Claudio Cavazza

Metabolic Approach to the Enhancement of Antitumor Effect of Chemotherapy: A Key Role of Acetyl-L-Carnitine

Purpose: Acetyl-l-carnitine (ALC) plays a relevant role in energy metabolism and stress response because of its function in the complex metabolic system regulating the acetyl-CoA levels that provide a source of acetyl groups for metabolic and acetylation-regulated processes. Because acetylation may influence p53 activity/stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with platinum compounds. Experimental Design: The antiproliferative and antitumor activity studies were done in a panel of human tumor cell lines with functional or defective p53. The antimetastatic drug efficacy was investigated in the s.c. growing H460/M tumor subline, which is able to generate lung metastases. Results: ALC enhanced the sensitivity to cisplatin of tumor cells with functional p53. The sensitization by ALC was reflected in an improved in vivo antitumor efficacy of the combination over cisplatin alone in wild-type p53 lung tumors. ALC did not increase the cisplatin efficacy in the p53-mutant SW620 tumor. ALC exhibited a significant antimetastatic activity, and this effect was better exploited in combination with the histone deacetylase inhibitor, ST3595. The in vivo ALC/cisplatin combination caused the activation of p53, associated with protein acetylation and induction of target genes. Conclusions: ALC was effective in enhancing the antitumor potential of platinum compounds in wild-type p53 tumors. ALC, alone and in combination with a histone deacetylase inhibitor, exhibited an outstanding antimetastatic activity. Both effects, likely mediated by protein acetylation, may have implications for platinum-based therapy and combinations with histone deacetylase inhibitors. Clin Cancer Res; 16(15); 3944–53. ©2010 AACR.

Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate

In the present study we have examined the effect of a single dose of the mitogen lead nitrate (75 mumols/kg body wt) on the methylation status of hepatic DNA in male Wistar rats. It was found that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect could be seen as early as 12 h after metal treatment and parallels the changes in liver weight. Probing with the methylation-sensitive enzymes HpaII, MspI, and HaeIII confirmed HPLC analyses and showed that methylation at these sites was affected by lead treatment. DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal nondividing liver. Thus the lowering of the DNA 5-methylcytosine content appears to be a property characteristic of cellular proliferation, regardless of whether it is caused by partial hepatectomy, carcinogen treatments, or mitogen administration.

Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.

Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitines effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial β-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins.

Abstract 566: Interaction of acetyl-L-carnitine with platinum compounds: A possible role of protein acetylation in modulation of antitumor activity

Several lines of evidence support that acetyl-L-carnitine (ALC) plays a relevant role as modulator of cellular stress response and may have a protective action in chemotherapy-induced neurotoxicity. ALC is implicated in the regulation of acetyl-CoA levels, a function mediated by carnitine acetyltransferase, thus providing a source of acetyl groups for histone acetylation. Since HDAC inhibitors may influence cellular response to cytotoxic drugs, including DNA damaging agents, it is conceivable that ALC may have a cooperative effect in combination with agents able to modulate protein acetylation. In particular, since the acetylation of wild-type p53, a substrate of HDAC, may influence its activity and stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with cisplatin and carboplatin. ALC (100 mg/kg;qdx14, p.o.), in combination with cisplatin (5 mg/kg, i.p., q4dx5), was able to enhance the life-span of mice bearing the EL-4 lymphoma tumor (ILS: ALC plus cisplatin >500%; cisplatin 105% vs control), resulting in 5/7 mice without evidence of disease. In addition, the combination of ALC plus cisplatin significantly increased the antitumor activity of cisplatin in three xenograft models of human non-small cell lung carcinoma (NCI-H460, A549 and NCI-H1650). Noteworthy, in a metastatic NCI-H460 model, an impressive reduction of lung metastasis in ALC and ALC+cisplatin groups was observed as compared with untreated mice. An increased antitumor activity was also observed in the combination of ALC with carboplatin. In addition, ALC produced an enhancement of antitumor activity of a novel HDAC inhibitor. To assess the relevance of acetyl moiety of ALC in the antitumor response, we investigated the activity of the combination of cisplatin with the related compound L-carnitine in NCI-H460 tumor model. In this experiment, L-carnitine did not showed any significative increases of antitumor effect when combined with cisplatin (TVI: 80% cisplatin vs 70% cisplatin plus carnitine), thus confirming a specific effect of ALC. In order to investigate the molecular mechanisms of the ALC-cisplatin interaction, several signal transduction pathways were analysed in H460 tumors by protein analysis and/or gene expression. The results revealed a significant increase of the p53 protein levels (p 373 acetylation, and the up-regulation of genes under control of p53. These observations may have implications for the therapeutic use of ALC in platinum-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 566.