Supplementary Figures 1-2, Table 1 from AMP-Activated Protein Kinase Signaling Results in Cytoplasmic Sequestration of p27

Supplementary Figure S1 from Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma

This work investigated the extraction efficacy of phenolic acids on the potato and its byproducts. Also, the compositions of bioactive compounds and antioxidants were evaluated in various parts of the potato, such as the tuber, microtuber, peel, and flesh. The chemical constituents were quantified by HPLC analysis, and the highest levels of phenolics (88.99 mg/L) were obtained in acetone extracts from a micropropagated potato. The micropropagated potato demonstrated that notable phenolic compounds were mainly a bound form of phenolic acids including caffeic acid and vanillic acid. The micropropagated extracts using acetone showed the higher radical scavenging activity, 94.3% and 95.5% at 5 mg/mL in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS ·+ ), respectively. In addition, the same extracts showed the highest (85.61%) β-carotene bleaching inhibition activity. A positive relationship existed between DPPH and either ABTS ·+ ( r = 0.58, p < 0.05), β-carotene bleaching ( r = 0.65, p < 0.05), or total phenolics ( r = 0.63, p < 0.05). However, ABTS ·+ did not show a significant correlation between both total phenolics and β-carotene bleaching. The effective phenolic compounds contributing to antioxidant activity were caffeic acid and vanillic acid, which could be extracted in high amounts by acetone from potato peels and micropropagated potatoes.

Nomilin is a bitter compound present in citrus and has been demonstrated as useful for various disease preventions through anti-proliferative, anti-inflammatory, and pro-apoptotic activities. Although in vitro disease models have shown that certain limonoids in the p38 mitogen-activated protein kinase signal cascade, the downstream signaling pathways remain unclear. In this study, the effects of nomilin on the proliferation and apoptotic pathways of human aortic smooth muscle cells (HASMCs) that forms the basis of progression of atherosclerotic diseases and restenosis was tested for the first time. The cellular uptake level and stability of nomilin were determined by high-performance liquid chromatography and high-resolution mass spectra. Pretreatment of HASMCs with nomilin stimulated extrinsic caspase-8, intrinsic caspase-9, and apoptotic caspase-3 and resulted in significant inhibition of TNF-α-induced proliferation. Additionally, results showed a decreased ratio of anti-apoptotic Bcl-2 protein to pro-apoptotic Bax (Bcl2/Bax), indicating mitochondrial dysfunction consistent with apoptosis. Furthermore, nomilin significantly decreased the phosphorylation of IκBα, an inhibitor of NF-κB and subsequently, reduced the downstream inflammatory signaling in TNF-α treated HASMCs. Our findings indicate that the anti-proliferative activity of nomilin on TNF-α-induced HASMCs results from apoptosis through a mitochondrial-dependent pathway and suppression of inflammatory signaling mediated through NF-κB.

BACKGROUND:Bioactives extracted from cactus (Opuntia ficus-indica) stems were investigated for their chemopreventive activities using human cancer cells in vitro. The bioactives present in crude extracts were detected and quantified using high-performance liquid chromatography. RESULTS:Among all the extracts, such as hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH), and MeOH:water (80:20), the MeOH extract had the highest amount of polyphenolic compounds and the acetone extract exhibited the most potent effect at scavenging the 2,2,-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(•+) ) radical. In addition, most of the extracts, with the exception of hexane, exhibited significant cytotoxicity in human SW480 colon and MCF7 breast cancer cells. Overall, the SW480 cells were more sensitive than the MCF7 cells to the cytotoxic effect of the O. ficus-indica extracts (OFEs). Cell death by OFE treatment caused significant inhibition of cyclooxygenase-2 and increased the Bax/Bcl2 ratio in both SW480 and MCF7 cell lines. However, degradation of poly (ADP-ribose) polymerase was significantly increased by OFE only in the MCF7 cells, thereby inducing apoptosis. CONCLUSION:These findings demonstrate the health-benefit roles, including anti-oxidative and anti-proliferative activities as well as pro-apoptotic effects, of bioactive compounds in OFEs, suggesting a chemopreventive role in human cancer cells.

