4-Hydroxyhexenal and 4-hydroxynonenal are mediators of the anti-cachectic effect of n-3 and n-6 polyunsaturated fatty acids on human lung cancer cells.

Cachexia, the most severe paraneoplastic syndrome, occurs in about 80% of patients with advanced cancer; it cannot be reverted by conventional, enteral, or parenteral nutrition. For this reason, nutritional interventions must be based on the use of substances possessing, alongside nutritional and energetic properties, the ability to modulate production of the pro-inflammatory factors responsible for the metabolic changes characterising cancer cachexia. In light of their nutritional and anti-inflammatory properties, polyunsaturated fatty acids (PUFAs), and in particular n-3, have been investigated for treating cachexia; however, the results have been contradictory. Since both n-3 and n-6 PUFAs can affect cell functions in several ways, this research investigated the possibility that the effects of both n-3 and n-6 PUFAs could be mediated by their major aldehydic products of lipid peroxidation, 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE), and by their anti-inflammatory properties. An "in vitro" cancer cachexia model, consisting of human lung cancer cells (A427) and murine myoblasts (C2C12), was used. The results showed that: 1) both n-3 and n-6 PUFAs reduced the growth of lung cancer cells without causing cell death, increased lipid peroxidation and Peroxisome Proliferator-Activated Receptor (PPAR)α, and decreased TNFα; 2) culture medium conditioned by A427 cells grown in the absence of PUFAs blocked myosin production and the differentiation of C2C12 muscle cells; conversely, muscle cells grown in culture medium conditioned by the same cells in the presence of PUFAs showed myosin expression and formed myotubes; 3) adding HHE or HNE directly to C2C12 cells maintained in culture medium conditioned by A427 cells in the absence of PUFAs stimulated myosin production and myotube formation; 4) putative consensus sequences for (PPARs) have been found in genes encoding fast isoforms of myosin heavy chain, by a bioinformatics approach. The overall results show, first, the ability of both n-3 and n-6 PUFAs and their lipid peroxidation products to prevent the blocking of myosin expression and myotube formation caused in C2C12 cells by medium conditioned by human lung tumour cells. The C2C12 cell differentiation can be due to direct effect of lipid peroxidation products, as evidenced by treating C2C12 cells with HHE and HNE, and to the decrease of pro-inflammatory TNFα in A427 cell culture medium. The presence of consensus sequences for PPARs in genes encoding the fast isoforms of myosin heavy chain suggests that the effects of PUFAs, HHE, and HNE are PPAR-mediated.

Restoration of peripheral blood natural killer and B cell levels in patients affected by rheumatoid and psoriatic arthritis during etanercept treatment.

Etanercept (ETN) is an anti-tumour necrosis factor (TNF)-α agent used in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Few studies focused on the effects of anti-TNF-α on peripheral blood cells. We aimed to evaluate peripheral blood cells in RA and PsA patients during ETN treatment and to explore their relationships with disease activity. RA (n = 82) and PsA (n = 32) patients who started ETN were included into the study and evaluated prospectively before the beginning of ETN therapy and after 14, 22, 54 and 102 weeks. Patients were studied in terms of disease activity score on 28 joints (DAS28), clinical response and laboratory findings. Natural killer (NK) cells, B cells and T cells were characterized by immunophenotyping. Both the RA and the PsA patients showed reduced NK and B cell count before ETN treatment compared with controls. A negative correlation was demonstrated between DAS28 and B cell count in RA patients at baseline. Sustained significant increase of NK and B cells up to normal levels was observed in RA and PsA patients along ETN treatment. Increase of NK cell count was associated with a good-moderate clinical response to ETN in both RA and PsA patients. During ETN treatment peripheral blood NK and B cells levels were restored in RA and PsA patients. Correlations between NK and B cells with disease activity were observed, suggesting that those effects could be mediated by ETN treatment.

Preventive oral surgery before bisphosphonate administration to reduce osteonecrosis of the jaws.

