Jean-Pierre Perchellet

Antioxidants and multistage carcinogenesis in mouse skin

The two-step initiation-promotion protocol for the induction of skin tumors in mice is a convenient model to elucidate what molecular events are involved in the multistage process of carcinogenesis and how they can be modulated. The current theories concerning the mechanisms of skin tumor initiation, stages 1 and 2 of tumor promotion, and tumor progression are reviewed. Because chemical carcinogens and tumor promoters may, directly or indirectly, generate reactive oxygen species (ROS) and because various antioxidants inhibit effectively some of the biochemical and biological events linked to tumor initiation, promotion and/or progression, it is conceivable that different sequences and levels of free radical-induced macromolecule damage may contribute to the evolution of the epidermal target cells from the preneoplastic stage to the malignant stage.

Inhibition of DMBA‐induced mouse skin tumorigenesis by garlic oil and inhibition of two tumor‐promotion stages by garlic and onion oils

A single 2-mg dose of garlic oil applied 30 minutes before a single carcinogenic dose of 7,12-dimethylbenz[a]-anthracene (DMBA) inhibited papilloma production in Sencar mice. The three groups were controls (Group 1), garlic oil applied 30 minutes before DMBA (Group 2), and garlic oil applied 30 minutes after DMBA (Group 3). The percents of mice with papillomas at 20 weeks were 94, 72, and 79, respectively. The decreases in Groups 2 and 3 were significant. The number of papillomas per mouse was 4.2 +/- 0.5 (Group 1), 2.3 +/- 0.8 (Group 2), and 3.4 +/- 0.6 (Group 3). The decrease in Group 2 was significant. A single 5-mg dose of garlic oil maximally inhibited DMBA-induced epidermal DNA synthesis by 86% when applied two hours before the carcinogen. Two-stage promotion in DMBA-initiated Sencar mice was achieved by twice-weekly applications of 8.5 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 weeks followed by twice-weekly applications of 8.5 nmol of mezerein for 18 weeks. The oils were applied 30 minutes after each promotion by TPA or mezerein. Single doses of 1 mg onion or garlic oil inhibited the first and second stages of promotion. The groups used were control (Group 1), garlic oil applied after stage 1 (Group 2), onion oil applied after stage 1 (Group 3), propenyl sulfide applied after stage 1 (Group 4), garlic oil applied after stage 2 (Group 5), onion oil applied after stage 2 (Group 6), and propenyl sulfide applied after stage 2 (Group 7). The percent of mice with papillomas was significantly decreased by all agents in Groups 2-7. The data are 81, 83, 91, 68, 96, and 86, respectively. The number of papillomas per mouse was significantly reduced by onion and garlic oils but not by propenyl sulfide. The data are 9.4 +/- 0.8, 6.3 +/- 0.7, 7.4 +/- 0.5, 9.2 +/- 1.2, 3.7 +/- 0.9, 6.2 +/- 0.6, and 9.1 +/- 1.4 for Groups 1-7, respectively. Onion and garlic oils inhibited the TPA-stimulated DNA synthesis when given as single doses of 5 mg one hour before TPA. The inhibition by garlic oil was most effective when given one hour before TPA but was evident when given from two hours before to two hours after TPA. These results, and those of others (AS Sadhana, Cancer Lett, 40, 193-197, 1988), who obtained inhibition of initiation, indicate that onion and garlic oils inhibit all stages of mouse skin tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and antiproliferative evaluation of 5-oxo and 5-thio derivatives of 1,4-diaryl tetrazoles

A series of 1,4-diaryl tetrazol-5-ones were synthesized by copper mediated N-arylation of 1-phenyl-1H-tetrazol-5(4H)-one with aryl boronic acids, o-R(1)C(6)H(4)B(OH)(2) where R(1)=H, OMe, Cl, CF(3), Br, CCH. The 1,4-diaryl tetrazol-5-ones substituted with OMe, Cl, CF(3), Br underwent thionation with Lawessons reagent to yield the corresponding 5-thio derivatives. The 1-(2-bromophenyl)-4-phenyl-1H-tetrazole-5(4H)-thione so obtained was subjected to lithiation/protonation and Sonogashira coupling to produce 1,4-diphenyl-1H-tetrazole-5(4H)-thione and 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione, respectively. The title compounds were found to be stable to strong Lewis acid conditions. Three of these novel compounds were found to inhibit L1210 leukemia cell proliferation and SK-BR-3 breast cancer cell growth over several days in culture in vitro. Shorter tetrazole derivative treatments also reduced the expression of the Ki-67 marker of cell proliferation in SK-BR-3 cells and the rate of DNA synthesis in L1210 cells.