Overexpression of the aromatase enzyme CYP19 has been implicated in the onset of estrogen-dependent breast carcinogenesis. Obacunone, a natural compound present in citrus fruits, has been demonstrated for various biological activities including anti-cancer and anti-inflammatory properties. In the present study, we have isolated obacunone and obacunone glucoside (OG) from lemon seeds, then fractionated these compounds using chromatographic techniques and characterized them by HPLC, LC-MS, and 2D NMR spectral analysis. To investigate the mechanism of anti-cancer and anti-aromatase activities of limonoids, their cytotoxic effect was tested on human breast cancer (MCF-7) and non-malignant (MCF-12F) breast cells. MTT assays confirmed that obacunone was strongly inhibited MCF-7 cell proliferation without affecting non-malignant breast cells. Treatment with obacunone increased apoptosis by up-regulating expression of the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl2, as well as inducing G1 cell cycle arrest. In addition, obacunone significantly inhibited aromatase activity in an in vitro enzyme assay. Exposure of MCF-7 breast cancer cells to obacunone down-regulated expression of inflammatory molecules including nuclear factor-kappa B (NF-κB) and cyclooxygenase-2 (COX-2). Furthermore, we found that obacunone inhibited COX-2 and NF-κB by activation of the p38 mitogen-activated protein kinase (MAPK). Finally, the uptake level of obacunone into MCF-7 cells was measured by HPLC and its structure was confirmed by LC-HR-MS. This study demonstrated that obacunone may have the potential to prevent estrogen-responsive breast cancer through inhibition of the aromatase enzyme and inflammatory pathways, as well as activation of apoptosis.

Lemons are a widely used citrus crop and have shown several potential health benefits. In the present study, the mechanism and effectiveness of the anti-cancer and anti-aromatase properties of limonoids were investigated for the first time. Defatted lemon (Citrus lemon L. Burm) seed powder was extracted with ethyl acetate (EtOAc) and methanol (MeOH) for 16 h each, successively. These extracts were fractionated using 1D (silica) and 2D (ion exchange and SP-70 columns) column chromatography to obtain nine limonoids. The compounds were identified by TLC, HPLC, and LC-MS techniques. A panel of 9 purified limonoids, including limonin, nomilin, obacunone, limonexic acid (LNA), isolimonexic acid (ILNA), nomilinic acid glucoside (NAG), deacetyl nomilinic acid glucoside (DNAG), limonin glucoside (LG) and obacunone glucoside (OG) as well as 4 modified compounds such as limonin methoxime (LM), limonin oxime (LO), defuran limonin (DL), and defuran nomilin (DN), were screened for their cytotoxicity on estrogen receptor (ER)-positive (MCF-7) or ER-negative (MDA-MB-231) human breast cancer cells. We further tested the mechanism of the anti-proliferative activity of limonoids using an in vitro aromatase enzyme assay and western blot with anti-caspase-7. Among the tested limonoids, 11 limonoids exhibited cytotoxicity on MCF-7 whereas 8 limonoids showed cytotoxicity against the MDA-MB-231 cell lines. Although most of the limonoids showed anti-aromatase activity, the inhibition of proliferation was not related to the anti-aromatase activity. On the other hand, the anti-proliferative activity was significantly correlated with caspase-7 activation by limonoids. Our findings indicated that the citrus limonoids may have potential for the prevention of estrogen-responsive breast cancer (MCF-7) via caspase-7 dependent pathways.

For the first time, three coumarins were isolated from the hexane extract of limes (Citrus aurantifolia) and purified by flash chromatography. The structures were identified by NMR (1D, 2D) and mass spectral analyses as 5-geranyloxy-7-methoxycoumarin, limettin, and isopimpinellin. These compounds inhibited human colon cancer (SW-480) cell proliferation, with 5-geranyloxy-7-methoxycoumarin showing the highest inhibition activity (67 %) at 25 µM. Suppression of SW480 cell proliferation by 5-geranyloxy-7-methoxycoumarin was associated with induction of apoptosis, as evidenced by annexin V staining and DNA fragmentation. In addition, 5-geranyloxy-7-methoxycoumarin arrested cells at the G0/G1 phase, and induction of apoptosis was demonstrated through the activation of tumour suppressor gene p53, caspase8/3, regulation of Bcl2, and inhibition of p38 MAPK phosphorylation. These findings suggest that 5-geranyloxy-7-methoxycoumarin has potential as a cancer preventive agent.

