinase C (PKC)-mediated activation of the cellular transcription factor NF-kB. PKC activation induces the dissociation of NF-kB from its inhibitor protein (IkB). The liberated NF-kB then binds to its proviral recognition sequence in the HIV-1 long terminal repeat (LTR) sequence. This step, however, is not sufficient to augment transcription. We demonstrate that NF-kB-mediated HIV-1 LTR activation is regulated by an additional event that is not dependent on IkB. A further phosphorylation event is proposed, since this step could be blocked by an inhibitor of a phospholipase C (PLC) type reaction. This inhibitor precludes the formation of diacylglycerols, which are required for activation of PKC isoenzymes. As an alternative pathway that is not dependent on PLC reactions, high-level transcription from the HIV-1 LTR is shown to require binding of both NF-kB and TAT.

Medium chain length fatty acids stimulate triacylglycerol synthesis in tissue culture cells.

In the presence of undecanoic acid (C11) or lauric acid (C12) the synthesis of triacylglycerols was stimulated up to 10-fold both in tumor cell lines and in normal cell lines. Monocarboxylic acids of shorter or longer chain length either had no effect at all or were less effective. The increased triacylglycerol production was demonstrated, on the one hand, by the incorporation of radiolabeled glycerol into triacylglycerols and, on the other, by the incorporation of radiolabeled monocarboxylic acids, the incorporation of all (1-14C)-labeled monocarboxylic acids (C6, C12, C16, C18) regardless of their chain length, being preferentially enhanced by C11 and C12. C12 stimulated the de novo synthesis of triacylglycerols to such a degree that a 7-fold increase in the total amount of triacylglycerols per cell was observed during the first 10 hr of incubation. After removal of C12 from the tissue culture medium levels of triacylglycerols reach initial values again within 6 hr, indicating that the stimulatory effect of C12 is dependent on its continued presence. This led to the speculation that medium chain length monocarboxylic acids might be involved in the control of triacylglycerol synthesis.

Activation of latent papillomavirus genomes by chronic mechanical irritation.

The skin of animals of a laboratory strain of Mastomys natalensis carrying endogenous, latent papillomavirus genomes was irritated by scratching with glasspaper. Hyperproliferation of the epidermis and amplification of viral DNA followed this treatment, and in approximately 27% of the animals virus-producing papillomas were induced.

Systemic treatment of a human epidermoid non-small cell lung carcinoma xenograft with a xanthate compound causes extensive intratumoral necrosis.

Therapeutic effects were obtained after systemic treatment of athymic mice bearing an epidermoid non-small cell human lung carcinoma (NSCLC) xenograft with tricyclodecan-9-yl xanthogenate (D609) and the potassium salt of a fatty (dodecanoic) acid. Extensive intratumoral necrosis was observed 3 days after the treatment.

Restoration of the responsiveness to growth factors in senescent cells by an embryonic cell extract.

In senescent fibroblast cell cultures which have approached a postmitotic stage in vitro, responsiveness to growth factors is restored upon exposure to an embryonic sheep cell extract. The extract contains molecules below a molecular weight of 1 x 10(5) Da in aqueous solution. Following a transient exposure to the extract, mitotic activity is resumed, and the cells keep dividing over several passages. The target cells which respond to the treatment were identified in a single-cell assay as those that still had the capacity to undergo at least several mitotic divisions before entering the final stage of senescence.

Tumor necrosis factor induces necrosis of human carcinoma xenografts in the presence of tricyclodecan-9-yl-xanthogenate and lauric acid.

Recombinant human tumor necrosis factor (rh TNF) when administered intravenously together with the phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate (D609) and lauric acid (C12), leads to the partial regression of various human tumor transplants in athymic mice. Extensive necrosis occurred after a single intravenous infusion, with no detectable side effects. TNF-mediated cytotoxicity was found to be correlated with the depletion of energy in HeLa cells. The activity of rh TNF was enhanced by the absence of glucose, while it was reduced by addition of extraneous ATP. In the presence of rh TNF, D609, and C12, cellular energy metabolism was almost completely switched to glycolysis. Under these conditions the cytocidal activity of rh TNF on HeLa cells was amplified at least 60-fold.

Antitumoral activity of a xanthate compound. I. Cytotoxicity studies with neoplastic cell lines in vitro.

Xanthate derivatives were shown previously to display antitumor activity against transformed fibroblasts and lymphoma cells in combination with monocarboxylic acids [1]. Various malignant cell lines of human origin were treated in vitro to explore the range of antitumoral activity of the compounds. The combination of tricyclodecan-9-yl-xanthogenate (D 609) with undecanoic acid (C11) exerted dose dependent cytotoxic and antiproliferative effects on cell lines both from solid tumors (glioblastomas, colon-carcinomas) and hematological diseases (lymphomas, CML/BC). Additionally, the combination of D 609/C11 was able to kill both methotrexate- and adriamycin-resistant L 1210 and S 180 cells, indicating that there is no cross-resistance for these drugs and D 609/C11 in vitro.

Antitumoral activity of a xanthate compound. II. Therapeutic studies in murine leukemia and tumor models in vivo.

The combinations of tricyclodecan-9-yl-xanthogenate (D 609) with undecanoic acid (C11) and D 609 with myristic acid (C14) were tested in 3 rodent tumor models in vivo. D 609 in combination with C11 or C14 did not show antitumoral efficacy in 3-Lewis lung carcinoma (3-LL) growing in syngeneic C57BL6-mice (primary tumor and metastasis) or in WEHI-3B myelomonocytic leukemia growing in Balb/c mice, when given in a dose range lower than the lethal dose for 10% of the treated animals (LD10). In L 1210 mouse lymphoid leukemia growing in CD2F1 mice the combination of D 609/C11 given intraperitoneally in a concentration of 100 mg/kg for more than 1 day effected a significant difference in the survival curves between the control and therapeutic groups in 1 out of 2 experiments. In conclusion, the treatment schedules of D 609/C11 or D 609/C14 used in this study has not revealed significant therapeutic effects in mouse tumors or leukemias in vivo.

Mechanistic aspects of the synergistic antiviral effect of xanthates and monocarbonic acids.

The xanthate tricyclodecan-9-yl-xanthogenate (D609) displays antiviral and antitumoral properties that are inversely proportional in vitro to the serum concentration. Accordingly, it has been found that D609 binds to serum albumin. Recently, we have reported that D609, in combination with undecanoic acid, has a synergistic antiviral effect, which appears, as shown here, to be due to competition for the same binding domain on serum albumin. Furthermore, undecanoic acid fosters the binding of D609 to the cell. Both the competition of D609 with monocarbonic acid for binding on serum albumin and the enhanced binding of xanthate to the cell are dependent, in accordance with previously reported results, on the chain length of the fatty acids. Eleven to 14 C-atoms (undecanoic, lauric and myristic acid) were found to be appropriate while shorter (C6) and larger (C18) monocarbonic acids were shown to lack synergistic properties.

Tumor prevention by a xanthate compound in experimental mouse-skin tumorigenesis.

The antiviral and antitumoral compound tricyclodecan-9-yl-xanthogenate (D609), which is an inhibitor of protein kinase C activation, has been used for tumor prevention in vivo. When applied chronically together with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the classic initiation-promotion mouse-skin model, D609 prevented tumor induction in a dose-dependent manner. At the concentration that inhibited tumor formation by 97%, no toxic effects were detected and the TPA-induced hyperplasia remained unaffected. As D609 failed to prevent the activity of a chronically applied carcinogen, it is concluded that the observed tumor prevention achieved with D609 is tumor-promotion-specific and is not due to killing of tumor cells.