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Curcumin disrupts uterine leiomyosarcoma cells through AKT-mTOR pathway inhibition
Gynecologic Oncology 122 (2011) 141–148
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Curcumin disrupts uterine leiomyosarcoma cells through AKT-mTOR pathway inhibition Tze Fang Wong a, Takashi Takeda a,b,⁎, Bin Li b, Kenji Tsuiji b, Mari Kitamura a, Akiko Kondo a, Nobuo Yaegashi a,b a b
Department of Obstetrics and Gynecology, Sendai, Japan Department of Traditional Asian Medicine, Sendai, Japan
a r t i c l e
i n f o
Article history: Received 25 November 2010 Available online 29 March 2011 Keywords: Curcumin Uterine leiomyosarcoma mTOR p70S6 S6 Apoptosis
a b s t r a c t Objective. Uterine leiomyosarcoma generally has an unfavorable response to standard chemotherapy. The loss of PTEN which results in constitutive AKT-mTOR activation causes an increase in leiomyosarcoma formation in mice. The active ingredient derived from the herb Curcuma longa, curcumin, shows antitumor properties in a variety of cancer cell lines by altering a number of oncogenic pathways. To explore the possibility of curcumin as an alternative to standard chemotherapy, we decided to investigate curcumin's antitumor effect on uterine leiomyosarcoma cells. Methods. Human leiomyosarcoma cell lines, SKN and SK-UT-1, were cultured for in vitro experiments. Rapamycin or curcumin was added in different doses and their effect on cell growth was detected by MTS assay. The influence of rapamycin or curcumin on AKT, mTOR, p70S6 and S6 phosphorylation and protein expression was detected by Western Blotting. The ability of rapamycin or curcumin to induce apoptosis was determined by Western blotting using cleaved-PARP specific antibody, Caspase-3 activity assay and TUNEL assay. Results. Both rapamycin and curcumin significantly reduced SKN cell proliferation. Curcumin inhibited mTOR, p70S6 and S6 phosphorylation similar with rapamycin. Cleaved PARP, caspase-3 activity and DNA fragmentation increased proportional with curcumin concentration. At a high concentration, curcumin significantly induced apoptosis in SKN cells, but not rapamycin. Conclusions. Curcumin inhibited uterine leiomyosarcoma cells' growth by targeting the AKT-mTOR pathway for inhibition. However, rapamycin, a specific mTOR inhibitor, did not induce apoptosis in SKN cells unlike curcumin that also has a pro-apoptotic potential in SKN cells. © 2011 Elsevier Inc. All rights reserved.
Introduction Uterine leiomyosarcoma (LMS) is a rare tumor of the female urogenital tract, affecting only approximately 1% of females with uterine malignancies [1]. Uterine LMSs are characterized by a high rate of local and distant recurrences, a propensity for early hematogenous spread [2], and poor response to chemotherapeutic regimens that usually work well on other tumors [3]. Of note, negligible activity (response rate, 3%) was observed in a phase II trial that tested cisplatin [4] in uterine LMS. The current mainstay of first-line chemotherapy for advanced uterine LMS is doxorubicin-based regimens that show the largest response rates, ranging from 19% to 30% [5–7]. In these studies, although improvement in response rates was observed in groups receiving doxorubicin in combination with other chemotherapeutic agents, no survival advantage was obtained despite a generally higher toxicity observed. This highlights ⁎ Corresponding author at: Seiryomachi 1-1, Aoba-ku, Sendai 980-8574, Miyagi, Japan. Fax: +81 22 717 7258. E-mail address: [email protected] (T. Takeda). 0090-8258/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2011.03.001
the intractability of uterine LMSs to cytotoxic agents, thus justifying effort in developing targeted therapies [8] to replace or combine with the current regimens. Human LMSs have a high frequency of loss of the chromosomal region 10q that contains the locus for PTEN (phosphatase and tensin homolog), whose transcriptional and translational product dephosphorylates the secondary messenger, PIP3 (phosphatidyl-inositoltriphosphate), to inhibit signal transduction from receptor tyrosine kinases [9]. The presence of growth factors or high nutrient states in association with the presence of insulin activates receptor tyrosine kinases (RTKs) phosphorylates and activates PI3K (phosphotidylinositol 3-kinases) and AKT (RAC-alpha serine-threonine-protein kinase), the upstream modulators of mTOR (mammalian target of rapamycin). The activation of mTOR via its phosphorylation activates the p70S6 (p70 ribosomal protein S6 kinase 1) that in turn phosphorylates the ribosomal protein S6 [10]. As a result, many upstream modulators of the PI3K-AKT-mTOR pathway are overexpressed at the mRNA level in different sarcoma types. In addition, conditional knock-out of PTEN from the smooth muscles of mice predisposes them to the development of
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LMSs in various organs [11]. This indicates that constitutive mTOR activity is crucial in the survival of LMS cells, as demonstrated by the high response rate of PTEN-deleted sarcomas to rapamycin [11], a specific inhibitor of mTOR. Inhibitors targeting various components of the PI3K-AKT-mTOR pathway are currently being tested at different stages of clinical trials and have shown promising potential against uterine sarcomas, including leiomyosarcomas [8]. Human populations consuming a considerable amount of curcumin, the biologically active component of turmeric (Curcuma longa), in their diets have lower rates of colorectal cancers [12]. In addition, curcumin possesses a broad spectrum of chemopreventive and anti-tumor properties in various in vivo mice models and tumor cells in vitro. Curcumin shows a broad range of molecular targets [13], one of which includes the suppression of the AKT-mTOR pathway. Unlike rapamycin, which specifically inhibits mTOR activity by disrupting its association with raptor (regulatory-associated protein of mTOR; forms mTOR complex 1 with mTOR, PRAS40 and mLST8); curcumin disrupts both raptor and rictor (rapamycin-insensitive companion of mTOR; forms mTOR complex 2 with mTOR, protor and mSin1) [14]. As rapamycin is unable to inhibit rictor, mTOR inhibition by rapamycin via disruption of raptor enables a negative-feedback activation of AKT [15] by the rictormTOR complex [16]. Hence, curcumin could be a more potent drug targeting the AKT-mTOR pathway compared with rapamycin. Furthermore, curcumin's low toxicity against normal non-tumor cells makes it an ideal candidate as an alternative to standard cytotoxic chemotherapy [17]. Converging these findings, we hypothesized that curcumin might demonstrate anti-tumor properties in human uterine LMS cells. Hence, we investigated curcumin's anti-tumor activity in human uterine LMS cell-lines in vitro, and its relationship to mTOR activation. Materials and methods Cell line and culture The human uterine LMS cell line SKN is a commercially available cell-line in Japan purchased from the Japan Health Sciences Foundation (Osaka, Japan). Cells were grown in complete HamF-12 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS (Biowest, Miami, FL, USA) and maintained at 37 °C in a humidified 5% CO2 atmosphere. SKN is a well-documented uterine LMS cell line that was established and well characterized since year 1977 [18]. SK-UT-1 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in Eagle's minimum essential medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and maintained at 37 °C in a humidified 5% CO2 atmosphere. Reagents Curcumin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at 1 M concentration as a stock solution that was stored at −20 °C. Rapamycin was purchased from Cell Signaling Technology (Beverly, MA, USA) and dissolved in DMSO at 100 μM concentration as a stock solution that was stored at −20 °C. Compound C was from Calbiochem (San Diego, CA) dissolved in DMSO prior to dilution in culture medium. As a vehicle control for all experiments, DMSO (0.1% v/v) was used because that is the final dilution of DMSO for all dosages of curcumin and rapamycin indicated.