Cactus (Opuntia spp) is widely cultivated as a vegetable, fruit, and forage crop and has been used in traditional medicine in American Indian, Mexican, and Korean cultures. Accumulative evidence from both in vitro and in vivo studies using cacti suggests their biological and pharmacological activities, such as their anti-cancer and anti-inflammatory roles in different cancer cells. In this study, the Opuntia humifusa stem (OHS) was extracted with different solvents and screened for radical scavenging activity using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS˙(+)) and 1,1-diphenyl-2-picryl hydrazyl (DPPH). In addition, the total phenolic and flavonoid contents of each extract were analyzed using the Folin-Ciocalteu method and high performance liquid chromatography, respectively. Further, the cacti's bioactive fractions were evaluated for cell cytotoxicity and to understand their mechanism of action on human colon cancer (SW480) and breast cancer (MCF7) cells. An ethyl acetate (EtOAc) extract exhibited the highest cytotoxicity and resulted in an up-regulated expression of the pro-apoptotic protein Bax (bcl-2 associated X protein) and a down-regulated expression of the anti-apoptotic protein Bcl2 in both SW480 and MCF7 cells. The apoptosis was mediated through activation of caspase 8, 9, and 3/7 activities as well as PARP cleavage in SW480 cells, while the same extract activated only a caspase 9 activity in MCF7 cells. Furthermore, incubation of cells with the EtOAc extract down-regulated the expression of inflammatory molecules such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) in SW480 cells but not in MCF7 cells. Taken together, these results suggest that SW480 colon cancer cells are more susceptible to bioactive compounds present in OHS and may have potential in the prevention of cancer through modulation of apoptosis markers and inhibition of inflammatory pathways.

Limonoids constitute one of the major phytochemicals along with flavonoids, coumarins, and carotenoids in citrus. Limonoids were shown to possess cancer preventive properties. In the past quarter century, a number of studies have accumulated considerable evidence, which advanced our understanding about the disease prevention mechanism of limonoids. It is speculated that limonoids suppress cell proliferation and induce apoptosis by inhibiting enzyme activities and signal transduction pathways. In addition, limonoids were also shown to inhibit cell metastasis in cell culture and animal studies. However, further research is required to identify the cellular targets of active limonoids. Identification of the target mechanism at cellular level may result in the development of limonoids as anticancer agents. In the present chapter, the diverse role and the biological activities of limonoids relating to their potential in cancer prevention are discussed.

Limonoids are triterpenoids found in citrus and possess cancer preventive properties in in vitro and in vivo assays. Although several mechanisms for the chemopreventive properties of limonoids have been postulated, the specific mechanisms involved in the anti-cancer effects have not been explored. In the present study, limonoids, including methyl nomilinate, isoobacunoic acid, isolimonexic acid (ILNA), and limonexic acid (LNA), were purified, identified by LC–MS and NMR spectral data and evaluated for their biological effects on SW480 human colon adenocarcinoma cells. Methyl nomilinate was the most potent inhibitor of cell metabolic activity in MTT and EdU incorporation assays. These limonoids did not affect apoptotic markers such as caspase-3 and PARP, but methyl nomilinate treatment resulted in significant induction of G0/G1 cell cycle arrest. Furthermore, methyl nomilinate suppressed CDK4/6 and cyclin D3 and the expression of CDK inhibitors. Taken together, the results suggest inhibition of cell proliferation by methyl nomilinate occurs due to G1 cell cycle arrest, indicating that methyl nomilinate has potential as a chemopreventive agent.