OBJECTIVES: The intravenous injection of bisphosphonates, currently used for osteoporosis, myeloma, or bone metastases, can cause ONJ especially in consequence of trauma. To avoid trauma during bisphosphonate treatment, preventive oral surgery is recommended. The research aimed to evidence whether inflammatory and osteoclastogenic factors are not induced in oral mucosa after bisphosphonate treatment in patients receiving oral preventive surgery procedure and whether proliferation factors are not inhibited. PATIENTS AND METHODS: Specimens of oral mucosa were removed from healthy subjects and from patients undergoing preventive oral surgery before bisphosphonate treatment. The expression of cytokines and factors involved in osteoclast activity, cell proliferation, and angiogenesis were examined. RESULTS: Cytokines and RANK-L levels decreased significantly in mucosa from patients undergoing preventive oral surgery procedure before bisphosphonate treatment in comparison with their levels at the beginning of procedure and also in comparison with the level in patients treated only with bisphosphonates and not developing ONJ; conversely, osteoprotegerin and hydroxymethylglutaryl coenzyme A reductase significantly increased or not changed. CONCLUSIONS: The results suggest that preventive oral surgery could be able to prevent ONJ due to bisphosphonate treatment: The mucosa is not stimulated by bisphosphonates to cause ONJ, as bisphosphonates are probably not released from the bone.

Colonization by human fibroblasts of polypropylene prosthesis in a composite form for hernia repair.

PURPOSE: Abdominal wall hernia is one of the commonest surgical disorders worldwide, and there is no single gold-standard operative technique to repair it. In an effort to improve techniques and technologies to reinforce hernia repair, synthetic meshes are employed. In this study, a new prosthesis (named composite) formed of two polypropylene layers, one macroporous (named mesh) and one transparent (named film), was examined to evaluate its capability to enable cell proliferation without inducing cell death. Inflammatory processes were also examined. METHODS: Human fibroblasts BJ were seeded on multiwells, on which composite or film had been placed. After 7, 14, and 21 days, cell growth and viability, deposition of collagen, and release of IL-6, IL-1ß, and TNF-α were evaluated. RESULTS: The "in vitro" protocol showed the composite to be colonized by human fibroblasts on the polypropylene macroporous mesh side; no cell growth occurred on the film. The slowdown of cell growth observed between 14 and 21 days was accompanied by an increase in type I collagen deposition and marked fibroblast activity. Inflammatory cytokines initially increased, followed by their reduction beginning at 14 days. CONCLUSIONS: The new prosthesis comprising two polypropylene layers of differing morphologies can be colonized by fibroblasts on the side facing the abdominal wall, whereas no cell growth occurs on the side facing the viscera. The transient inflammation, observed at early experimental times, is probably important for the healing process.

Hydroxyapatite paste Ostim, without elevation of full-thickness flaps, improves alveolar healing stimulating BMP- and VEGF-mediated signal pathways: an experimental study in humans.

OBJECTIVE: Tooth extraction is considered as the starting point of jaw atrophy via osteoclast activity stimulation. The maintenance of dental alveolar bone depends on surgery procedure and use of materials to maintain prior space favoring bone regeneration. Among substitutes used in dentistry to fill bone defects, Ostim-Pastes (Ostim) is a nanocrystalline paste tested for treatment of severe clinical conditions. This research first investigated the effect of Ostim on alveolar healing, comparing in the same healthy subjects, an Ostim-filled socket with a not-filled one. Moreover, it also proposed a new surgical protocol for the post-extractive socket treatment using the graft materials without elevation of full-thickness flaps. MATERIAL AND METHODS: Fourteen patients were enrolled to bilateral maxillary or mandibular extraction that was performed without elevation of full-thickness flaps. In each patient, one socket was filled using Ostim, and the other one was allowed to undergo natural healing. No suture was carried out. Clinical and biologic parameters were screened at 1, 7, and 14 days. RESULTS: Obtained results evidenced that nanocrystalline hydroxyapatite supports bone regeneration, increasing the synthesis of pro-osteogenic factors as bone morphogenetics protein (BMP)-4, BMP-7, alkaline phosphatase, and osteocalcin. Moreover, filling post-extractive socket with nanocrystalline hydroxyapatite paste leads to a complete epithelialization already at 7 days after extraction, despite the fact that the teeth were extracted without elevation of full-thickness flaps . The improved epithelialization is mediated by increased vascular endothelial growth factor (VEGF) expression. No significant change was observed in inflammatory parameters, with exception of an early and transient IL-1ß induction, that could trigger and improve alveolar healing. CONCLUSIONS: Clinical and biomolecular observations of this explorative study evidenced that nanocrystalline hydroxyapatite improves alveolar socket healing, increasing angiogenesis, epithelialization, and osteogenesis, also in absence of elevation of full-thickness flaps.

