Melatonin and doxorubicin synergistically enhance apoptosis via autophagy-dependent reduction of AMPKα1 transcription in human breast cancer cells.

Doxorubicin is one of the most effective agents used to treat various cancers, including breast cancer, but its usage is limited by the risk of adverse effects, including cardiotoxicity. Melatonin, a natural hormone that functions as a major regulator of circadian rhythms, has been considered a supplemental component for doxorubicin due to its potential to improve its effectiveness. However, the mechanisms and biological targets of the combination of melatonin and doxorubicin with respect to cancer cell death are not well understood. In the present study, we found that melatonin synergized with doxorubicin to induce apoptosis of breast cancer cells by decreasing the expression of AMP-activated protein kinase α1 (AMPK α1), which acts as a critical survival factor for cancer cells. This cotreatment-induced reduction in AMPKα1 expression occurred at the transcriptional level via an autophagy-dependent mechanism. The synergistic effects of the combined treatment were evident in many other cancer cell lines, and melatonin was also highly effective in inducing cancer death when combined with other cancer drugs, including cisplatin, 5-fluorouracil, irinotecan, and sorafenib. AMPKα1 expression was decreased in all of these cases, suggesting that reducing AMPKα1 can be considered an effective method to increase the sensitivity of cancer cells to doxorubicin treatment.

Melatonin prevents doxorubicin-induced cardiotoxicity through suppression of AMPKα2-dependent mitochondrial damage.

The clinical application of doxorubicin, one of the most effective anticancer drugs, has been limited due to its adverse effects, including cardiotoxicity. One of the hallmarks of doxorubicin-induced cytotoxicity is mitochondrial dysfunction. Despite intensive research over recent decades, there are no effective approaches for alleviating doxorubicin-induced cytotoxicity. Melatonin, a natural hormone that is primarily secreted by the pineal gland, is emerging as a promising adjuvant that protects against doxorubicin-induced cytotoxicity owing to its pharmaceutical effect of preserving mitochondrial integrity. However, the underlying mechanisms are far from completely understood. Here, we provide novel evidence that treatment of H9c2 cardiomyoblasts with doxorubicin strongly induced AMP-activated protein kinase α2 (AMPKα2), which translocated to mitochondria and interfered with their function and integrity, ultimately leading to cellular apoptosis. These phenomena were significantly blocked by melatonin treatment. The levels of AMPKα2 in murine hearts were tightly associated with cardiotoxicity in the context of doxorubicin and melatonin treatment. Therefore, our study suggests that the maintenance of mitochondrial integrity is a key factor in reducing doxorubicin-induced cytotoxicity and indicates that AMPKα2 may serve as a novel target in the design of cytoprotective combination therapies that include doxorubicin.

Anti-obesity Effect of Fermented Persimmon Extracts via Activation of AMP-Activated Protein Kinase.

There is significant cultivation of persimmon (Diospyros kaki) in East Asia, a plant whose fruit has abundant nutrients, including vitamins, polyphenols, and dietary fiber. Persimmon dietary supplements can benefit health by amelioration of diabetes, cardiovascular disease, and obesity. There are also persimmon-based beverages produced via fermentation, such as wines and vinegars, and increasing consumption of these products in East Asia. Although there is great interest in functional foods, the health effects of fermented persimmon extract (FPE) are completely unknown. We examined the effects of FPE on the metabolic parameters of mice fed a high-fat diet (HFD). Our results indicated that FPE supplementation led to an approx. 15% reduction of body weight, reduced abdominal and liver fat, and reduced serum levels of triglycerides, total cholesterol, and glucose. FPE also blocked the differentiation of murine 3T3-L1 pre-adipocyte cells into mature adipocytes. We suggest that gallic acid is a major bioactive component of FPE, and that AMP-activated protein kinase mediates the beneficial effects of FPE and gallic acid.

Krill Oil Supplementation Improves Dyslipidemia and Lowers Body Weight in Mice Fed a High-Fat Diet Through Activation of AMP-Activated Protein Kinase.