Extracts from lemon seed were investigated for the radical scavenging activity and apoptotic effects in human breast adenocarcinoma (MCF-7) cells and non-malignant breast (MCF-12F) cells for the first time. Defatted seed powder was successively extracted with ethyl acetate (EtOAc), acetone, methanol (MeOH), and MeOH:water (80:20). The chemical constituents were identified and quantified by LC-MS and HPLC analysis, respectively. The highest radical scavenging activity of 62.2% and 91.3% was exhibited by MeOH:water (80:20) at 833μg/mL in 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(+)), respectively. In addition, the MeOH:water (80:20) extract showed the highest (29.1%, P<0.01) inhibition of MCF-7 cells in MTT assay. Treatment of the MeOH:water (80:20) extract induced DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Increased levels of Bax and cytosolic cytochrome C and decreased levels of Bcl2 were also observed in MeOH:water (80:20) treated MCF-7 cells. In conclusion, the MeOH:water (80:20) extract from lemon seed has potent antioxidant activity and induces apoptosis in MCF-7 cells, leading to the inhibition of proliferation. These results suggest that aglycones and glucosides of the limonoids and flavonoid present in MeOH:water (80:20) extract may potentially serve as a chemopreventive agent for breast cancer.

Limonoids, abundantly present in citrus fruits, have potential role in reducing risk of different type of cancer. In the present study, we hypothesized that seven structurally different limonoids would involve in inflammatory pathway via modulating p38 MAP kinase activity at various extent in vascular smooth muscle cells. Results demonstrated that the different functional groups containing limonoids had differential effects on the p38 MAP kinase activity in human aortic smooth muscle cells. Among seven limonoids, nomilin exhibited the highest (38%) inhibition of p38 MAP kinase activity, followed by limonin (19%), deacetyl nomilin (19%), and defuran nomilin (17%). While defuran limonin and methyl nomilinate showed no significant decrease in p38 MAP kinase activity, obacunone significantly increased the p38 MAP kinase activity by 38%. Furthermore, TNF-α induced p38 MAP kinase activity in the smooth muscle cells was completely inhibited by nomilin. Thus our data provide the first evidence that nomilin is the potent natural inhibitor for p38 MAP kinase activity in human aortic smooth muscle cells. These data also suggest that a seven-membered A ring with acetoxy group, present in nomilin, seems to be essential for its inhibitory activity on p38 MAP kinase.

A number of studies in the recent years have evaluated the anti-proliferative activity of flavonoids. Although certain studies investigated the structure–activity based on the phenotypic assays, no study has correlated the flavonoids structure with the ability to alter gene/protein expression. Present study was focused to understand the structure–function relationship of citrus flavonoids in terms of their ability to alter the gene expression in the colon adenocarcinoma cells. Eight structurally related flavonoids found in citrus were evaluated for their ability to inhibit colon cancer (SW480) cells, as well as change the expression of apoptosis related genes/proteins. Apigenin and quercetagetin demonstrated most significant inhibition of cell proliferation with 63.6% and 45.7% inhibition of cell growth at 200 μM after 48 h of incubation, respectively. The cell death was also confirmed by images of fluorescently tagged cells. Furthermore, up-regulation of Bax/Bcl2 protein ratio as well as activation of Caspase3 at 200 μM at 48 h confirmed the induction of apoptosis by apigenin and quercetagetin. In addition, results suggest that the change in Bax/Bcl2 ratio by apigenin and quercetagetin seems to be due to their ability to alter the expression of bax and bcl2 transcription. Results of the currents study suggest that among the citrus flavonoids, double bond between C2 and C3 and hydroxyl group at C3, C6 are highly decisive for the proliferation inhibition and apoptosis induction ability. Taken together, these results demonstrate that among the major flavonoids of citrus, apigenin and quercetagetin have potent anti-cancer activity through inducing apoptosis in SW480 human colon cancer cells.

