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ANTICANCER RESEARCH 26: 1177-1182 (2006)

The Antiproliferative Effect of Quercetin in Cells is Mediated via Inhibition of the PI3K-Akt/PKB Pathway

NICHOLAS GULATI, BEATRICE LAUDET, VAHE MICHAEL ZOHRABIAN, RAJ MURALI and MEENA JHANWAR-UNIYAL

Department of Neurosurgery, New York Medical College, Valhalla, NY, U.S.A.

Abstract. Background: The tumor suppressor PTEN, Phosphatidylinositol (PtdIns) 3-kinase(PI3K) is an enzyme mutated in 40-50% of patients with brain tumors, especially those involved in proliferation and survival in response to with glioblastomas, maps to 10q23.3 and encodes a growth factors. Akt/ (PKB), a dual-specificity phosphatase. PTEN exerts its effects partly via serine/threonine protein kinase, has been implicated in inhibition of protein tyrosine kinase B (Akt/Protein Kinase B), mediating a variety of biological responses. Cell survival which is involved in the phosphatidylinositol (PtdIns) 3-kinase signals can be elicited by the activation of PI3K and (PI3K)-mediated cell-survival pathway. The naturally occurring downstream Akt/PKB, both potent regulators of . bioflavonoid Quercetin (Qu) shares structural homology with the The tumor suppressor gene PTEN (phosphatase and tensin commercially available selective PI3K inhibitor, LY 294002 (LY). homolog), which maps to chromosome 10q23.3 and encodes a Here, the effects of Qu on the Akt/PKB pathway were evaluated. dual-specificity phosphatase, has been shown to be a negative Materials and Methods: The human breast cell lines, regulator of the PI3K-Akt/PKB cell-survival pathway (1). The HCC1937, with homozygous deletion of the PTEN gene, and PTEN gene product acts on phosphatidylinositol-3, 4, T47D, with intact PTEN, were time-treated with Qu or LY and 5-triphosphate [PtdIns(3,4,5)P3](PIP3), which lies upstream of analyzed for activated levels of Akt by measuring phospho-Akt the Akt/PKB pathway. Among high-grade gliomas, activation (p-Akt) levels using immunoblotting analysis. To detect p-Akt, the of the PI3K-Akt/PKB signaling pathway through loss of PTEN T47D cells were treated with EGF prior to treatment with or function is common. Loss of PTEN is also prevalent in many without Qu or LY. after 24-h treatment with Qu other tumor types (2). PTEN de-phosphorylates PIP3, leading or LY was quantified by the 3-(4, 5-dimethylthiazol-2-yl)-2, to its decreased level and, thus, diminishes Akt/PKB activity 5-diphenyltetrazolium bromide (MTT) assay. Results: Treatment (3). Lack of the PTEN gene product is associated with high with Qu (25 ÌM) for 0.5, 1 and 3 h completely suppressed Akt/PKB activation (3), and re-expression of PTEN reverts constitutively activated Akt/PKB phosphorylation at Ser-473 in some of the growth-activated function. HCC1937 cells. Pre-exposing T47D cells to Qu (25 ÌM) or LY The commercially available inhibitor of PI3K, LY 294002 (10 ÌM) abrogated EGF-induced Akt/PKB phosphorylation at (LY), retards the activated Akt/PKB pathway. LY is Ser-473. Both Qu (100 ÌM) and LY (50 ÌM) treatments for 24 h structurally very similar to Quercetin (Qu) (2-(3,4- significantly decreased cell proliferation, as shown by the MTT dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4- assay. Conclusion: Pharmacologically safe doses of the naturally one), a naturally occurring bioflavonoid generally found in occurring bioflavonoid Qu inhibit the PI3K-Akt/PKB pathway, in red wine, apple, berries and other components of the human a manner similar to that of the commercially available LY. diet (4). Qu has been shown to have anti-carcinogenic and Overall, our results indicated that Qu inhibited the constitutively anti-tumorigenic properties. Studies have revealed Qu to be activated-Akt/PKB pathway in PTEN-null cancer cells, and effective against progression in many organs and suggest that this compound may have therapeutic benefit against the anti-tumorigenic activity of Qu has been evidenced by tumorigenesis and cancer progression. its ability to suppress the growth and of cells (5). Although one study found no anti- tumor effects of Qu in in vivo mice models of colon cancer (6), the administration of the bioflavonoid was quite Correspondence to: Meena Jhanwar-Uniyal, Departments of different in the two studies. At the same time, experiments Neurosurgery/Pathology, New York Medical College, Valhalla, have shown that Qu inhibits DNA synthesis and New York 10595, U.S.A. Tel: 914-594-3186, Fax: 914-594-3641, e- mail: [email protected] of cells via the suppression of and the epidermal (EGF) involved in cell Key Words: PTEN, Akt/PKB, LY 294002, quercetin. cycle regulation (7). These studies have argued that Qu has

