Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K

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Toxicol Appl Pharmacol. 2008 Jan 15;226(2):178-91. Epub 2007 Sep 21.
Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells.

Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH.
Institute of Biochemistry and Biotechnology, Chung Shan Medical University Hospital, Taichung, Taiwan.
Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration and anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of beta 4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin beta 4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-beta 4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin beta 4 function.

PMID: 17961621 [PubMed - indexed for MEDLINE]


Mol Cancer Ther. 2008 Jul;7(7):2096-108.
Apigenin inhibits antiestrogen-resistant breast cancer cell growth through estrogen receptor-alpha-dependent and estrogen receptor-alpha-independent mechanisms.

Long X, Fan M, Bigsby RM, Nephew KP.
Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401, USA.
Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 mumol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 mumol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-alpha (ERalpha); transactivation activity, mobility, stability, and ERalpha-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERalpha and its coactivator amplified in breast cancer-1. However, higher doses (>10 mumol/L) of apigenin inhibited ERalpha mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERalpha and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERalpha-dependent and ERalpha-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.

PMID: 18645020 [PubMed - indexed for MEDLINE]




Oncol Rep. 2009 Dec;22(6):1533-7.
5-Fluorouracil combined with apigenin enhances anticancer activity through induction of apoptosis in human breast cancer MDA-MB-453 cells.

Choi EJ, Kim GH.
Plant Resources Research Institute, Duksung Women's University, Seoul, Korea. ejchoi@duksung.ac.kr
We investigated the effects of combined treatment with 5-fluorouracil and apigenin on proliferation and apoptosis, as well as the underlying mechanism, in human breast cancer MDA-MB-453 cells. The MDA-MB-453 cells, which have been shown to overexpress ErbB2, were resistant to 5-fluorouracil; 5-fluorouracil exhibited a small dose-dependent anti-proliferative effect, with an IC50 of 90 microM. Interestingly, combined treatment with apigenin significantly decreased the resistance. Cellular proliferation was significantly inhibited in cells exposed to 5-fluorouracil at its IC50 and apigenin (5, 10, 50 and 100 microM), compared with proliferation in cells exposed to 5-fluorouracil alone. This inhibition in turn led to apoptosis, as evidenced by an increased number of apoptotic cells and the activation of caspase-3. To investigate the mechanism by which the combination of 5-fluorouracil and apigenin induces apoptosis, ErbB2 expression was analyzed. The level of ErbB2 was unchanged by 5-fluorouracil alone but was drastically reduced in cells treated with 5-fluorouracil plus apigenin. Moreover, compared with 5-fluorouracil alone, 5-fluorouracil in combination with apigenin at concentrations >10 microM exerted a pro-apoptotic effect via the inhibition of Akt expression. Taken together, our results suggest that 5-fluorouracil acts synergistically with apigenin inhibiting cell growth and inducing apoptosis via the down-regulation of ErbB2 expression and Akt signaling.

PMID: 19885610 [PubMed - in process]





Carcinogenesis vol.28 no.4 pp.858–864, 2007
doi:10.1093/carcin/bgl205

Apigenin inhibits tumor angiogenesis through decreasing HIF-1a and VEGF expression

FULL TEXT(PDF)

Jing Fang1, Qiong Zhou1, Ling-Zhi Liu1, Chang Xia2,
Xiaowen Hu1, Xianglin Shi1 and Bing-Hua Jiang1,2,
1The Institute for Nutritional Sciences, Shanghai Institute for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China and
2Mary Babb Randolph Cancer Center, Department of Microbiology,
Immunology and Cell Biology, West Virginia University, Morgantown,
WV 26506, USA
To whom correspondence should be addressed.
Email: bhjiang@hsc.wvu.edu
Apigenin is a non-toxic dietary flavonoid with anti-tumor properties. We recently showed that apigenin-inhibited hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in human ovarian
cancer cells under normoxic condition. However, the effect of apigenin in angiogenesis remains to be elucidated. Angiogenesis is the formation of new blood vessels and is required for tumor growth and metastasis. In this study, we showed that apigenin-inhibited expression of HIF-1 and VEGF in different cancer cells under both normoxic and hypoxic conditions. We demonstrated that apigenin significantly inhibited tumor angiogenesis in vivo, by using both the chicken chorioallantoic membrane and Matrigel
plug assays. The inhibition of tumor angiogenesis was associated with the decrease of HIF-1 and VEGF in tumor tissues. Taken together, our results show that apigenin suppresses tumor angiogenesis through HIF-1 and VEGF
expression.