Krill oil is a novel, commercially available marine oil rich in long-chain polyunsaturated omega-3 fatty acids, particularly eicosapentaenoic acid and docosahexaenoic acid. Compared with fish oil, the effects of krill oil supplementation on human health and its underlying action mechanisms are currently poorly understood. In the present study, we examined the effect of krill oil supplementation on metabolic parameters of mice fed a high-fat diet (HFD). Krill oil supplementation in mice fed a HFD for 10 weeks resulted in an ∼15% lower body weight gain and a dramatic suppression of hepatic steatosis. These effects were associated with significantly lower serum triglyceride and low-density lipoprotein-cholesterol levels. We further uncovered a novel underlying mechanism, showing that AMP-activated protein kinase, a master regulator of glucose and lipid metabolism, mediates the beneficial effects of krill oil.

Expression levels of the hypothalamic AMPK gene determines the responsiveness of the rats to electroacupuncture-induced analgesia.

BACKGROUND: Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5'-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia ("responder"), as compared to the rats that were insensitive to EA analgesia ("non-responder"). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia. METHODS: Sprague-Dawley (SD) rats were divided into the responder and the non-responder groups, based on EA-induced analgesic effects in the tail flick latency (TFL) test, which measures the latency of the tail flick response elicited by radiant heat applied to the tail. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the expression levels of AMPK mRNA in the hypothalamus of the responder and non-responder rats. Further, we examined whether viral manipulation of the AMPK expression in the hypothalamus modulates EA analgesia in rats. RESULTS: The real-time RT-PCR analysis showed that mRNA expression levels of AMPK in the hypothalamus of the responder rats are significantly higher than those of the non-responder rats, validating the previous microarray results. Microinjection of dominant negative (DN) AMPK adenovirus, which inhibits AMPK activity, into the rat hypothalamus significantly attenuates EA analgesia (p < 0.05), whereas wild type (WT) AMPK virus did not affect EA analgesia (p > 0.05). CONCLUSIONS: The present results demonstrated that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia. Thus, our findings provide a clinically useful evidence for the application of acupuncture or EA for analgesia.

Berberine inhibits human colon cancer cell migration via AMP-activated protein kinase-mediated downregulation of integrin ß1 signaling.

Colon cancer is associated with a poor prognosis, motivating strategies to prevent its development. An encouraging preventative strategy is the use of nutraceuticals; however, scientific verification of therapeutic functions and mechanisms of biological activity are necessary for the acceptance of dietary supplements in cancer treatment. Berberine is a benzylisoquinoline alkaloid extracted from many kinds of medicinal plants that has been extensively used as a Chinese traditional medicine. Recently, berberine has been reported to possess antitumoral activities. Among the various cellular targets of berberine is AMP-activated protein kinase (AMPK), which regulates tumor progression and metastasis. However, the specific role of berberine-induced AMPK activation and its effects on the metastatic potential of colon cancer remain largely unknown. The present study investigated berberine-induced activation of AMPK and its effects on colon cancer cell migration. Berberine decreased the migration of SW480 and HCT116 cells. We found that berberine activated AMPK in human colon cancer cell lines. Notably, berberine-induced activation of AMPK reduced the integrin ß1 protein levels and decreased the phosphorylation of integrin ß1 signaling targets. Knockdown of AMPKα1 subunits using small interfering RNA significantly attenuated berberine-induced downregulation of integrin ß1 and inhibition of tumor cell migration. Collectively, our results suggest that berberine-induced AMPK activation inhibits the metastatic potential of colon cancer cells by decreasing integrin ß1 protein levels and downstream signaling.

The antioxidant effects of genistein are associated with AMP-activated protein kinase activation and PTEN induction in prostate cancer cells.

