Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine.

CLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cells.

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor "in vivo", excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1ß and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.

Effect of n-3 fatty acids on patients with advanced lung cancer: a double-blind, placebo-controlled study.

PUFA from fish oil appear to have anti-inflammatory and anti-oxidative effects and improve nutritional status in cancer patients. With this as background, the aim of the present study was to investigate the effect of EPA plus DHA on inflammatory condition, and oxidative and nutritional status in patients with lung cancer. In our multicentre, randomised, double-blind trial, thirty-three patients with a diagnosis of advanced inoperable non-small-cell lung cancer and undergoing chemotherapy were divided into two groups, receiving four capsules/d containing 510 mg of EPA and 340 mg of DHA, or 850 mg of placebo, for 66 d. At the start of chemotherapy (T0), after 8 d (T1), 22 d (T2) and 66 d (T3), biochemical (inflammatory and oxidative status parameters) and anthropometric parameters were measured in both groups. A significant increase of body weight in the n-3 group at T3 v. T0 was observed. Concerning inflammation, C-reactive protein and IL-6 levels differed significantly between the n-3 and placebo groups at T3, and progressively decreased during chemotherapy in the n-3 group, evidencing n-3 PUFA anti-inflammatory action. Concerning oxidative status, plasma reactive oxygen species levels increased in the placebo group v. the n-3 group at the later treatment times. Hydroxynonenal levels increased in the placebo group during the study, while they stabilised in the n-3 group. Our data confirm that the continual assumption of EPA plus DHA determined an anti-inflammatory and anti-oxidative action which could be considered a preliminary goal in anti-cachectic therapy.

Conjugated linoleic acid prevents cell growth and cytokine production induced by TPA in human keratinocytes NCTC 2544.

Conjugated linoleic acid (CLA) is reported to have anti-cancer activity, based on animal and in vitro studies. Since it has been suggested that CLA anti-carcinogenic effect stems from its anti-inflammatory properties, this study investigated whether CLA can prevent cell proliferation induced by TPA in human keratinocytes NCTC 2544 contemporary to inhibition of inflammation. Results obtained showed that CLA prevents increased cell proliferation and production of pro-inflammatory molecules determined by TPA, being this effect due to modulation of PPARs and NFkB activity. The involvement of PPARalpha in CLA effect was demonstrated by adding to the cells an antagonist of PPARalpha.

Decreased polyunsaturated Fatty Acid content contributes to increased survival in human colon cancer.

Among diet components, some fatty acids are known to affect several stages of colon carcinogenesis, whereas others are probably helpful in preventing tumors. In light of this, our aim was to determine the composition of fatty acids and the possible correlation with apoptosis in human colon carcinoma specimens at different Duke's stages and to evaluate the effect of enriching human colon cancer cell line with the possible reduced fatty acid(s). Specimens of carcinoma were compared with the corresponding non-neoplastic mucosa: a significant decrease of arachidonic acid, PPARalpha, Bad, and Bax and a significant increase of COX-2, Bcl-2, and pBad were found. The importance of arachidonic acid in apoptosis was demonstrated by enriching a Caco-2 cell line with this fatty acid. It induced apoptosis in a dose- and time-dependent manner via induction of PPARalpha that, in turn, decreased COX-2. In conclusion, the reduced content of arachidonic acid is likely related to carcinogenic process decreasing the susceptibility of cancer cells to apoptosis.

Superpulsed laser irradiation increases osteoblast activity via modulation of bone morphogenetic factors.

