Luciana Tessitore

Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma

Anthocyanins extracted from the berries of Phillyrea latifolia L., Pistacia lentiscus L., and Rubia peregrina L., three evergreen shrubs widely distributed in the Mediterranean area, were examined for their antioxidant and anticancer activity. The P. lentiscus anthocyanins showed the highest H2O2 and 1,1-diphenyl-2-picryl-hydrazil radical scavenging effects, indicating that these compounds can be considered as an alternative source of natural antioxidants for food and pharmaceutical products. Here, we also report a novel function of anthocyanins: the induction of autophagy, a process of subcellular turnover involved in carcinogenesis. Autophagy was characterized by the up-regulation of eIF2α, an autophagy inducer, and down-regulation of mTOR and Bcl-2, two autophagy inhibitors. This led to the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Anthocyanin-induced autophagy switched to apoptosis, as shown by the activation of Bax, cytochrome c and caspase 3, terminal deoxynucleotide transferase–mediated dUTP nick-end labeling–positive fragmented nuclei, and cells with sub-G1 DNA content, which were prevented by z-VAD. Inhibition of autophagy by either 3-methyladenine or Atg5 small interfering RNA enhanced anthocyanin-triggered apoptosis. This provided evidence that autophagy functions as a survival mechanism in liver cancer cells against anthocyanin-induced apoptosis and a rationale for the use of autophagy inhibitors in combination with dietary chemopreventive agents. [Mol Cancer Ther 2008;7(8):2476–85]

Understanding Autophagy in Cell Death Control

Autophagy is an evolutionarily conserved degradation pathway which primary functions as a cell survival adaptive mechanism during stress conditions. Autophagy is a tumor suppressor process and induction of the autophagic machinery can cause cell demise in apoptosis-resistant cancer. Thus, this metabolic pathway can act either to prevent or to promote carcinogenesis, as well as to modulate the response to anticancer therapies, included drug-induced apoptosis. Conventional therapies exert their cytotoxic activity mainly by inducing apoptosis. Massive activation of the apoptotic program in a tissue can result in cell loss providing a selective advantage for growth to displastic cells and tumor cell subpopulations with high levels of malignancy. This suggests that the activation of autophagy can counteract malignancy. On the contrary, therapeutic intervention-induced apoptosis can eliminate cells with pro-mutational biochemical alterations at risk for initiation, initiated cells and cells of focal and advanced preneoplastic and neoplastic lesions. Thus, pharmacological inhibition of autophagy may enhance apoptosis. Autophagy and apoptosis share common stimuli and signaling pathways, so that the final fate, life or death, depends on the cell response. Recently, accumulating data fuel novel potential therapeutic interventions to modulate autophagy to be beneficial in cancer therapy. This review highlights current knowledges aimed at unraveling the molecular interplay between autophagy and cell death as well as the possible therapeutic exploitation in cancer.

P-glycoprotein mediates celecoxib-induced apoptosis in multiple drug-resistant cell lines.

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein-mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 micromol/L celecoxib. We found that 10 micromol/L celecoxib reduced P-glycoprotein, Bcl-x(L), and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 micromol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-x(L) and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression.

Expression of Phosphatidylethanolamine N-Methyltransferase in Human Hepatocellular Carcinomas

Objective: Hepatic phosphatidylethanolamine is converted into phosphatidylcholine by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT) when the dietary choline supply is inadequate. Our previous reports implicated PEMT in the regulation of hepatocyte growth and transformation. In the present study, we analyzed PEMT activity, Pempt gene status and its mRNA expression in 29 human hepatocellular carcinomas (HCC). Methods: The status of the Pempt gene and PEMT2 mRNA expression were evaluated with Southern and Northern blotting, respectively, in HCC and the noninvolved liver. PEMT activity was assessed by biochemical assay. Cell proliferation markers were defined by immunohistochemical or histoautoradiographic methods. Results: PEMT activity was lower in HCC than in the noninvolved liver and it was negligible in 62% of the tumors. No deletions or mutations of the Pempt gene were found and PEMT2 mRNA expression was absent or reduced in HCC compared with peritumoral liver tissue. PEMT2 mRNA expression was inversely related to tumor proliferation and to histologic grade. Patients whose HCC did not express PEMT2 mRNA showed poorer outcomes for cancer-related survival than those with PEMT2-positive HCC. Conclusions: The present findings suggest that (1) clones lacking PEMT2 expression may have been selected during liver tumorigenesis and progression, and (2) PEMT2 expression seems to be associated with clinical progression.