Antitumor-Promoting Activities of Tannic Acid, Ellagic Acid, and Several Gallic Acid Derivatives in Mouse Skin

Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the biochemical and biological effects of the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse epidermis in vivo. When applied topically to mouse skin, tannic acid (TA), ellagic acid, and several gallic acid derivatives all inhibit TPA-induced ornithine decarboxylase activity, hydroperoxide production, and DNA synthesis, three biochemical markers of skin tumor promotion. Moreover, in the two-step initiation-promotion protocol, the same phenolic compounds also inhibit the incidence and yield of skin tumors promoted by TPA. TA is the most effective of these treatments. Since they are already known to inhibit tumor initiation, the plant phenols protecting against skin tumor promotion by TPA may be universal inhibitors of multistage carcinogenesis. TA and other polyphenols, therefore, might be valuable in cancer therapy and/or prevention.

Inhibition of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase and ornithine decarboxylase activities by glutathione level-raising agents and selenium-containing compounds

The present study was undertaken to determine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter known to inhibit superoxide dismutase (SOD) (superoxide: superoxide oxidoreductase, EC 1.15.1.1) and catalase (CAT) (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) activities, on mouse epidermal glutathione (GSH) peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) activity in vivo and in vitro. TPA led to a rapid and transient increase in GSH peroxidase specific activity within 30 min followed by a decrease from 1 to 12 h. Incubation of isolated epidermal cells with GSH level-raising agents and/or selenium-containing compounds increased remarkably basal GSH peroxidase activity, and thus, abolished totally the prolonged inhibitory effects of TPA on this enzyme. The inhibitory effects of 0.2 mM cysteine (Cys) or 0.5 mM GSH and 2.5 microM Na2 SeO3 or 50 microM selenocystamine on TPA-decreased GSH peroxidase activity were additive, in relation with their additive inhibitory effects on TPA-induced ornithine decarboxylase (ODC) (L-ornithine carboxylase, EC 4.1.1.17) activity. These data support the hypothesis that the stimulators of the GSH-dependent antioxidant protective system of the epidermal cells may inhibit the oxidative challenge linked to skin tumor promotion by TPA.

Triptycenes: a novel synthetic class of bifunctional anticancer drugs that inhibit nucleoside transport, induce DNA cleavage and decrease the viability of leukemic cells in the nanomolar range in vitro.

In contrast to their inactive parent compound triptycene (code name TT0), several triptycene (TT) analogs (code names TT1 to TT13), most of them new compounds, were synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. The most potent rigid tetracyclic quinones synthesized so far are TT2 and its C2-brominated derivative, TT13. The antitumor activity of TT2 has been compared to that of daunomycin (DAU), a clinically valuable anthracycline antibiotic which is structurally different from TT2 but also contains a quinone moiety. TT2 inhibits the proliferation (IC50: 300 nM at day 2 and 150 nM at day 4) and viability (IC50: 250 nM at day 2 and 100 nM at day 4) of L1210 cells to the same maximal degree as DAU, suggesting that the cytostatic and cytotoxic activities of TT2 are a combination of drug concentration and duration of drug exposure. Since TT2 does not increase the mitotic index of L1210 cells at 24 h like vincristine, it is unlikely to be an antimitotic drug that disrupts microtubule dynamics. Like DAU, a 1.5-3 h pretreatment with TT2 is sufficient to inhibit the rates of DNA, RNA and protein syntheses determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro (IC50: 6 microM). In contrast to DAU, which is inactive, a 15 min pretreatment with TT2 has the advantage of also inhibiting the cellular transport of nucleosides occuring over a 30 s period in vitro (IC50: 6 microM), suggesting that TT2 prevents the incorporation of [3H]thymidine into DNA because it rapidly blocks the uptake of [3H]thymidine by the tumor cells. After 24 h, TT2 induces as much DNA cleavage as camptothecin and DAU, two anti-cancer drugs producing DNA strand breaks and known to respectively inhibit DNA topoisomerase I and II activities. Interestingly, the abilities of TT2 to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because inhibition of nucleoside transport is highly unusual among DNA-damaging drugs, the use of bifunctional TTs with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Regioselective lactonization of naphthoquinones: synthesis and antitumoral activity of the WS-5995 antibiotics

Abstract An acid promoted quinolactonization of naphthoquinones has been developed, providing direct access to either ortho or para isomers as desired. Application of this methodology in syntheses of the antibiotics WS-5995A, WS-5995C and functional analogs is demonstrated. Preliminary antitumoral activity of the analogs is presented together with electrochemical analysis.

Microtubule-disrupting effects of gallium chloride in vitro.