Exposing human epithelial cells to zoledronic acid can mediate osteonecrosis of jaw: an in vitro model.

BACKGROUND: Osteonecrosis of the jaw (ONJ) is a chronic complication of bisphosphonate therapy, mainly when intravenous, in cancer patients with bone metastases and myeloma. Its pathophysiology is not yet fully elucidated; in particular, the molecular/cellular events triggering ONJ remain unclear. This complication could result from the effect of bisphosphonates released from bone into the soft-tissues, or from osteolysis induced by soft-tissues directly exposed to bisphosphonates. This research investigated the possibility that ONJ may be evocated by changes induced in osteoblast activity by factors released by soft-tissue cells exposed to zoledronic acid. METHODS: An 'in vitro' model was used, in which human osteoblast-like MG-63 cells were grown in medium conditioned by human keratinocytes NCTC 2544, exposed or not to zoledronic acid (5 or 50 µM); 5 µM zoledronic acid was also directly administered to MG-63 cells. RESULTS: In NCTC 2544 cells, zoledronic acid decreased proliferation via decreased hydroxy-3-methyl-glutaryl-CoA reductase, suggesting that a decrease in healing capability can occur in case of injury. An increased pro-inflammatory potential was also observed. Osteoblasts grown in medium conditioned in the presence of zoledronic acid showed decreased proliferation and osteogenic properties, and increased ability to induce osteoclast differentiation and inflammatory process. Zoledronic acid directly administered to MG-63 modulated only some parameters and in a lesser extent. CONCLUSIONS: The research evidenced, for the first time, the direct involvement of epithelial cells in zoledronic acid-triggered molecular mechanisms leading to osteonecrosis of the jaw, by modulating both osteoblast and osteoclast properties.

Complement system in psoriatic arthritis: a useful marker in response prediction and monitoring of anti-TNF treatment.

OBJECTIVES: Treatment with anti-TNF agents is well established in psoriatic arthritis (PsA). Anti-TNF agents are capable of modulating complement activity in vitro but there are no data on the in vivo effect. Anti-TNF have high costs and potential risks, thus, there is an urgent need for accurate predictors of response. We aimed at studying the usefulness of erythrocyte-sedimentation-rate (ESR), C-reactive protein (CRP), and complement for response prediction and monitoring of anti-TNF treatment in PsA patients. METHODS: Fifty-five patients were included consecutively before starting etanercept or adalimumab. ESR, CRP, plasma complement C3, C4, and C3 and B cleavage fragments were evaluated at baseline and after 22 weeks of anti-TNF treatment. Disease activity was measured with DAS28 and response to therapy with EULAR criteria. Complement was evaluated at baseline in 30 healthy subjects as well. RESULTS: At baseline, C3 and C4 levels were significantly higher than in controls (C3 126.9±22 vs. 110±25 mg/dl, p=0.000002; C4 31.2±9.2 vs. 22.7±8.3 mg/dl, p=0.0003). After anti-TNF therapy, C3 and C4 levels were significantly reduced to normalization (p=0.0009 and 0.0005, respectively) and ESR, CRP and DAS28 showed a significant reduction (p=0.002, 0.004 and 0.0001, respectively). Split products of C3 and B were not observed at baseline and after 22 weeks. Higher baseline C3 levels were associated with EULAR non-response (p=0.011). CONCLUSIONS: PsA patients with moderate to severe disease show elevated C3 and C4 levels, reverted by anti-TNF treatment. High C3 may be considered a hallmark of inflammation and C3 revealed the highest predictive value for response to anti-TNF.