0250-7005/2006 $2.00+.40 1177 ANTICANCER RESEARCH 26: 1177-1182 (2006) no effect on the expression of other and apoptosis regulators, such as , , , bcl-2 and bax (7). Qu is known to inhibit PI3K, as well as mediating the inhibition directed towards the ATP-binding site of PI3K (8). The BRCA1 mutant human breast carcinoma cell line HCC1937 displays constitutive activation of Akt/PKB due to homozygous deletion of the PTEN gene. The aim of this study was to suppress Akt/PKB activity in HCC1937 using the bioflavonoid Qu. In order to qualify the efficacy of Qu, T47D human breast cancer cells with intact PTEN and Figure 1. Chemical structures of Qu (left) and LY (right). Chemical suppressed Akt/PKB pathways were treated with a growth structural analysis revealed that both Qu and LY are members of the factor so as to stimulate the cell survival pathway. LY and benzopyranone class of compounds. Qu were both found to significantly curb Akt/PKB activity.

Materials and Methods The cells were incubated in a humidified 5% CO2 atmosphere at 37ÆC for 24 h. The cells were made quiescent by incubating with Cell culture. The human breast cancer cell lines, HCC1937 with serum-free RPMI for 24 h. The quiescent cells were then treated homozygous deletion of the PTEN gene, and T47D cells with intact with either the bioflavonoid Qu or LY in serum-free media for PTEN, were obtained from the American Type Culture Collection 24 h. At the end of the assay, all media was removed and replaced (ATCC, VA, USA). Both cell lines were cultured in RPMI 1640, with 100 ÌL/well fresh serum-free RPMI. The MTT Cell Growth supplemented with 1% L-Glutamine, 7.5% fetal bovine serum, and Assay Kit (Chemicon International, CA, USA) was utilized to 1% penicillin-streptomycin, and incubated in a humidified 5% CO 2 evaluate cell proliferation. In brief, 10 ÌL AB solution (MTT, (3-(4, atmosphere at 37ÆC. Serum-free RPMI (0.5% FBS) was given to 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, 50 mg), 80-100% confluent cells 24 h prior to Qu or LY treatments to and PBS pH 7.4, 15 mL) were added to each well. The culture induce quiescence (G phase). The HCC1937 cells were treated 0 plates were mixed gently and incubated at 37ÆC for 4 h in order for with 25 ÌM Qu (Calbiochem, CA, USA) or 10 ÌM LY cleavage of MTT to occur. One hundred microliters of Solution C (Calbiochem) for 1 and 3 h. The T47D cells were treated with either (isopropanol with 0.04 N HCl) were added to each well and mixed Qu or LY prior to EGF (Calbiochem) or TPA (Sigma, MO, USA) by pipetting. The absorbance was measured within 1 h on an treatment. Each experiment was repeated 3 times. ELISA plate reader at 595 nm. Western blot analysis. After treatment, the cells were harvested in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, Results 1 mM EDTA and 1% (V/V) Triton X-100 plus protease and phosphate inhibitors (Sigma). The protein content was measured by Structural similarity between LY and Qu. Chemical structural the Bradford procedure, with bovine serum albumin as a