plate and incubated at 37 °C in a humidified chamber with 5% CO2 for 24 h before treatment with curcumin, rapamycin or compound C. 20 μl CellTiter 96® AQueous One Solution reagent was added to each well 72 h after the treatment with various reagents as indicated. Absorbance was recorded at 490 nm after 2 h. Western blot analysis Eighty to ninety percent of confluent cells cultured for 24 h in 100 mm dish containing full medium were exposed to different indicated doses of reagents before being harvested for Western blotting. Whole-cell lysates were generated in the buffer containing 20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% protease inhibitor cocktail (Sigma-Aldrich) and 0.5% Nonidet P-40 (Sigma-Aldrich). Cell lysate proteins were fractionated by SDS-PAGE at 40 μg per well and blotted to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) in Tris/Glycine buffer (Bio-Rad) as previously described [19]. The membranes were treated at room temperature for 1 h in 5% non-fat dried milk dissolved in phosphate-buffered saline with 0.1% Tween 20 (PBS-T). Next, the membranes were washed in PBS-T at room temperature twice each for 10 min. For determination of AKT-mTOR pathway activity, the proteins were probed with AKT antibody, phospho-AKT (Thr308) antibody, mTOR antibody, phospho-mTOR (Ser2448) antibody, phospho-p70S6 (Thr389) antibody, S6 ribosomal protein antibody and phospho-S6 ribosomal protein (Ser235/236) antibody at 4 °C overnight. For detection of AMPK phosphorylation, the blots were incubated at 4 °C overnight with phospho-AMPKα (Thr172) antibody. For detection of apoptosis, the blots were probed with total-PARP and human-specific cleaved PARP (Asp214) antibody. These antibodies were purchased from Cell Signaling Technology. The loading control, beta-actin antibody was purchased from Sigma-Aldrich. Immunoreactive proteins were detected using the manufacturer's protocol (Millipore Corporation, Billerica, MA, USA). Determination of apoptosis Curcumin-induced apoptosis was determined using CaspACETM Assay System (Promega, Madison, WI, USA). Curcumin was added to the cells in a 100 mm dish at different concentrations for 3 h and then collected and stored at −80 °C. Whole-cell lysates were prepared in the buffer containing 20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% protease inhibitor cocktail (Sigma-Aldrich) and 0.5% Nonidet P-40 (Sigma-Aldrich). 40 μg protein from each sample was used to measure caspase-3 activity and other reagents were added following the manufacturer's instructions. TUNEL staining using the In situ Cell Death Detection Kit (Roche, Basel, Switzerland) was performed on adherent SKN cells cultured in an 8-well Lab-TekTM chamber slide (Thermo Scientific, Rochester, NY, USA). SKN cells were seeded at 5000 cells per well and incubated at 37 °C in a humidified chamber with 5% CO2 for 24 h. Then, the cells were treated with different concentrations of curcumin for 48 h and TUNEL staining was performed according to the manufacturer's protocol. Three independent microscopic fields were observed for each sample and stained nuclei were counted as positive. Results Rapamycin reduces uterine leiomyosarcoma cell viability in a dose-dependent manner
Cell viability analysis Analysis of cell viability was performed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay system (MTS assay) (Promega, Madison, WI, USA). All reagents were added according to the manufacturer's instructions. SKN cells were seeded at 5000 cells per well; SK-UT-1 cells were seeded at 10,000 cells per well in a 96-well
In order to confirm the role of AKT-mTOR pathway activation in the proliferation of uterine leiomyosarcoma cells, we performed MTS assay on SKN cells treated with increasing doses of rapamycin, a specific mTOR inhibitor (as illustrated in Fig. 1A). Rapamycin significantly inhibited cell proliferation in SKN cells from 1 nM concentration (p b 0.05) and achieved almost 50% inhibition at 100 nM (p b 0.01).
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As the constitutive activation of growth factor and nutrientdependent AKT-mTOR pathway was implicated in tumor proliferation and progression, we investigated the effect of rapamycin on the
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phosphorylation of mTOR and its downstream targets: p70S6 and S6 ribosomal proteins. SKN cells treated with 10 nM and 100 nM rapamycin for 24 h showed a dramatic reduction in mTOR phosphorylation and an
Fig. 1. Validation of SKN cells' dependence on AKT-mTOR pathway activation. (A) The cells were exposed to rapamycin for 72 h at concentrations ranging from 0.1 to 100 nM, cell viability assessed by MTS assay. Control cells were treated with DMSO (0.1% v/v). Independent t-tests were used for all statistical comparisons. (B) SKN cells were treated with rapamycin for 24 h and harvested for Western blotting. Control cells were treated with DMSO (0.1% v/v). Phospho-mTOR, phospho-p70S6 and phospho-S6 antibodies were used to detect the downstream modulators of AKT-mTOR pathway. Multiple bands were observed with phospho-p70S6 immunoblotting and the arrow indicates the correct band. (C) Induction of apoptosis was determined using PARP and human-specific cleaved PARP antibodies. (D) The effect of prolonged exposure to rapamycin on AKT activity and expression was confirmed in the presence of 100 nM rapamycin for 0, 6, 24 and 48 h.
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Fig. 2. Curcumin's effect on uterine leiomyosarcoma cells. (A) SKN cells and (B) SK-UT-1 cells were exposed to curcumin for 72 h at concentrations ranging from 5 to 200 μM. Control cells were treated with DMSO (0.1% v/v). Cell viability was assessed by MTS assay. (C) SKN cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Phospho-AKT, AKT, phospho-mTOR, mTOR, phospho-p70S6, phospho-S6 and S6 antibodies were used to detect the effectors of the AKT-mTOR pathway. Multiple bands were observed with phospho-p70S6 and S6 immunoblotting and the arrows indicate the correct band. (D) SK-UT-1 cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Phospho-AKT, AKT, phospho-mTOR and mTOR antibodies were used to detect the effect of curcumin on the AKT-mTOR pathway. Independent t-tests were used for all statistical comparisons.
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Fig. 2 (continued).
almost complete abrogation of p70S6 and S6 ribosomal proteins' phosphorylation compared with the vehicle control as shown in Fig. 1B. However, mTOR inhibition by rapamycin did not seem to increase the cleavage of PARP, an early apoptotic event (as shown in Fig. 1C).
In order to investigate the effect of rapamycin on AKT, we conducted a time-course study on SKN cells. The exposure to 100 nM rapamycin showed a sustained suppression of the phosphorylation of mTOR with a corresponding slight elevation of AKT phosphorylation compared with the control at 0 h (Fig. 1D).
Fig. 3. Determination of apoptosis induction by curcumin in SKN cells. (A) SKN cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Induction of apoptosis was determined using PARP and human-specific cleaved PARP antibodies. (B) Caspase-3 activity was measured in SKN cells treated with curcumin at increasing doses after 3 h using the luminescence based caspase-3 kit.
