Colon cancer is a serious health problem in most developed countries and is the main cause of cancer mortality throughout the world [¹]. Epidemiological studies have shown a significant difference in colon cancer incidence among different ethnic groups. The incidence of colon cancer is much higher in the United States and European countries compared with Asian countries, such as Japan and China, which is believed to be partially due to dietary habits [¹, ²]. One of the major differences in diet between these populations is that Americans and Europeans consume larger amounts of fats than Japanese and Chinese. Animal fats, especially saturated fats, have been regarded as the most important nutritional influence on the development of colon cancer [³].

Lipid peroxidation is a free-radical-initiated chain oxidation of polyunsaturated fatty acids (PUFAs). PUFAs are converted into hydroperoxides and aldehydes under oxidative stress. To date, there has been no overall systemic investigation concerning the influences of lipid hydroperoxides and aldehydes on the growth of colorectal cancer.

The present study demonstrated that among lipid hydroperoxides and aldehydes, 4-oxononenal (4-ONE) strongly inhibited the growth of human colorectal cancer cell line HT-29. Furthermore, the present study indicated that 4-ONE induced apoptosis of HT-29 cells through the formation of activated caspase-3 from pro-caspase-3 by the decreased expression of Bcl-2.

Fig.?1 shows the results of cell viability after 48?h treatment with n-6 (LA and AA) and n-3 (EPA) PUFAs, and hydroperoxy adducts of LA (13-HPODE) and EPA (15-HPEPE). These fatty acids and hydroperoxides had no significant effect on the growth of HT-29 cells at concentrations of 1 and 10??M. On the other hand, Fig.?2 illustrates the efficacies of unsaturated aldehydes generated from 13-HPODE (4-HNE and 4-ONE) and from 15-HPEPE (4-HHE) [⁶] on the growth of HT-29 cells. No significant alteration was observed with 4-HNE and 4-HHE at concentrations ranging from 1 to 10??M, whereas 4-ONE potently reduced HT-29 cell growth at concentrations of 5 and 10??M (59 and 85% inhibition).

To clarify the mechanism by which 4-ONE inhibited HT-29 cell growth, its effects on the DNA fragmentation (Fig.?3), the expression levels of Bcl-2 and Bax mRNA (Fig.?4), the activity of caspase-3 and protein expression levels of pro-caspase-3 and caspase-3 (Fig.?5) were observed. Using the TUNEL method, DNA fragmentation was found as the fluorescence of small DNA particles in nuclei, when 4-ONE (10??M) was added to HT-29 cells (Fig.?3a). The fragmentation of DNA in nuclei by incubation with 4-ONE (10??M) was also visualized by agarose-gel electrophoresis of nuclear extracts (Fig.?3b); 4-ONE treatment resulted in small-sized DNA debris electrophoresed from the origin. Thus, 4-ONE showed DNA fragmentation in HT-29 cells in the same manner as TNF-? (positive control [⁷, ⁸]). As shown in Fig.?4, 4-ONE (10??M) did not have any effect on Bax mRNA expression level, whereas it dramatically reduced the expression level of Bcl-2. Furthermore, 4-ONE (10??M) enhanced the activity of caspase-3 in HT-29 cells (Fig.?5a). This might be partially due to a tentative increase in activated caspase-3 concomitant with a tentative decrease in pro-caspase-3 (Fig.?5b).

It has been reported that overexpression of Bcl-2 in transgenic models leads to the protection of many cell types against apoptosis, suggesting that Bcl-2 exerts an anti-apoptotic function [⁹, ¹⁰]. Anti-apoptotic activity of Bcl-2 was found to be antagonized by a homologous Bax protein, which was able to form heterodimers with Bcl-2. Proapoptotic Bax forms the channels for cytochrome c release that may initiate the activation of caspase-3 identified as the major caspase that contributes to the hallmark of apoptosis, while anti-apoptotic Bcl-2 prevents the opening of mitochondrial transition pores by binding Bax [¹¹, ¹²]. Thus, the present study indicated that 4-ONE induces cell apoptosis by affecting the ratio of Bax to Bcl-2, activating cytochrome c release, and inducing proteolytic cleavage of pro-caspase-3 to activated caspase-3.

Also, is there any specificity to a particular cell, tumor or histological type in the growth inhibitory and cytotoxic effects of 4-ONE? When 4-ONE (10??M) was added to a human colorectal cancer cell CACO-2, human hepatocellular carcinoma cell HepG2, mouse preadipocyte cell 3T3-L1 and rat primary hepatocyte cell, the growth inhibition was detected in all these cells (67?93% inhibition). So, it seems likely that 4-ONE can be an inhibitor of cell proliferation or cell growth, independent of cell types.

Lipid peroxidation is a paradox of aerobic life, affecting human health and quality of life [¹³]. Biological systems are lipid-rich matrices susceptible to autoxidation unless protected by either endogenous enzymatic or non-enzymatic mechanisms. On the other hand, various lipid peroxidative products are generated physiologically and some of them are known as haemostatic regulators in cells [¹³]. Lipid peroxidation has been suggested to be involved in the control of cell division [¹⁴, ¹⁵]. Dreher and Junod [¹⁶] also showed that low levels of oxygen free radicals stimulate cell proliferation, whereas high levels induce cytotoxicity and cell death in later stages of carcinogenesis. Pillai et al. [¹⁷] and Srihari et al. [¹⁸] have reported that, using 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis, levels of lipid peroxidation byproducts, thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD), in the rat liver are higher, whereas those levels in the colon and caecum are significantly lowered, as compared with control rats. Kamaleeswari and Nalini [¹⁹] have also shown that, in addition to TBARS and CD, lipid peroxide levels in the colon cancer tissue are lower in DMH-treated rats, as compared with control rats. Furthermore, in vivo studies on human colon adenocarcinoma have shown decreased TBARS and 4-HNE contents in the tumor tissue related to malignancy progression [²⁰, ²¹]. The chemical reactions that lead to 4-ONE production have been the subject of considerable investigation. A likely pathway involves the breakdown of n-6 derived polyunsaturated fatty acid hydropeoxides (e.g., 13-HPODE) to 4-hydroperoxynonenal (4-HPNE), which can dehydrate to 4-ONE or be reduced to 4-HNE [⁶]. Both 4-HPNE and 4-ONE have been isolated as products of lipid peroxidation, and 4-ONE has been demonstrated to be a powerful electrophile, capable of reacting with protein and DNA at rates significantly greater than 4-HNE [²², ²³]. The physiological concentration of 4-HNE in rat tissues can reach up to 3??M [⁶], and therefore it might be possible that 4-ONE also appears in the tissues at micromolar concentration range under normal physiological states. Cancer cells acquire particular characteristics that benefit their proliferation [²⁴]. Namely, the present study might suggest that decreased 4-ONE, which is speculated to be associated with tumor progression, favors the proliferation of colon cancer.