Gallium chloride (GaCl3), an antitumor agent with antagonistic action on iron, magnesium and calcium, was tested for its ability to alter the polymerization of purified tubulin (2.2 mg/ml) in a cell-free system in vitro. GaCl3 (250 microM) does not mimic the effect of 10 microM paclitaxel and, therefore, is not a microtubule (MT)-stabilizing agent that can promote tubulin polymerization in the absence of glycerol and block MT disassembly. In contrast, GaCl3 mimics the effect of 1 microM vincristine (VCR) and inhibits glycerol-induced tubulin polymerization in a concentration-dependent manner (IC50: 125 microM), indicating that GaCl3 is a MT de-stabilizing agent that prevents MT assembly. However, 150 microM GaCl3 must be used to match or surpass the inhibitions of tubulin polymerization caused by 0.25 microM of known MT de-stabilizing agents, such as colchicine (CLC), nocodazole, podophyllotoxin, tubulozole-C and VCR. The inhibitory effect of 250 microM GaCl3 persists in the presence of up to 9 mM MgCl2, suggesting that the exogenous Mg2+ cations absolutely required for the binding of GTP to tubulin and MT assembly cannot overcome the antitubulin action of Ga3+ ions of a higher valence. The binding of [3H]vinblastine (VBL) to tubulin (0.5 mg/ml) is inhibited by unlabeled VBL but enhanced by concentrations of GaCl3 > 200 microM. However, increasing concentrations of GaCl3 mimic the ability of cold CLC to reduce the amount of [3H]CLC bound to tubulin, suggesting that GaCl3 may interact with the CLC binding site to inhibit tubulin polymerization. The binding of [3H]GTP to tubulin is decreased by unlabeled GTP but markedly enhanced by GaCl3, especially when concentrations of this metal salt of 32 microM or higher are added to the reaction mixture before rather than after the radiolabeled nucleotide. These data suggest that changes in protein conformation following GaCl3 binding might increase the interactions of tubulin with nucleotides and Vinca alkaloids. After a 24 h delay, the viability of GaCl3-treated L1210 leukemic cells is reduced in a concentration-dependent manner at days 2 (IC50: 175 microM), 3 (IC50: 35 microM) and 4 (IC50: 16 microM). Since GaCl3 (100-625 microM) increases the percentage of mitotic cells at 2-4 days, it might arrest tumor cell progression in M phase, but its antimitotic activity is much weaker than that of 0.25 microM VCR. Because the concentrations of GaCl3 that inhibit tubulin polymerization also increase the mitotic index and decrease the viability of L1210 cells in vitro, the antitubulin and antimitotic effects of GaCl3 might contribute, at least in part, to its antitumor activity.

1,4-anthraquinone: an anticancer drug that blocks nucleoside transport, inhibits macromolecule synthesis, induces Dna fragmentation, and decreases the growth and viability of L1210 leukemic cells in the same nanomolar range as daunorubicin in vitro

1,4-Anthraquinone (AQ) was synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. In contrast, its dihydroxy-9,10-anthraquinone precursor, quinizarin, was inactive. The antitumor activity of AQ was compared to that of daunorubicin (DAU), which is structurally different from AQ but also contains a quinone moiety. AQ is equipotent to DAU against L1210 tumor cell proliferation (IC50: 25 nM at day 2 and 9 nM at day 4) and viability (IC50: 100 nM at day 2 and 25 nM at day 4), suggesting that its cytostatic and cytotoxic activities are a combination of drug concentration and duration of drug exposure. Since AQ does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression. Like DAU, a 1.5-3 h pretreatment with AQ is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC50: 2 μM) determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro. In contrast to DAU, which is inactive, a 15 min pretreatment with AQ has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides (IC50: 2.5 μM) over a 30 s period in vitro. Hence, AQ may prevent the incorporation of [3H]adenosine and [3H]thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells. After 24 h, AQ induces as much DNA cleavage as camptothecin and DAU, two anticancer drugs producing DNA strand breaks and known to, respectively, inhibit topoisomerase I and II activities. However, the concentration-dependent induction of DNA cleavage by AQ, which peaks at 1.6-4 μM and disappears at 10-25 μM, resembles that of DAU. The mechanism by which AQ induces DNA cleavage is inhibited by actinomycin D, cycloheximide and aurintricarboxylic acid, suggesting that AQ activates endonucleases and triggers apoptosis. The abilities of AQ to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because of its potency and dual effects on nucleoside transport and DNA cleavage, the use of bifunctional AQ with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling

Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 μM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 μM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 μM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 μM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 μM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but caspase-2 is already maximally activated 6 h after 4 μM DAU or TT13 treatments, whereas DAU- or TT-induced caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 μM of the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally block DAU- and TT13-induced caspase-2, -8 and -9 activities, whereas pre-treatments with 15 μM of the caspase-8 inhibitor z-Ile-Glu-Thr-Asp (IETD)-fmk prevent DAU and TT13 from inducing caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream caspases, including caspase-9 and the mitochondrial amplification loop through caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be caspase-independent since they are both insensitive to similar pre-treatments with 100 μM of these specific caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 μg/ml of the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and caspase-2, -8 and -9 activities, suggesting that the Fas–FasL signaling pathway is not involved in the mechanism by which these quinone antitumor drugs trigger apoptosis in HL-60 cells.