Epidemiological evidence suggests a lower incidence of prostate cancer in Asian countries, where soy products are more frequently consumed than in Western countries, indicating that isoflavones from soy have chemopreventive activities in prostate cells. Here, we tested the effects of the soy isoflavone genistein on antioxidant enzymes in DU145 prostate cancer cells. Genistein significantly decreased reactive oxygen species levels and induced the expression of the antioxidant enzymes manganese (Mn) superoxide dismutase (SOD) and catalase, which were associated with AMP-activated protein kinase (AMPK) and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) pathways. The induced expression of catalase, MnSOD, and PTEN were attenuated by pretreatment with a pharmacological inhibitor for AMPK, indicating the effects of genistein primarily depend on AMPK. Furthermore, PTEN is essential for genistein activity, as shown by PTEN transfection in PTEN-deficient PC3 cells. Thus, genistein induces antioxidant enzymes through AMPK activation and increased PTEN expression.

Inulin increases glucose transport in C2C12 myotubes and HepG2 cells via activation of AMP-activated protein kinase and phosphatidylinositol 3-kinase pathways.

Inulin, a naturally occurring, functional food ingredient found in various edible plants, has been reported to exert potential health benefits, including decreased risk of colonic diseases, non-insulin-dependent diabetes, obesity, osteoporosis, and cancer. However, the mechanism of the antidiabetic activity of inulin has not yet been elucidated. In this study, we showed that inulin increased the uptake of glucose in C2C12 myotubes, which was associated with both AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways, but both of these pathways appeared to transmit their signals in an independent manner. Moreover, we found that inulin was able to increase the uptake of glucose in C2C12 myotubes in which insulin resistance was induced by exposing cells to high glucose concentrations. The identical effects of inulin were also observed in HepG2 hepatoma cells. Collectively, we report the antidiabetic activity of inulin and further demonstrate for the first time that such activity is associated with AMPK and PI3-K activation.

Green tea catechin controls apoptosis in colon cancer cells by attenuation of H2O2-stimulated COX-2 expression via the AMPK signaling pathway at low-dose H2O2.

This study investigated the apoptotic regulation by green tea catechin epigallcatechin-3-gallate (EGCG) on colon cancer cells in the presence of low-dose H(2)O(2) known to exert the activation of signal pathways leading to cell proliferation. In the presence of low-dose H(2)O(2), EGCG induced apoptosis and abolished the cell-proliferative effect exhibited by low-dose H(2)O(2). This reduction of growth was accompanied by an activation of AMP-activated kinase (AMPK), a decrease in cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) levels, and the induction of apoptotic markers such as p53 and poly(ADP-ribose) polymerase (PARP) cleavage. The low-dose H(2)O(2) stimulated COX-2 expression, and treating cells with synthetic AMPK activator AICAR (5-aminoimiazole-4-carboxamide-1-beta-d-ribofuranoside) resulted in greater suppression of COX-2 expression and PGE(2). By treating cells with high concentrations of the reactive oxygen species (ROS) scavenger NAC (N-acetyl-1-cysteine), the apoptotic effect of EGCG was abolished and led to suppression of AMPK and COX-2, indicating that the liberation of excessive ROS might be the upstream signal of the AMPK-COX-2 signaling pathway even in the presence of low-dose H(2)O(2).

The association of serum leptin with the reduction of food intake and body weight during electroacupuncture in rats.

Previous studies indicate that acupuncture or electroacupuncture (EA) treatment reduces body weight and food intake in rats by increasing the level of anoretic peptides and decreasing that of orexigenic peptides in the hypothalamus. Considering a well-established role of leptin as a major regulator for feeding behavior in the hypothalamus, we hypothesized that EA might exert its effect via increasing serum leptin levels. In this study, we tested our hypothesis by evaluating the effects of EA on food intake and body weight, as well as on serum leptin levels in rats. Rats were randomly divided into 3 groups: AL (fed ad libitum with no treatments), Holder (fed ad libitum with daily holder restraint) and EA (fed ad libitum with daily holder restraint and 100 Hz EA stimulation) groups. During the four-week experimental period, daily food intake and body weight were measured. At the end of the experiment, levels of serum leptin and corticosterone, and plasma epinephrine (Epi) and norepinephrine (NE) were determined. Here we demonstrate that EA treatment indeed led to reduction of food intake and body weight, and to an increase of serum leptin levels. The level of Epi, NE, and corticosterone increased in the Holder group, but such increase in the level of aforementioned stress hormones was not observed in the EA group. Overall, our results suggest that EA treatment reduces food intake and body weight in rats possibly through increasing leptin levels, and that this effect of EA is not due to the stress caused by the daily holder restraint.

