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Hibiscus Sabdariffa (HS) is a plant commonly used in folk medicine (1). In recent years HS has gained great interest due to its important antioxidant, anti-inflammatory and antitumoral properties. In our work, we evaluated the in vitro anticancer effects of HS extract against two different human breast cancer cell lines: estrogen receptor (ER) positive MCF-7 cells and ER negative MDA-MB-231 cells. We tested both total extract (HSE) and one fraction obtained by ethyl acetate extraction (HSEC). MTT assay and Trypan Blue vital count showed a dose and time dependent reduction of the viability in both cell lines treated with different concentrations of HSE or HSEC compared to untreated control cells. A significantly marked reduction was observed in MCF-7 cells treated with HSEC. On the basis of our results we used the concentrations of 7.5mg/ml and 3.5mg/ml respectively for HSE and HSEC. In order to evaluate ER involvement in HS effect, we analyzed the cellular localization of the receptor (ERα isotype) by immunofluorescence experiments. Untreated MDA-MB-231 cells showed a low expression of the receptor mostly localized at the cytoplasmic level and treatment with HSE or HSEC didn’t change this state. Untreated MCF-7 cells showed a greater expression of the receptor, with nuclear and cytoplasmic localization. Following HSE or HSEC treatment ERα localization became more cytoplasmic and this effect was more evident after HSEC induction. These data were also confirmed by ERα western blot analysis. Subsequently, we studied HSE and HSEC ability to alter migration and invasion capacity of ER positive MCF-7 cells. Using a scratch wound healing assay we did not observe any change in the migration of cells compared to untreated cells. On the contrary, in a Boyden chamber invasion assay, HSE, and especially HSEC, induced reduction of MCF-7 cell invasion. In conclusion, we have demonstrated that HS is able to reduce cell viability of ER positive MCF-7 and ER negative MDA-MB-231 cells. This effect is more evident in MCF-7 cells in which ER localization and reduced cell invasion were observed. These results are more evident after HSEC treatment. Further studies will be needed to better elucidate the involved mechanisms of action., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Cholangiocarcinoma (CCA) remains a therapeutic challenge. The small-molecule Rigosertib can selectively synchronize cancer cells to G2/M phase improving the efficacy of radiation. In our study, we evaluated in vitro Rigosertib (gifted by Onconova Therapeutics Inc) effects on two human CCA cell lines: EGI-1 and TFK-1. Rigosertib was compared with Gemcitabine (GEM) and 5-Fluorouracil (5-FU), two antineoplastic and radio-sensitizer agents used in the treatment of CCA. Rigosertib impaired cell viability (evaluated by Tripan-blue vital count) in both cell lines in a dose- and time-dependent manner (IC50 of 100nM at 24h). GEM and 5-FU had a IC50 of 30µM and 7µM after 24h, respectively. Cell migration and invasion tests was performed by scratch wound healing and Boyden chamber assay respectively. Rigosertib caused a 50% inhibition of the EGI-1 cell migration (10µM) and invasion (100nM), while the inhibitory effects on TFK-1 cells were observed with doses of 100µM and 10µM, respectively. GEM 30µM and 5-FU 7µM had no effect on cell migration and invasion. Evaluation of cell cycle by FACS cytometry showed a G2/M arrest in both cell lines after Rigosertib 100nM for 24h. Radio-sensitizing test was performed by clonogenic survival assay after irradiation. 24h Rigosertib pre-treatment (100nM for EG-1 and 1 µM for TFK-1) when followed by 2, 4 or 6 Gy irradiation, reduced survival in both CAA cell lines when compared with radiation alone. The Rigosertib radio-sensitizer effect was similar to that seen after GEM or 5-FU 24 pre-treatment both plus irradiation. However, 48h Rigosertib pre-treatment was more effective than radiation alone as well as GEM for 48h. Our study highlights the preliminary but promising preclinical activity of Rigosertib both as antitumoral and as a radio-sensitizer agent in CCA and provides a background for further investigations., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Axonal transport of mitochondria (Mt) controlled by specialized motor and docking proteins that distribute Mt throughout the axon where they provide energy for metabolic and synaptic activity is a vulnerable target in neuronal pathology (1). Bortezomib (BZ) is a proteasome inhibitor active in multiple myeloma (2). One of its key toxicities is painful peripheral neuropathy (BIPN), which frequently requires treatment discontinuation (3). BIPN is dose-related and predominantly sensory, resulting from axonal degeneration. Recent results indicate that BZ modifies axonal tubulin dynamic and we hypothesize that BZ alters fast axonal transport. Here we studied using time-lapse imaging the effect of different BZ concentration on axonal Mt transport in isolated dorsal root ganglion (DRG) neurons from adult male mice. We used kymograph to quantify the total number of Mt and to discriminate antero and retrogradely moving Mt from stationary Mt. Twenty-four hours of BZ treatment (0.1 to 15 µM) induced a dose-dependent reduction in Mt trafficking. Moreover, BZ had no impact on MT motion directions, but it induced a progressive reduction of both anterograde and retrograde axonal transport velocities. These events were associated with increase in tubulin polymerization and of MAP2 expression, but they occurred only after 72h of chronic BZ treatment. We have developed an in vitro model of BIPN demonstrating that transport impairment is already present before evident tubulin polymerization, suggesting that transport deficit represents an early stage of axonal dysfunction. Perpetuated transport dysfunction could impair distal organelle supply and play a critical role in advanced stages of BIPN.This work was supported by the University of Milan-Bicocca and University of Michigan research grants, Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

A step forward in the identification of compounds interfering with the Embryonic Lethal Abnormal Vision (ELAV) protein - RNA complexes

Sigma 1 receptor modulators as a new weapon against multiple sclerosis

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. 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.

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability reduction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS extract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin respectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved in its action, we proposed HS as a potential chemopreventive agent., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese traditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we investigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200μM) in limiting EGI-1 viability using MTT assay. After 24h and 48h treatment, 5 and 10μM BA had no effect while rising from 25μM to 200μM (i.e. 25, 50, 100 and 200μM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA exposure. This reduction well correlated with the adherent absolute cell number decrease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200μM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concentrations but 5μM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10μM BA showed similar migration inhibition extent at 24 and 48h whilst 200μM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant way. Taken together our preliminary results demonstrate that BA impairs CCA cell viability and migration suggesting a promising adjuvant therapeutic use for BA as antitumoral agent., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014