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Fig. 4. (A) TUNEL staining to detect DNA fragmentation in SKN cells. SKN cells were treated with indicated doses of curcumin for 48 h. Control cells were treated with DMSO (0.1% v/v). Dark nuclei indicate positive staining. (B) Quantification of TUNEL-positive SKN cells. Independent t-tests were used for all statistical comparisons.
Curcumin reduces uterine leiomyosarcoma cell viability via mTOR pathway inhibition Curcumin is a drug that targets numerous cellular pathways including the AKT-mTOR pathway. To test the hypothesis that curcumin can decrease uterine LMS cell viability by inhibiting mTOR activity, MTS assay was performed in three independent experiments each. Curcumin was able to significantly inhibit SKN cell growth from 10 μM (p b 0.01) and onwards and achieved almost 80% inhibition of cell growth at 200 μM curcumin (p b 0.01) as shown in Fig. 2A. In order to demonstrate the universality of curcumin's antitumor effect on uterine leiomyosarcoma cells, we used another cell line, SK-UT-1 in our experiment. As demonstrated by our MTS assay, curcumin significantly reduced SK-UT1 cell proliferation from 20 μM (p b 0.05) (Fig. 2B). Similar with rapamycin, our Western blotting results in SKN cells showed that curcumin inhibited mTOR, p70S6 and S6 ribosomal proteins phosphorylation in a dose-dependent manner (Fig. 2C). AKT was also completely abrogated at 200 μM curcumin. The protein expression of AKT, mTOR, and S6 was not significantly altered by curcumin. Similar with SKN, curcumin was able to reduce the phosphorylation of AKT and mTOR in SK-UT-1 cells. AKT protein expression seemed unaffected, but mTOR protein expression was slightly reduced by curcumin (Fig. 2D).
SKN cells as it was documented that curcumin induces apoptosis in other types of tumor before. As shown in Fig. 3A, curcumin increased PARP cleavage in a dose-dependent manner, especially at 200 μM concentration. Caspase-3 activity also increased in direct proportion with curcumin concentration (as illustrated in Fig. 3B). To investigate whether the last stage of apoptosis, DNA fragmentation, also occurs in the presence of curcumin, TUNEL staining was performed (Fig. 4A). Consistent with the results so far, curcumin significantly induced DNA fragmentation in SKN cells (as shown in Fig. 4B), of which 200 μM curcumin showed the most dramatic effect (pb 0.001). mTOR inhibition in SKN cells is independent from curcumin-induced AMPK phosphorylation To confirm the role of AMPK activation in SKN cells, we checked AMPK phosphorylation in response to curcumin. We found out that there was a dramatic increase of AMPK phosphorylation against 200 μM curcumin in SKN cells. When we added compound C, a specific inhibitor of AMPK, only partial inhibition of AMPK phosphorylation was observed. There was no rescue of mTOR activity, as observed through mTOR, p70S6 and S6 phosphorylation (Fig. 5A). Similarly, SKN cell proliferation did not recover with the addition of compound C to inhibit AMPK activity (Fig. 5B).
Curcumin induces apoptosis in uterine leiomyosarcoma cells
Discussion
As mTOR inhibition by rapamycin did not induce apoptosis in SKN cells, it was important to investigate whether curcumin is pro-apoptotic in
Curcumin and its anti-tumor properties have attracted substantial attention during the recent years. However, to the best of our knowledge,
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Fig. 5. (A) SKN cells were treated with the presence or absence of 10 μM compound C for 1 h before being exposed to 200 μM curcumin. Control cells were treated with DMSO (0.1% v/v). After harvesting, cells were lysed and prepared for Western blotting with antibodies against phospho-AMPK, phospho-mTOR and phospho-S6. (B) For MTS assay, SKN cells were treated with the presence or absence of 10 μM compound C for 1 h before being exposed to 200 μM curcumin. Control cells were treated with DMSO (0.1% v/v). Independent t-tests were used for statistical comparisons.
this is the first study that investigated curcumin's antitumor potential in uterine LMS cells. We are also the first to demonstrate that mTOR activity is essential in uterine LMS cell survival and that curcumin exerts its antitumor effect by inhibiting the phosphorylation of the effectors of the mTOR pathway. As predicted, inhibition of the AKT-mTOR pathway in uterine LMS SKN cells by either rapamycin or curcumin resulted in a dose-dependent decrease in cell proliferation. Consistent with the in vivo murine model [20], human uterine LMS indeed requires constitutive activation of the AKT-mTOR pathway for cell growth. Both rapamycin and curcumin were able to reduce the phosphorylation of mTOR and its downstream effectors, p70S6 and S6. Therefore, curcumin inhibits uterine LMS cell growth in the same manner as rapamycin by inhibiting mTOR and its effectors. The mechanisms associated with the antineoplastic activity of various agents vary. Similar with doxorubicin in one previous study [21], rapamycin did not induce apoptotic cell death in uterine LMS cells. Furthermore, apoptosis is a complex process that involves the activation of the caspase enzymes and PARP (poly-ADP ribose polymerase) cleavage in the early stage [22] and culminates in DNA fragmentation or laddering in the final stages [23]. By contrast, we demonstrated that
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curcumin was able to induce both the early and late apoptotic events in uterine LMS cells from a concentration as low as 25 μM. Curcumin's effect at 6 h seems to be weaker than 3 h (Fig. 3A). As PARP cleavage is an early apoptotic event that peaks between three to 6 h, 200 μM curcumin may seem less effective at 6 h, looking at PARP alone. However, if we observe the inhibition of mTOR's downstream targets, p70S6 and S6, curcumin at a lower concentration was sufficient to inhibit their phosphorylation at 6 h compared with 3 h (Fig. 2C). Thinking along the same line regarding apoptosis, DNA fragmentation, a late apoptotic event, was markedly elevated using 200 μM curcumin at 48 h (Fig. 4A, B). This difference in pro-apoptotic activity between curcumin and rapamycin led us to investigate the role of AMPK in SKN cells. AMPK is a conserved serine/threonine protein kinase that is activated in response to nutrient deprivation and pathological stresses. Metformin, an antidiabetic drug, inhibits mTOR activity via AMPK activation in breast cancer [20] and ovarian cancer [24] cells. AMPK activation in response to curcumin has also been investigated in prostate cancer [25] and rhabdomyosarcoma cells [14] but was found to be independent from curcumin's inhibitory effect on mTOR. Similarly, our results showed that AMPK phosphorylation by curcumin in SKN cells was not necessary for mTOR inhibition. At 200 μM curcumin, AKT phosphorylation was completely abrogated, which corresponds with a dramatic increase in PARP cleavage. As mTOR inhibition via the dissociation of raptor from the mTOR complex 1 (mTORC1) results in a negative-feedback activation of AKT (Fig. 1D) [15], which is located upstream of the AKT-mTOR pathway, curcumin's dual ability [14] to inhibit both complexes that constitute mTOR, mTORC1 (that contains raptor) and mTORC2 (that contains rictor), may contribute to curcumin's proapoptic effect. This phenomenon could account for curcumin's advantage over rapamycin as a drug targeting the AKT-mTOR pathway. Curcumin is safe at high doses but shows low bioavailability [26]. Therefore, various efforts have been made to improve curcumin's bioavailability, such as the coadministration of curcumin with piperine [27], and the development of curcumin analogs with much higher potency. For example, one such compound, GO-Y030, was found to be 8–40 times more potent than curcumin, thus requiring much lower concentrations to achieve similar effects [28,29]. Therefore, curcumin combined with other agents or potent curcumin derivatives can also be a useful option for further clinical development. Curcumin induces apoptosis in various tumor cell lines, including colon [30], ovarian [31], lung [32] and breast cancer cells [33], and rhabdomyosarcoma cells [14]. In many instances, curcumin even shows the ability to overcome chemotherapy resistance [30,33]. Hence, it is not surprising that curcumin could induce apoptosis in uterine LMS cells even though doxorubicin [21] and rapamycin failed to do so as curcumin has a wider range of molecular targets. In summary, curcumin showed a potent antitumor effect in human uterine leiomyosarcoma SKN cells. Growth inhibition was associated with downregulation of the AKT-mTOR pathway activity. Unlike rapamycin, curcumin has a potent pro-apoptotic effect on uterine LMS cells. Curcumin's ability to induce apoptosis as well as inhibit cell growth in human uterine LMS cells supports the development of curcumin as a candidate for further pre-clinical development in animal models. Conflict of interest statement The authors declare that there are no conflicts of interest.