Inflammation is the hallmark of cardiovascular disease (CVD) and several inflammatory pathways act in concert to contribute to its pathogenesis. Although several studies report an increase in fruit consumption reduces the risk of CVD, the specific mechanisms are poorly understood. We have previously purified and identified several limonoids from citrus fruits using novel chromatographic and spectroscopic techniques. In our present study, the effect was determined of one such citrus limonoid, nomilin, on the MAP kinase activity in cardiovascular cells. Human aortic smooth muscle cells (HASMCs), human umbilical vein endothelial cells (HUVECs) and rat lymphatic muscle cells (LMCs) were used. These cells were treated with nomilin at different concentrations followed by the treatment with either substance P (SP) or TNF-alpha to activate MAPK pathway. Results demonstrate that nomilin completely reduced TNF-alpha induced p38 MAP kinase activity in HASMCs. Furthermore, nomilin plays a significant role in the regulation of SP or TNF-alpha mediated ERK1/2 and p38 MAP kinase activation in HUVECs and LMCs in a dose and time dependant manner. Taken together, we demonstrate for the first time that the citrus limonoid, nomilin, play a vital role in the modulation of pro-inflammatory pathways in cardiovascular cells. Funding: USDA-NIFA No. 2010-34402-20875 and “Designing Foods for Health,” VAFIC to BP, and NIH HL80526 and NIH KO2 HL86650 to MM.

Abstract Plant bioactive compounds found in many plant species have been used in Chinese, Unani, and Indian ayurvedic medicine. Accumulative evidences in both in vitro and in vivo studies using berberine seem to play anti-cancer and anti-inflammatory roles in different cancer cells. Previous research suggested berberine to exhibit potential anticancer activities in the various cancer cell lines. In the present study, we have isolated a putative compound from Phellodendron amurense using methanol and purified by chromatographic techniques. The structure of the isolated compound was identified as berberine by 2D NMR and mass spectral data. The isolated berberine was tested against MCF-7 breast cancer cell line and the MCF-12f normal breast epithelial cell line for 24, 48 and 72h at various concentrations (0, 12.5, 25, 50, 100, and 200µM). Using the MTT cell viability assay, berberine has demonstrated a significant cytotoxic effect on the MCF-7 cell line (p <0.05) in a time and concentration dependent manner. However, the same alkaloid did not show an effect on the proliferation of the MCF-12f normal cells. The induction of apoptosis or debilitation of cancer cells without causing excessive damage to normal cells by any natural putative compound is a key target for identifying methods of cancer prevention and therapy. Based on these results, MCF-7 cells treated with 25µM berberine for 48 and 72h were used for further studies to illustrate a time dependent induction of apoptosis with flow cytometry and agarose gel electrophoresis to identify visible DNA fragmentation. Western blotting with treated cells revealed that berberine induces apoptosis in MCF-7 cells through a mitochondrion-dependent pathway by increasing levels of pro-apoptotic protein, Bax, cytoplasmic cytochrome c, caspase-9 activity and cleavage of PARP while decreasing levels of anti-apoptotic protein, Bcl-2. Immunoblotting also suggests that p53 plays a pro-apoptotic role in berberine treated cells. We conclude that berberine inhibits the proliferation of MCF-7 breast cancer cells through an apoptotic pathway which may serve as a potential compound for cancer prevention or therapy. Further studies are needed to elucidate specific mechanism of action of this putative compound in breast cancer cells. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B70.

Bioactive compounds found in many plant species have been used in Chinese, Unani, and Indian ayurvedic medicine. Accumulative evidences in both in vitro and in vivo studies using berberine demonstrated anti-cancer and anti-inflammatory properties in different cancer cells. In the present study, a putative compound from commercial sample was purified by chromatographic techniques. The structure of the pure compound was confirmed by spectroscopic studies. The purified berberine was tested against breast cancer (MCF-7) and normal human breast epithelial (MCF-12F) cells for 24, 48 and 72h at various concentrations. Using MTT assay, berberine exhibited a significant cytotoxic effect on the MCF-7 cells (P<0.01) without affecting the breast normal epithelial cell growth at 25μM concentration. Based on these results, MCF-7 cells were treated with 25μM berberine for 48 and 72h for further studies to illustrate induction of apoptosis through cell cycle distribution and DNA fragmentation with agarose gel electrophoresis. Western blotting with treated cells revealed that berberine induces apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytoplasmic cytochrome c, caspase-9 activity and cleavage of PARP while decreasing levels of Bcl-2. Furthermore, immunoblotting results demonstrated that p53 and p27 were up-regulated suggesting that barberine seems to play a pro-apoptotic role in cancer cells. In conclusion, berberine inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway. It is possible that berberine may serve as a potential naturally occurring compound for breast cancer therapy.

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy.Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated.Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not.Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.