Rosa Angela Canuto

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.

An overview of the effect of linoleic and conjugated-linoleic acids on the growth of several human tumor cell lines.

Both n‐6 and n‐3 polyunsaturated fatty acids are dietary fats important for cell function, being involved in several physiologic and pathologic processes, such as tumorigenesis. Linoleic acid and conjugated linoleic acid, its geometrical and positional stereoisomer, were tested on several human tumor cell lines originating from different tissues and with different degrees of malignancy. This was to provide the widest possible view of the impact of dietary lipids on tumor development. While linoleic acid exerted different effects, ranging from inhibitory to neutral, even promoting growth, conjugated linoleic acid inhibited growth in all lines tested and was particularly effective against the more malignant cells, with the exception of mammary tumor cells, in which behavior was the opposite, the more malignant cell line being less affected. The inhibitory effect of conjugated linoleic acid on growth may be accompanied by different contributions from apoptosis and necrosis. The effects of conjugated linoleic acid on growth or death involved positive or negative variations in PPARs. The important observation is that a big increase of PPARα protein occurred in cells undergoing strong induction of apoptosis, whereas PPARβ/δ protein decreased. Although PPARα and PPARβ/δ seem to be correlated to execution of the apoptotic program, the modulation of PPARγ appears to depend on the type of tumor cell, increasing as protein content, when inhibition of cell proliferation occurred. In conclusion, CLA may be regarded as a component of the diet that exerts antineoplastic activity and its effect may be antiproliferative or pro‐apoptotic.

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.

Cholesterol and phospholipid composition of mitochondria and microsomes isolated from morris hepatoma 51 23 and rat liver

The major component of membranous lipids is represented by phospholipids [l] . It is well known that this class of lipids plays an important role in regulating the biochemical properties of subcellular particles [2] . Moreover, there exists a cholesterol:phospholipid ratio peculiar for several kinds of membranes [3]. Cholesterol is important in stabilizing arrays of phospholipids in cellular and cytoplasmic membranes [4,5] , and in regulating the permeability to small molecules of artificial [6,7] and natural [8] membranes. Lipid-lipid interactions play an important role in the interaction between lipid and protein [3] ; the protein conformation is greatly influenced by their association with the lipid environment [9]. Alterations in the protein components of mitochondrial membranes have been observed in hepatoma mitochondria [ 10,l l] . They could be, at least in part, explained by changes in the lipid composition of membranes. In hepatoma mitochondria, we have also found functional changes, which could be related to high fragility and to an increased resistance to deformation and stretching [ 12,131 . Similar alterations have been observed also in cholesterol-enriched mitochondria [5] . The knowledge of lipid composition of membranes isolated from tumors is lacking and, sometimes, contrasting [ 14,151 . As a first approach to this problem we have studied the phospholipid and cholesterol content of mitochondria and microsomes isolated from rat liver and Morris hepatoma 5 123. 2. Methods

TNF‐α TGF‐β2 and IL‐1β levels in gingival and peri‐implant crevicular fluid before and after de novo plaque accumulation

AIMS The aim of this split-mouth study was to investigate levels of tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta2) and interleukin-1 beta (IL-1beta) in gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) after a 21-day-period of de novo plaque accumulation in the same patient. MATERIAL AND METHODS In 25 patients, samples of GCF and PICF were collected in the sulcus of the tooth and of the implant after professional hygiene. After the no-hygiene phase (21 days), second samples of GCF and PICF were taken. Third samples were collected after 69 days of re-establishment oral hygiene techniques. The crevicular fluids were used to determine the volume and the levels of TNF-alpha, TGF-beta2 and IL-1beta. RESULTS The volume of the crevicular fluids increased significantly after 21 days of plaque accumulation around teeth and implants and decreased significantly by 69 days. TNF-alpha and TGF-beta2 did not change significantly among the three different samples. A significant increase of IL-1beta was observed after plaque accumulation around the teeth GCF, whereas in the PICF the increase was not statistically significant. CONCLUSIONS These data suggest that increased volumes of GCF and PICF could be useful markers of early inflammation in gingival and peri-implant tissues. In the presence of de novo plaque, implants showed lower, and nearly significant, levels of IL-1beta compared with teeth.