In this study, we tested several human cancer cells including prostate cancer cells PC-3, DU145 and LNCaP, breast cancer cell MCF-7, and colon cancer cell HCT-8. PC-3 and DU145 cells produced high levels of HIF-1a and VEGF under normoxic conditions (Figure 1A and B). Apigenin inhibited expression of HIF-1a and VEGF in these cells (Figure 1A and B). We found that apigenin inhibited VEGF transcriptional activation through HIF-1a expression (Figure 1D and E). During tumor growth, there is low oxygen environment inside the tumor. Therefore, it is interesting to know whether apigenin could inhibit HIF-1a and VEGF expression induced by hypoxia. Under hypoxia condition, accumulation of HIF-1a and VEGF proteins was observed in PC-3 cells and addition of apigenin suppressed hypoxia-induced HIF-1a and VEGF expression (Figure 1C). Similarly, the induction of HIF-1a and VEGF by hypoxia was abrogated in several different cancer cells by
apigenin treatment (Figure 2). These results suggest that apigenin inhibits HIF-1a and VEGF expression under both normoxic and hypoxic conditions in different cancer cells.

To assess the effects of apigenin on tumor angiogenesis, we performed angiogenesis assay in vivo using two different assay systems. The results showed that apigenin is a potent inhibitor of tumor-induced angiogenesis (Figures 4 and 5). Apigenin also inhibited expression of HIF-1a and VEGF in tumor tissues (Figure 4C and D), suggesting that apigenin inhibits angiogenesis through HIF-1a and VEGF expression.

Mol Carcinog. 2008 Sep;47(9):686-700.
Inhibition of HIF-1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3-M cells.

Mirzoeva S, Kim ND, Chiu K, Franzen CA, Bergan RC, Pelling JC.
Department of Pathology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, USA.
Progression of cancer leads to hypoxic solid tumors that mount specific cell signaling responses to low oxygen conditions. An important objective of anti-cancer therapy is the development of new drugs that suppress hypoxic responses in solid tumors. Apigenin is a natural flavone that has been shown to have chemopreventive and/or anti-cancer properties against a number of tumor types. However, the mechanisms underlying apigenin's chemopreventive properties are not yet completely understood. In this study, we have investigated the effects of apigenin on expression of hypoxia-inducible factor-1 (HIF-1) in human metastatic prostate PC3-M cancer cells. We found that hypoxia induced a time-dependent increase in the level of HIF-1alpha subunit protein in PC3-M cells, and treatment with apigenin markedly decreased HIF-1alpha expression under both normoxic and hypoxic conditions. Further, apigenin prevented the activation of the HIF-1 downstream target gene vascular endothelial growth factor (VEGF). We then showed that apigenin inhibited expression of HIF-1alpha by reducing stability of the protein as well as by reducing the level of HIF-1alpha mRNA. We also found that apigenin inhibited Akt and GSK-3beta phosphorylation in PC3-M cells. Further experiments demonstrated that constitutively active Akt blunted the effect of apigenin on HIF-1alpha expression. Taken together, our results identify apigenin as a bioflavonoid that inhibits hypoxia-activated pathways linked to cancer progression in human prostate cancer, in particular the PI3K/Akt/GSK-3 pathway. Further studies on the mechanism of action of apigenin will likely provide new insight into its applicability for pharmacologic targeting of HIF-1alpha for cancer therapeutic or chemopreventive purposes.

PMID: 18240292 [PubMed - indexed for MEDLINE]




Biochem Pharmacol. 2010 Feb 11. [Epub ahead of print]
Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism.

Ansó E, Zuazo A, Irigoyen M, Urdaci MC, Rouzaut A, MartÃ*nez-Irujo JJ.
Department of Biochemistry and Molecular Biology, University of Navarra, Pamplona, Spain.
Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure-activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4' position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4',7-dihydroxiflavone, reduced the expression of HIF-1alpha under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1alpha expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1alpha translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process. Copyright © 2010 Elsevier Inc. All rights reserved.

