Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.

Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA.

OBJECTIVENicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome.RESEARCH DESIGN AND METHODSWe used β-lapachone (βL), a natural substrate of NADH:quinone oxidoreductase 1 (NQO1), to stimulate NADH oxidation. The βL-induced pharmacological effect on cellular energy metabolism was evaluated in cells derived from NQO1-deficient mice. In vivo therapeutic effects of βL on metabolic syndrome were examined in diet-induced obesity (DIO) and ob/ob mice.RESULTSNQO1-dependent NADH oxidation by βL strongly provoked mitochondrial fatty acid oxidation in vitro and in vivo. These effects were accompanied by activation of AMP-activated protein kinase and carnitine palmitoyltransferase and suppression of acetyl-coenzyme A (CoA) carboxylase activity. Consistently, systemic βL administration in rodent models of metabolic syndrome dramatically ameliorated their key symptoms such as increased adiposity, glucose intolerance, dyslipidemia, and fatty liver. The treated mice also showed higher expressions of the genes related to mitochondrial energy metabolism (PPARγ coactivator-1α, nuclear respiratory factor-1) and caloric restriction (Sirt1) consistent with the increased mitochondrial biogenesis and energy expenditure.CONCLUSIONSPharmacological activation of NADH oxidation by NQO1 resolves obesity and related phenotypes in mice, opening the possibility that it may provide the basis for a new therapy for the treatment of metabolic syndrome.

Compounds bearing an acyl group of a various size at 1'-OH of shikonin were synthesized as acyl analogues of shikonin, which was isolated from the root of Lithospermum erythrorhizon, and evaluated for inhibitory effect on topoisomerase-I activity. A selective acylation at 1'-OH of shikonin in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine gave rise to a good yield of corresponding acylshikonin derivatives. In general, analogues with an acyl group of shorter chain lengths (C2-C6) exerted a stronger inhibitory action than those with longer chain lengths (C7-C20). While the halogen substitution at C-2 of the acetyl moiety failed to increase the inhibitory potency, the placement of double bonds in the acyl group (C5-C7) augmented the potency remarkably. Of the 32 derivatives evaluated, 15 compounds exhibited a higher inhibitory effect than shikonin. Noteworthy, the inhibitory potency of acetylshikonin, propanoylshikonin, and 4-pentenoylshikonin was approximately 4-fold greater than that of camptothecin. All these data suggest that the size of acyl moiety is important for the enhancement of potency, and the presence of olefinic double bonds is also beneficial.

The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.