Antimitotic chemos promote adhesion in detached and circulating tumor cells

[Rich66]

(Broad implications and possible relation to other chemo concerns, Taxol studied, filed patent shows reversal strategies: Vimentin, glu tubulin, kinesin inhibition, other theoretical remedies: green tea, Ashwaghanda etc)

1: Breast Cancer Res Treat. 2009 Jul 11. [Epub ahead of print]

Antimitotic chemotherapeutics promote adhesive responses in detached and circulating tumor cells.

(DOI: 10.1007/s10549-009-0457-3)

View Highlights from full text Here

Contact lead researcher (Martin) here

Balzer EM, Whipple RA, Cho EH, Matrone MA, Martin SS.
Program in Molecular Medicine, University of Maryland School of Medicine, Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD, 21201, USA.
In the clinical treatment of breast cancer, antimitotic cytotoxic agents are one of the most commonly employed chemotherapies, owing largely to their antiproliferative effects on the growth and survival of adherent cells in studies that model primary tumor growth. Importantly, the manner in which these chemotherapeutics impact the metastatic process remains unclear. Furthermore, since dissemination of tumor cells through the systemic circulation and lymphatics necessitates periods of detached survival, it is equally important to consider how circulating tumor cells respond to such compounds. To address this question, we exposed both nontumorigenic and tumor-derived epithelial cell lines to two antitumor compounds, jasplakinolide and paclitaxel (Taxol), in a series of attached and detached states. We report here that jasplakinolide promoted the extension of microtubule-based projections and microtentacle protrusions in adherent and suspended cells, respectively. These protrusions were specifically enriched by upregulation of a stable post-translationally modified form of alpha-tubulin, and this occurred prior to, and independently of any reductions in cellular viability. Microtubule stabilization with Taxol significantly enhanced these effects. Additionally, Taxol promoted the attachment and spreading of suspended tumor cell populations on extracellular matrix. While the antiproliferative effects of these compounds are well recognized and clinically valuable, our findings that microfilament and microtubule binding chemotherapeutics rapidly increase the mechanisms that promote endothelial adhesion of circulating tumor cells warrant caution to avoid inadvertently enhancing metastatic potential, while targeting cell division.
PMID: 19593636 [PubMed - as supplied by publisher]

Some highlights:

Because the cytoskeleton is a key regulator of cellular function and viability, some of the most clinically successful chemotherapies operate by directly targeting cytoskeletal filaments [3–6]. The most prominent examples of this approach are the taxanes, docetaxel, and paclitaxel, a family of microtubule (MT) stabilizing agents

The vast majority of studies to date have examined these effects exclusively with regard to adherent cell populations. As a result, the effects of these chemotherapeutics on suspended cell populations have been largely overlooked. Since dissemination of tumor cells through the lymphatics or vasculature requires release from extracellular matrix (ECM) and indefinite periods of suspension, it is equally important to consider the cytoskeletal dynamics of circulating tumor cells (CTCs) and their contributions to survival and metastatic spread. Interestingly, taxane treatment has been shown to increase the concentration of CTCs in the bloodstream more than 1000-fold when applied in advance of surgery [16]. Additionally, taxanes can worsen progression-free survival compared to anthracycline treatment when used as a monotherapy [17], though a mechanism to explain such findings has not been delineated. Our recent work describing microtubule-based microtentacles (McTNs) in detached and circulating breast tumor cells [18, 19] provides a mechanism by which cytoskeletally directed chemotherapies could influence CTCs and highlights the need to investigate these effects.

studies have focused principally on early steps of tumor progression that occur prior to release ECM release, transit through the circulation, extravasation, and colonization of secondary tissues [1], and therefore fail to address the manner in which these chemotherapies impact the behavior of circulating tumor cells, or those that are not actively circulating, but disseminated and resisting apoptosis.
taxane treatment has been shown to increase the concentration of CTCs in the bloodstream more than 1000-fold when applied in advance of surgery [16]. Additionally, taxanes can worsen progression-free survival compared to anthracycline treatment when used as a monotherapy [17], though a mechanism to explain such findings has not been delineated. Our recent work describing microtubule-based microtentacles (McTNs) in detached and circulating breast tumor cells [18, 19] provides a mechanism by which cytoskeletally directed chemotherapies could influence CTCs and highlights the need to investigate these effects.

surgical resection of a primary tumor increases the concentration of epithelial cells in systemic circulation by 1000-fold, and tumor cells can still be detected in the bloodstream of patients many years after entering what is clinically recognized as remission [16, 30]

periods of dormant survival require tumor cells to be resistant to apoptotic stimuli, and are thus ineffectively targeted by cytotoxic chemotherapies due to a combination of mitotic inactivity and apoptotic resistance [29, 33]. Circulating and dormant tumor cells may fail to die in response to MT and microfilament-binding chemotherapeutics, but the cytoskeletal behavior of these tumor cells is likely to be affected by continued exposure to these drugs.

a two-step model for extravasation, wherein initial adhesion is tubulin-dependent and subsequent escape into surrounding tissue is driven by actin-mediated contractile forces. Our results support this model, and indicate that the initial phase of this adhesive transition depends upon microtentacles to facilitate endothelial or ECM contact. Therefore, exposure of CTCs to MT-stabilizing agents such as Taxol could enhance this initial MT-dependent adhesion and promote extravasation into secondary sites. In this study, Taxol served to enhance the adhesion and efficient cell spreading of MDA-436 tumor cells, relative to cells under vehicle control conditions.

Though no apparent increase in McTN frequency was recorded in response to Taxol alone, there was a very significant increase in ECM adhesion and degree of cell spreading after 45 min. This suggests that while Taxol is insufficient to stimulate the formation of additional McTNs, it stabilizes existing structures and therefore promotes ECM contact and adhesion via a MT-dependent mechanism. It is likely because of the fact that the highly invasive MDA-436 cell line produces high levels of McTNs basally and that this enhancement was so pronounced in these cells[19].

In light of our recent observations that microtentacles were present in a high percentage of circulating tumor cells isolated from the blood of breast cancer patients with advanced metastatic disease (Balzer, EM and Chumsri, S unpublished data), the work described here highlights the importance of determining whether Taxane treatment can promote extravasation of disseminated tumor cells. So, while stabilizing microtubules and disrupting actin filaments can each decrease primary tumor volume, it is important to consider the effects of these treatments on circulating tumor cells to avoid inadvertently enhancing the metastatic potential of disseminated tumor cells, while targeting cell division.

http://lifesciences.umaryland.edu/Pa...nt.aspx?SID=12
Research

Epithelial cells perform a critical barrier function in vivo, and the integrity of this barrier is maintained by rigid cell-cell and cell-ECM contacts. If such contacts are disrupted and the detached cells fail to re-adhere quickly, apoptosis is initiated. Our lab has shown that suspended epithelial cells extend microtubule-based plasma membrane protrusions (microtentacles) that promote reattachment to neighboring cells and extracellular substrates.


While this phenomenon appears to be a feature of healthy epithelial tissues, it is significantly enhanced in highly invasive tumor cells, and has been directly observed in circulating tumor cells isolated from blood samples of patients with advanced metastatic disease (Balzer, E. and Chumsri, S unpublished data). Our lab is seeking to test the hypothesis that microtentacles promote metastasis by facilitating the extravasation and/or survival of circulating tumor cells. Specifically, my research focuses on defining the molecular interplay between the actin and tubulin cytoskeletons that forms the mechanistic basis for the extension of these microtentacles, in an effort to understand how this process is naturally exploited by cancer cells.