Protective activity of Theobroma cacao L. phenolic extract on AML12 and MLP29 liver cells by preventing apoptosis and inducing autophagy.

Theobroma cacao L. is known to have potential cardiovascular and cancer chemopreventive activities because of its high content of phenolic phytochemicals and their antioxidant capacities. In this work, we show for the first time that cocoa inhibits drug-triggered liver cytotoxicity by inducing autophagy. Phenolic-rich extracts of both unroasted and roasted cocoa prevented Celecoxib-induced cell viability inhibition in MLP29 liver cells because of the accumulation of G1 cells and cell death. Death prevented by cocoa had hallmarks of apoptosis such as the sub-G1 peak at flow cytometry and activation of Bax expression, together with down-regulation of Bcl-2, released cytochrome c in the cytosol with activation of Caspase 3, indicating that components of the apoptotic pathway such as Bax or upstream are major targets of cocoa phytochemicals. The protective effect of cocoa against liver cytotoxicity by Celecoxib was probably accounted for by inducing the autophagic process, as shown by enhanced Beclin 1 expression and accumulation of monodansylcadaverine in autolysosomes. This fact suggests that apoptosis was prevented by inducing autophagy. Finally, considering all these findings, we suggest that cocoa can be added to the list of natural chemopreventive agents whose potential in hepatopathy prevention and therapy should be evaluated.

Mucin‐depleted foci are modulated by dietary treatments and show deregulation of proliferative activity in carcinogen‐treated rodents

The correlation between mucin‐depleted foci (MDF) and colon carcinogenesis was studied in F344 rats initiated with 1,2‐dimethylhydrazine and treated with a chemopreventive regimen (polyethylene glycol, PEG) or with a promoting diet (high‐corn oil). High corn oil diet increased MDF, while PEG reduced them. The expression of p27 and p16, inhibitors of cyclin‐dependent kinases, which inhibit the progression of the cell cycle, was studied by immunohistochemistry in MDF and in aberrant crypt foci (ACF) of control rats. In both MDF and ACF, the nuclear expression of p27 was markedly reduced, while p16 was reduced to a lower extent. Mitotic activity was higher in MDF and ACF than in normal mucosa of control rats. MDF were also identified in azoxymethane‐initiated SWR/J mice. These results further confirm that MDF are preneoplastic lesions and could be useful biomarkers of colon carcinogenesis.

Inactivation of phosphatidylethanolamine N-methyltransferase-2 in aflatoxin-induced liver cancer and partial reversion of the neoplastic phenotype by PEMT transfection of hepatoma cells

Phosphatidylethanolamine N‐methyltransferase(PEMT) is an enzyme in liver that catalyzes the stepwise methylation of phosphatidylethanolamine to phosphatidylcholine, in addition to the main pathway that synthesizes phosphatidylcholine directly from choline. We have reported that PEMT is permanently inactivated in liver cancer induced by the Solt and Farber model. Here we studied, (i) whether similar changes also occur in the progression of hepatocarcinoma triggered by aflatoxin B1 (AFB1) in rats; (ii) whether the hepatoma phenotype could be reversed by over‐expression of PEMT2. We found that PEMT2 protein decreased in pre‐neoplastic nodules and virtually disappeared in hepatocellular carcinoma induced by AFB1 due to decreased levels of mRNA without any deletion or mutation of the DNA sequence. PEMT activity, which reflects the function of both PEMT1 and PEMT2, was lower in nodules and negligible in the tumor, consistent with its regulation at the level of gene transcription. McArdle hepatoma cells transfected with PEMT2 failed to form anchorage‐independent colonies in soft agar, while the vector‐transfected control line grew efficiently. Moreover, PEMT2‐transfected cells were also poorly tumorigenic in vivo in athymic mice, as shown by the lower tumor incidence, the longer cancer‐free‐time and the lower tumor volume and weight. Together, these data indicate that the loss of PEMT function may contribute to malignant transformation of hepatocytes. Int. J. Cancer 86:362–367, 2000.