Aldehyde dehydrogenases and cell proliferation.

Aldehyde dehydrogenases (ALDHs) oxidize aldehydes to the corresponding carboxylic acids using either NAD or NADP as a coenzyme. Aldehydes are highly reactive aliphatic or aromatic molecules that play an important role in numerous physiological, pathological, and pharmacological processes. ALDHs have been discovered in practically all organisms and there are multiple isoforms, with multiple subcellular localizations. More than 160 ALDH cDNAs or genes have been isolated and sequenced to date from various sources, including bacteria, yeast, fungi, plants, and animals. The eukaryote ALDH genes can be subdivided into several families; the human genome contains 19 known ALDH genes, as well as many pseudogenes. Noteworthy is the fact that elevated activity of various ALDHs, namely ALDH1A2, ALDH1A3, ALDH1A7, ALDH2*2, ALDH3A1, ALDH4A1, ALDH5A1, ALDH6, and ALDH9A1, has been observed in normal and cancer stem cells. Consequently, ALDHs not only may be considered markers of these cells, but also may well play a functional role in terms of self-protection, differentiation, and/or expansion of stem cell populations. The ALDH3 family includes enzymes able to oxidize medium-chain aliphatic and aromatic aldehydes, such as peroxidic and fatty aldehydes. Moreover, these enzymes also have noncatalytic functions, including antioxidant functions and some structural roles. The gene of the cytosolic form, ALDH3A1, is localized on chromosome 17 in human beings and on the 11th and 10th chromosome in the mouse and rat, respectively. ALDH3A1 belongs to the phase II group of drug-metabolizing enzymes and is highly expressed in the stomach, lung, keratinocytes, and cornea, but poorly, if at all, in normal liver. Cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons or chlorinated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, in rat liver cells and increases during carcinogenesis. It has been observed that this increased activity is directly correlated with the degree of deviation in hepatoma and lung cancer cell lines, as is the case in chemically induced hepatoma in rats. High ALDH3A1 expression and activity have been correlated with cell proliferation, resistance against aldehydes derived from lipid peroxidation, and resistance against drug toxicity, such as oxazaphosphorines. Indeed, cells with a high ALDH3A1 content are more resistant to the cytostatic and cytotoxic effects of lipidic aldehydes than are those with a low content. A reduction in cell proliferation can be observed when the enzyme is directly inhibited by the administration of synthetic specific inhibitors, antisense oligonucleotides, or siRNA or indirectly inhibited by the induction of peroxisome proliferator-activated receptor γ (PPARγ) with polyunsaturated fatty acids or PPARγ transfection. Conversely, cell proliferation is stimulated by the activation of ALDH3A1, whether by inhibiting PPARγ with a specific antagonist, antisense oligonucleotides, siRNA, or a medical device (i.e., composite polypropylene prosthesis for hernia repair) used to induce cell proliferation. To date, the mechanisms underlying the effects of ALDHs on cell proliferation are not yet fully clear. A likely hypothesis is that the regulatory effect is mediated by the catabolism of some endogenous substrates deriving from normal cell metabolism, such as 4-hydroxynonenal, which have the capacity to either stimulate or inhibit the expression of genes involved in regulating proliferation.

Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent.

Scaffolds based on gelatin (G) are considered promising for tissue engineering, able to mimic the natural extracellular matrix. G drawback is its poor structural consistency in wet conditions. Therefore, crosslinking is necessary to fabricate stable G scaffolds. In this work, a comparative study between the performance of two different crosslinkers, genipin (GP) and γ-glycidoxypropyltrimethoxysilane (GPTMS), is presented. Flat membranes by solvent casting and porous crosslinked scaffolds by freeze-drying were prepared. Infrared spectroscopy and thermal analysis were applied to confirm G chain crosslinking. Moreover, GP and GPTMS increased the stability of G in aqueous media and improved the mechanical properties. Crosslinking reduced the wettability, especially in the case of G_GPTMS samples, due to the introduction of hydrophobic siloxane chains. Both G_GP and G_GPTMS scaffolds supported MG-63 osteoblast-like cell adhesion and proliferation.