Laboratory Techniques

We utilize a wide range of traditional cell and molecular genetic techniques. We also employ a variety of imaging applications, including live-cell microscopy, laser scanning confocal analysis of detached cells, epifluorescence microscopy of tumor-endothelial interactions (under flow conditions and in intact blood vessels), and whole-animal optical tracking of circulating tumor cells.



Publications

Balzer, E.M., Whipple, R.A., Cho, E.H., and Martin, S.S. (2009). Antimitotic chemotherapeutics promote adhesive responses in detached and circulating tumor cells. Breast Cancer Res Treat. epub July 11. (DOI: 10.1007/s10549-009-0457-3)

Whipple, R.A., Balzer, E.M., Cho, E.H., Matrone, M.A., Yoon, J.R., and Martin, S.S. (2007) Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cell lines. Cancer Res. 2008. 68, 5678-5688

Cheng, Q., Balzer, E., Yoshida, M., Wong, J.C., and Miller, S.M. (2006) Effect of histone deacetylase inhibitors on tubulin acetylation in Volvox carteri (Volvocales). J. Phycol. 2006. 42, 417-422.

Balzer, E.M., et al.(2009). Microtentacles and Invadopodia: functionally distinct plasma membrane protrusions of metastatic breast tumor cells separated mechanistically by c-Src. (submitted to Cancer Research)

Whipple, R.A., Cho, E.H., Balzer, E.M., Matrone, M.A., Yang, J., and Martin, S.S. Metastasis-associated microtentacles are promoted by Epithelial-Mesenchymal Transition in human mammary epithelial cells and breast tumor cells. (submitted)



http://www.worldstemcellsummit.com/2009_abstracts.html
Epithelial-to-Mesenchymal Transition Promotes Breast Tumor Stem Cell Characteristics, Tubulin Detyrosination and Microtentacles

Stuart S. Martin, Rebecca Whipple Bettes, Monica Charpentier, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, Kimberly C. Tuttle, Michele I. Vitolo, Jennifer R. Yoon, Jing Yang, Olga B. Ioffe
University of Maryland Baltimore, USA

Understanding the biology of breast tumor stem cells during passage through the bloodstream could provide new therapeutic opportunities to destroy circulating tumor cells. When detached from extracellular matrix, breast tumor cells with increased stem cell characteristics produce unique microtentacles composed of coordinated vimentin intermediate filaments and detyrosinated microtubules. These microtentacles promote the reattachment of circulating tumor cells and are associated with increased invasiveness in vitro and metastatic potential in vivo. Our previous work has shown that microtentacles are increased in Basal B breast tumor cell lines that display hallmarks of epithelial-to-mesenchymal transition (EMT) and stem cell markers (CD44+/CD24-). We report here that the direct induction of EMT by expression of the transcription factor Twist promotes increased stem cell characteristics (CD44+/CD24-) and microtentacle formation in detached human mammary epithelial cells (HMECs). Mechanistically, EMT results in an elevation of alpha-tubulin in which the c-terminal tyrosine residue has been removed (Glu-tubulin), and reorganization of such Glu-tubulin into microtentacle extensions. Conversely, siRNA-mediated downregulation of endogenous Twist in (CD44+/CD24-) breast carcinoma cells decreased Glu-tubulin levels, organization and the frequency of microtentacles. Twist siRNA decreased levels of Glu-tubulin before any changes in either vimentin or E-Cadherin expression, indicating that Twist can have a primary effect on tubulin detyrosination without requiring additional elements of the EMT program. This model is further supported by immunohistochemical staining of 66 patient tissue samples that show a high concordance between Glu-tubulin and Twist staining in invasive ductal carcinomas. In addition, tumor cells co-staining for Twist and detyrosinated tubulin are enriched at the invasive front where tumor cells encounter a changing microenvironment. Given the role of detyrosinated tubulin in cell motility and orientation, these EMT-induced microtubule alterations and stem cell induction may occur as part of an inherent program for cell movement during development or wound healing. However, this program may also prime tumor cells for metastatic success by increasing stem cell charactersitics, stabilizing microtubules, and promoting microtentacles. Functionally, expression of Twist led to an enhanced rate of tumor cell reattachment as monitored over time by electrical impedence or measured by fluorescence over an endothelial layer. Confocal microscopy captures microtentacles engaging and invading through an endothelial layer to facilitate reattachment. These data support a novel model in which the EMT that occurs during tumor invasion increases stem cell characteristics, alpha-tubulin detyrosination and promotes microtentacles which enhance the reattachment of circulating tumor cells during metastasis.


Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells

FULL TEXT

Rebecca A. Whipple¹, Eric M. Balzer^1,2, Edward H. Cho^1,2, Michael A. Matrone^1,2, Jennifer R. Yoon^1,2 and Stuart S. Martin^{1,2 1} University of Maryland School of Medicine, Marlene and Stewart Greenebaum Cancer Center, Department of Physiology and ² Graduate Program in Life Sciences Baltimore, Maryland
Requests for reprints: Stuart S. Martin, Bressler Building, Room 10-29, 655 W. Baltimore Street, Baltimore, MD 21201. Phone: 410-706-6601; Fax: 410-706-6600; E-mail: ssmartin@som.umaryland.edu.

Key Words: breast • microtentacle • vimentin

Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulin-based protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated -tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated -tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread. [Cancer Res 2008;68(14):5678–88]

/Live cell imaging and membrane microtentacle scoring. Cells were trypsinzed at f80% confluency after 24 h expression and resuspended in
serum/phenol red (PR)–free media over ultralow attachment plates in the presence or absence of 5 AM Latrunculin-A (LA; BioMol) for 1 h. For
microtentacle scoring involving phosphatase inhibitor treatment, MCF10A
and MDA-MB-436 were pretreated with either 1 AM okadiac acid
(OKA; Sigma) or 5 nM Calyculin-A (Cal-A; Chemicon) in serum-containing
media for 1 h and resuspended in serum/PR-free media in the respective
phosphatase inhibitor with or without 5 AM L A./

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Microtubule-destabilizing agents significantly reduced the frequency with which microtentacles were observed and impaired both cell cell
interaction and reattachment of cells to surfaces. Interestingly,
microtubule-destabilizing drugs prevent circulating colon carcinoma
cells from attaching to the microvascular endothelium in vivo,
although the precise mechanism is currently unclear (11). Strikingly,
actin depolymerization significantly increases binding of circulating
tumor cells to the vascular endothelium (11) and also strongly
promotes the extension of tubulin microtentacles (10).

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We report for the first time that vimentin-expressing invasive breast carcinomas display a comparatively higher frequency of tubulin microtentacles following detachment than non–vimentin-expressing, noninvasive cell lines.
Microtentacle preservation following detachment indicates that
vimentin coaligns with Glu-tubulin in microtentacles, whereas
cytokeratin does not. Time-lapse video shows that membrane
microtentacles are rapidly disrupted by treatment with PP1/PP2A
inhibitors that disassemble vimentin, decreasing microtentacle
frequency and impairing reattachment of vimentin-expressing
tumor cells.

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Detachment of invasive breast tumor cell lines Hs578t, HCC1395, MDA-MB-436, and MDA-MB-157 produced unusually long, semiflexible, and motile microtentacles compared with noninvasive MCF10A,
ZR-75-1, Bt-20, SkBr3, and MDA-MB-468 lines, which display relatively short, rigid, and semimotile microtentacles.
Our previous research has shown that inhibiting actin polymerization with LA (5 AM) enhances microtentacle frequency, motility, and length in both human and mouse mammary epithelial cells following detachment (10). LA similarly increases the length, flexibility, and motility of microtentacles in breast tumor cells (Fig. 1B and Supplementary Movies
S4–S6). However, the invasive breast tumor cell lines were clearly able to form dynamic and flexible microtentacles even in the absence of LA treatment.