Anti-inflammatory and anti-nociceptive effects of the extract from Kalopanax pictus, Pueraria thunbergiana and Rhus verniciflua.

The combined extracts obtained from three Chinese herb medicine, Kalopanax pictus, Pueraria thunbergiana and Rhus verniciflua, have been used as therapeutics for diabetes mellitus in Korea. In the present study, we have investigated their possible anti-inflammatory effects by comparing the potency of individual extracts with that of the combined extracts. An individual water extract prepared from Kalopanax pictus, Pueraria thunbergiana, and Rhus verniciflua was named K-1, P-1, and R-1, respectively. Simultaneously, we also prepared the combined extracts from above three plant materials by identical methods and named KPR-1. These four extracts were further fractionated into the EtOAc extracts, and these were designated as K-2, P-2, R-2, and KPR-2, respectively. These eight samples were subjected to the nitrite assays in LPS-induced macrophage 264.7 cells. KPR-2 exhibited the most pronounced effect on the inhibition of NO production among all the extracts. KPR-2 also significantly decreased PGE2, and TNF-alpha release. In addition, KPR-2 showed in vivo anti-inflammatory activity against acute paw edema induced by carrageenan in rats. When analgesic activity was measured by the acetic acid-induced abdominal constriction and hot plate test, KPR-2 showed a dose-dependent inhibition in animal models. These results suggested that the mixture extract and successive fractionation could lead to the better use of anti-inflammatory medicinal crude drugs.

Induction of apoptosis by yomogin in human promyelocytic leukemic HL-60 cells.

Yomogin is an active compound isolated from Artemisia princep, a traditional Oriental medicinal herb, which has been shown to inhibit tumor cell proliferation. In this study, we investigated the effects of yomogin on the cytotoxicity, induction of apoptosis, and putative pathways of its actions in human promyelocytic leukemia cells. Yomogin-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders in agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine residues. We observed that yomogin caused activation of caspase-8, caspase-9, and caspase-3. A general caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk), almost completely suppressed the yomogin-induced DNA fragmentation. We further demonstrated that yomogin induced Bid cleavage, mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria in a caspase-8-dependent manner. Taken together, our data indicate that yomogin is a potent inducer of apoptosis and facilitates its activity via caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, and subsequent release of cytochrome c into the cytoplasm, providing a potential mechanism for the anticancer activity of yomogin.

Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) functions as a fuel sensor in the cell and is activated when cellular energy is depleted. Here we report that alpha-lipoic acid (alpha-LA), a cofactor of mitochondrial enzymes, decreases hypothalamic AMPK activity and causes profound weight loss in rodents by reducing food intake and enhancing energy expenditure. Activation of hypothalamic AMPK reverses the effects of alpha-LA on food intake and energy expenditure. Intracerebroventricular (i.c.v.) administration of glucose decreases hypothalamic AMPK activity, whereas inhibition of intracellular glucose utilization through the administration of 2-deoxyglucose increases hypothalamic AMPK activity and food intake. The 2-deoxyglucose-induced hyperphagia is reversed by inhibiting hypothalamic AMPK. Our findings indicate that hypothalamic AMPK is important in the central regulation of food intake and energy expenditure and that alpha-LA exerts anti-obesity effects by suppressing hypothalamic AMPK activity.