Complement system and rheumatoid arthritis: relationships with autoantibodies, serological, clinical features, and anti-TNF treatment.

Autoantibodies (rheumatoid factor, RF; anti-citrullinated-protein antibodies, ACPA) and complement system are involved in rheumatoid arthritis (RA). ACPA and anti-TNF agents are capable of in vitro modulating complement activity. We investigated the relationships between complement, autoantibodies, and anti-TNF treatment in vivo. One-hundred fourteen RA patients (89F/25M), diagnosed according to 1987 ACR criteria, and 30 healthy controls were enrolled. Serological analysis included ESR, CRP, complement C3, C4 and CH50, RF and ACPA (ELISA, cut-off>20 U/ml). Split-products (SP) of C3 and B were studied by immunoelectrophoresis/counterimmunoelectrophoresis. Seventy-six patients started anti-TNF treatment and were studied at baseline and after 22 weeks. Disease activity was measured with DAS28 and response to therapy with EULAR criteria. At baseline, RA patients showed significantly higher levels of C3 and C4 than controls (C3 127.9±26.5 vs 110±25 mg/dl, P=0.0012; C4 29.7±10.2 vs 22.7±8.3mg/dl, P=0.0003). No differences in C3, C4 and CH50 levels were observed between ACPA+ (n=76) and ACPA- (n=38) patients. After 22 weeks of anti-TNF, C3, C4 and RF were significantly reduced (P<0.003, <0.005 and <0.04, respectively) and RF changes showed negative correlation with CH50. SP of C3 and B were observed neither at baseline nor after 22 weeks. DAS28 significantly improved after 22 weeks. Patients showing higher baseline C3 or lower reduction of C3 levels after 22 weeks had a worse EULAR outcome (X2=22.793, P<0.001). RF levels seem to correlate with complement CH50. The presence of high levels of C3 in RA patients may reflect a pro-inflammatory status and represent a negative prognostic factor for anti-TNF therapy.

Mechanisms of clofibrate-induced apoptosis in Yoshida AH-130 hepatoma cells.

Peroxisome proliferators (PPs) are a class of compounds that exert their nominal effects through the peroxisome proliferator-activated receptors. PPs, among which clofibrate (CF), have been extensively studied for their hepatocarcinogenic properties in rodents, generally ascribed to their antiapoptotic action. However, previous results demonstrated that various PPs may also have apoptogenic properties. CF, in particular, promptly induces a massive apoptotic death in cell lines established from murine or human hepatomas and from breast or lung cancers as well. The present study was aimed at elucidating the apoptotic pathway(s) triggered by CF in AH-130 cells. The results show that CF-induced cell death is completely blocked by the poly-caspase inhibitor z-VAD-fmk and that caspases 3, 8, and 9 are early activated. Consistently, cytochrome c is released from mitochondria, and CF cytotoxicity is inhibited by cyclosporine A, partially at least. In addition, the occurrence of endoplasmic reticulum (ER) stress is suggested by the observation that the levels of phosphorylated eIF2alpha and JNK increase in CF-treated cells, while the caspase 2 precursor protein levels are concurrently reduced. Finally, some degree of calpain activation also takes place, as suggested by the appearance of fodrin cleavage products. The present findings demonstrate that CF-induced apoptosis in the Yoshida AH-130 cells basically is a caspase-dependent process that involves more than a single mechanisms. Activation of the intrinsic apoptotic pathway and ER stress both play a major and concurrent role, while calpain activation seems to have only a marginal part in the process.

Involvement of PPARs in Cell Proliferation and Apoptosis in Human Colon Cancer Specimens and in Normal and Cancer Cell Lines.