Expression of vimentin is much higher in breast tumor cells which display long and numerous microtentacles, whereas cytokeratin expression predominated in those cell lines that displayed fewer and shorter microtentacles (Fig. 1D).
Vimentin extends into microtentacles. Given the correlation of vimentin expression in those cells that exhibit more extensive and frequent microtentacles, we examined intermediate filament localization with indirect immunofluorescence, finding that vimentin, and not cytokeratin, extends into microtentacles (Fig. 2).

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Treatment with OKA resulted in a more dramatic change in vimentin morphology than Cal-A in MCF10A cells marked by cell rounding and vimentin fragmentation (Fig. 4A, d). MDA-MB-436 displayed similar morphologic changes with OKA as seen with Cal-A with a slight increase in retraction of the vimentin network to the perinuclear region (Fig. 4A, h)

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While MCF10A attachment efficiency to either uncoated or laminin-coated surfaces was not significantly affected by Cal-A treatment, attachment was significantly sensitive to treatment with OKA (Fig. 6A). This result matches the less-pronounced vimentin disruption in MCF10A cells after Cal-A compared with OKA (Fig. 4A, b and d). Interestingly, MDA-MB-436 attachment was significantly impaired by the presence of either Cal-A or OKA (Fig. 5C). Figure 5.

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Vimentin disassembly with PP1/PP2A inhibitors reduces microtentacles and attachment.

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Our data showing colocalization of vimentin with Glu-tubulin in microtentacles, the extensive microtentacles formed in cells with high vimentin expression, and the inhibition of microtentacles with dominant negative vimentin or compounds targeting vimentin assembly, all support a model in which structural coordination of Glu-tubulin
and vimentin underlie the striking morphologic abnormalities we
observe in detached breast tumor cells.
Our data also indicate that microtentacles in detached cells are distinct from other actin-based structures in attached cells, such as invadopodia or podosomes (30), which are inhibited by actin destablizing agents, such as LA or Cytochalasin-D. In contrast, microtentacles are enhanced by actin disruption and seem to become more flexible, motile, and longer (Fig. 1B).

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Vimentin expression is a marker of the aggressive ‘‘basal cell’’ subtype of breast tumor (33, 34), and up-regulation of vimentin predicts metastatic progression and poor patient prognosis (35).
Beyond breast cancer, vimentin is an established independent predictor of poor patient prognosis in many tumors, including cervical (36), thyroid (37), hepatocellular (38), pancreatic (39), and renal carcinomas (40). Our results indicate that cell lines with the highest microtentacle counts display reduced expression of cytokeratins and increased vimentin, a change which is also observed during the epithelial-to-mesenchymal transition (EMT) that is associated with tumor progression and poor patient prognosis (41, 42). Interestingly, vimentin is also strongly upregulated when tumor cells are exposed to hypoxia (43). Our current evidence supports a direct role for vimentin in the
mechanism underlying microtentacles in detached breast tumor cells.

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vimentin is rapidly hyperphosphorylated and disassembled after treatment with specific inhibitors of types 1 and 2A phosphatases,
providing evidence that vimentin is a major target of PP1 and PP2A
activity (23, 24).

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we propose that, upon detachment, vimentin aligns with stable microtubules via associated proteins to provide a flexible scaffold to Glu-enriched microtubules, which allows microtentacles to protrude at great length from the cell body. The shorter, less frequent microtentacles observed in non–vimentin-expressing, less invasive cell lines likely rely on the population of stable microtubules but lack the increased resilience
and strength of vimentin filaments (48). Disruption of vimentin filaments with either PP1/PP2A inhibitors or dominant-negative mutant vimentin likely promotes the collapse of Glu-microtubules and ultimate deformation of the microtentacles.

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While we continue to pursue the functional role of tubulin microtentacles in metastasis, the dramatic morphologic differences observed in detached breast tumor cell lines are compelling. Intriguing in vivo evidence exists that a yet-unidentified, tubulin-based (not actin-based) mechanism promotes the binding of circulating tumor cells to the vascular endothelium (11)

Available Vimentin Inhibitors:

The Intermediate Filament Protein Vimentin Is a New Target for Epigallocatechin Gallate

FULL TEXT

Hormel Institute, University of Minnesota, Austin, Minnesota 55912

To whom correspondence should be addressed: Hormel Institute, University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-9600; Fax: 507-437-9606; E-mail: zgdong@hi.umn.edu.
Abstract

Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [³H]EGCG demonstrate binding of EGCG to vimentin with a K_d of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect.




Chem Biol. 2007 Jun;14(6):623-34.
The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin.

FULL TEXT

Bargagna-Mohan P, Hamza A, Kim YE, Khuan Abby Ho Y, Mor-Vaknin N, Wendschlag N, Liu J, Evans RM, Markovitz DM, Zhan CG, Kim KB, Mohan R.
Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40536, USA.
Correspondence: royce.mohan@uky.edu

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

PMID: 17584610 [PubMed - indexed for MEDLINE]



Cancer Sci. 2006 Jul;97(7):658-64.
Chemotherapeutic efficacy of paclitaxel in combination with Withania somnifera on benzo(a)pyrene-induced experimental lung cancer.

Senthilnathan P, Padmavathi R, Magesh V, Sakthisekaran D.
Department of Medical Biochemistry, ALM Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600-113, India.
Lung cancer is one of the leading causes of cancer death in the world and is notoriously difficult to treat effectively. In the present study, male Swiss albino mice were divided into five groups of six animals each: group I animals received corn oil orally and served as a control; group II cancer-induced animals received benzo(a)pyrene (50 mg/kg bodyweight dissolved in corn oil, orally) twice weekly for four successive weeks; group III cancer-bearing animals (after 12 weeks of induction) were treated with paclitaxel (33 mg/kg bodyweight, i.p.) once weekly for 4 weeks; group IV cancer-bearing animals were treated with paclitaxel along with Withania somnifera (400 mg/kg bodyweight) orally once weekly for 4 weeks; and group V animals constituted the drug control treated with paclitaxel along with W. somnifera. The serum, lung and liver were investigated biochemically for aryl hydrocarbon hydroxylase, gamma-glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase and protein-bound carbohydrate components (hexose, hexosamine and sialic acid). These enzyme activities were increased significantly in cancer-bearing animals compared with control animals. The elevation of these in cancer-bearing animals was indicative of the persistent deteriorating effect of benzo(a)pyrene in cancer-bearing animals. Our data suggest that paclitaxel, administered with W. somnifera, may extend its chemotherapeutic effect through modulating protein-bound carbohydrate levels and marker enzymes, as they are indicators of cancer. The combination of paclitaxel with W. somnifera could effectively treat the benzo(a)pyrene-induced lung cancer in mice by offering protection from reactive oxygen species damage and also by suppressing cell proliferation.

PMID: 16827807 [PubMed - indexed for MEDLINE]







Chem Biol Interact. 2006 Feb 25;159(3):180-5. Epub 2005 Dec 22.
Enhancement of antitumor effect of paclitaxel in combination with immunomodulatory Withania somnifera on benzo(a)pyrene induced experimental lung cancer.