References [1] Brooks SE, Zhan M, Cote T, Baquet CR. Surveillance, Epidemiology, and End Results analysis of 2677 cases of uterine sarcoma 1989–1999. Gynecol Oncol 2004;93:204–8. [2] Leitao MM, Sonoda Y, Brennan MF, Barakat RR, Chi DS. Incidence of lymph node and ovarian metastases in leiomyosarcoma of the uterus. Gynecol Oncol 2003;91: 209–12. [3] Giuntoli RL, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL, et al. Retrospective review of 208 patients with leiomyosarcoma of the uterus: prognostic
148
[4]
[5]
[6]
[7]
[8] [9]
[10] [11]
[12] [13]
[14] [15]
[16] [17] [18]
T.F. Wong et al. / Gynecologic Oncology 122 (2011) 141–148 indicators, surgical management, and adjuvant therapy[small star, filled]. Gynecol Oncol 2003;89:460–9. Thigpen JT, Blessing JA, Beecham J, Homesley H, Yordan E. Phase II trial of cisplatin as first-line chemotherapy in patients with advanced or recurrent uterine sarcomas: a Gynecologic Oncology Group study. J Clin Oncol 1991;9:1962–6. Muss HB, Bundy B, Disaia PJ, Homesley HD, Fowler WC, Creasman W, et al. Treatment of recurrent or advanced uterine sarcoma. A randomized trial of doxorubicin Versus doxorubicin and cyclophosphamide (a phase III trial of the gynecologic oncology group). Cancer 1985;55:1648–53. Omura GA, Major FJ, Blessing JA, Sedlacek TV, Thigpen JT, Creasman WT, et al. A randomized study of adriamycin with and without dimethyl triazenoimidazole carboxamide in advanced uterine sarcomas. Cancer 1983;52:626–32. Sutton G, Blessing JA, Malfetano JH. Ifosfamide and doxorubicin in the treatment of advanced leiomyosarcomas of the uterus: a Gynecologic Oncology Group Study. Gynecol Oncol 1996;62:226–9. Amant F, Coosemans A, Debiec-Rychter M, Timmerman D, Vergote I. Clinical management of uterine sarcomas. Lancet Oncol 2009;10:1188–98. Hu J, Rao UNM, Jasani S, Khanna V, Yaw K, Surti U. Loss of DNA copy number of 10q is associated with aggressive behavior of leiomyosarcomas: a comparative genomic hybridization study. Cancer Genet Cytogen 2005;161:20–7. Inoki K, Corradetti MN, Guan K-L. Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005;37:19–24. Hernando E, Charytonowicz E, Dudas ME, Menendez S, Matushansky I, Mills J, et al. The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nat Med 2007;13:748–53. Kaefer CM, Milner JA. The role of herbs and spices in cancer prevention. J Nutr Biochem 2008;19:347–61. Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB. Curcumin and cancer: an “old-age” disease with an “age-old” solution. Cancer Lett 2008;267: 133–64. Beevers CS, Chen L, Liu L, Luo Y, Webster NJG, Huang S. Curcumin disrupts the mammalian target of rapamycin-raptor complex. Cancer Res 2009;69:1000–8. Breuleux M, Klopfenstein M, Stephan C, Doughty CA, Barys L, Maira S-M, et al. Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther 2009;8:742–53. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex. Science 2005;307:1098–101. Sharma RA, Euden SA, Platton SL, Cooke DN, Shafayat A, Hewitt HR, et al. Phase I Clinical Trial of Oral Curcumin. Clin Cancer Res 2004;10:6847–54. Ishiwata I, Nozawa S, Nagal S, Kurihara S, Mikata A, Okumura H. Establishment of a Human Leiomyosarcoma Cell Line. Cancer Res 1977;37:658–64.
[19] Kuesap J, Li B, Satarug S, Takeda K, Numata I, Na-Bangchang K, Shibahara S. Prostaglandin D2 induces heme oxygenase-1 in human retinal pigment epithelial cells. Biochem Bioph Res Co 2008;367:413–9. [20] Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin Is an AMP Kinase–Dependent Growth Inhibitor for Breast Cancer Cells. Cancer Res 2006;66: 10269–73. [21] Lambert LA, Qiao N, Hunt KK, Lambert DH, Mills GB, Meijer L, Keyomarsi K. Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model. Cancer Res 2008;68:7966–74. [22] Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, et al. Yama/CPP32², a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 1995;81:801–9. [23] Nagata S. Apoptotic DNA Fragmentation. Exp Cell Res 2000;256:12–8. [24] Gotlieb WH, Saumet J, Beauchamp M-C, Gu J, Lau S, Pollak MN, Bruchim I. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer. Gynecol Oncol 2008;110:246–50. [25] Yu S, Shen G, Khor TO, Kim J-H, Kong A-NT. Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein phosphatase-dependent mechanism. Mol Cancer Ther 2008;7:2609–20. [26] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;6:807–18. [27] Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med 1998;64:353–6. [28] Cen L, Hutzen B, Ball S, DeAngelis S, Chen CL, Fuchs JR, et al. New structural analogues of curcumin exhibit potent growth suppressive activity in human colorectal carcinoma cells. BMC Cancer 2009;9:99. [29] Shibata H, Yamakoshi H, Sato A, Ohori H, Kakudo Y, Kudo C, et al. Newly synthesized curcumin analog has improved potential to prevent colorectal carcinogenesis in vivo. Cancer Sci 2009;100:956–60. [30] Li L, Ahmed B, Mehta K, Kurzrock R. Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis, and angiogenesis in colorectal cancer. Mol Cancer Ther 2007;6:1276–82. [31] Wahl H, Tan L, Griffith K, Choi M, Liu JR. Curcumin enhances Apo2L/TRAIL-induced apoptosis in chemoresistant ovarian cancer cells. Gynecol Oncol 2007;105:104–12. [32] Radhakrishna Pillai G, Srivastava AS, Hassanein TI, Chauhan DP, Carrier E. Induction of apoptosis in human lung cancer cells by curcumin. Cancer Lett 2004;208: 163–70. [33] Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, Bueso-Ramos CE, et al. Curcumin suppresses the paclitaxel-induced nuclear factor-κB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clin Cancer Res 2005;11:7490–8.