PPAR involvement in cell growth was investigated "in vivo" and "in vitro" and was correlated with cell proliferation and apoptotic death. "In vivo" PPARgamma and alpha were evaluated in colon cancer specimens and adjacent nonneoplastic colonic mucosa. PPARgamma increased in most cancer specimens versus mucosa, with a decrease in c-Myc and in PCNA proteins, suggesting that colon cancer growth is due to increased cell survival rather than increased proliferation. The prevalence of survival over proliferation was confirmed by Bcl-2 or Bcl-X(L) increase in cancer versus mucosa, and by decreased PPARalpha. "In vitro" PPARgamma and PPARalpha were evaluated in human tumor and normal cell lines, treated with natural or synthetic ligands. PPARgamma was involved in inhibiting cell proliferation with a decrease in c-Myc protein, whereas PPARalpha was involved in inducing apoptosis with modulation of Bcl-2 and Bad proteins. This involvement was confirmed using specific antagonists of two PPARs. Moreover, the results obtained on treating cell lines with PPAR ligands confirm observations in colon cancer: there is an inverse correlation between PPARalpha and Bcl-2 and between PPARgamma and c-Myc.

Effect of superpulsed laser irradiation on bone formation in a human osteoblast-like cell line.

AIM: The effect superpulsed of low-level laser therapy (SLLLT) on bone regeneration has been the focus of recent research. This preliminary study investigated the effect of superpulsed laser irradiation on proliferation and bone formation in human osteoblast-like cells MG-63. METHODS: Human osteoblast-like cells MG-63 were exposed every 24 h to superpulsed low-level laser produced by the device Lumix 2 HFPL Dental (Fisioline s.n.c., Verduno, Cuneo, Italy); the experimental protocol comprised 4 days of treatment. At each experimental time, cell proliferation and some markers of osteoblast activity were evaluated. RESULTS: Numbers of laser-treated cells increased starting from day 2 of treatment. The ability of SLLLT irradiation to stimulate bone production was evaluated by determining the expression of osteocalcin and alkaline phosphatase, proteins involved in calcium nodule formation. These proteins increased markedly after 3 days of laser treatment. CONCLUSIONS: These preliminary results show that repeated SLLLT irradiation stimulates cell proliferation in human osteoblast-like cells and, importantly, increases the expression of proteins essential for bone formation.

Development of glass-ceramic scaffolds for bone tissue engineering: characterisation, proliferation of human osteoblasts and nodule formation.

Glass-ceramic macroporous scaffolds for tissue engineering have been developed using a polyurethane sponge template and bioactive glass powders. The starting glass (CEL2) belongs to the system SiO(2)-P(2)O(5)-CaO-MgO-Na(2)O-K(2)O and has been synthesised by a conventional melting-quenching route. A slurry of CEL2 powder, polyvinyl alcohol and water has been prepared in order to coat, by impregnation, the polymeric template. An optimised thermal treatment was then use to remove the sponge and to sinter the glass powders, leading to a glass-ceramic replica of the template. Morphological observations, image analyses, mechanical tests and in vitro tests showed that the obtained devices are good candidates as scaffolds for bone-tissue engineering, in terms of pore-size distribution, pore interconnection, surface roughness, and both bioactivity and biocompatibility. In particular, a human osteoblast cell line (MG-63) seeded onto the scaffold after a standardised preconditioning route in simulated body fluid showed a high degree of cell proliferation and a good ability to produce calcium nodules. The obtained results were enhanced by the addition of bone morphogenetic proteins after cell seeding.

Impact of peritoneal tears on the outcome and late results (4 years) of endoscopic totally extra-peritoneal inguinal hernioplasty.