Senthilnathan P, Padmavathi R, Banu SM, Sakthisekaran D.
Department of Medical Biochemistry, Dr. ALM Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India. senthilnpm@rediffmail.com
The current experimental work deals with the immunomodulatory studies on the extract of Withania somnifera (L.) Dunal root powder against benzo(a)pyrene induced lung cancer in male Swiss albino mice. In our previous study, we reported the antioxidant and anticarcinogenic effect of W. somnifera (L.) Dunal along with paclitaxel. Immune dysfunction has been found to be associated with cancer and chemotherapy. Benzo(a)pyrene induced cancer animals were treated with 400mg/kg bodyweight of W. somnifera (L.) Dunal extract for 30 days significantly alters the levels of immunocompetent cells, immune complexes and immunoglobulins. Based on the data, the carcinogen as well as the paclitaxel affects the immune system, the toxic side effects on the immune system is more reversible and more controllable by W. somnifera (L.) Dunal. These results concluded the immunomodulatory activity of W. somnifera (L.) Dunal extract, which is a known immunomodulator in indigenous medicine.

PMID: 16375880 [PubMed - indexed for MEDLINE]


Altern Med Rev. 2006 Dec;11(4):269-77.
Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology.

Winters M.
Withania somnifera Dunal, commonly known as ashwagandha, has been used for centuries in Ayurvedic medicine to increase longevity and vitality. Western research supports its polypharmaceutical use, confirming antioxidant, anti-inflammatory, immune-modulating, and antistress properties in the whole plant extract and several separate constituents. This article reviews the literature pertaining to Withania somnifera and its botanical constituents as antitumor agents and in conjunction with radiation and chemotherapy treatment. Following a search of MEDLINE and EBSCO databases, it can be concluded that Withania somnifera reduces tumor cell proliferation while increasing overall animal survival time. Furthermore, it has been shown to enhance the effectiveness of radiation therapy while potentially mitigating undesirable side effects. Withania somnifera also reduces the side effects of chemotherapeutic agents cyclophosphamide and paclitaxel without interfering with the tumor-reducing actions of the drugs. These effects have been demonstrated in vitro on human cancer cell lines, and in vivo on animal subjects, but there have been no human trials to date. Given its broad spectrum of cytotoxic and tumor-sensitizing actions, Withania somnifera presents itself as a novel complementary therapy for integrative oncology care.

PMID: 17176166 [PubMed - indexed for MEDLINE]




Indian J Physiol Pharmacol. 2001 Apr;45(2):253-7.
Reversal of paclitaxel induced neutropenia by Withania somnifera in mice.

Gupta YK, Sharma SS, Rai K, Katiyar CK.
Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110 029.
The effect of aqueous extract of Withania somnifera (L. Solanaceae) was studied against paclitaxel induced neutropenia in mice. After paclitaxel 1 mg/kg, i.v. administration significant fall in total WBC and absolute neutrophil count was observed on day 3 and day 5. W. Somnifera (200 mg/kg, p.o.) per se produced significant increase in neutrophil counts. W. somnifera (200 mg/kg, p.o.) when administered for 4 days before paclitaxel treatment and continued for 12 days caused significant reversal of neutropenia of paclitaxel. The findings of the study suggest the potential of W. somnifera as an adjuvant during cancer chemotherapy for the prevention of bone marrow depression associated with anticancer drugs.

PMID: 11480235 [PubMed - indexed for MEDLINE]




Clin Cancer Res. 2007 Apr 1;13(7):2298-306.
Selective killing of cancer cells by leaf extract of Ashwagandha: identification of a tumor-inhibitory factor and the first molecular insights to its effect.

Widodo N, Kaur K, Shrestha BG, Takagi Y, Ishii T, Wadhwa R, Kaul SC.
Research Institute for Cell Engineering, GENE Therapeutics, Inc., National Institute of Advanced Industrial Science and Technology, Higashi, Tsukuba, Japan.
PURPOSE: Ashwagandha is regarded as a wonder shrub of India and is commonly used in Ayurvedic medicine and health tonics that claim its variety of health-promoting effects. Surprisingly, these claims are not well supported by adequate studies, and the molecular mechanisms of its action remain largely unexplored to date. We undertook a study to identify and characterize the antitumor activity of the leaf extract of ashwagandha. EXPERIMENTAL DESIGN: Selective tumor-inhibitory activity of the leaf extract (i-Extract) was identified by in vivo tumor formation assays in nude mice and by in vitro growth assays of normal and human transformed cells. To investigate the cellular targets of i-Extract, we adopted a gene silencing approach using a selected small hairpin RNA library and found that p53 is required for the killing activity of i-Extract. RESULTS: By molecular analysis of p53 function in normal and a variety of tumor cells, we found that it is selectively activated in tumor cells, causing either their growth arrest or apoptosis. By fractionation, purification, and structural analysis of the i-Extract constituents, we have identified its p53-activating tumor-inhibiting factor as with a none. CONCLUSION: We provide the first molecular evidence that the leaf extract of ashwagandha selectively kills tumor cells and, thus, is a natural source for safe anticancer medicine.

PMID: 17404115 [PubMed - indexed for MEDLINE]


Cancer Lett. 2008 Jan 9. [Epub ahead of print]
Selective killing of cancer cells by leaf extract of Ashwagandha: Components, activity and pathway analyses.

Widodo N, Takagi Y, Shrestha BG, Ishii T, Kaul SC, Wadhwa R.
National Institute of Advanced Industrial Science & Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan; Department of Molecular and Cellular Physiology, University of Tsukuba, Ibaraki 305-8575, Japan.
Ashwagandha, also called as "Queen of Ayurveda" and "Indian ginseng", is a commonly used plant in Indian traditional medicine, Ayurveda. Its roots have been used as herb remedy to treat a variety of ailments and to promote general wellness. However, scientific evidence to its effects is limited to only a small number of studies. We had previously identified anti-cancer activity in the leaf extract (i-Extract) of Ashwagandha and demonstrated withanone as a cancer inhibitory factor (i-Factor). In the present study, we fractionated the i-Extract to its components by silica gel column chromatography and subjected them to cell based activity analyses. We found that the cancer inhibitory leaf extract (i-Extract) has, at least, seven components that could cause cancer cell killing; i-Factor showed the highest selectivity for cancer cells and i-Factor rich Ashwagandha leaf powder was non-toxic and anti-tumorigenic in mice assays. We undertook a gene silencing and pathway analysis approach and found that i-Extract and its components kill cancer cells by at least five different pathways, viz. p53 signaling, GM-CFS signaling, death receptor signaling, apoptosis signaling and G2-M DNA damage regulation pathway. p53 signaling was most common. Visual analysis of p53 and mortalin staining pattern further revealed that i-Extract, fraction F1, fraction F4 and i-Factor caused an abrogation of mortalin-p53 interactions and reactivation of p53 function while the fractions F2, F3, F5 work through other mechanisms.

PMID: 18191020 [PubMed - as supplied by publisher]


J Ethnopharmacol. 2006 May 24;105(3):336-41. Epub 2006 Jan 10.
Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis.