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Curcumin disrupts uterine leiomyosarcoma cells through AKT-mTOR pathway inhibition Tze Fang Wong a, Takashi Takeda a,b,⁎, Bin Li b, Kenji Tsuiji b, Mari Kitamura a, Akiko Kondo a, Nobuo Yaegashi a,b a b
Department of Obstetrics and Gynecology, Sendai, Japan Department of Traditional Asian Medicine, Sendai, Japan
a r t i c l e
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Article history: Received 25 November 2010 Available online 29 March 2011 Keywords: Curcumin Uterine leiomyosarcoma mTOR p70S6 S6 Apoptosis
a b s t r a c t Objective. Uterine leiomyosarcoma generally has an unfavorable response to standard chemotherapy. The loss of PTEN which results in constitutive AKT-mTOR activation causes an increase in leiomyosarcoma formation in mice. The active ingredient derived from the herb Curcuma longa, curcumin, shows antitumor properties in a variety of cancer cell lines by altering a number of oncogenic pathways. To explore the possibility of curcumin as an alternative to standard chemotherapy, we decided to investigate curcumin's antitumor effect on uterine leiomyosarcoma cells. Methods. Human leiomyosarcoma cell lines, SKN and SK-UT-1, were cultured for in vitro experiments. Rapamycin or curcumin was added in different doses and their effect on cell growth was detected by MTS assay. The influence of rapamycin or curcumin on AKT, mTOR, p70S6 and S6 phosphorylation and protein expression was detected by Western Blotting. The ability of rapamycin or curcumin to induce apoptosis was determined by Western blotting using cleaved-PARP specific antibody, Caspase-3 activity assay and TUNEL assay. Results. Both rapamycin and curcumin significantly reduced SKN cell proliferation. Curcumin inhibited mTOR, p70S6 and S6 phosphorylation similar with rapamycin. Cleaved PARP, caspase-3 activity and DNA fragmentation increased proportional with curcumin concentration. At a high concentration, curcumin significantly induced apoptosis in SKN cells, but not rapamycin. Conclusions. Curcumin inhibited uterine leiomyosarcoma cells' growth by targeting the AKT-mTOR pathway for inhibition. However, rapamycin, a specific mTOR inhibitor, did not induce apoptosis in SKN cells unlike curcumin that also has a pro-apoptotic potential in SKN cells. © 2011 Elsevier Inc. All rights reserved.
Introduction Uterine leiomyosarcoma (LMS) is a rare tumor of the female urogenital tract, affecting only approximately 1% of females with uterine malignancies [1]. Uterine LMSs are characterized by a high rate of local and distant recurrences, a propensity for early hematogenous spread [2], and poor response to chemotherapeutic regimens that usually work well on other tumors [3]. Of note, negligible activity (response rate, 3%) was observed in a phase II trial that tested cisplatin [4] in uterine LMS. The current mainstay of first-line chemotherapy for advanced uterine LMS is doxorubicin-based regimens that show the largest response rates, ranging from 19% to 30% [5–7]. In these studies, although improvement in response rates was observed in groups receiving doxorubicin in combination with other chemotherapeutic agents, no survival advantage was obtained despite a generally higher toxicity observed. This highlights ⁎ Corresponding author at: Seiryomachi 1-1, Aoba-ku, Sendai 980-8574, Miyagi, Japan. Fax: +81 22 717 7258. E-mail address: [email protected] (T. Takeda). 0090-8258/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2011.03.001
the intractability of uterine LMSs to cytotoxic agents, thus justifying effort in developing targeted therapies [8] to replace or combine with the current regimens. Human LMSs have a high frequency of loss of the chromosomal region 10q that contains the locus for PTEN (phosphatase and tensin homolog), whose transcriptional and translational product dephosphorylates the secondary messenger, PIP3 (phosphatidyl-inositoltriphosphate), to inhibit signal transduction from receptor tyrosine kinases [9]. The presence of growth factors or high nutrient states in association with the presence of insulin activates receptor tyrosine kinases (RTKs) phosphorylates and activates PI3K (phosphotidylinositol 3-kinases) and AKT (RAC-alpha serine-threonine-protein kinase), the upstream modulators of mTOR (mammalian target of rapamycin). The activation of mTOR via its phosphorylation activates the p70S6 (p70 ribosomal protein S6 kinase 1) that in turn phosphorylates the ribosomal protein S6 [10]. As a result, many upstream modulators of the PI3K-AKT-mTOR pathway are overexpressed at the mRNA level in different sarcoma types. In addition, conditional knock-out of PTEN from the smooth muscles of mice predisposes them to the development of
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LMSs in various organs [11]. This indicates that constitutive mTOR activity is crucial in the survival of LMS cells, as demonstrated by the high response rate of PTEN-deleted sarcomas to rapamycin [11], a specific inhibitor of mTOR. Inhibitors targeting various components of the PI3K-AKT-mTOR pathway are currently being tested at different stages of clinical trials and have shown promising potential against uterine sarcomas, including leiomyosarcomas [8]. Human populations consuming a considerable amount of curcumin, the biologically active component of turmeric (Curcuma longa), in their diets have lower rates of colorectal cancers [12]. In addition, curcumin possesses a broad spectrum of chemopreventive and anti-tumor properties in various in vivo mice models and tumor cells in vitro. Curcumin shows a broad range of molecular targets [13], one of which includes the suppression of the AKT-mTOR pathway. Unlike rapamycin, which specifically inhibits mTOR activity by disrupting its association with raptor (regulatory-associated protein of mTOR; forms mTOR complex 1 with mTOR, PRAS40 and mLST8); curcumin disrupts both raptor and rictor (rapamycin-insensitive companion of mTOR; forms mTOR complex 2 with mTOR, protor and mSin1) [14]. As rapamycin is unable to inhibit rictor, mTOR inhibition by rapamycin via disruption of raptor enables a negative-feedback activation of AKT [15] by the rictormTOR complex [16]. Hence, curcumin could be a more potent drug targeting the AKT-mTOR pathway compared with rapamycin. Furthermore, curcumin's low toxicity against normal non-tumor cells makes it an ideal candidate as an alternative to standard cytotoxic chemotherapy [17]. Converging these findings, we hypothesized that curcumin might demonstrate anti-tumor properties in human uterine LMS cells. Hence, we investigated curcumin's anti-tumor activity in human uterine LMS cell-lines in vitro, and its relationship to mTOR activation. Materials and methods Cell line and culture The human uterine LMS cell line SKN is a commercially available cell-line in Japan purchased from the Japan Health Sciences Foundation (Osaka, Japan). Cells were grown in complete HamF-12 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS (Biowest, Miami, FL, USA) and maintained at 37 °C in a humidified 5% CO2 atmosphere. SKN is a well-documented uterine LMS cell line that was established and well characterized since year 1977 [18]. SK-UT-1 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in Eagle's minimum essential medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and maintained at 37 °C in a humidified 5% CO2 atmosphere. Reagents Curcumin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at 1 M concentration as a stock solution that was stored at −20 °C. Rapamycin was purchased from Cell Signaling Technology (Beverly, MA, USA) and dissolved in DMSO at 100 μM concentration as a stock solution that was stored at −20 °C. Compound C was from Calbiochem (San Diego, CA) dissolved in DMSO prior to dilution in culture medium. As a vehicle control for all experiments, DMSO (0.1% v/v) was used because that is the final dilution of DMSO for all dosages of curcumin and rapamycin indicated.