Inhibition of methanol extract from the fruits of Kochia scoparia on lipopolysaccharide-induced nitric oxide, prostaglandin [correction of prostagladin] E2, and tumor necrosis factor-alpha production from murine macrophage RAW 264.7 cells.

In an attempt to search for bioactive natural products exerting antiinflammatory activity, we have evaluated the effects of the methanol extract of the fruits of Kochia scoparia (L.) CHARD. (Chenopodiaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor (TNF)-alpha release by the macrophage cell line RAW 264.7. Our data indicate that this extract is a potent inhibitor of NO production and it also significantly decreased PGE(2) and TNF-alpha release. Consistent with these observations, the protein and mRNA expression level of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 was inhibited by MeOH extracts of Kochia scoparia (KSM) in a dose-dependent manner. Furthermore, KSM inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was associated with prevention of the inhibitor kappaB degradation. These results suggest that the methanol extract of K. scoparia inhibits LPS-induced iNOS and COX-2 expression by blocking NF-kappaB activation.

Saucernetin-7 isolated from Saururus chinensis inhibits proliferation of human promyelocytic HL-60 leukemia cells via G0/G1 phase arrest and induction of differentiation.

In the present study, we investigated the in vitro effect of saucernetin-7, which is a dineolignan isolated from Saururus chinensis, on the proliferation, cell cycle-regulation and differentiation of HL-60 human promyelocytic leukemia cells. Saucernetin-7 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an IC50, approximately 5 microM. DNA flow-cytometry indicated that saucernetin-7 markedly induced a G1 phase arrest of HL-60 cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent kinase (CDK)6 and cyclin D1 were reduced by saucernetin-7, whereas the steady-state levels of CDK2, CDK4, cyclin D2, cyclin D3 and cyclin E were unaffected. The protein and mRNA levels of a CDK inhibitor p21CIP1/WAF1, but not p27KIP1, were markedly increased by saucernetin-7 and p21CIP1/WAF1 induction is likely to occur at the transcriptional level because actinomycin D blocked this induction. In addition, saucernetin-7 markedly enhanced the binding of p21CIP1/WAF1 with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. We furthermore suggest that saucernetin-7 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD14 and CD66b surface antigens. In conclusion, the onset of saucernetin-7-induced the G0/G1 arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the p21CIP1/WAF1 level and a decrease in the CDK2 and CDK6 activities. This is the first report demonstrating that saucernetin-7 potently inhibits the proliferation of human promyelocytic HL-60 cells via the G1 phase cell cycle arrest and differentiation induction.

Antirheumatoid arthritis effect of Rhus verniciflua and of the active component, sulfuretin.

Oral administration of the MeOH extract of Rhus verniciflua or of an EtOAc fraction containing an EtOAc-soluble portion of the MeOH extract slightly decreased rheumatoid arthritis (RA) and C-reactive protein (CRP) factors in Freund's complete adjuvant reagent FCA-treated rats, indicating that they are active extracts for rheumatoid arthritis, the EtOAc extract being more active. Treatment with these two extracts prevented histological changes such as synovial cell proliferation, inflammatory cell infiltration and fat necrosis compared with an FCA-treated group. Oral administration (30 mg/kg) of sulfuretin and fustin, which were isolated from the EtOAc extract by activity-guided separation, significantly decreased RA and CRP factors, the former being more active than the latter. Treatment with the EtOAc fraction ( p. o.) containing sulfuretin significantly decreased malondialdehyde (MDA) formation, and highly increased the activities of superoxide dismutase, catalase and glutathione peroxidase. Inhibition of xanthine oxidase and aldehyde oxidase in FCA-treated rats was also evident. Since treatment with sulfuretin and the EtOAc extract decreased the concentration of infiltrated mast cells in the rat knee exhibiting rheumatoid arthritis, we suggest that the Rhus verniciflua extract, which contains sulfuretin as an active component, may prevent rheumatoid syndromes by inhibiting reactive oxygen species.

Saucernetin-7 and saucernetin-8 isolated from Saururus chinensis inhibit the LPS-induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 cells.