Mathur R, Gupta SK, Singh N, Mathur S, Kochupillai V, Velpandian T.
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
In the Indian System of Medicine, the medicinal plant, Withania somnifera Dunal (Solanaceae) finds application for numerous ailments including cancer. This study explores the mechanism(s) underlying this property. The hydroalcoholic extract of the roots (WS) was partitioned between chloroform (WS-chloroform) and water (WS-water). Further, WS-chloroform was fractionated (A1-A12) by reverse-phase column chromatography and their withanolide content was quantified by high-performance liquid chromatography (HPLC). Preliminarily, the anti-proliferative activity of all the extracts and fractions was screened against human laryngeal carcinoma (Hep2) cells by microculture tetrazolium assay (MTT). Two extracts (WS and WS-chloroform) and three fractions (A4, A5 and A6) negatively affected Hep2 viability at the concentration of 25 microg/ml and these were further investigated pharmacologically. Flow cytometry revealed cell cycle block and accumulation of hypoploid (sub G1) cells as the mode of anti-proliferative activity of all but A4. Their anti-angiogenic potential was investigated by a chickchorio-allantoic membrane (CAM) wherein a significant inhibition (p<0.0001) of vascular endothelium growth factor (VEGF), induced neovascularization was recorded. The effect was confirmed in vivo by mouse sponge implantation method. These findings suggest that the roots of Withania somnifera possess cell cycle disruption and anti-angiogenic activity, which may be a critical mediator for its anti-cancer action.

PMID: 16412596 [PubMed - indexed for MEDLINE]




Cancer Res. 2008 Sep 15;68(18):7661-9.


Nutr Cancer. 2008;60 Suppl 1:51-60.
Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells.

Stan SD, Zeng Y, Singh SV.
University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Withaferin A (WA) is derived from the medicinal plant Withania somnifera that has been safely used for centuries in the Indian Ayurvedic medicine for treatment of various ailments. We now demonstrate that WA treatment causes G2 and mitotic arrest in human breast cancer cells. Treatment of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) cell lines with WA resulted in a concentration- and time-dependent increase in G2-M fraction, which correlated with a decrease in levels of cyclin-dependent kinase 1 (Cdk1), cell division cycle 25C (Cdc25C) and/or Cdc25B proteins, leading to accumulation of Tyrosine15 phosphorylated (inactive) Cdk1. Ectopic expression of Cdc25C conferred partial yet significant protection against WA-mediated G2-M phase cell cycle arrest in MDA-MB-231 cells. The WA-treated MDA-MB-231 and MCF-7 cells were also arrested in mitosis as judged by fluorescence microscopy and analysis of Ser10 phosphorylated histone H3. Mitotic arrest resulting from exposure to WA was accompanied by an increase in the protein level of anaphase promoting complex/cyclosome substrate securin. In conclusion, the results of this study suggest that G2-M phase cell cycle arrest may be an important mechanism in antiproliferative effect of WA against human breast cancer cells.

PMID: 19003581 [PubMed - indexed for MEDLINE]



Phytother Res. 2009 Jul;23(7):987-92.
Withanolide sulfoxide from Aswagandha roots inhibits nuclear transcription factor-kappa-B, cyclooxygenase and tumor cell proliferation.

Mulabagal V, Subbaraju GV, Rao CV, Sivaramakrishna C, Dewitt DL, Holmes D, Sung B, Aggarwal BB, Tsay HS, Nair MG.
Bioactive Natural Products and Phytoceuticals, Department of Horticulture and National Food, Safety and Toxicology Center, Michigan State University, East Lansing, Michigan, USA.
Investigation of the methanol extract of Aswagandha (Withania somnifera) roots for bioactive constituents yielded a novel withanolide sulfoxide compound (1) along with a known withanolide dimer ashwagandhanolide (2) with an S-linkage. The structure of compound 1 was established by extensive NMR and MS experiments. Compound 1 was highly selective in inhibiting cyclooxygenase-2 (COX-2) enzyme by 60% at 100 microm with no activity against COX-1 enzyme. The IC(50) values of compound 1 against human gastric (AGS), breast (MCF-7), central nervous system (SF-268) and colon (HCT-116) cancer cell lines were in the range 0.74-3.63 microm. Both S-containing dimeric withanolides, 1 and 2, completely suppressed TNF-induced NF-kappaB activation when tested at 100 microm. The isolation of a withanolide sulfoxide from W. somnifera roots and its ability to inhibit COX-2 enzyme and to suppress human tumor cell proliferation are reported here for the first time. In addition, this is the first report on the abrogation of TNF-induced NF-kappaB activation for compounds 1 and 2. Copyright 2009 John Wiley & Sons, Ltd.

PMID: 19152372 [PubMed - indexed for MEDLINE]




Pharmazie. 2007 Apr;62(4):305-7.
Study of the anticancer potential of Yemeni plants used in folk medicine.

Mothana RA, Grünert R, Lindequist U, Bednarski PJ.
Department of Pharmacognosy, Faculty of Pharmacy, Sana'a-University, Yemen. r_mothana@yahoo.com
The present work evaluated the anticancer activity of methanol extracts from 24 plants used in Yemeni traditional medicine. To evaluate the in vitro cytotoxic potency of the investigated extracts, an established microtiter plate assay based on cellular staining with crystal violet was used with 5 human cancer cell lines: two lung cancer (A-427 and LCLC-103H), two urinary bladder carcinoma (5637 and RT-112) and one breast cancer (MCF-7) line. The methanolic extracts of Dendrosicyos socotrana, Withanina aduensis, Withania riebeckii, Dracena cinnabari and Buxus hildebrandtii exhibited the highest toxicity on all tumor cell lines with IC50 values ranging between 0.29 and 5.54 microg/ml. The extracts of Jatropha unicostata and Punica protopunica showed a moderate potency on the most tumor cell lines.

PMID: 17484289 [PubMed - indexed for MEDLINE]



Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo.

Stan SD, Hahm ER, Warin R, Singh SV.
Department of Pharmacology and Chemical Biology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human bre


Studies Suggest Potent Anti-Cancer Activity

http://www.lef.org/magazine/mag2006/...t_ashwa_01.htm


In addition to ashwagandha’s documented neuroprotective effects, exciting recent evidence suggests that it also has the potential to stop cancer cells in their tracks. For example, a recent analysis showed that ashwagandha extract inhibited the growth of human breast, lung, and colon cancer cell lines in the laboratory. This inhibition was comparable to that achieved with the common cancer chemotherapy drug doxorubicin (Caelyx®, Myocet®). In fact, researchers reported that withaferin A, a specific compound extracted from ashwagandha, was more effective than doxorubicin in inhibiting breast and colon cancer cell growth.11,14
Scientists in India recently conducted cell studies showing that ashwagandha extract disrupts cancer cells’ ability to reproduce—a key step in fighting cancer. Additionally, laboratory analysis indicates that ashwagandha extract possesses anti-angiogenic activity, also known as the ability to prevent cancer from forming new blood vessels to support its unbridled growth. These findings lend further support to ashwagandha’s potential role in fighting cancer.15 Based on these studies, research in this area continues.
In another study, orally administered ashwagandha extract significantly inhibited experimentally induced stomach cancer in laboratory animals. Tumor incidence was reduced by 60% and tumor multiplicity (number) by 92%. Similarly, in a rodent model of skin cancer, ashwagandha inhibited tumor incidence and multiplicity by 45% and 71%, respectively.16 Ashwagandha’s protective effect against skin cancer has been shown in other studies as well.17
A recent experiment demonstrated that ashwagandha extract produced a marked increase in life span and a decrease in tumor weight in animals with experimentally induced cancer of the lymphatic system.18 This is an exciting finding, suggesting that ashwagandha could enhance survival in individuals with cancer.