standard. analysis revealed that both the naturally occurring The cell lysate (50 Ìg) was separated on a 10% sodium dodecyl bioflavonoid Qu and the commercially available selective sulfate (SDS)-polyacrylamide gel. After electrophoresis, the PI3K inhibitor LY are members of the benzopyranone class proteins were transferred to PVDF or nitrocellulose membranes. of compounds (Figure 1). LY was synthesized using Qu as a The sheets were pre-incubated in a mixture of TBS (20 mM Tris- HCl [pH 7.5], 150 mM NaCl), 0.05% Tween 20 and 5% de-fatted model (9). Studies have shown that LY has a greater milk powder for 1 h at room temperature, and were then incubated potency than Qu, which is considered a broad-spectrum with TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl), 0.05% Tween inhibitor. Binding of LY and Qu to PI3K differ (8). The 20 and 5% de-fatted milk powder with the following antibodies, as chrome moiety of Qu occupies the space filled by the appropriate: anti-phospho-Akt (Cell Signaling Technology, MA, morpholino ring of LY 294002. The hydrogen bond between USA), anti-Akt (Cell Signaling Technology). After being washed in the 3-OH of Qu and the side chain of LYS-833 mimics the TBS-0.05% Tween 20, the membranes were incubated in peroxide- interaction made by the ketone moiety of LY, indicating coupled secondary antibodies (1/2000 dilution; Cell Signaling Technology) for 1 h at room temperature. Following incubation, the that the chrome moiety of LY is flipped 180 degrees with membranes were washed in TBS (20 mM Tris-HCl [pH 7.5], respect to the mode of binding in the Qu/PI3K complex. 150 mM NaCl), 0.05% Tween 20, 3 times for 5 min each. The membranes were incubated with secondary antibodies following the Qu and LY similarly inhibit Akt/PKB activation. HCC1937 manufacturer’s instructions (Cell Signaling Technology). Following cells with deletion of the PTEN gene and activated Akt/PKB, the immunoanalysis, the blots were stripped and re-probed with un- and T47D cells lacking constitutively activated Akt/PKB, phosphorylated Ab-Akt to ensure equal loading. were utilized in order to determine whether Qu inhibits Cell proliferation assay. The HCC1937 cells were cultured and Akt/PKB in a manner comparable to LY. As shown in incubated as above. Approximately 15,000 HCC1937 cells/well were Figures 2A and B, phosphorylated-Akt (p-Akt) levels were seeded in 96-well flat-bottomed microtiter culture plates (Sigma). seen in vehicle-treated controls in HCC1937 cells. However,

1178 Gulati et al: Inhibition of the Akt/PKB Pathway

Figure 2. Western blot analysis illustrating the levels of activated Akt/PKB (p-Akt) in HCC1937 cells with homozygous PTEN deletion (2A top panel; 2B). The vehicle-treated control shows p-Akt levels. Treatment of these cells with Qu (25 ÌM) or LY (10 ÌM) suppressed Akt/PKB activation, noted by lack of detectable p-Akt/PKB levels. The same blot was stripped of the p-Akt antibody and then re-probed with anti-Akt antibody to ensure that basic level of Akt/PKB was the same (2A bottom panel; 2C).

