Manuela Oraldi

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.

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.

PPARα and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1

Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA‐induced apoptosis in human hepatoma SK‐HEP‐1 cells. The cells exposed to CLA were evaluated for PPARα, PP2A, pro‐apoptotic proteins Bak, Bad and caspases, and anti‐apoptotic proteins Bcl‐2 and Bcl‐XL. Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy‐14643, a specific PPARα agonist. The CLA‐induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPARα, PP2A and pro‐apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPARα agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl‐2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPARα, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK‐HEP‐1 cells, by increasing PPARα, PP2A and pro‐apoptotic proteins.

Oral mucosa produces cytokines and factors influencing osteoclast activity and endothelial cell proliferation, in patients with osteonecrosis of jaw after treatment with zoledronic acid

ObjectivesThe intravenous injection of bisphosphonates, currently used as treatment for osteoporosis, bone Paget’s disease, multiple myeloma, or bone metastases, can cause jaw bone necrosis especially in consequence of trauma. The present research aimed to clarify the mechanisms underlying bone necrosis, exploring involvement of the oral mucosa “in vivo.”Patients and methodsSpecimens of oral mucosa were removed from bisphosphonate-treated patients with or without jaw bone necrosis. In mucosa specimens, expression was evaluated of: cytokines involved in the inflammatory process, factors involved in osteoclast activity, i.e., receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin, a factor involved in cell proliferation, namely hydroxymethylglutaryl coenzyme A reductase, and a factor involved in angiogenesis, namely vascular endothelial growth factor (VEGF).ResultsInterleukin (IL)-6 and the RANK/osteoprotegerin ratio were significantly elevated in mucosa from patients with versus without jaw necrosis, whereas hydroxymethylglutaryl coenzyme A reductase and VEGF were significantly decreased.ConclusionsOur results suggest that mucosa, stimulated by bisphosphonate released from the bone, can contribute to the development of jaw necrosis, reducing VEGF, and producing IL-6 in consequence of hydroxymethylglutaryl coenzyme A reductase reduction. In turn, IL-6 stimulates osteoclast activity, as shown by the increased RANKL/osteoprotegerin ratio.Clinical relevanceThe results of this study suggest the importance of evaluating during bisphosphonate treatment the production of IL-6, RANKL, osteoprotegerin, and VEGF, in order to monitor the jaw osteonecrosis onset. To avoid repeated mucosa excisions, the determination of these factors could be carried out in crevicular fluid.

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 Dukes 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, PPARα, 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 PPARα 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.

Involvement of PPARα and PPARγ in apoptosis and proliferation of human hepatocarcinoma HepG2 cells.

Peroxisome proliferator‐activated receptors (PPARs) mediate the effects of various ligands, known as peroxisome proliferators, a heterogeneous class of compounds including industrial chemicals, pharmaceuticals, and biomolecules such as fatty acids and eicosanoids. Among peroxisome proliferators, fibrate derivatives are considered specific ligands for PPARα, whereas eicosanoids, such as PGJ2, for PPARγ. The study aimed to clarify the relation between PPARs and apoptosis or proliferation on the same type of cells, using clofibrate as specific ligand of PPARα and PGJ2 as specific ligand of PPARγ. The cells used were human hepatocarcinoma HepG2 cells. The results showed that PPARα protein content increased in HepG2 cells treated with clofibrate, causing apoptosis in a time‐ and concentration‐dependent way, as evidenced by the citofluorimetric assay and determination of BAD, myc and protein phosphatase 2A protein content. It also emerged that PPARγ increased in the same cells when treated with a specific ligand of this PPAR; in this case the increase of PPARγ did not cause an increase of apoptosis, but a time‐ and concentration‐dependent inhibition of cell proliferation, evidenced by decreased cell numbers and increased number of cells in the G0/G1 phase of the cycle. It may be concluded that PPARα is chiefly related to apoptosis and PPARγ to cell proliferation. Copyright

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.

Biocompatibility versus peritoneal mesothelial cells of polypropylene prostheses for hernia repair, coated with a thin silica/silver layer

Hernias are generally repaired using synthetic prostheses. Infection may already be present or develop during implantation. Based on the increasing resistance to antibiotics, and the well-known antimicrobial properties of silver (Ag), the possibility of coating hernia prostheses with a nanostructured layer containing Ag was explored. Prostheses (Clear Mesh Composite [CMC]) made up of two polypropylene layers (macroporous light mesh and thin transparent film) were tested with human mesothelial cells from omentum biopsies. Mesotheliocytes modulate abdominal wall healing producing cytokines, growth factors, and adhesion molecules. Evaluating the growth of these cells on CMC or film alone showed that cell numbers on CMC increased over time, and were higher than those on film alone. Vimentin immunostaining confirmed the cells to be mesotheliocytes. Subsequently, the biocompatibility of mesh layer, coated or not with a thin layer of Ag/SiO2 -nanoclusters, was analyzed, showing no difference in absence or presence of Ag/SiO2 . Differently, TGF-β2 production, involved in tissue repair and fibrosis, increased in the presence of Ag/SiO2 . Moreover, Ag/SiO2 -coated mesh showed antibacterial properties. In conclusion, the mesh layer coated with Ag/SiO2 afforded cell growth, and showed antibacterial activity. Coating only the mesh layer did not decrease film transparency, and did not favor the formation of adhesions on the visceral side.