Ashwagandha's Pharmacological Activity Scientists speculate that some of ashwagandha’s benefits stem from its antioxidant properties and ability to scavenge free radicals.10 Two main classes of compounds—steroidal alkaloids and steroidal lactones—may account for its broad range of beneficial effects. Steroidal lactones comprise a class of constituents called withanolides. To date, scientists have identified and studied at least 12 alkaloids and 35 withanolides. Much of ashwagandha’s pharmacological activity has been attributed to two primary withanolides, withaferin A and withanolide D.11 Other studies reveal that ashwagandha has antimicrobial properties, with antibacterial activity against potentially dangerous bacteria, including Salmonella, an organism associated with food poisoning. This activity was demonstrated in cell cultures as well as in infected laboratory animals.12 Additional studies show that ashwagandha root extract enhances the ability of macrophage immune cells to “eat” pathogens, as compared to macrophages from a control group that did not receive ashwagandha.13

Ashwagandha extract may also have applications as an adjunct to cancer chemotherapy treatment. One of the consequences of chemotherapy is neutropenia, a decrease in white blood cells called neutrophils that can leave patients dangerously vulnerable to infection. A study of animals demonstrated that orally administered ashwagandha extract protected against this decline in infection-fighting neutrophils. While further human studies are needed, these findings suggest that ashwagandha may be an excellent adjunctive therapy to chemotherapy.19
Another animal study investigated ashwagandha extract’s effects in normalizing the immune-suppressing effects of chemotherapy. When test animals received a common chemotherapy drug, levels of the desirable immune factors interferon-gamma and interleukin-2 decreased.
When the animals also received orally administered ashwagandha extract, however, their immune system parameters remained normal. These findings add support to the idea that ashwagandha may help protect immune function during chemotherapy treatment.20



Bad with cyclophosphamide??:


J Ethnopharmacol. 1998 Oct;62(3):209-14.
Suppressive effect of cyclophosphamide-induced toxicity by Withania somnifera extract in mice.

Davis L, Kuttan G.
Professor Amala Cancer Research Centre, Amala Nagar P.O., Kerala, India.
Administration of Withania somnifera extract (Solanaceae) was found to significantly reduce leucopenia induced by cyclophosphamide (CTX) treatment. The total WBC count on the 12th day of the CTX-treated group was 3720 cells/mm3 and that of CTX along with Withania was 6120 cells/mm3. Treatment of Withania along with CTX was found to significantly (P < 0.001) increase the bone marrow cellularity (13.1 x 10(6) cells/femur) compared to CTX alone treated group (8 x 10(6) cells/femur). Administration of Withania extract increased the number of alpha-esterase positive cells (1130/4000 cells) in the bone marrow of CTX treated animals, compared to the CTX-alone treated group (687/4000 cells). The major activity of Withania somnifera may be the stimulation of stem cell proliferation. These studies indicate that Withania somnifera could reduce the cyclophosphamide induced toxicity and its usefulness in cancer therapy.

PMID: 9849630 [PubMed - indexed for MEDLINE]








Biochem Pharmacol. 2010 Feb 15;79(4):542-51.
Withaferin A targets heat shock protein 90 in pancreatic cancer cells.

Yu Y, Hamza A, Zhang T, Gu M, Zou P, Newman B, Li Y, Gunatilaka AA, Zhan CG, Sun D.
Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA.
The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC(50)s of 1.24, 2.93 and 2.78 microM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 microM) disrupted Hsp90-Cdc37 complexes from 1 to 24h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

PMID: 19769945 [PubMed - in process]


Angiogenesis. 2004;7(2):115-22.
Withaferin A is a potent inhibitor of angiogenesis.

Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, Zhang L, Hanson AD, Conner BP, Rougas J, Pribluda VS.
Discovery Research, EntreMed, Inc., Rockville, Maryland, USA. Royce.Mohan@uky.edu
The medicinal plant Withania somnifera is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda , the major Traditional Indian medicine system, extracts from W. somnifera are distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somnifera root extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50 =12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.

PMID: 15516832 [PubMed - indexed for MEDLINE]




http://www.raysahelian.com/ashwagandha.html

Ashwagandha, Withania somnifera, is an herb that is extensively used in Ayurveda, the traditional health care system in India. This herb is used as a general tonic and "adaptogen," helping the body adapt to stress. In addition, this herb has been shown to possess antioxidant activity as well as an ability to support a healthy immune system. Ashwagandha - also known as Indian Winter Cherry - is a shrub cultivated in India and North America whose roots have been used for thousands of years by Ayurvedic practitioners. The root contains flavonoids and many active ingredients of the withanolide class. Several studies over the past few years have looked into whether this herb has anti-inflammatory, anti-tumor, anti-stress, antioxidant, mind-boosting, immune-enhancing, and rejuvenating properties (see studies at bottom of page). Historically ashwagandha root has also been noted to have sex-enhancing properties. Many people notice a relaxing effect within hours of taking a 500 mg pill.

Anticarcinogenic activity of Withania somnifera Dunal against Dalton's Ascitic Lymphoma.
J Ethnopharmacol. 2004 Aug;93(2-3):359-61.
The effect of ethanolic extract of ashwagandha root against Dalton's Ascitic Lymphoma has been evaluated in Swiss albino mice. A significant increase in the life span and a decrease in the cancer cell number and tumour weight were noted in the tumour-induced mice after treatment with ashwagandha extract. These observations are suggestive of the protective effect of ashwagandha extract in Dalton's Ascitic Lymphoma.


http://www.ethnoleaflets.com/leaflets/withafer.htm
Conclusion: The studies so far indicate that W. somnifera could prove to be a good natural source of a potent and relatively safe radiosensitizer/chemotherapeutic agent.


http://www.drugs.com/npp/ashwaganda.html
Cancer
Withaferin A was first isolated as a cytotoxic agent, 3 and a considerable amount of investigation followed. The compound produced mitotic arrest in Ehrlich ascites carcinoma cells in vitro 31 while in vivo effects were mediated by macrophage activation. 26 , 32 Further investigations on peripheral blood lymphocytes determined that withaferin A destroyed spindle microtubules of cells in metaphase. 33

<
Despite a large volume of basic scientific studies on the plant, there is minimal evidence for a proper dose of this herb. A single study in which W. somnifera was the principal component of a polyherbal mixture administered 450 mg of root powder 4 times per day for arthritis. 49

The protective effect of a purified extract of Withania somnifera against doxorubicin-induced cardiac toxicity in rats 2007 LINK





www.raysahelian.com/silibinin.html
Silibinin is a flavonolignan found in certain herbs, particularly milk thistle

Acta Pharmacol Sin. 2009 Aug;30(8):1162-8. Epub 2009 Jul 6.
Silibinin inhibits prostate cancer invasion, motility and migration by suppressing vimentin and MMP-2 expression.

Wu KJ, Zeng J, Zhu GD, Zhang LL, Zhang D, Li L, Fan JH, Wang XY, He DL.
Department of Urology, First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an 710061, China.
AIM: Silibinin is known to exert growth inhibition and cell death together with cell cycle arrest and apoptosis in human prostate cancer cells. Whether silibinin could inhibit the invasion, motility and migration of prostate cancer cells remains largely unknown. This study was designed to evaluate this efficacy and possible mechanisms using a novel highly bone metastatic ARCaP(M) cell model. METHODS: Four prostate cancer cell lines, LNCaP, PC-3, DU145, and ARCaP(M), were used in this study. These cells were treated with increasing concentrations of silibinin (50, 100, and 200 micromol/L) for different periods of time. After treatment, cell viabilities of four prostate cancer cells were compared by MTT assay. Alterations of ARCaP(M) cell invasion, motility and migration were assessed by cell invasion, motility and wound healing assays. The changes of vimentin expression were observed by Western blotting and immunofluorescence staining, and the expression of MMP-2, MMP-9, and uPA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: ARCaP(M) cells showed less sensitivity to the growth inhibition of pharmacological doses of silibinin than LNCaP, PC-3, and DU145 cells. However, silibinin exerted significant dose- and time-dependent inhibitory effects on the invasion, motility and migration of ARCaP(M) cells. Furthermore, the expression of vimentin and MMP-2, but not MMP-9 or uPA, was down-regulated in a dose- and time-dependent manner after treatment of silibinin. CONCLUSION: This study shows that silibinin could inhibit the invasion, motility and migration of ARCaP(M) cells via down-regulation of vimentin and MMP-2 and therefore may be a promising agent against prostate cancer bone metastasis.