treatment with LY (10 ÌM) and Qu (25 ÌM) for 0.5 h, 1 h Discussion and 3 h completely eliminated Akt/PKB activity, as evidenced by the lack of detectable p-Akt levels. Both LY In the absence of the tumor suppressor gene PTEN, we expect and Qu were similarly effective. The same blot was stripped a constitutive activation of the Akt/PKB pathway. In HCC1937 of the p-Akt antibody and then re-probed with anti-Akt cells that carry homozygous deletions of PTEN, we found that antibody to ensure that the basic level of Akt/PKB was the Akt/PKB was constitutively activated. This activation was same. As shown in Figures 3A and B, the T47D cell line evidenced by increased p-Akt levels in the control cells. displayed p-Akt only after EGF treatment. The control Furthermore, the results of this study demonstrated that Qu revealed no detectable p-Akt levels. However, EGF failed to and LY were equally effective in suppressing Akt/PKB activity induce Akt/PKB phosphorylation in cells pre-treated with in PTEN-deficient cells within the same span of time. Qu may LY (10 ÌM) or Qu (25 ÌM). Treatment with phorbol ester, be more effective, since activation of ERK was seen 1 h after TPA, insignificantly increased Akt/PKB activity, which was treatment, as compared to the 24 h that were required for suppressed in the Qu- or LY-treated T47D cells. activation with LY treatment (data not shown). The colorimetric MTT assay for cell proliferation and survival Qu and LY and cell proliferation. In support of the proposed revealed significant decreases in cell growth in Qu- or LY- efficacy of Qu and LY, the results from the colorimetric treated HCC1937 cells. This study clearly demonstrated that MTT assay for cell survival and proliferation revealed Qu suppresses cell proliferation and, thus, may have anti- significant decreases in cell proliferation in Qu- or LY- tumorigenic properties due to its ability to suppress the treated HCC1937 cells. The control values were set to 100. Akt/PKB pathway. Qu may be particularly useful in such cells The LY-treated cells displayed cell growth value decreases where the PTEN gene is inactivated, and Akt/PKB pathways of 57% at 24 h. The results of Qu on the HCC1937 cells constitutively activated. also showed approximately 50% decreases in cell Phosphatidylinositol second messengers play an important proliferation (Figures 4A and B). role in the pathways that regulate cell

1179 ANTICANCER RESEARCH 26: 1177-1182 (2006)

Figure 3. Western blot analysis revealing the levels of activated Akt/PKB (p-Akt) in T47D cells. The vehicle-treated control showed no activated Akt/PKB (p-Akt); the EGF-treated cells showed p-Akt/PKB expression (3A top panel; 3B). Pre-treatment with either Qu or LY prevented the activation of Akt/PKB induced by EGF. TPA treatment caused an insignificant increase in Akt/PKB activity, which was also abolished by pre-treatment with Qu or LY. The same blot was stripped of the p-Akt antibody and then re-probed with anti-Akt antibody to ensure that the basic level of Akt/PKB was the same (3A bottom panel; 3C).

Figure 4. Colorimetric MTT assay depicting LY and Qu inhibition of the Akt/PKB pathway in HCC1937 cells. The cells were exposed to either LY (50 ÌM) or Qu (100 ÌM) for 24 h, and cell proliferation measured by the MTT assay. Control wells contained untreated cells. Both Qu (100 ÌM) and LY (50 ÌM) treatments caused approximately 50% decreases in cell proliferation, compared to the control set to 100 (4A). Photomicrographs of Quercetin-induced apoptotic cells following 24-h treatment, and vehicle-treated control showing intact cells, are depicted in (4B).