plate and incubated at 37 °C in a humidified chamber with 5% CO2 for 24 h before treatment with curcumin, rapamycin or compound C. 20 μl CellTiter 96® AQueous One Solution reagent was added to each well 72 h after the treatment with various reagents as indicated. Absorbance was recorded at 490 nm after 2 h. Western blot analysis Eighty to ninety percent of confluent cells cultured for 24 h in 100 mm dish containing full medium were exposed to different indicated doses of reagents before being harvested for Western blotting. Whole-cell lysates were generated in the buffer containing 20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% protease inhibitor cocktail (Sigma-Aldrich) and 0.5% Nonidet P-40 (Sigma-Aldrich). Cell lysate proteins were fractionated by SDS-PAGE at 40 μg per well and blotted to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) in Tris/Glycine buffer (Bio-Rad) as previously described [19]. The membranes were treated at room temperature for 1 h in 5% non-fat dried milk dissolved in phosphate-buffered saline with 0.1% Tween 20 (PBS-T). Next, the membranes were washed in PBS-T at room temperature twice each for 10 min. For determination of AKT-mTOR pathway activity, the proteins were probed with AKT antibody, phospho-AKT (Thr308) antibody, mTOR antibody, phospho-mTOR (Ser2448) antibody, phospho-p70S6 (Thr389) antibody, S6 ribosomal protein antibody and phospho-S6 ribosomal protein (Ser235/236) antibody at 4 °C overnight. For detection of AMPK phosphorylation, the blots were incubated at 4 °C overnight with phospho-AMPKα (Thr172) antibody. For detection of apoptosis, the blots were probed with total-PARP and human-specific cleaved PARP (Asp214) antibody. These antibodies were purchased from Cell Signaling Technology. The loading control, beta-actin antibody was purchased from Sigma-Aldrich. Immunoreactive proteins were detected using the manufacturer's protocol (Millipore Corporation, Billerica, MA, USA). Determination of apoptosis Curcumin-induced apoptosis was determined using CaspACETM Assay System (Promega, Madison, WI, USA). Curcumin was added to the cells in a 100 mm dish at different concentrations for 3 h and then collected and stored at −80 °C. Whole-cell lysates were prepared in the buffer containing 20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% protease inhibitor cocktail (Sigma-Aldrich) and 0.5% Nonidet P-40 (Sigma-Aldrich). 40 μg protein from each sample was used to measure caspase-3 activity and other reagents were added following the manufacturer's instructions. TUNEL staining using the In situ Cell Death Detection Kit (Roche, Basel, Switzerland) was performed on adherent SKN cells cultured in an 8-well Lab-TekTM chamber slide (Thermo Scientific, Rochester, NY, USA). SKN cells were seeded at 5000 cells per well and incubated at 37 °C in a humidified chamber with 5% CO2 for 24 h. Then, the cells were treated with different concentrations of curcumin for 48 h and TUNEL staining was performed according to the manufacturer's protocol. Three independent microscopic fields were observed for each sample and stained nuclei were counted as positive. Results Rapamycin reduces uterine leiomyosarcoma cell viability in a dose-dependent manner
Cell viability analysis Analysis of cell viability was performed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay system (MTS assay) (Promega, Madison, WI, USA). All reagents were added according to the manufacturer's instructions. SKN cells were seeded at 5000 cells per well; SK-UT-1 cells were seeded at 10,000 cells per well in a 96-well
In order to confirm the role of AKT-mTOR pathway activation in the proliferation of uterine leiomyosarcoma cells, we performed MTS assay on SKN cells treated with increasing doses of rapamycin, a specific mTOR inhibitor (as illustrated in Fig. 1A). Rapamycin significantly inhibited cell proliferation in SKN cells from 1 nM concentration (p b 0.05) and achieved almost 50% inhibition at 100 nM (p b 0.01).
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As the constitutive activation of growth factor and nutrientdependent AKT-mTOR pathway was implicated in tumor proliferation and progression, we investigated the effect of rapamycin on the
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phosphorylation of mTOR and its downstream targets: p70S6 and S6 ribosomal proteins. SKN cells treated with 10 nM and 100 nM rapamycin for 24 h showed a dramatic reduction in mTOR phosphorylation and an
Fig. 1. Validation of SKN cells' dependence on AKT-mTOR pathway activation. (A) The cells were exposed to rapamycin for 72 h at concentrations ranging from 0.1 to 100 nM, cell viability assessed by MTS assay. Control cells were treated with DMSO (0.1% v/v). Independent t-tests were used for all statistical comparisons. (B) SKN cells were treated with rapamycin for 24 h and harvested for Western blotting. Control cells were treated with DMSO (0.1% v/v). Phospho-mTOR, phospho-p70S6 and phospho-S6 antibodies were used to detect the downstream modulators of AKT-mTOR pathway. Multiple bands were observed with phospho-p70S6 immunoblotting and the arrow indicates the correct band. (C) Induction of apoptosis was determined using PARP and human-specific cleaved PARP antibodies. (D) The effect of prolonged exposure to rapamycin on AKT activity and expression was confirmed in the presence of 100 nM rapamycin for 0, 6, 24 and 48 h.
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Fig. 2. Curcumin's effect on uterine leiomyosarcoma cells. (A) SKN cells and (B) SK-UT-1 cells were exposed to curcumin for 72 h at concentrations ranging from 5 to 200 μM. Control cells were treated with DMSO (0.1% v/v). Cell viability was assessed by MTS assay. (C) SKN cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Phospho-AKT, AKT, phospho-mTOR, mTOR, phospho-p70S6, phospho-S6 and S6 antibodies were used to detect the effectors of the AKT-mTOR pathway. Multiple bands were observed with phospho-p70S6 and S6 immunoblotting and the arrows indicate the correct band. (D) SK-UT-1 cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Phospho-AKT, AKT, phospho-mTOR and mTOR antibodies were used to detect the effect of curcumin on the AKT-mTOR pathway. Independent t-tests were used for all statistical comparisons.
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Fig. 2 (continued).
almost complete abrogation of p70S6 and S6 ribosomal proteins' phosphorylation compared with the vehicle control as shown in Fig. 1B. However, mTOR inhibition by rapamycin did not seem to increase the cleavage of PARP, an early apoptotic event (as shown in Fig. 1C).
In order to investigate the effect of rapamycin on AKT, we conducted a time-course study on SKN cells. The exposure to 100 nM rapamycin showed a sustained suppression of the phosphorylation of mTOR with a corresponding slight elevation of AKT phosphorylation compared with the control at 0 h (Fig. 1D).
Fig. 3. Determination of apoptosis induction by curcumin in SKN cells. (A) SKN cells were treated with indicated doses of curcumin for 3 and 6 h. Control cells were treated with DMSO (0.1% v/v). Induction of apoptosis was determined using PARP and human-specific cleaved PARP antibodies. (B) Caspase-3 activity was measured in SKN cells treated with curcumin at increasing doses after 3 h using the luminescence based caspase-3 kit.
