Misaki Ono

Anti‐proliferative and apoptosis‐inducing activity of lycopene against three subtypes of human breast cancer cell lines

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF‐7, HER2‐positive SK‐BR‐3 and triple‐negative MDA‐MB‐468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time‐dependent and dose‐dependent anti‐proliferative activity against these cell lines by arresting the cell cycle at the G0/G1 phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA‐MB‐468 where the sub‐G0/G1 apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti‐apoptotic Bcl‐xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA‐MB‐468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.

Mechanism of the Anticancer Effect of Lycopene (Tetraterpenoids)

Increasing evidence suggests that lycopene, a major carotenoid detected in human plasma, may be preventive against the formation and the development of different types of human cancers including prostate, breast, and lung cancer. Experimental studies demonstrated that lycopene inhibits the growth of various cancer cells of different organs and prevent chemically induced carcinogenesis in animal models. Although the excellent antioxidant property of lycopene is most likely the basis for its preventive role toward cancer, the direct anticancer activities of lycopene through multiple mechanisms are disclosed, including regulation of growth factor signaling, cell cycle arrest and/or apoptosis induction, and changes in antioxidant and phase II detoxifying enzymes. The anti-inflammatory activity of lycopene is also considered as an important determinant that suppresses the promotion and progression of carcinogenesis. Moreover, lycopene inhibits cell invasion, angiogenesis, and metastasis. Importantly, those activities have been shown to be exhibited at the physiologically attainable concentration in humans. Although the preclinical data strongly suggest an antitumor activity of lycopene, a number of epidemiological and intervention studies indicate that there is still no clear clinical evidence that supports its use for the prevention of those cancers. More well controlled clinical intervention trials are needed to further clarify the exact role of lycopene in the cancer prevention. Nonetheless, because of its multiple tumor-inhibitory activities, lycopene still remains to be an attractive and promising carotenoid that will potentially contribute to the prevention and treatment of human cancers. This chapter reviews data on the cancer preventive activities of lycopene, possible mechanisms involved, and the relationship between lycopene consumption and human cancer risk.

Differential anti-tumor activities of curcumin against Ras- and Src-activated human adenocarcinoma cells

Although curcumin has been studied as a potential anticancer drug targeting multiple signaling molecules, the role of oncogenic Src and Ras in curcumin sensitivity remains unknown. Using HAG-1 human adenocarcinoma cells transfected with either activated Src or Ras, we investigated here the functional role of these oncogenes in curcumin sensitivity. Activation of either Src or Ras did not confer resistance to curcumin, compared to vehicle-transfected cells. Curcumin enhanced Erk1/2 predominantly in Ras-activated cells, but inhibited Akt and its downstream molecules (mTOR and S6K1) regardless of these oncogene activations. The sub-G0/G1 apoptotic populations were substantially increased with demonstrable cleavage of PARP, but this increase was most prominent in Src-activated cells. Suppression of Bcl-xL level and enhanced expression of Bax were demonstrated in Src-activated, but not Ras-activated cells. By contrast, drastic increases of G2/M cell populations were seen in Ras-activated cells rather than Src-activated cells, suggesting a potential role of Ras/Erk1/2 activation in curcumin-induced G2/M arrest. These data indicate that curcumin-induced growth inhibition would be mediated mainly by G2/M arrest in Ras-driven cells but by apoptosis induction in Src-driven cells, providing a mechanistic rationale for the potential use of curcumin in the treatment of human cancers with activated Src or Ras.

Effects of Dietary Genistein on Hormone-Dependent Rat Mammary Carcinogenesis Induced by Ethyl Methanesulphonate

Genistein, a major soy isoflavone having weak estrogenic activities, has been suggested to reduce the risk of breast cancer incidence. However, many studies have yielded inconsistent results. We investigated the effects of dietary genistein on the development of breast cancer using ethyl methanesulphonate (EMS) chemically induced rat model of hormone-dependent mammary carcinoma. Female Wistar King A rats were orally given EMS for 12 wk and fed isoflavone-free NIH-07PLD diets with or without genistein, beginning immediately after weaning period. All EMS-treated rats fed either diet developed estrogen and/or progesterone receptor-positive mammary carcinoma by 24 wk. The addition of either low or high genistein, which produced the plasma concentrations comparable with those observed in humans consuming high soy diets, did not show any preventive activity. Soy-containing pellet food, exhibiting substantial plasma concentrations of isoflavones such as genistein, daidzein, equol, and glycitein, significantly increased the latency periods, compared to either NIH-07PLD diet with low (P = 0.027) or high (P = 0.034) genistein. Body weights, total EMS uptakes, and urinary estradiol concentrations were not significantly different among groups. These data indicate that genistein does not exert clear preventive effects and that isoflavone components other than genistein might be preventive against hormone-dependent mammary carcinogenesis.

