Francine Gossé

Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells.

Resveratrol, a natural polyphenolic phytoalexine present in grapes and wines, has been reported to exert a variety of important pharmacological effects. We investigated the effects of resveratrol on the growth and polyamine metabolism of CaCo-2 human colon cancer cells. Treatment of the CaCo-2 cells with 25 microM resveratrol caused a 70% growth inhibition. The cells accumulated at the S/G2 phase transition of the cell cycle. No signs of cytotoxicity or apoptosis were detected. Resveratrol caused a significant decrease of ornithine decarboxylase (ODC) activity, a key enzyme of polyamine biosynthesis, which is enhanced in cancer growth. ODC inhibition resulted in the reduction of the intracellular putrescine content, indicating that polyamines might represent one of several targets involved in the anti-proliferative effects of resveratrol.

Resveratrol analog (Z)-3,5,4′-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization

Resveratrol (3,5,4′‐trihydroxystilbene) a natural polyphenol present in medicinal plants, grapes and wines, has potent chemopreventive properties on intestinal carcinogenesis. A methylated derivative (Z‐3,5,4′‐trimethoxystilbene: R3) was synthesized. R3 at 0.3 μM exerted a 80% growth inhibition of human colon cancer Caco‐2 cells and arrested growth completely at 0.4 μM (R3 was 100‐fold more active than resveratrol). The cis conformation of R3 was also 100‐fold more potent than the trans isomer. R3 (0.3 μM) caused cell cycle arrest at the G2/M phase transition. The drug inhibited tubulin polymerization in a dose‐dependent manner (IC50= 4 μM), and it reduced also by 2‐fold ornithine decarboxylase and s‐adenosylmethionine decarboxylase activities. This caused the depletion of the polyamines, putrescine and spermidine, which are growth factors for cancer cells. R3 inhibited partially colchicine binding to its binding site on tubulin, indicating that R3 either partially overlaps with colchicine binding or that R3 binds to a specific site of tubulin that is not identical with the colchicine binding site modifying colchicine binding by allosteric influences. The resveratrol derivative (Z)‐3,5,4′‐trimethoxystilbene (R3) is an interesting anti‐mitotic drug that exerts cytotoxic effects by depleting the intracellular pool of polyamines and by altering microtubule polymerization. Such a drug may be useful for the treatment of neoplastic diseases.

Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620)

Summary. The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth – most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2).

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N1-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded.

Polyamines and the Recovery of Intestinal Morphology and Function after Ischemic Damage in Rats

We have followed the time-course of the morphological and functional recovery of intestinal mucosa after 90 min of mesenteric vascular occlusion. At the end of the ischemic period the villi were smashed, but crypts were preserved. Microvillous hydrolase activities showed a dramatic drop when compared with sham-operated controls. Reperfusion was followed by an immediate upsurge of ornithine decarboxylase activity and a significant (p < 0.01) enhancement of putrescine and N1-acetyl-spermidine concentrations, while spermidine and spermine concentrations in mucosal cells decreased. This indicated that, both, de novo synthesis and degradation rates of the polyamines were increased. Treatment with alpha-difluoromethyl-ornithine, a selective inactivator of ornithine decarboxylase prevented the accumulation of active enzyme, but did not prevent morphological healing. It delayed however the recovery of sucrase and aminopeptidase-specific activities. Our results suggest that in addition to de novo synthesis, other sources of polyamines are mobilized to an extent that growth at a normal rate is supported. This indicates that the presence of active ornithine decarboxylase enzyme is not a prerequisite for the restitution of intestinal integrity after ischemia. We suggest that in a situation of inadequate polyamine supply the restoration of vital processes (mucosal regeneration) has priority over the restoration of specific functions.

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N1-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N1-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions.

Effect of the polyamine oxidase inactivator MDL 72527 on N1-(n-octanesulfonyl)spermine toxicity

N(1)-(n-octanesulfonyl)spermine (N(1)OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N(1)OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N(1)OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N(1)OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N(1), N(12)-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N(1)OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N(1)OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.

Prenatal exposure to alcohol in rat: Effect on intestinal enzymes in offspring

Intestinal hydrolase activities were studied during postnatal development in the offspring of rats exposed to 20% ethanol during gestation; alcohol was withdrawn at birth. Controls received water during gestation. Sucrase, lactase, glucoamylase and aminopeptidase activities were determined 2 and 4 weeks after birth in the proximal jejunum. Offspring prenatally exposed to ethanol showed a deficit in body weight and lower aminopeptidase activity during the suckling period (2 weeks). These effects were reversible by 4 weeks when alcohol was withdrawn at birth. The prenatal exposure to ethanol did not change the pattern of sucrase maturation in the intestine of offsprings. The activities of lactase and glucoamylase were not modified following prenatal exposure to ethanol. In conclusion, exposure to ethanol during gestation caused decreased abilities for the intestine of the offspring to digest protein.

Performance of Pyridylthiourea-Polyethylenimine Polyplex for siRNA-Mediated Liver Cancer Therapy in Cell Monolayer, Spheroid, and Tumor Xenograft Models

Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea‐polyethylenimine (πPEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The πPEI‐assisted delivery of a siRNA targeting the polo‐like kinase 1 into Huh‐7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh‐7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh‐7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA‐mediated gene knockdown, and their clinical promise in cancer therapeutics.