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Fig. 4. (A) TUNEL staining to detect DNA fragmentation in SKN cells. SKN cells were treated with indicated doses of curcumin for 48 h. Control cells were treated with DMSO (0.1% v/v). Dark nuclei indicate positive staining. (B) Quantification of TUNEL-positive SKN cells. Independent t-tests were used for all statistical comparisons.
Curcumin reduces uterine leiomyosarcoma cell viability via mTOR pathway inhibition Curcumin is a drug that targets numerous cellular pathways including the AKT-mTOR pathway. To test the hypothesis that curcumin can decrease uterine LMS cell viability by inhibiting mTOR activity, MTS assay was performed in three independent experiments each. Curcumin was able to significantly inhibit SKN cell growth from 10 μM (p b 0.01) and onwards and achieved almost 80% inhibition of cell growth at 200 μM curcumin (p b 0.01) as shown in Fig. 2A. In order to demonstrate the universality of curcumin's antitumor effect on uterine leiomyosarcoma cells, we used another cell line, SK-UT-1 in our experiment. As demonstrated by our MTS assay, curcumin significantly reduced SK-UT1 cell proliferation from 20 μM (p b 0.05) (Fig. 2B). Similar with rapamycin, our Western blotting results in SKN cells showed that curcumin inhibited mTOR, p70S6 and S6 ribosomal proteins phosphorylation in a dose-dependent manner (Fig. 2C). AKT was also completely abrogated at 200 μM curcumin. The protein expression of AKT, mTOR, and S6 was not significantly altered by curcumin. Similar with SKN, curcumin was able to reduce the phosphorylation of AKT and mTOR in SK-UT-1 cells. AKT protein expression seemed unaffected, but mTOR protein expression was slightly reduced by curcumin (Fig. 2D).
SKN cells as it was documented that curcumin induces apoptosis in other types of tumor before. As shown in Fig. 3A, curcumin increased PARP cleavage in a dose-dependent manner, especially at 200 μM concentration. Caspase-3 activity also increased in direct proportion with curcumin concentration (as illustrated in Fig. 3B). To investigate whether the last stage of apoptosis, DNA fragmentation, also occurs in the presence of curcumin, TUNEL staining was performed (Fig. 4A). Consistent with the results so far, curcumin significantly induced DNA fragmentation in SKN cells (as shown in Fig. 4B), of which 200 μM curcumin showed the most dramatic effect (pb 0.001). mTOR inhibition in SKN cells is independent from curcumin-induced AMPK phosphorylation To confirm the role of AMPK activation in SKN cells, we checked AMPK phosphorylation in response to curcumin. We found out that there was a dramatic increase of AMPK phosphorylation against 200 μM curcumin in SKN cells. When we added compound C, a specific inhibitor of AMPK, only partial inhibition of AMPK phosphorylation was observed. There was no rescue of mTOR activity, as observed through mTOR, p70S6 and S6 phosphorylation (Fig. 5A). Similarly, SKN cell proliferation did not recover with the addition of compound C to inhibit AMPK activity (Fig. 5B).
Curcumin induces apoptosis in uterine leiomyosarcoma cells
Discussion
As mTOR inhibition by rapamycin did not induce apoptosis in SKN cells, it was important to investigate whether curcumin is pro-apoptotic in
Curcumin and its anti-tumor properties have attracted substantial attention during the recent years. However, to the best of our knowledge,
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Fig. 5. (A) SKN cells were treated with the presence or absence of 10 μM compound C for 1 h before being exposed to 200 μM curcumin. Control cells were treated with DMSO (0.1% v/v). After harvesting, cells were lysed and prepared for Western blotting with antibodies against phospho-AMPK, phospho-mTOR and phospho-S6. (B) For MTS assay, SKN cells were treated with the presence or absence of 10 μM compound C for 1 h before being exposed to 200 μM curcumin. Control cells were treated with DMSO (0.1% v/v). Independent t-tests were used for statistical comparisons.
this is the first study that investigated curcumin's antitumor potential in uterine LMS cells. We are also the first to demonstrate that mTOR activity is essential in uterine LMS cell survival and that curcumin exerts its antitumor effect by inhibiting the phosphorylation of the effectors of the mTOR pathway. As predicted, inhibition of the AKT-mTOR pathway in uterine LMS SKN cells by either rapamycin or curcumin resulted in a dose-dependent decrease in cell proliferation. Consistent with the in vivo murine model [20], human uterine LMS indeed requires constitutive activation of the AKT-mTOR pathway for cell growth. Both rapamycin and curcumin were able to reduce the phosphorylation of mTOR and its downstream effectors, p70S6 and S6. Therefore, curcumin inhibits uterine LMS cell growth in the same manner as rapamycin by inhibiting mTOR and its effectors. The mechanisms associated with the antineoplastic activity of various agents vary. Similar with doxorubicin in one previous study [21], rapamycin did not induce apoptotic cell death in uterine LMS cells. Furthermore, apoptosis is a complex process that involves the activation of the caspase enzymes and PARP (poly-ADP ribose polymerase) cleavage in the early stage [22] and culminates in DNA fragmentation or laddering in the final stages [23]. By contrast, we demonstrated that
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curcumin was able to induce both the early and late apoptotic events in uterine LMS cells from a concentration as low as 25 μM. Curcumin's effect at 6 h seems to be weaker than 3 h (Fig. 3A). As PARP cleavage is an early apoptotic event that peaks between three to 6 h, 200 μM curcumin may seem less effective at 6 h, looking at PARP alone. However, if we observe the inhibition of mTOR's downstream targets, p70S6 and S6, curcumin at a lower concentration was sufficient to inhibit their phosphorylation at 6 h compared with 3 h (Fig. 2C). Thinking along the same line regarding apoptosis, DNA fragmentation, a late apoptotic event, was markedly elevated using 200 μM curcumin at 48 h (Fig. 4A, B). This difference in pro-apoptotic activity between curcumin and rapamycin led us to investigate the role of AMPK in SKN cells. AMPK is a conserved serine/threonine protein kinase that is activated in response to nutrient deprivation and pathological stresses. Metformin, an antidiabetic drug, inhibits mTOR activity via AMPK activation in breast cancer [20] and ovarian cancer [24] cells. AMPK activation in response to curcumin has also been investigated in prostate cancer [25] and rhabdomyosarcoma cells [14] but was found to be independent from curcumin's inhibitory effect on mTOR. Similarly, our results showed that AMPK phosphorylation by curcumin in SKN cells was not necessary for mTOR inhibition. At 200 μM curcumin, AKT phosphorylation was completely abrogated, which corresponds with a dramatic increase in PARP cleavage. As mTOR inhibition via the dissociation of raptor from the mTOR complex 1 (mTORC1) results in a negative-feedback activation of AKT (Fig. 1D) [15], which is located upstream of the AKT-mTOR pathway, curcumin's dual ability [14] to inhibit both complexes that constitute mTOR, mTORC1 (that contains raptor) and mTORC2 (that contains rictor), may contribute to curcumin's proapoptic effect. This phenomenon could account for curcumin's advantage over rapamycin as a drug targeting the AKT-mTOR pathway. Curcumin is safe at high doses but shows low bioavailability [26]. Therefore, various efforts have been made to improve curcumin's bioavailability, such as the coadministration of curcumin with piperine [27], and the development of curcumin analogs with much higher potency. For example, one such compound, GO-Y030, was found to be 8–40 times more potent than curcumin, thus requiring much lower concentrations to achieve similar effects [28,29]. Therefore, curcumin combined with other agents or potent curcumin derivatives can also be a useful option for further clinical development. Curcumin induces apoptosis in various tumor cell lines, including colon [30], ovarian [31], lung [32] and breast cancer cells [33], and rhabdomyosarcoma cells [14]. In many instances, curcumin even shows the ability to overcome chemotherapy resistance [30,33]. Hence, it is not surprising that curcumin could induce apoptosis in uterine LMS cells even though doxorubicin [21] and rapamycin failed to do so as curcumin has a wider range of molecular targets. In summary, curcumin showed a potent antitumor effect in human uterine leiomyosarcoma SKN cells. Growth inhibition was associated with downregulation of the AKT-mTOR pathway activity. Unlike rapamycin, curcumin has a potent pro-apoptotic effect on uterine LMS cells. Curcumin's ability to induce apoptosis as well as inhibit cell growth in human uterine LMS cells supports the development of curcumin as a candidate for further pre-clinical development in animal models. Conflict of interest statement The authors declare that there are no conflicts of interest.