Interannual study of spot urine–evaluated sodium excretion in young Japanese women

The authors investigated interannual differences in the sodium excretion levels of young healthy Japanese women as estimated from spot urine analysis at Nakamura Gakuen University from 1995 to 2015. Participants included 4931 women aged 18 to 20 years who were classified into three time periods according to year of health check: first (1995–2001), second (2002–2007), and third (2008–2015). Estimated daily urinary sodium and potassium excretion levels and the sodium to potassium ratio were 120.6±31.9 mmol, 35.2±8.1 mmol, and 3.5±0.9, respectively. Adjusted for body weight, sodium excretion, and potassium excretion significantly decreased in the second and third period compared with the first period (P<.001). Systolic blood pressure also decreased in the same way between time periods (P<.001). Estimated urinary excretion levels of sodium and potassium in young Japanese women have decreased over the past 20 years independently of body weight.

Equol Enhances Apoptosis-inducing Activity of Genistein by Increasing Bax/Bcl-xL Expression Ratio in MCF-7 Human Breast Cancer Cells

ABSTRACT Anticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50 μM equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.

Abstract 909: Mechanistic study of synergistic interaction between genistein and equol in MCF-7 human breast cancer cells in vitro

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Soy isoflavones, such as genistein, daidzein, glycitein and equol, a metabolite of daidzein, have been suggested to affect the growth of estrogen-receptor positive breast cancer cells in vitro, and thus considered to be involved in the prevention of breast cancer. We have recently reported that soy isoflavone component mixture has a greater preventive activity against breast cancer rather than genistein alone using chemically induced rat model of hormone-dependent mammary carcinoma. Therefore, we investigated here whether a synergistic interaction exists between genistein and other isoflavone components in estrogen receptor positive MCF-7 cells. Effects of genistein in combination with other isoflavone components on proliferation, apoptosis, cell cycle perturbation, and signal proteins for survival, proliferation, and apoptosis were evaluated by WST-1 assay, FACS analyses, and Western blotting. Synergistic anti-proliferative activity of genistein was observed in combination with three respective isoflavone components, with the most efficacious interaction observed in combination with equal. In FACS study, cells were arrested at the G2/M by genistein but G1/S by equol. However, when these components were combined, cells were arrested at the G2/M, indicating a dominant effect of genistein over equol. Although apoptosis was not evident when cells were treated with either genistein or equol alone, combination treatment markedly induced apoptosis, with demonstrable cleavage of PARP. Upon combination treatment, constitutive activities of Erk1/2 were enhanced, but those of Akt were significantly inhibited, with subsequent reductions of activities of downstream mTOR. Reduced expression of anti-apoptotic Bcl-xL and enhanced expression of proapoptotic Bax were also demonstrated. These data indicate that synergic mechanism of genistein in combination with equol would be mediated by enhanced apoptosis induction through inactivation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of Bax and downregulation of Bcl-xL, thus providing mechanistic rationale for soy isoflavone for the prevention of human breast cancer. Citation Format: MISAKI ONO, KAORU EJIMA, MIKAKO TAKESHIMA, TAKAKO HIGUCHI, REI WAKIMOTO, SHUJI NAKANO. Mechanistic study of synergistic interaction between genistein and equol in MCF-7 human breast cancer cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 909. doi:10.1158/1538-7445.AM2015-909

PO-057 Genistein, a major isoflavone component, suppresses Src-induced proliferative activity by arresting cell cycle at G2/M through increasing the P53 and P21 levels

Introduction Src oncogene have been strongly implicated in the development, growth, progression, and metastasis of a variety of human cancers. Although soy isoflavones have been shown to have potential anticancer activity, the role of isoflavones in the oncogenic activity of Src remains unknown. Using HAG-1 human adenocarcinoma cells transfected with v-src, we investigated the functional role of Src in anti-proliferative activity of isoflavones such as genistein, daidzein, glycitin and equol. Material and methods The growth inhibitory activities of those isoflavones against Src- and vehicle-transfected cells (HAG/src and HAG/neo) were investigated using WST-1 cell proliferation assay. Effects of those isoflavones on apoptosis and cell cycle perturbation were evaluated by FACS analyses. Results and discussions The growth of HAG/neo cells was inhibited potently by genistein and equol, but modestly by daidzein and glycitin. In contrast, activated Src conferred resistance to either daidzein, glycitin or equol, but rendered the cells more sensitive to genistein, compared to HAG/neo; Genistein strongly inhibited the growth of HAG/src cells in a dose–dependent manner with IC50 value of 25 µM, whereas in other three isoflavones, the inhibitory effects were minimal without reaching an IC50 even at a dose of 100 µM. Upon treatment with 50 µM genistein for 72 hour, HAG/src cells were significantly arrested at the G2/M compared to HAG/neo cells (37.7% versus 7.0%). By contrast, the same concentration of either daidzein, glycitin or equol could not arrest HAG/src cells at any checkpoint of the cell cycle. The sub-G0/G1 apoptotic cell populations were not increased following 72 hour exposure with either isoflavones. Therefore, it appears that growth inhibition by genistein in Src-activated cells would be mediated mainly by the G2/M arrest of cell cycle rather than apoptosis induction. Genistein increased the expression levels of p53 and p21 with decreased phosphorylated p21. The levels of other main cell cycle-related proteins such as cyclin B, cyclin E, CDK2, and cdc2 were not altered. These data suggest that genistein would be the only isoflavone component that may potentially suppress oncogenic activity driven by Src through increasing the p53 and p21 levels. Conclusion These data suggest that genistein would be the only isoflavone component that may potentially suppress oncogenic activity driven by Src, providing a mechanistic rationale for the potential use of genistein in the prevention and treatment of human cancers with activated Src.