PMID: 20153296 [PubMed - as supplied by publisher]

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How Eating Fruit and Vegetables Can Improve Cancer Patients’ Response to Chemotherapy

UC Riverside study describes how naturally occurring apigenin facilitates the death of cancer cells
(October 22, 2008)

NEWS MEDIA CONTACT

Name: Iqbal Pittalwala
Tel: (951) 827-6050
E-mail: iqbal@ucr.edu

RIVERSIDE, Calif. – The leading cause of death in all cancer patients continues to be the resistance of tumor cells to chemotherapy, a form of treatment in which chemicals are used to kill cells.

Now a study by UC Riverside biochemists that focuses on cancer cells reports that ingesting apigenin – a naturally occurring dietary agent found in vegetables and fruit – improves cancer cells’ response to chemotherapy.

Xuan Liu, a professor of biochemistry, and Xin Cai, a postdoctoral researcher working in her lab, found that apigenin localizes tumor suppressor p53, a protein, in the cell nucleus – a necessary step for killing the cell that results in some tumor cells responding to chemotherapy.

The study, published this week in the online early edition of the Proceedings of the National Academy of Sciences, provides a novel approach to conquer tumor resistance to chemotherapy, and suggests an avenue for developing safe chemotherapy via naturally occurring agents.

Normally, cells have low levels of p53 diffused in their cytoplasm and nucleus. When DNA in the nucleus is damaged, p53 moves to the nucleus where it activates genes that stop cell growth and cause cell death. In this way, p53 ensures that cells with damaged DNA are killed.

In many cancers, p53 is rendered inactive by a process called cytoplasmic sequestration. Apigenin is able to activate p53 and transport it into the nucleus, resulting in a stop to cell growth and cell death.

“In therapy you want to kill cancer cells,” explained Cai, the first author of the research paper. “But to stop cell growth and to kill the cell, p53 first needs to be moved to the cell’s nucleus to function. Apigenin is very effective in localizing p53 this way.”

Apigenin is mainly found in fruit (including apples, cherries, grapes), vegetables (including parsley, artichoke, basil, celery), nuts and plant-derived beverages (including tea and wine). It has been shown by researchers to have growth inhibitory properties in several cancer lines, including breast, colon, skin, thyroid and leukemia cells. It has also been shown to inhibit pancreatic cancer cell proliferation.

“Our study advocates the inclusion of vegetables and fruit in our daily diet to help prevent cancer,” said Liu, the research paper’s coauthor.

The National Institutes of Health supported the five-year study.

Next in their research Liu and Cai plan to design therapies for cancer by finding compounds that are like, but perform better than, apigenin.

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Apigenin supplement: http://www.naturatherapeutics.com/apigen/faq

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J Cell Physiol. 2006 Apr;207(1):23-9.
Flavonoids inhibit melanoma lung metastasis by impairing tumor cells endothelium interactions.

Piantelli M, Rossi C, Iezzi M, La Sorda R, Iacobelli S, Alberti S, Natali PG.
Department of Oncology and Neurosciences, G.d'Annunzio University of Chieti-Pescara, Chieti, Italy. mpiantel@unich.it
Flavonoids comprise a class of low molecular weight compounds displaying a variety of biological activities including inhibition of tumor growth and metastasis. To gain insight into the mechanisms underlying metastasis inhibition, we have employed the B16-BL6 murine melanoma metastasis model. B57BL/6N mice were injected i.v. with tumor cells and Apigenin, Quercetin, or Tamoxifen, each at 50 mg/kg given i.p., and lung tumor cell colonies counted 14-6 days thereafter. Three different injection schedules were used for each drug: (a) daily injection, starting 24 h before injection of the tumor cells; (b) single dose, 24 h preceding tumor challenge; (c) daily injection, starting 24 h after the injection of the tumor cells. All three compounds significantly reduced tumor lung deposits (Apigenin = Quercetin > Tamoxifen). However, when treatment was delayed by 24 h after tumor cells (schedule c), multiple daily doses of Apigenin or Quercetin were less effective that a single dose of the same compound given 24 h before tumor challenge (schedule b). Apigenin and Quercetin, but not Tamoxifen, were found to inhibit VCAM-1 expression in a dose-dependent manner in HUVEC and in murine pulmonary endothelial cells. In ex vivo experiments, the number of tumor cells adhering to lung vessels was significantly diminished in animals treated with a single dose of Apigenin and Quercetin. These findings indicate that the inhibition of tumor cell metastasis by Apigenin or Quercetin may significantly depend on the ability of these compounds to alter the host's microenvironment, further substantiating the role of the intravascular processes in the metastatic cascade.

PMID: 16222712 [PubMed - indexed for MEDLINE]