Curcumin targets the AKT-mTOR pathway for uterine leiomyosarcoma tumor growth suppression.

2013 May 11. [Epub ahead of print]
Curcumin targets the AKTmTOR pathway for uterine leiomyosarcoma tumor
growth suppression.
Wong TF, Takeda T, Li B, Tsuiji K, Kondo A, Tadakawa M, Nagase S, Yaegashi

Department of Obstetrics and Gynecology, Tohoku University Graduate School
of Medicine, Sendai, Miyagi, Japan.


Uterine leiomyosarcomas generally do not respond well to standard
chemotherapy. We previously demonstrated that curcumin, the active
ingredient derived from the herb Curcuma longa, inhibits uterine
leiomyosarcoma cells in vitro via the inhibition of the AKT-mammalian
target of rapamycin (mTOR) pathway. As a preclinical investigation, we
performed an in vivo study using female nude mice to confirm the
therapeutic potential of curcumin against uterine leiomyosarcoma.
Human leiomyosarcoma cells, SK-UT-1, were inoculated in female nude mice
to establish subcutaneous tumors. Either vehicle control or 250 mg/kg
curcumin was administered intraperitoneally every day for 14 consecutive
days, and the mice were then killed. The tumors were measured every 2-3
days. The tumors were processed for immunohistochemical analyses to detect
total AKT, phosphorylated AKT, total mTOR, phosphorylated mTOR, and
phosphorylated S6. To detect apoptosis, the tumors were stained for cleaved
PARP and TUNEL. Ki-67 immunohistochemistry was performed to determine cell
viability of the tumors.
Compared with the control, curcumin reduced uterine leiomyosarcoma tumor
volume and mass significantly with a concordant decrease in mTOR and S6
phosphorylation. However, AKT phosphorylation was not significantly
altered. Cleaved PARP and TUNEL staining increased significantly with
curcumin administration, indicating the induction of apoptosis. There was
no difference in Ki-67 staining between the two groups.
Curcumin inhibited uterine leiomyosarcoma tumor growth in vivo by
targeting the AKT-mTOR pathway for inhibition.



[PubMed – as supplied by publisher]

Metformin, aging and cancer

Metformin, aging and cancer

Olga Moiseeva, Xavier-Deschênes-Simard, Michael Pollak, and Gerardo Ferbeyre
Many cancers are associated with aging [1]. Metformin, a widely used antidiabetic drug, has been linked to a reduced cancer incidence in some retrospective, hypothesis-generating studies [2]. Since cancer and aging may share certain molecular processes, it is plausible that metformin may prevent cancer by acting on the aging process. Consistent with this idea, several studies report a life span extension in animal models after treatment with metformin [3].
What is the mechanism by which aging may increase cancer incidence? Although many molecular changes correlate with aging, the presence of senescent cells capable of secreting inflammatory cytokines may be involved. This senescence associated secretory phenotype (SASP) consists of multiple cytokines, chemokines, growth factors and extracellular matrix degrading enzymes that can potentially affect normal tissue structure [4]. The SASP probably evolved as a gene expression program to assist the senescent tumor suppression response and tissue repair after damage and should be viewed as an initial adaptive response [5]. However, like acute inflammation, the SASP should be turned off to avoid maladaptive consequences. In some contexts, senescent cells are cleared by professional phagocytic cells [6] and this mechanism avoids any further complications. On the other hand, if senescent cells escape clearance, mechanisms that prevent the SASP should operate to avoid chronic inflammation and tissue disruption. Such endogenous mechanisms for clearing senescent cells or suppressing the SASP may fail with age. As a consequence, chronic SASP may cause a microenvironment in old tissues that facilitates tumor initiation and then stimulates cancer cell growth, motility and angiogenic activity. This unfortunate interaction between senescent cells and cancer cells has been reproduced in experimental mouse models where senescent fibroblasts stimulated tumor progression [4]). The mechanisms of senescent cell clearance and SASP control are not yet known. However, during experiments to study the potential cancer prevention activity of metformin, we found serendipitously that the drug prevented the expression of many proteases, cytokines and chemokines in senescent cells [7].
At the molecular level, we found that metformin interfered with the activation of protein kinases IKK a and b, which are responsible for activating NF-kB, an essential transcription factor for SASP activation. Intriguingly, metformin did not reduce the expression of anticancer cytokines such as interferon and interferon target genes in senescent cells, suggesting that it modulates SASP to reduce its inflammatory potential but retaining its antitumor activity. In addition, metformin did not affect the senescent cell cycle arrest caused by oncogenic ras in primary human cells, suggesting again that it can modulate the SASP without allowing proliferation of potentially malignant cells. The primary site of action of metformin is considered to be the complex I of the electron transport chain [2]. However, molecular details of the interaction between metformin and complex I remain to be identified. Complex I is one of the main cellular sources for reactive oxygen species (ROS) and we have shown that metformin can prevent ROS production by senescent cells [8]. It is thus plausible that ROS links senescence to NF-kB activation and that metformin interferes with this mechanism by acting on complex I (Fig 1). Metformin is not immunosuppressive so its ability to inhibit NF-kB is likely confined to certain pro-inflammatory contexts such as senescence. We thus propose that metformin prevents cancer by modulating the SASP in tissues where senescent cells were not naturally cleared.
Figure 1. Metformin inhibits the activation of IKK kinases in senescent cells. The model proposes that metformin reduces ROS generation by mitochondria preventing the activation of IKK kinases a step that is ROS-sensitive. Metformin does not affect the activation of the interferon response in senescent cells suggesting that it modulates the senescence associated secretory phenotype in a way that reduces chronic inflammation but not tumor suppression.
Many questions remain to be addressed in order to fully characterize metformin actions. Our results were obtained using cultured senescent fibroblasts and macrophages; other cell types should be studied as well. In addition, it remains to be determined if metformin can achieve this anti-SASP activity in vivo or whether it can influence the clearance of senescent cells by modulating the SASP. Anisimov and colleagues reported that metformin extends life span in female mice but not males [3] and it would be interesting to study whether NF-kB and SASP inhibition by metformin is gender dependent. Additional epidemiological data and laboratory experiments may justify well-designed clinical studies to evaluate metformin as a cancer preventive agent in specific contexts where its recently described actions would be hypothesized to be useful.
Olga Moiseeva, Xavier-Deschênes-Simard, Michael Pollak, and Gerardo Ferbeyre
Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada 
Received: 5/8/13; Published: 5/9/13