Abstract 841: Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS)

Soy isoflavones such as daidzein and its bioactive metabolite equol converted from daidzein by human intestinal bacterial flora, have been proposed to reduce the risk of breast cancer incidence in Asian women probably due to higher number of the inhabitants capable of producing equol in these countries. Although several in vitro studies shows the anti-cancer activity of equol against human breast cancer cells, there have been few reports that have directly showed the preventive role of equol in the experimental animal model of mammary carcinoma. We investigated here the effects of equol on the development of breast cancer using the EMS chemically induced rat model of hormone-dependent mammary carcinoma. Female WKA rats (n = 30) were equally divided into 2 groups, subsequently given EMS orally for 12 weeks and fed the CE2 diets with or without 50 mg equol/g diet throughout the experiments. All EMS-treated rats fed either diet developed ER- and/or PgR- positive hormone-dependent mammary carcinoma by 24 weeks. Compared with the equol-free diet, the diet supplemented with equol could not significantly delay the development of mammary carcinoma, showing any preventive activity on the development of EMS-induced hormone-dependent mammary carcinoma. The body weight, total EMS uptakes, and urine estradiol concentrations were not significantly different between these 2 groups. These data indicate that high dose administration of equol alone does not exert clear preventive effects on hormone-dependent EMS-induced mammary carcinogenesis, and may require other soy isoflavone components such as genistein that has previously been shown to exhibit a great synergy when combined with equol. The in vivo combination studies using genistein and equol are underway. Citation Format: MISAKI ONO, Takako Higuchi, Mikako Takeshima, Shuji Nakano. Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 841.

Abstract 2575: Differential anti-tumor activities of curcumin against apoptosis and cell Cycle progression in Src- and Ras-activated human gallbladder carcinoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Curcumin, a phytochemical in turmeric, has been studied as a potential anticancer drug targeting multiple signaling molecules. However, the role of Src and Ras signaling pathways in curcumin sensitivity remains unknown. We investigated therefore the molecular mechanism of anti-proliferative and apoptosis-inducing activities of curcumin in HAG-1 human gallbladder adenocarcinoma cells transfected with either activated Src (HAG/src3-1) or Ras (HAG/ras5-1). Effects of curcumin on proliferation, apoptosis, cell cycle perturbation, and signal proteins for survival, proliferation, and apoptosis were evaluated by WST-1 assay, FACS analyses and Western blotting. Activation of either Src or Ras did not confer resistance to curcumin compared to HAG/neo3-5 vehicle-transfected cells, producing similar IC50 concentrations of curcumin among these 3 cell lines. Upon treatment with curcumin, constitutive activities of Erk1/2 were enhanced, but those of Akt were significantly inhibited in these three cell lines, with subsequent reductions of activities of downstream mTOR and S6K1. The sub-G0/G1 apoptotic cell populations were substantially increased in all cell lines with demonstrable cleavage of PARP, but this increase was most prominent in Src-activated cells. Reduced expression and phosphorylation of Bcl-2 and enhanced expression of Bax were demonstrated to have a causative role for the curcumin-induced apoptosis in Src-driven, but not Ras-driven cells. By contrast, drastic increases of the proportion of cells in a G2/M phase were seen in Ras-activated cells, suggesting a potential role of Ras/Erk1/2 activation in curcumin-induced G2/M cell cycle arrest. These data indicate that curcumin-induced growth declines would be mediated mainly by G2/M arrest of the cell cycle in Ras-driven cells but by apoptosis induction in Src-driven cells. Taken together, the results indicate that curcumin exhibits differential activities against apoptosis and cell cycle progression, depending on activation mechanisms of signal transduction, and that curcumin can overcome Src- and Ras-driven activation of downstream signaling pathways, thus providing evidence of potential application of curcumin for the treatment of human cancers with activated Src or Ras. Citation Format: Misaki Ono, Takako Higuchi, Mikako Takeshima, Chen Chen, Shuji Nakano. Differential anti-tumor activities of curcumin against apoptosis and cell Cycle progression in Src- and Ras-activated human gallbladder carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2575. doi:10.1158/1538-7445.AM2013-2575