BACKGROUND AND OBJECTIVE: Laser therapy is a new approach applicable in different medical fields when bone loss occurs, including orthopedics and dentistry. It has also been used to induce soft-tissue healing, for pain relief, bone, and nerve regeneration. With regard to bone synthesis, laser exposure has been shown to increase osteoblast activity and decrease osteoclast number, by inducing alkaline phosphatase (ALP), osteopontin, and bone sialoprotein expression. Studies have investigated the effects of continuous or pulsed laser irradiation, but no data are yet available on the properties of superpulsed laser irradiation. This study thus aimed to investigate the effect of superpulsed laser irradiation on osteogenic activity of human osteoblast-like cells, paying particular attention to investigating the molecular mechanisms underlying the effects of this type of laser radiation. STUDY DESIGN/MATERIALS AND METHODS: Human osteoblast-like MG-63 cells were exposed to 3, 7, or 10 superpulsed laser irradiation (pulse width 200 nanoseconds, minimum peak power 45 W, frequency 30 kHz, total energy 60 J, exposure time 5 minutes). The following parameters were evaluated: cell growth and viability (light microscopy, lactate dehydrogenase release), calcium deposits (Alizarin Red S staining), expression of bone morphogenetic factors (real-time PCR). RESULTS: Superpulsed laser irradiation decreases cell growth, induces expression of TGF-beta2, BMP-4, and BMP-7, type I collagen, ALP, and osteocalcin, and increases the size and the number of calcium deposits. The stimulatory effect is maximum on day 10, that is, after seven applications. CONCLUSIONS: Reported results show that superpulsed laser irradiation, like the continuous and pulsed counterparts, possesses osteogenic properties, inducing the expression of molecules known to be important mediators of bone formation and, as a consequence, increasing calcium deposits in human MG-63 cells. Moreover, the data suggest a new potential role for PPARgamma as a regulator of osteoblast proliferation.

Biocompatible glass-ceramic materials for bone substitution.

A new bioactive glass composition (CEL2) in the SiO(2)-P(2)O(5)-CaO-MgO-K(2)O-Na(2)O system was tailored to control pH variations due to ion leaching phenomena when the glass is in contact with physiological fluids. CEL2 was prepared by a traditional melting-quenching process obtaining slices that were heat-treated to obtain a glass-ceramic material (CEL2GC) that was characterized thorough SEM analysis. Pre-treatment of CEL2GC with SBF was found to enhance its biocompatibility, as assessed by in vitro tests. CEL2 powder was then used to synthesize macroporous glass-ceramic scaffolds. To this end, CEL2 powders were mixed with polyethylene particles within the 300-600 microm size-range and then pressed to obtain crack-free compacted powders (green). This was heat-treated to remove the organic phase and to sinter the inorganic phase, leaving a porous structure. The biomaterial thus obtained was characterized by X-ray diffraction, SEM equipped with EDS, density measurement, image analysis, mechanical testing and in vitro evaluation, and found to be a glass-ceramic macroporous scaffold with uniformly distributed and highly interconnected porosity. The extent and size-range of the porosity can be tailored by varying the amount and size of the polyethylene particles.

Cytokines and growth factors involved in the osseointegration of oral titanium implants positioned using piezoelectric bone surgery versus a drill technique: a pilot study in minipigs.

BACKGROUND: Most dental implants are positioned using a drilling surgery technique. However, dentistry recently experienced the implementation of piezoelectric surgery. This technique was introduced to overcome some of the limitations involving rotating instruments in bone surgery. This study used biomolecular and histologic analyses to compare the osseointegration of porous implants positioned using traditional drills versus the piezoelectric bone surgery technique. METHODS: Porous titanium implants were inserted into minipig tibias. Histomorphology and levels of bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-beta2, tumor necrosis factor-alpha, and interleukin-1beta and -10 were evaluated in the peri-implant osseous samples. RESULTS: Histomorphological analyses demonstrated that more inflammatory cells were present in samples from drilled sites. Also, neo-osteogenesis was consistently more active in bone samples from the implant sites that were prepared using piezoelectric bone surgery. Moreover, bone around the implants treated with the piezoelectric bone surgery technique showed an earlier increase in BMP-4 and TGF-beta2 proteins as well as a reduction in proinflammatory cytokines. CONCLUSION: Piezoelectric bone surgery appears to be more efficient in the first phases of bone healing; it induced an earlier increase in BMPs, controlled the inflammatory process better, and stimulated bone remodeling as early as 56 days post-treatment.

Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs.

Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.

Arachidonic acid suppresses growth of human lung tumor A549 cells through down-regulation of ALDH3A1 expression.

Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) in certain normal and tumor cells is associated with protection against the growth inhibitory effect of reactive aldehydes generated during membrane lipid peroxidation. We found that human lung tumor (A549) cells, which express high levels of ALDH3A1 protein, were significantly less susceptible to the antiproliferative effects of 4-hydroxynonenal compared to human hepatoma HepG2 or SK-HEP-1 cells that lack ALDH3A1 expression. However, A549 cells became susceptible to lipid peroxidation products when they were treated with arachidonic acid. The growth suppression of A549 cells induced by arachidonic acid was associated with increased levels of lipid peroxidation and with reduced ALDH3A1 enzymatic activity, protein, and mRNA levels. Furthermore, arachidonic acid treatment of the A549 cells resulted in an increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), whereas NF-kappaB binding activity was inhibited. Blocking PPARgamma using a selective antagonist, GW9662, prevented the arachidonic acid-mediated reduction of ALDH3A1 expression as well as the growth inhibition of A549 cells, suggesting the central role of PPARgamma in these phenomena. The increase in PPARgamma and the reduction in ALDH3A1 were also prevented by exposing cells to vitamin E concomitant with arachidonic acid treatment. In conclusion, our data show that the arachidonic acid-induced suppression of A549 cell growth is associated with increased lipid peroxidation and decreased ALDH3A1 expression, which may be due to activation of PPARgamma.

Conjugated linoleic acid induces apoptosis in MDA-MB-231 breast cancer cells through ERK/MAPK signalling and mitochondrial pathway.

We investigated the molecular mechanisms involved in the anti-proliferative activity exerted by conjugated linoleic acid (CLA) on the estrogen unresponsive MDA-MB-231 human breast cancer cell line. The effects on cell proliferation, cell cycle progression and induction of apoptosis were examined. CLA caused the reduction of cell proliferation along with the accumulation of cells in the S phase of the cycle. The occurrence of apoptosis in these cells was indicated by flow cytometry data and further confirmed by the onset of cells with morphological features typical of apoptosis. ERK1/2 reduction and upregulation of pro-apoptotic protein Bak were induced. These events were associated with: (a) reduced levels of the anti-apoptotic protein Bcl-x(L), (b) the translocation of cytochrome c from the mitochondria to the cytosol, (c) the cleavage of pro-caspase-9 and pro-caspase-3. From the above data, we are induced to think that CLA may trigger apoptosis in the estrogen unresponsive MDA-MB-231 cell line via mechanisms involving above all the mitochondrial pathway.

Differences in cell proliferation in rodent and human hepatic derived cell lines exposed to ciprofibrate.

Humans appear to be refractory to some effects of peroxisome proliferators including alterations in cell proliferation, whereas rodents are susceptible. In this study, differences between the human and rat response to peroxisome proliferators were evaluated using rat and human tumour liver cell lines. Rat 7777 cells were more responsive than human HepG2 cells to ciprofibrate as they exhibited a higher decrease in cell number than HepG2, and underwent apoptosis. Results from these studies reveal a surprising response in tumour cell lines as the typical in vivo response of increased cell proliferation and reduced apoptosis was not observed in rat tumour cell lines at concentrations greater than those used to elicit the former response.

Aldehyde dehydrogenase 3 expression is decreased by clofibrate via PPAR gamma induction in JM2 rat hepatoma cell line.