PMID: 19578386 [PubMed - indexed for MEDLINE]
> >
Clin Cancer Res. 2009 May 15;15(10):3557-67. Epub 2009 May 15.




Clin Cancer Res. 2008 Dec 1;14(23):7773-80.
Silibinin inhibits established prostate tumor growth, progression, invasion, and metastasis and suppresses tumor angiogenesis and epithelial-mesenchymal transition in transgenic adenocarcinoma of the mouse prostate model mice.

Singh RP, Raina K, Sharma G, Agarwal R.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Denver, Colorado 80262, USA.
Comment in:

• Clin Cancer Res. 2008 Dec 1;14(23):7581-2.

PURPOSE: The chronic nature of prostate cancer growth and progression leading to metastasis provides a large window for intervention. Herein, for the first time, we investigated the effect and associated mechanisms of silibinin phosphatidylcholine (silybin-phytosome) on established prostate tumors in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. EXPERIMENTAL DESIGN: Twenty-week-old TRAMP male mice having palpable prostate tumor were fed with control or 0.5% and 1%, w/w, silybin-phytosome diets for 11 weeks and then sacrificed. RESULTS: Dietary silibinin inhibited the growth of prostate tumors (up to 60%, P < 0.001) and suppressed tumor progression from prostatic intraepithelial neoplasia to differentiated adenocarcinoma and poorly differentiated adenocarcinoma, with a complete absence of poorly differentiated adenocarcinoma at higher doses. It also inhibited the incidence of tumor invasion of seminal vesicle (up to 81%, P < 0.001) with complete absence of distant metastasis. Silibinin moderately inhibited tumor cell proliferation and induced apoptosis, but strongly suppressed tumor microvessel density (up to 60%, P < 0.001), vascular endothelial growth factor, and vascular endothelial growth factor receptor-2 expression. Antibody array analysis of plasma showed a decrease in the circulatory levels of vascular endothelial growth factor and basic fibroblast growth factor. Decreased levels of matrix metalloproteinases (MMP), snail-1, and vimentin, and an increased level of E-cadherin were also observed, indicating the anti-epithelial-mesenchymal transition effect of silibinin in tumors. CONCLUSIONS: Overall, silibinin treatment of TRAMP mice bearing prostate tumor inhibited tumor growth, progression, local invasion, and distant metastasis involving suppression of tumor angiogenesis and epithelial-mesenchymal transition. These findings would have greater relevance for the ongoing phase II clinical trial with silibinin-phytosome in prostate cancer patients.

PMID: 19047104 [PubMed - indexed for MEDLINE]




Nuclear factor-kappaB-mediated transforming growth factor-beta-induced expression of vimentin is an independent predictor of biochemical recurrence after radical prostatectomy.

Zhang Q, Helfand BT, Jang TL, Zhu LJ, Chen L, Yang XJ, Kozlowski J, Smith N, Kundu SD, Yang G, Raji AA, Javonovic B, Pins M, Lindholm P, Guo Y, Catalona WJ, Lee C.
Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. q-zhang2@northwestern.edu .
PURPOSE: Transforming growth factor-beta (TGF-beta)-mediated epithelial-to-mesenchymal transition (EMT) has been shown to occur in some cancers; however, the pathway remains controversial and varies with different cancers. In addition, the mechanisms by which TGF-beta and the EMT contribute to prostate cancer recurrence are largely unknown. In this study, we elucidated TGF-beta-mediated EMT as a predictor of disease recurrence after therapy for prostate cancer, which has not been reported before. EXPERIMENTAL DESIGN: We analyzed TGF-beta-induced EMT using nuclear factor-kappaB (NF-kappaB) as an intermediate mediator in prostate cancer cell lines. A total of 287 radical prostatectomy specimens were evaluated using immunohistochemistry in a high-throughput tissue microarray analysis. Levels of TGF-beta signaling components and EMT-related factors were analyzed using specific antibodies. Results were expressed as the percentage of cancer cells that stained positive for a given antibody and were correlated with disease recurrence rates at a mean of 7 years following radical prostatectomy. RESULTS: In prostate cancer cell lines, TGF-beta-induced EMT was mediated by NF-kappaB signaling. Blockade of NF-kappaB or TGF-beta signaling resulted in abrogation of vimentin expression and inhibition of the invasive capability of these cells. There was high risk of biochemical recurrence associated with tumors that displayed high levels of expression of TGF-beta1, vimentin, and NF-kappaB and low level of cytokeratin 18. This was particularly true for vimentin, which is independent of patients' Gleason score. CONCLUSIONS: The detection of NF-kappaB-mediated TGF-beta-induced EMT in primary tumors predicts disease recurrence in prostate cancer patients following radical prostatectomy. The changes in TGF-beta signaling and EMT-related factors provide novel molecular markers that may predict prostate cancer outcomes following treatment.







Vimentin inhibitors:


http://www.faqs.org/patents/app/20090137473

Patent application title:
INHIBITION OF MICROTUBULE PROTRUSION IN CANCER CELLS Inventors: Stuart Martin Rebecca Whipple-Bettes
Agents: FULBRIGHT & JAWORSKI, LLP
Assignees:
Origin: HOUSTON, TX US
IPC8 Class: AA61K3817FI
USPC Class: 514 12


Read more: http://www.faqs.org/patents/app/2009...#ixzz0c0JwIB7l

Abstract:


The present invention generally concerns microtubule protrusions in cancer cells, including detached cancer cells, and inhibition of the protrusions. In particular aspects, the inhibition of the protrusions interferes with attachment of the cell to a vessel wall, and in further aspects the cell is killed by forcing it to enter capillaries and be destroyed, for example by shearing. Inhibition by a variety of agents and methods is contemplated.

In embodiments of the invention, one or more components of a microtubule protrusion are inhibited, directly or indirectly. In particular aspects of the invention, the components comprise Glu-tubulin, kinesin and/or vimentin. Therefore, in specific embodiments of methods of the invention, one targets Glu-tubulin, kinesin and/or vimentin to inhibit one or more microtubule protrusions, for example from a cancer cell. In certain embodiments, the protrusions may be targeted by inhibiting Glu-tubulin, such as by one or more of the following, for example: 1) increasing activity and/or expression of tubulin tyrosine ligase (TTL), which postranslationally converts Glu-tubulin back to Tyr-tubulin; 2) inhibition of a carboxypeptidase that produces Glu-tubulin from Tyr-tubulin, such as by delivery of okadaic acid, 3-nitrotyrosine, 1-nor-okadone, Cantharidin, Phoslactomycin B (Fostriecin), DL-benzylsuccinic acid, Sodium orthovanadate, or a combination thereof, and in specific embodiments the carboxypeptidase is hAGBL3 (Rodriguez de la Vega et al., 2007). In certain embodiments, the protrusions may be targeted by inhibiting vimentin, such as by one or more of the following, for example: a dominant negative mutant, such as R113C and/or .DELTA.C2B mutants; antisense or siRNA; and/or calyculin-A. In other embodiments, the protrusions may be targeted by inhibiting kinesin, such as by one or more of the following, for example: siRNA, including KIF5b siRNA, for example, lidocaine, and/or tetracaine.







