Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Abstract Rhodiola rosea is a medicinal plant, mainly used in Asia and Scandinavia. It is characterized as an adaptogen and is reported to have many pharmacological properties, which are ascribed to the glycosides of cinnamyl alcohol and tyrosol. As natural habitats are already overharvested and the cultivation of this plant needs 4–6 years, the production of the pharmacologically important compounds in in vitro cultures could be an alternative. In the work presented here, the production of these glycosides in compact callus aggregate cultures of roseroot was addressed. Biotransformation of exogenously added cinnamylalcohol and tyrosol was studied. Glucosylation of the precursors yielded high amounts of rosin and salidroside and low amounts of rosavin. During the course of this work, four new glycosides of cinnamyl alcohol were found and identified. The optimal concentration of the precursors and the time needed for the biotransformation was also determined. For enhancing the biotransformation rate, glucose was added to the culture medium alongside with sucrose, which doubled the production of cinnamyl alcohol glycosides but did not affect the production of salidroside. A pilot experiment using air-lift bioreactor was performed. A cDNA fragment encoding tyrosine decarboxylase was isolated and described. The expression of this gene was analysed in the leaves and roots of two chemotypes. The results demonstrate the important role of tyrosine decarboxylase in the production of salidroside. The results revealed production of the pharmacologically important glycosides of Rhodiola rosea; however the successful pilot bioreactor experiment remains to be scaled-up. New information was obtained on the biosynthesis of salidroside, which substantiate the metabolic engineering of roseroot.

Melanoma remains an aggressive form of skin cancer with limited treatment options. Novel methods to treat primary tumors and prevent metastatic disease can lead to improved survival for those diagnosed with melanoma. Through this work, we have evaluated the antineoplastic effects of Rhodiola crenulata (R. crenulata) root extracts on B16-F10 melanoma both in vitro and in vivo. In this study, we observed that R. crenulata treatment resulted in an increased cell death as well as a reduced cell growth, proliferation and migration in vitro. Additionally, we observed that R. crenulata decreased the expression of integrin β1 and vimentin, and increased expression of E-cadherin upon in vitro treatment. Further, we observed in a topical R. crenulata based cream therapy, a more radial growth pattern of tumors as well as a reduced mitotic activity and increased tumor necrosis. Markedly, we observed that mice supplemented with R. crenulata orally in their drinking water also displayed reduced establishment of metastatic foci in a disseminated model of melanoma. Collectively, these findings reveal that R. crenulata exhibits striking anti-tumorigenic and anti-metastatic properties, and that this extract may increase survival and harbor potential novel adjuvant therapy for the treatment of melanoma.

The root of the Tibetan plant Rhodiola crenulata is part of eastern traditional medicine. Studies have suggested that members of the Rhodiola genus display anticancer properties. In this study we examine the effect of R. crenulata in a cellular model of invasive breast cancer, this disease being the second cause of cancer death among women in the US. Deregulation of the Wnt/β-catenin pathway has been frequently observed in breast cancers and appears to have a key role in the transformation of benign cells to a malignant form. Although mutations of the Wnt growth factor are rarely observed in cancer, the Wnt signaling pathway is often up-regulated by either mutations that result in stabilization of β-catenin or by hypermethylation and subsequent loss of expression of Wnt signaling antagonists like secreted Frizzled-Related Protein 1 (SFRP1) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008). We used an engineered cell line in which SFRP1 expression has been knocked down. These cells were derived from 76NTert cell line, an immortalized human mammary epithelium cell line. The resulting 76NTert-siSFRP1 cells display a mesenchymal-like phenotype, invasive behavior and are more resistant to apoptosis triggered by anchorage independent conditions, or anoikis. Additionally we used a highly invasive estrogen receptor negative (ER-), progesterone receptor negative (PR-), Her2/neu negative (triple negative) breast cancer cells line MDA-MB-231. Treatment of MDA-MB-231 and 76NTert-siSFRP1 cells with an extract of R. crenulata inhibited migration and invasion of both cell types, as compared to untreated cells. Furthermore, R. crenulata sensitizes cells to anoikis but does not increase γ-irradiation induced cell death. We provide evidence that death induced by R. crenulata does not occur through the inhibition of an epithelial-to-mesenchymal transition (EMT). Taken together, our initial results suggest R. crenulata as a potential therapeutic agent for breast cancer patients with mutations in the Wnt/β-Catenin signaling pathway. Additionally, we present evidence that R. crenulata exerts its anti-metastasis effect by inhibiting cell migration and increasing cell attachment to a substrate. We demonstrate that this effect occurs by R. crenulata’s modulation of the Rho GTPase effector ROCK1. We further show evidence that R. crenulata’s effect on ROCK is not limited to cancer cells (in vitro), but also affects isolated and intact embryonic tissues. We discovered that treatment of X. laevis embryos with R. crenulata can cause a spina bifida phenotype, suggesting (1) that compounds in R. crenulata may prove detrimental to a developing embryo, and (2) the active compounds in R. crenulata may prove useful in the study of developmental anomalies that lead to conditions such as spina bifida. More importantly, our results suggest that pregnant women should avoid taking R. crenulata-containing supplements during pregnancy. Compounds in R. crenulata may be contraindicative to the pregnancy and cause injury to a developing fetus. The information provided may help health providers offer better advice on natural supplements to expecting mothers. We also characterized and identified multiple R. crenulata compounds and report predicted protein targets for these compounds. Finally, we provide evidence that R. crenulata affects cancer cell metabolism and suggest potential protein targets of the chemical components of this extract. This study provides new information that will help dissect the mechanisms of action of the R. crenulata compounds and possible synergies amongst them.

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