In normal liver aldehyde dehydrogenase 3 (ALDH3) is poorly expressed. In hepatoma cells, its expression increases in direct correlation with the degree of deviation and increased ALDH3 activity is one cause of resistance to the toxicity of drugs and lipid peroxidation aldehydes. Hepatoma cells with high ALDH3 content are more resistant to the cytotoxic effect of aldehydes than those with low ALDH3, and inhibition of the enzyme with aldehydes, specific inhibitors or antisense oligonucleotides (AS-ODN), decreases cell growth. It remains open how ALDH3 influences cell growth or cell phenotype. Recently, we have shown that enrichment of a highly deviated rat hepatoma cell line, JM2, with arachidonic acid, a natural ligand of peroxisome proliferator activated receptors (PPARs), inhibits growth, partially restores ALDH2 and ALDH3 to their normal levels and induces PPAR expression. In the present study we address the effect of clofibrate, a hypolipidemic drug and synthetic PPAR ligand on ALDH gene expression. We show that treatment of JM2 cells with clofibrate inhibits cell growth, induces PPARgamma and decreases ALDH3 expression. To determine the relationship between PPARgamma and ALDH3 expression, we exposed JM2 cells to AS-ODN against PPARgamma. AS-ODN reduced PPARgamma content and prevented the inhibitory effect of clofibrate on cell proliferation and ALDH3 expression. Since these results indicate that ALDH3 expression is under PPAR control, we examined the 5' flanking sequence of the ALDH3 gene, but were unable to find any sequence similar to any known peroxisome proliferator response element. We thus believe that the effect of PPARgamma on ALDH3 occurs via other transcription factors, whose identity remain to be determined. The results indicate that PPARgamma plays a key role in regulation of growth and differentiation of hepatoma cells, and that ALDH3 collaborates in modulating cell proliferation and in determining some aspects of the hepatoma phenotype, i.e. resistance to drugs and to lipid peroxidation products.

Antisense oligonucleotides against aldehyde dehydrogenase 3 inhibit hepatoma cell proliferation by affecting MAP kinases.

The increased activity of enzymes that eliminate anti-tumour drugs or their metabolites is one of the important limiting factors in therapeutic protocols. Among these enzymes, aldehyde dehydrogenase 3 (ALDH3) is considered a mechanism by which tumour cells evade the cytotoxic effects exerted by cyclophosphamide and drugs acting by free radical generation. It is also important in metabolising cytostatic aldehydes derived from lipid peroxidation. Therefore, ALDH3 may play a role in regulating cell proliferation in tumour cells with high activity of this enzyme. We previously reported that antisense oligonucleotides (AS-ODN) against ALDH3 strongly inhibit hepatoma cell growth, suggesting that this effect could be due to the accumulation of cytostatic aldehydes in the cells. In this research we demonstrate that AS-ODN against ALDH3 increase the quantity of malondialdehyde in the cells, and inhibit cell proliferation by affecting the MAPK pathway: a reduction of pRaf-1 and pERK1,2 was observed. These results confirm the importance of aldehydes derived from lipid peroxidation and of ALDH3 in regulating hepatoma proliferation. Moreover, the results indicate the use of AS-ODN against ALDH3 as a possible strategy to reduce growth in tumours overexpressing this enzyme.

Apoptosis induced by clofibrate in Yoshida AH-130 hepatoma cells: role of HMG-CoA reductase.

Clofibrate is a hypolipidemic drug belonging to the peroxisome proliferator (PP) family. PPs are well-recognized hepatocarcinogens, though only for rodents and not for humans. Their oncogenicity is usually ascribed to mitogenic or antiapoptotic action. However, we have reported that clofibrate can trigger fast and extensive apoptosis in rodent and human tumor cell lines. The present study examines the possible mechanisms involved in clofibrate-induced apoptosis in AH-130 hepatoma cells. The results show that the apoptogenic effect of clofibrate does not depend on induction of peroxisome proliferator activated receptors (PPARs), but on interference with HMG-CoA reductase (HMGR), a key enzyme that regulates cholesterol biosynthesis and production of isoprenoid units for protein farnesylation. The level and activity of HMGR mRNA are reduced in clofibrate-treated AH-130 cells and apoptosis can be partially prevented by addition of mevalonate. Moreover, cholesterol and cholesterol ester content decreases early in mitochondria, and cytocrome c is released in the cytosol. On the contrary, perturbations at the level of protein farnesylation are not important in determining the fast apoptogenic effect, since treatment of AH-130 cells with an inhibitor of farnesyltransferase induces apoptosis only after 4 h. In conclusion, inhibition of HMGR and decreased cholesterol content are crucial events in clofibrate-induced apoptosis in AH-130 hepatoma cells.