References [1] Brooks SE, Zhan M, Cote T, Baquet CR. Surveillance, Epidemiology, and End Results analysis of 2677 cases of uterine sarcoma 1989–1999. Gynecol Oncol 2004;93:204–8. [2] Leitao MM, Sonoda Y, Brennan MF, Barakat RR, Chi DS. Incidence of lymph node and ovarian metastases in leiomyosarcoma of the uterus. Gynecol Oncol 2003;91: 209–12. [3] Giuntoli RL, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL, et al. Retrospective review of 208 patients with leiomyosarcoma of the uterus: prognostic
148
[4]
[5]
[6]
[7]
[8] [9]
[10] [11]
[12] [13]
[14] [15]
[16] [17] [18]
T.F. Wong et al. / Gynecologic Oncology 122 (2011) 141–148 indicators, surgical management, and adjuvant therapy[small star, filled]. Gynecol Oncol 2003;89:460–9. Thigpen JT, Blessing JA, Beecham J, Homesley H, Yordan E. Phase II trial of cisplatin as first-line chemotherapy in patients with advanced or recurrent uterine sarcomas: a Gynecologic Oncology Group study. J Clin Oncol 1991;9:1962–6. Muss HB, Bundy B, Disaia PJ, Homesley HD, Fowler WC, Creasman W, et al. Treatment of recurrent or advanced uterine sarcoma. A randomized trial of doxorubicin Versus doxorubicin and cyclophosphamide (a phase III trial of the gynecologic oncology group). Cancer 1985;55:1648–53. Omura GA, Major FJ, Blessing JA, Sedlacek TV, Thigpen JT, Creasman WT, et al. A randomized study of adriamycin with and without dimethyl triazenoimidazole carboxamide in advanced uterine sarcomas. Cancer 1983;52:626–32. Sutton G, Blessing JA, Malfetano JH. Ifosfamide and doxorubicin in the treatment of advanced leiomyosarcomas of the uterus: a Gynecologic Oncology Group Study. Gynecol Oncol 1996;62:226–9. Amant F, Coosemans A, Debiec-Rychter M, Timmerman D, Vergote I. Clinical management of uterine sarcomas. Lancet Oncol 2009;10:1188–98. Hu J, Rao UNM, Jasani S, Khanna V, Yaw K, Surti U. Loss of DNA copy number of 10q is associated with aggressive behavior of leiomyosarcomas: a comparative genomic hybridization study. Cancer Genet Cytogen 2005;161:20–7. Inoki K, Corradetti MN, Guan K-L. Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005;37:19–24. Hernando E, Charytonowicz E, Dudas ME, Menendez S, Matushansky I, Mills J, et al. The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nat Med 2007;13:748–53. Kaefer CM, Milner JA. The role of herbs and spices in cancer prevention. J Nutr Biochem 2008;19:347–61. Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB. Curcumin and cancer: an “old-age” disease with an “age-old” solution. Cancer Lett 2008;267: 133–64. Beevers CS, Chen L, Liu L, Luo Y, Webster NJG, Huang S. Curcumin disrupts the mammalian target of rapamycin-raptor complex. Cancer Res 2009;69:1000–8. Breuleux M, Klopfenstein M, Stephan C, Doughty CA, Barys L, Maira S-M, et al. Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther 2009;8:742–53. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex. Science 2005;307:1098–101. Sharma RA, Euden SA, Platton SL, Cooke DN, Shafayat A, Hewitt HR, et al. Phase I Clinical Trial of Oral Curcumin. Clin Cancer Res 2004;10:6847–54. Ishiwata I, Nozawa S, Nagal S, Kurihara S, Mikata A, Okumura H. Establishment of a Human Leiomyosarcoma Cell Line. Cancer Res 1977;37:658–64.
[19] Kuesap J, Li B, Satarug S, Takeda K, Numata I, Na-Bangchang K, Shibahara S. Prostaglandin D2 induces heme oxygenase-1 in human retinal pigment epithelial cells. Biochem Bioph Res Co 2008;367:413–9. [20] Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin Is an AMP Kinase–Dependent Growth Inhibitor for Breast Cancer Cells. Cancer Res 2006;66: 10269–73. [21] Lambert LA, Qiao N, Hunt KK, Lambert DH, Mills GB, Meijer L, Keyomarsi K. Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model. Cancer Res 2008;68:7966–74. [22] Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, et al. Yama/CPP32², a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 1995;81:801–9. [23] Nagata S. Apoptotic DNA Fragmentation. Exp Cell Res 2000;256:12–8. [24] Gotlieb WH, Saumet J, Beauchamp M-C, Gu J, Lau S, Pollak MN, Bruchim I. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer. Gynecol Oncol 2008;110:246–50. [25] Yu S, Shen G, Khor TO, Kim J-H, Kong A-NT. Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein phosphatase-dependent mechanism. Mol Cancer Ther 2008;7:2609–20. [26] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;6:807–18. [27] Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med 1998;64:353–6. [28] Cen L, Hutzen B, Ball S, DeAngelis S, Chen CL, Fuchs JR, et al. New structural analogues of curcumin exhibit potent growth suppressive activity in human colorectal carcinoma cells. BMC Cancer 2009;9:99. [29] Shibata H, Yamakoshi H, Sato A, Ohori H, Kakudo Y, Kudo C, et al. Newly synthesized curcumin analog has improved potential to prevent colorectal carcinogenesis in vivo. Cancer Sci 2009;100:956–60. [30] Li L, Ahmed B, Mehta K, Kurzrock R. Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis, and angiogenesis in colorectal cancer. Mol Cancer Ther 2007;6:1276–82. [31] Wahl H, Tan L, Griffith K, Choi M, Liu JR. Curcumin enhances Apo2L/TRAIL-induced apoptosis in chemoresistant ovarian cancer cells. Gynecol Oncol 2007;105:104–12. [32] Radhakrishna Pillai G, Srivastava AS, Hassanein TI, Chauhan DP, Carrier E. Induction of apoptosis in human lung cancer cells by curcumin. Cancer Lett 2004;208: 163–70. [33] Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, Bueso-Ramos CE, et al. Curcumin suppresses the paclitaxel-induced nuclear factor-κB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clin Cancer Res 2005;11:7490–8.