In the present study, we demonstrate that saucernetin-8 ( 1) and saucernetin-7 ( 2), isolated from the underground parts of Saururus chinensis (Saururaceae), exerted potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Both compounds 1 and 2, known as dineolignans, also suppressed the expression of iNOS and COX-2 protein in a dose-dependent manner. Thus, this study suggests that compounds 1 and 2-mediated inhibition of iNOS and COX-2 expression may be one of the mechanisms responsible for the anti-inflammatory effects of the underground parts of Saururus chinensis.

Antinociceptive and antiinflammatory effects of Niga-ichigoside F1 and 23-hydroxytormentic acid obtained from Rubus coreanus.

As an attempt to search for bioactive natural constituents exerting antinociceptive and antiinflammatory activities, we examined the potency of the extract of Rubus coreanus fruits by the activity-guided fractionation. The EtOAc- and BuOH fraction and those alkaline hydrolysates showed significant antinociceptive effects as assessed by writhing-, hot plate- and tail flicks tests in mice and rats as well as antiinflammatory effect in rats with carrageenan-induced edema. BuOH extract was subjected to column chromatography to obtain a large amount of niga-ichigoside F(1) (1,23-hydroxytormentic acid 28-O-glc), which was again hydrolyzed in NaOH solution to yield an aglycone 23-hydroxytormentic acid (1a). The aglycone, 23-hydroxytormentic acid, was much more potent in both antinociceptive and antiinflammatory tests than the glycoside, niga-ichigoside F(1). The antiinflammatory effects of these compounds were further supported by the reduction of carrageenan-induced lipid peroxidation and hydroxyl radical in serum. These results suggested that 23-hydroxytormentic acid might be an active moiety of niga-ichigoside F(1) present in R. coreanus.

In vivo anti-inflammatory and antinociceptive effects of liriodendrin isolated from the stem bark of Acanthopanax senticosus.

In the present study, liriodendrin isolated by activity-guided fractionation from the ethyl acetate (EtOAc) extracts of the stem bark of Acanthopanax senticosus, was evaluated for anti-inflammatory and antinociceptive activities. Liriodendrin (5, 10 mg/kg/day, p. o.) significantly inhibited the increase of vascular permeability induced by acetic acid in mice and reduced an acute paw edema induced by carrageenan in rats. When the analgesic activity was measured by the acetic acid-induced writhing test and hot plate test, liriodendrin showed a dose-dependent inhibition in animal models. In addition, syringaresinol, the hydrolysate of liriodendrin, more potently inhibited the LPS-induced production of NO, PGE 2 and TNF-alpha production of macrophages than liriodendrin. Consistent with these observations, the expression level of iNOS and COX-2 enzyme was decreased by syringaresinol in a concentration-dependent manner. These results suggest that the anti-inflammatory and antinociceptive effects of liriodendrin after oral administration were attributable to the in vivo transformation to syringaresinol, which may function as the active constituent.

Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells.

Cinnamaldehyde is an active compound isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which has been shown to inhibit tumor cell proliferation. In this study, we investigated the effects of cinnamaldehyde on the cytotoxicity, induction of apoptosis and the putative pathways of its actions in human promyelocytic leukemia cells. Using apoptosis analysis, measurement of reactive oxygen species (ROS), and assessment of mitochondrial membrane potentials (DeltaPsim), we show that cinnamaldehyde is a potent inducer of apoptosis and that it transduces the apoptotic signal via ROS generation, thereby inducing mitochondrial permeability transition (MPT) and cytochrome c release to the cytosol. ROS production, mitochondrial alteration, and subsequent apoptotic cell death in cinnamaldehyde-treated cells were blocked by the antioxidant N-acetylcystein. Taken together, our data indicate that cinnamaldehyde induces the ROS-mediated mitochondrial permeability transition and resultant cytochrome c release. This is the first report on the mechanism of the anticancer effect of cinnamaldehyde.