1180 Gulati et al: Inhibition of the Akt/PKB Pathway growth, actin rearrangement and differentiation, as well as 2 Wang SI, Puc J, Li J, Bruce JN, Cairns P, Sidransky D and apoptosis. Abundant data have demonstrated that PIP3 is Parsons R: Somatic of PTEN in glioblastoma required for the activation of Akt/PKB, a serine/threonine multiforme. Cancer Res 57: 4183-4186, 1997. 3 Hyun T, Yam A, Pece S, Xie X, Zhang J, Miki T, Gutkind JS protein kinase that plays an important role in cell survival. and Li W: Loss of PTEN expression leading to high Akt PTEN antagonizes the PI3K-Akt/PKB pathway by de- activation in human multiple myelomas. Blood 96(10): 3560- phosphorylating PIP3, resulting in the translocation of 3568, 2000. Akt/PKB to the cellular membranes, and subsequent down- 4 Careri M, Corradini C, Elviri L, Nicoletti I and Zagnoni I: regulation of Akt/PKB activation. Furthermore, it has been Direct HPLC analysis of quercetin and trans-resveratrol in red established that expression of PTEN in cells leads to decreased wine, grape, and winemaking byproducts. J Agric Food Chem levels of phospho-Akt, and to increased apoptosis (10). In 51: 5226-5231, 2003. 5 Caltagirone S, Rossi C, Poggi A, Ranelletti FO, Natali PG, addition, it has been shown that constitutively active Akt/PKB Brunetti M, Aiello FB and Piantelli M: Flavonoids apigenin and can rescue cells from PTEN-mediated G1 arrest and apoptosis. Qu inhibit melanoma growth and metastatic potential. Int J The selective PI3K inhibitor LY has been shown to mimic the Cancer 87(4): 595-600, 2000. effects of PTEN (11). The effect of PTEN on PI3K is 6 Mahmoud NN, Carothers AM, Grunberger D, Bilinski RT, phosphatase-dependent. Studies have shown that PTEN Churchill MR, Martucci C, Newmark HL and Bertagnolli MM: coordinates G1 arrest through the up-regulation of p27 and Plant phenolics decrease intestinal tumors in an animal model concomitant down-regulation of cyclin D1 (12, 13). of familial adenomatous polyposis. 21(5): 921- 927, 2000. High Akt/PKB activity in breast cancer cells is associated 7 Kaneuchi M, Sasaki M, Tanaka Y, Sakuragi N, Fujimoto S with a poor patho-phenotype, as well as and and Dahiya R: Qu regulates growth of Ishikawa cells through resistance. Moreover, the PTEN gene is the suppression of EGF and cyclin D1. Int J Oncol 22: 159- commonly mutated or deleted in glioblastoma multiforme 164, 2003. (GBM) and endometrial cancer. Therefore, a 8 Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, chemotherapeutic agent directed specifically towards poor Wymann MP and Williams RL: Structural determinants of prognosis tumors, especially those with high Akt/PKB activity, phosphoinositide 3-kinase inhibition by wortmannin, LY294002, Qu, myricetin, and staurosporine. Mol Cell 6(4): 909-919, 2000. would be a worthy drug treatment model to explore. 9 Vlahos CJ, Matter WF, Hui KY and Brown RF: A specific Furthermore, rapamycin, an inhibitor of the mammalian inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8- target of rapamycin (mTOR), downstream of Akt/PKB, has phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269: also been demonstrated to have an anti-proliferative effect on 5241-5248, 1994. cancer cells (14). Future studies may demonstrate whether or 10 Davies MA, Lu Y, Sano T, Fang X, Tang P, LaPushin R, Koul not Qu inhibits mTOR in a similar manner and, thus, has D, Bookstein R, Stokoe D, Yung WK, Mills GB and Steck PA: potential as a therapeutic target for glioblastoma patients. It Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis. Cancer may be possible that long-term inhibition of the Akt/PKB Res 58(23): 5285-5290, 1998. pathway affects cell proliferation and growth, whereas short- 11 Li DM and Sun H: PTEN/MMAC1/TEP1 suppresses the term effects may modulate cell motility, leading to the invasive tumorigenicity and induces G1 cell cycle arrest in human nature of cancer cells devoid of PTEN. Further investigation is glioblastoma cells. Proc Natl Acad Sci USA 95(26): 15406- needed to consider these differences in the response of cancer 15411, 1998. cells to agents such as Qu that influence Akt/PKB signaling 12 Persad S, Troussard AA, McPhee TR, Mulholland DJ and pathways. For now, our results suggest that the inhibition of Dedhar S: Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer the constitutively activated Akt/PKB pathway in cancer cells, factor 1-mediated transcriptional activation. J Cell Biol 153(6): due to inactivation of the PTEN gene, can be suppressed by 1161-1174, 2001. the naturally occurring compound Qu in a manner similar to 13 Cheney IW, Neuteboom ST, Vaillancourt MT, Ramachandra the structurally related, commercially available compound LY. M and Bookstein R: Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits entry by Acknowledgements the recruitment of p27Kip1 into /CDK2 complexes. Cancer Res 59(10): 2318-2323, 1999. This work was supported by grants from The Tow Foundation and 14 Altomare DA, Wang HQ, Skele KL, De Rienzo A, Klein- DAMD-00-1-0675. We thank Ms. Venus Williams for her technical Szanto AJ, Godwin AK and Testa JR: AKT and mTOR help. phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. References 23(34): 5853-5857, 2004. 1 Cantley LC and Neel BG: New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci Received October 3, 2005 USA 96(8): 4240-4245, 1999. Accepted December 5, 2005

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