Related?:

Cancer Cell. 2008 Sep 9;14(3):263-73.
Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents.

Shaked Y, Henke E, Roodhart JM, Mancuso P, Langenberg MH, Colleoni M, Daenen LG, Man S, Xu P, Emmenegger U, Tang T, Zhu Z, Witte L, Strieter RM, Bertolini F, Voest EE, Benezra R, Kerbel RS.
Molecular and Cellular Biology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON M4N 3M5, Canada. yshaked@tx.technion.ac.il
Comment in:

• Cancer Cell. 2008 Sep 9;14(3):195-6.

Several hypotheses have been proposed to explain how antiangiogenic drugs enhance the treatment efficacy of cytotoxic chemotherapy, including impairing the ability of chemotherapy-responsive tumors to regrow after therapy. With respect to the latter, we show that certain chemotherapy drugs, e.g., paclitaxel, can rapidly induce proangiogenic bone marrow-derived circulating endothelial progenitor (CEP) mobilization and subsequent tumor homing, whereas others, e.g., gemcitabine, do not. Acute CEP mobilization was mediated, at least in part, by systemic induction of SDF-1alpha and could be prevented by various procedures such as treatment with anti-VEGFR2 blocking antibodies or paclitaxel treatment in CEP-deficient Id mutant mice, both of which resulted in enhanced antitumor effects mediated by paclitaxel, but not by gemcitabine.

PMID: 18772115


Especially interesting since Paclitaxel is an antimitotic, Gemcitabine is an antimetabolite.

But can metronomic approach reduce this?:

Maximum Tolerable Dose and Low-Dose Metronomic Chemotherapy Have Opposite Effects on the Mobilization and Viability of Circulating Endothelial Progenitor Cells¹

http://cancerres.aacrjournals.org/cg...ull/63/15/4342

Francesco Bertolini², Saki Paul, Patrizia Mancuso, Silvia Monestiroli, Alberto Gobbi, Yuval Shaked and Robert S. Kerbel Division of Hematology-Oncology, Department of Medicine [F. B., S. P., P. M.] and Department of Experimental Oncology, IFOM-Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology [S. M., A. G.], European Institute of Oncology, 20141 Milan, Italy; Molecular and Cell Biology Research, Sunnybrook and Women’s College Health Sciences Centre, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4N 3M5 Canada [Y. S., R. S. K.]
There is growing evidence that vasculogenesis (progenitor cell-derived generation of new blood vessels) is required for the growth of some neoplastic diseases. Here we show that the administration of cyclophosphamide (CTX) at the maximum tolerable dose with 21-day breaks or at more frequent low-dose (metronomic) schedules have opposite effects on the mobilization and viability of circulating endothelial progenitors (CEPs) in immunodeficient mice bearing human lymphoma cells. Animals treated with the maximum tolerable dose CTX experienced a robust CEP mobilization a few days after the end of a cycle of drug administration, and tumors rapidly became drug resistant. Conversely, the administration of metronomic CTX was associated with a consistent decrease in CEP numbers and viability and with more durable inhibition of tumor growth. Our findings suggest that metronomic low-dose chemotherapy regimens are particularly promising for avoiding CEP mobilization and, hence, to potentially reduce vasculogenesis-dependent mechanisms of tumor growth.


To Evade Chemotherapy, Some Cancer Cells Mimic Stem Cells

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http://www.aacr.org/home/public--med...ses.aspx?d=822

Using the CellSearch System techique that quantifies circulating tumor cells, researchers had shown that chemotherapy with Taxol causes a massive release of cells into the circulation, while at the same time reducing the size of the tumor, explaining that complete pathologic responses do not correlate well with improvements in survival.

Circulating tumor cells (CTCs) are cancer cells that have detached from solid tumors and entered the blood stream. This can begin the process of metastasis, the most life-threatening aspect of cancer. To metastasize, or spread cancer to other sites in the body, CTCs travel through the blood and can take root in another tissue or organ.

In this stem cell research, anti-cancer treatments often effectively shrink the size of tumors, but some might have the opposite effect, actually expanding the small population of cancer stem cells that then are capable of metastasizing.

Even before the advent of the CellSearch technique, it had been observed in "cell death" cell culture assays, that there was an increase in the number of metabolic activity of mitochondria of the surviving cells from Taxol therapy, even in cases where the majority of the cells are being killed by Taxol. It may indeed give clinical response (tumor shrinkage), however, these are mostly short-lived and relapses after a response are often dramatic.



To evade chemotherapy, some cancer cells mimic stem cells

September 2007 ATLANTA - Anti-cancer treatments often effectively shrink the size of tumors, but some might have an opposite effect, actually expanding the small population of cancer stem cells believed to drive the disease, according to findings presented today in Atlanta, Georgia at the American Association for Cancer Research's second International Conference on Molecular Diagnostics in Cancer Therapeutic Development.

"Our experiments suggest that some treatments could be producing more cancer stem cells that then are capable of metastasizing, because these cells are trying to find a way to survive the therapy," said one of the study's investigators, Vasyl Vasko, M.D. Ph.D., a pathologist at the Uniformed Services University of the Health Sciences in Bethesda, Md.

"This may help explain why the expression of stem cell markers has been associated with resistance to chemotherapy and radiation treatments and poor outcome for patients with cancers including prostate, breast and lung cancers," Dr. Vasko said. "That tells us that understanding how to target these markers and these cells could prove useful in treating these cancers."

The cancer stem cell markers include Nanog and BMI1, both of which contribute to stem cells' defining ability to renew themselves and differentiate into different cell types, Dr. Vasko said. These same molecules are found in embryonic stem cells.

Researchers have recently debated the notion that some therapies are not capable of eradicating cancer because they do not target the cancer stem cells responsible for tumor development. To test this hypothesis, Dr. Vasko, along with scientists from the CRTRC Institute for Drug Development in San Antonio and from the Johns Hopkins University, set out to measure both stem cells markers and tumor volume before and after treatment in a mouse model.

They selected a rare form of cancer, mesenchymal chondrosarcoma (MCS), which has not been well described and for which there is no effective treatment. The researchers first determined that Nanog and BMI1 stem cell markers were more highly expressed in metastatic tumors compared to primary tumors. "This suggests that expression of the marker plays some role in development of metastasis," Dr. Vasko said.

They then applied various therapies - from VEGF inhibitors such as Avastin to the proteasome inhibitor Velcade - in mice implanted with human MSC, and analyzed the effects on tumors. Some of the treatments seemed to work, because they led to a dramatic decrease in the size of the tumors, Dr. Vasko said. But analysis of stem cell expression before and after treatment revealed that even as some anti-cancer treatments shrank tumors, they increased expression of Nanog and BMI1. "These treatments were not enough to completely inhibit tumor growth, and the cancer stem cell markers were still present," Dr. Vasko said.

Use of the agents Velcade and Docetaxel led to the most significant increase in stem cell markers within the treated tumor, while ifosfamide and Avastin inhibited expression of the markers in this cancer subtype.

"We hypothesize that the tumor escapes from chemotherapy by induction of stem cell marker expression," he said. "The small number of cells that survive the treatment could then generate another tumor that metastasizes."

Dr. Vasko doesn't know how this happens, but theorizes that "dying cells could secrete a lot of factors that induce expression of stem cell markers in other cancer cells. I think they are trying to survive and they use a mechanism from their experience of embryonic life."

American Association for Cancer Research. September 2007.