Compound in Mediterranean Diet Makes Cancer Cells “Mortal”

Compound in Mediterranean Diet Makes Cancer Cells ‘Mortal’

May 20, 2013 — New research suggests that a compound abundant in the Mediterranean diet takes away cancer cells’ “superpower” to escape death.

By altering a very specific step in gene regulation, this compound essentially re-educates cancer cells into normal cells that die as scheduled.
One way that cancer cells thrive is by inhibiting a process that would cause them to die on a regular cycle that is subject to strict programming. This study in cells, led by Ohio State University researchers, found that a compound in certain plant-based foods, called apigenin, could stop breast cancer cells from inhibiting their own death.
Much of what is known about the health benefits of nutrients is based on epidemiological studies that show strong positive relationships between eating specific foods and better health outcomes, especially reduced heart disease. But how the actual molecules within these healthful foods work in the body is still a mystery in many cases, and particularly with foods linked to lower risk for cancer.
Parsley, celery and chamomile tea are the most common sources of apigenin, but it is found in many fruits and vegetables.
The researchers also showed in this work that apigenin binds with an estimated 160 proteins in the human body, suggesting that other nutrients linked to health benefits — called “nutraceuticals” — might have similar far-reaching effects. In contrast, most pharmaceutical drugs target a single molecule.
“We know we need to eat healthfully, but in most cases we don’t know the actual mechanistic reasons for why we need to do that,” said Andrea Doseff, associate professor of internal medicine and molecular genetics at Ohio State and a co-lead author of the study. “We see here that the beneficial effect on health is attributed to this dietary nutrient affecting many proteins. In its relationship with a set of specific proteins, apigenin re-establishes the normal profile in cancer cells. We think this can have great value clinically as a potential cancer-prevention strategy.”
Doseff oversaw this work with co-lead author Erich Grotewold, professor of molecular genetics and director of Ohio State’s Center for Applied Plant Sciences (CAPS). The two collaborate on studying the genomics of apigenin and other flavonoids, a family of plant compounds that are believed to prevent disease.
The research appears this week in the online early edition of the journal Proceedings of the National Academy of Sciences.
Though finding that apigenin can influence cancer cell behavior was an important outcome of the work, Grotewold and Doseff point to their new biomedical research technique as a transformative contribution to nutraceutical research.
They likened the technique to “fishing” for the human proteins in cells that interact with small molecules available in the diet.
“You can imagine all the potentially affected proteins as tiny fishes in a big bowl. We introduce this molecule to the bowl and effectively lure only the truly affected proteins based on structural characteristics that form an attraction,” Doseff said. “We know this is a real partnership because we can see that the proteins and apigenin bind to each other.”
Through additional experimentation, the team established that apigenin had relationships with proteins that have three specific functions. Among the most important was a protein called hnRNPA2.
This protein influences the activity of messenger RNA, or mRNA, which contains the instructions needed to produce a specific protein. The production of mRNA results from the splicing, or modification, of RNA that occurs as part of gene activation. The nature of the splice ultimately influences which protein instructions the mRNA contains.
Doseff noted that abnormal splicing is the culprit in an estimated 80 percent of all cancers. In cancer cells, two types of splicing occur when only one would take place in a normal cell — a trick on the cancer cells’ part to keep them alive and reproducing.
In this study, the researchers observed that apigenin’s connection to the hnRNPA2 protein restored this single-splice characteristic to breast cancer cells, suggesting that when splicing is normal, cells die in a programmed way, or become more sensitive to chemotherapeutic drugs.
“So by applying this nutrient, we can activate that killing machinery. The nutrient eliminated the splicing form that inhibited cell death,” said Doseff, also an investigator in Ohio State’s Davis Heart and Lung Research Institute. “Thus, this suggests that when we eat healthfully, we are actually promoting more normal splice forms inside the cells in our bodies.”
The beneficial effects of nutraceuticals are not limited to cancer, as the investigators previously showed that apigenin has anti-inflammatory activities.
The scientists noted that with its multiple cellular targets, apigenin potentially offers a variety of additional benefits that may even occur over time. “The nutrient is targeting many players, and by doing that, you get an overall synergy of the effect,” Grotewold explained.
Doseff is leading a study in mice, testing whether food modified to contain proper doses of this nutrient can change splicing forms in the animals’ cells and produce an anti-cancer effect.