PPARγ-dependent effects of conjugated linoleic acid on the human glioblastoma cell line (ADF)

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator‐activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR α, β/δ and γ, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARγ results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARγ ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high‐grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARγ ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARγ agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARγ activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the γ‐agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.

Superpulsed Laser Irradiation Increases Osteoblast Activity Via Modulation of Bone Morphogenetic Factors

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.

New Data on Kinetics of Lipid Peroxidation in Experimental Hepatomas and Preneoplastic Nodules

Lipid peroxidation has been found decreased in several hepatomas. The decline has been shown already at the level of preneoplastic nodules obtained after DEN treatment of rats. A substantial exception is represented by the hepatoma cell line MH1C1, deriving from a slightly deviated Morris tumor. Most of the described experiments estimated lipid peroxidation levels in terms of malonaldehyde production by the thiobarbituric acid test. It is now clear that this test does not account for several other aldehydes produced during lipid peroxidation. We now investigated by high performance liquid chromatography (HPLC) the whole range of non-polar aldehydes produced by tumor homogenates and by preneoplastic nodules both in basal conditions and after stimulation with ADP-iron or ascorbate. It was reduced in the preneoplastic nodules as well as in the DEN-induced hepatoma. The susceptibility to the prooxidant effect of ADP-iron or ascorbate was strongly decreased in all hepatomas as well as in preneoplastic nodules. It has been recently published that hepatoma cells are more susceptible than normal liver to the toxic action of aldehydes. This was attributed at least in part to the decreased activity of aldehyde dehydrogenases, as well as to their different distribution in tumor cells. A deeper study on aldehyde metabolism in hepatomas has shown that alcohol dehydrogenase and NADPH-aldehyde reductase also are markedly decreased in Yoshida hepatoma cells and the MH1C1 cell line. However, glutathione transferase, that can use hydroxynonenal as a substrate, is strongly decreased in Yoshida hepatoma cells but not in MH1C1 cells.

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” PPARγ and α were evaluated in colon cancer specimens and adjacent nonneoplastic colonic mucosa. PPARγ 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-XL increase in cancer versus mucosa, and by decreased PPARα. “In vitro” PPARγ and PPARα were evaluated in human tumor and normal cell lines, treated with natural or synthetic ligands. PPARγ was involved in inhibiting cell proliferation with a decrease in c-Myc protein, whereas PPARα 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 PPARα and Bcl-2 and between PPARγ and c-Myc.

The impact of plasma rich in growth factors on clinical and biological factors involved in healing processes after third molar extraction

Extraction of an impacted mandibular third molar is a common surgical procedure, although it still leads to several postoperative symptoms and complications. The study assessed the efficacy of autologous plasma rich in growth factors (PRGF) in the healing process by checking the difference of tissue cytokines and other healing factors produced by the mucosa after extraction between sites treated with PRGF and control sites and, at the same time, by evaluating the clinical efficacy of PRGF in terms of reduced pain and facial swelling. This study was a split-mouth study, in which the patient becomes his/her own control, to eliminate any individual response differences toward PRGF treatment. The parameters regarding inflammation and subsequent wound healing were all significantly higher at PRGF sites than at control sites. The increase at PRGF sites of the two proinflammatory cytokines evaluated, interleukin (IL)-1β and IL-6, was accompanied by the increase of two anti-inflammatory cytokines, IL-10 and transforming growth factor-β. Furthermore, IL-1β and IL-6 induce fibroblast and keratinocyte proliferation, important events in wound healing. Postoperative pain and the swelling, measured at all experimental times, were reduced in the presence of PRGF.