BACKGROUND: The reported rate of intra-operative peritoneal laceration during endoscopic extra-peritoneal hernioplasty (TEP) ranges from 10 to 64%. AIMS: To evaluate in a prospective study the predictive factors of peritoneal tears, their consequences in terms of outcome and late results. PATIENTS AND METHODS: Between July 1994 and December 2000, we performed 467 endoscopic extra-peritoneal hernia repairs (TEP). In 14.8% of the cases, single or multiples recurrences after conventional open herniotomy were treated. One hundred and forty-nine patients (38%) had had previous surgery (appendectomy); 277 procedures (70.8%) were performed by experienced surgeons and 114 (29.2%) by surgical trainees. We used a diathermic hook in 26.3% of the procedures. The mean follow-up period was 68 months (48-100). RESULTS: Peritoneal tears occurred in 43 patients (10.9%). Six of them (13%) required operative closure, and six a conversion (four Lichtenstein, one Shouldice, and one TAPP). In 37 cases (86%), the tears were not closed. Peritoneal tears were significantly correlated with surgical experience, Nyhus classification, scar adhesion from previous surgery and the use of sharp instruments. Peritoneal tears interfere significantly (P=0.001) with the operating time (82 vs. 63 min) and conversion rate (13.9 vs. 1.7%). It does not affect the outcome and late results in terms of recurrences, pain, or small bowel obstruction. CONCLUSION: Our data suggest that peritoneal tears in the vast majority of cases may be safely managed without peritoneal closure. In case of peritoneal laceration, the operative time was significantly longer, and the conversion rate was increased. These situations do not affect the outcome and late complications compared with the procedures without peritoneal tears.

HMG-CoA reductase and PPARalpha are involved in clofibrate-induced apoptosis in human keratinocytes.

Contrasting data have been reported on the effects of clofibrate, a PPARalpha agonist and hypolipidemic drug. The carcinogenic and anti-apoptotic effects have been demonstrated especially in rodents in both "in vivo" and "in vitro" experiments. In contrast, in rat and human hepatoma cell lines, several reports have shown its concentration-dependent pro-apoptotic effect. No epidemiological data exist about its carcinogenetic effect in man. This study shows that clofibrate also induced apoptosis in a human non-tumour cell line, NCTC 2544, which shares the characteristic of proliferation with tumour cells. Both HMG-CoA reductase and PPARalpha were found to be involved in the signal transduction pathway inducing apoptosis, the former being the principal target: HMG-CoA reductase decreased and PPARalpha increased. Changes in HMG-CoA reductase expression caused activation of parameters leading to apoptosis via the mitochondria pathway. Clofibrate must be considered a pro-apoptotic molecule at concentrations of 0.25 mM and above: the effect is exercised not only on tumour cells but also on normal human proliferating cells. Clofibrate should thus be regarded as a potential drug to reduce the number of proliferating cells in pathological conditions.

Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells.

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells.

Aldehyde dehydrogenases (ALDHs) are a superfamily of several isoenzymes widely expressed in bacteria, yeast, plant and animals. Three major classes of ALDHs have been traditionally identified, classes 1, 2 and 3. Both exogenous and endogenous aldehydes, including aldehydes derived from lipid peroxidation, are oxidized by the ALDH superfamily. Several changes in ALDH isoenzyme expression take place in hepatoma cells, in particular cytosolic class 3 ALDH (ALDH3), not expressed in normal hepatocytes, appears and increases with the degree of deviation. It has been demonstrated that cytosolic ALDH3 is important in determining the resistance of tumor cells to antitumor drugs, such as cyclophosphamide. Moreover, hepatoma-associated ALDH3 seems to be important in metabolizing aldehydes derived from lipid peroxidation, and in particular the cytostatic aldehyde 4-hydroxynonenal (4-HNE). We demonstrated previously that restoring endogenous lipid peroxidation in hepatoma cells by enriching them with arachidonic acid causes a decrease of mRNA, protein and enzyme activity of ALDH3 and that this decrease reduces cell growth and/or causes cell death, depending on basal class 3 ALDH activity. To confirm the correlation between inhibition of class 3 ALDH and reduction of cell proliferation, we exposed hepatoma cells to antisense oligonucleotides (ODNs) against ALDH3. In JM2 hepatoma cell line, with high ALDH3 activity, the exposure to antisense ODNs significantly decreases mRNA and enzyme activity (90%). At the same time, cell growth was reduced by about 70%. The results confirm that in hepatoma cells ALDH3 expression is closely related with cell growth, and that its inhibition is important in reducing the proliferation of hepatoma cells overexpressing ALDH3.

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation.

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.