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Biochimie. 2013 Jun;95(6):1297-309. doi: 10.1016/j.biochi.2013.02.010. Epub  2013 Feb 26.
Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin.
Choudhury D, Ganguli A, Dastidar DG, Acharya BR, Das A, Chakrabarti G.

Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, West Bengal, India.


Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.
Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor-mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue-isolated human leiomyosarcoma xenografts

J Pineal Res. 2009 Aug;47(1):32-42. doi: 10.1111/j.1600-079X.2009.00686.x. Epub 2009 May 27.
Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor-mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue-isolated human leiomyosarcoma xenografts.
Dauchy RT, Blask DE, Dauchy EM, Davidson LK, Tirrell PC, Greene MW, Tirrell RP, Hill CR, Sauer LA.

Laboratory of Chrono-Neuroendocrine Oncology, Department of Structural and Cellular Biology, Tulane University School of Medicine, Tulane Cancer Center, Louisiana Cancer Research Consortium, New Orleans, LA 70112-2699, USA.


Melatonin provides a circadian signal that regulates linoleic acid (LA)-dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth-stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). This study tested the hypothesis that both acute and long-term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue-isolated human leiomyosarcoma (LMS), a rare, mesenchymally-derived smooth muscle tissue sarcoma, via melatonin receptor-mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue-isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 +/- 0.02 g/day) versus control xenografts that continued to grow at 0.22 +/- 0.03 g/day over a 10-day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose-dependent suppression of tumor cAMP production, LA uptake, 13-HODE release, extracellular signal-regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [(3)H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-Br-cAMP. These results demonstrate that, as observed in epithelially-derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long-term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor-mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.



[PubMed – indexed for MEDLINE]

Curcumin Induces Cross-Regulation Between Autophagy and Apoptosis in Uterine Leiomyosarcoma Cells

Curcumin Induces Cross-Regulation Between Autophagy and Apoptosis in Uterine Leiomyosarcoma Cells.
Li B, Takeda T, Tsuiji K, Wong TF, Tadakawa M, Kondo A, Nagase S, Yaegashi N.

*Division of Women’s Health, Research Institute of Traditional Asian Medicine, Kinki University School of Medicine, Osaka; and †Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan.


Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapy. A natural occurring compound, curcumin, has been shown to have inhibitory effects on cancers. We previously demonstrated that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway and activating apoptosis. To further explore the anticancer effect of curcumin, we investigated the efficacy of curcumin on autophagy in LMS cells.
Cell proliferation in human uterine LMS cell lines, SKN and SK-UT-1, was assessed after exposure to rapamycin or curcumin. Autophagy was detected by Western blotting for light chain 3 and sequestosome 1 (SQSTM1/p62) expression. Apoptosis was confirmed by Western blotting for cleaved poly (ADP-ribose) polymerase (PARP).
Both rapamycin and curcumin potently inhibited SKN and SK-UT-1 cell proliferation in a dose-dependent manner. Curcumin induced autophagy and apoptosis in SKN and SK-UT-1 cells, whereas rapamycin, a specific mTOR inhibitor, did not. Curcumin increased extracellular signal-regulated kinase 1/2 activity in both SKN and SK-UT-1 cells, whereas PD98059, an MEK1 inhibitor, inhibited both the extracellular signal-regulated kinase 1/2 pathway and curcumin-induced autophagy.
These experimental findings suggest that curcumin is a potent inhibitor of cell proliferation in uterine LMS and provide new insights about ongoing signaling events leading to the possible development of a new therapeutic agent.



[PubMed – as supplied by publisher]