EVALUATION OF ANTITUMORAL EFFECTS OF HIBISCUS SABDARIFFA ON MULTIPLE MYELOMA CELLS

Hibiscus Sabdariffa (HS) is a plant of the Malvacee family commonly cultured in tropical and subtropical countries. It is mainly known as the main ingredient for the preparation of cold drink called Karkadè. Calices and leaves of HS plant are also used in folk medicine thanks to their antioxidant and anti-inflammatory properties. In recent years, HS has also gained great interest as a possible antitumoral agent. In the present PhD project, we evaluated the antitumoral effects of HS against multiple mye-loma cells in vitro. Multiple myeloma is the most frequent hematological malignancy world-wide. In recent years, new drugs have increased the survival expectancy of patients. Despite this, new therapeutic approaches are necessary, especially for high multiple myeloma hetero-geneity and for relapsed or refractory multiple myeloma. The project was organized in three distinct phases: 1- Evaluation of antitumoral effects of HS against RPMI 8226 human multiple myeloma cells. We demonstrated by MTT and Trypan blue assays that a total HS extract (HSE) and one of its fraction obtained by liquid-liquid extraction (HSEC) were able to impair cell viability of human multiple myeloma RPMI 8226 in a dose and time dependent manner. HSE cell viability reduction was due to a cytostatic action, while HSEC was more cytotoxic and induced a caspase dependent apoptosis. Moreover, both HSE and HSEC impaired cell migration and invasion of RPMI 8226 cells in a Boyden chamber as-say. We also demonstrated in in vitro model of neurotoxicity (dorsal root ganglia model) that HSE and HSEC concentrations used in our experiments were not neurotoxic. In RPMI 8226 cells autophagy and proteasome activity were impaired by both HSE and HSEC. MAPK p38 activation was observed in the first 6h of treatment, while ERK 1 and ERK 2 activation occurred between 16 and 48h. 2- Evaluation of combinations between Bortezomib (BTZ) and HSE or HSEC against RPMI 8226 multiple myeloma cells. We evaluated several combinations between BTZ and HSE or HSEC (simultaneous, not-simultaneous treatment and pretreatment) using MTT assay to assess their effectiveness. Among all evaluated combinations, only the 24h BTZ 1nM pretreatment followed by HSE or HSEC treatment resulted more effective than BTZ or HSE or HSEC single treatment in reducing cell viability. This combination was not neurotoxic in the dorsal root ganglia model. 3- Isolation and characterization of HSEC molecules responsible of the antitumoral ef-fect. Using a bioguided method, we isolated several fractions from HSEC. Fractions were obtained by flash column chromatography or by resin use. Molecular characterization was performed by HPLC, NMR or mass spectrometry, while biological activity was screened in human multiple myeloma RPMI 8226 cells by MTT and proteasome activity assay. We isolated three fractions with a first fractionating process that keep their activ-ity on RPMI 8226 cells, but that are not completely pure. In a second fractionating, we isolated a highly pure fraction with a significant activity on RPMI 8226 cell viability. We also characterized this fraction by both a molecular and biological point of view. In conclusion, we demonstrated the antitumoral effect of HS in vitro against RPMI 8226 multiple myeloma cells. We also found a new therapeutic combination between BTZ and HS that enhanced their antitumoral effect when compared to single treatments. Moreover, we also isolated one of the molecules that are involved in the antitumoral effect of HS. The results of my PhD project showed that HS could be a promising chemotherapeutic agent, but further studies are still needed.