26 research outputs found
Antitumoral effects of Hibiscus Sabdariffa on human breast cancer cells
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
Antitumoral effects of Hibiscus sabdarifa on human oral squamous cell carcinoma and multiple myeloma cells
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
Tubulin involvement in Bortezomib peripheral neurotoxicity
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 grant
Innovative 3D proteome-wide scale identification of ALKBH5 target for MV1035 small molecule able to reduce migration and invasiveness in U87 glioblastoma cell lines by SPILLO-PBSS
The innovative in silico technologies developed at SPILLOproject,1 e.g., the SPILLO potential binding sites searcher (SPILLO-PBSS) software,2,3 allow to identify targets and off-targets of any small molecule on a multiple-organism proteomewide scale, and to perform an accurate multilevel cross-organism transferability analysis (MCOTA) aimed at rationalising animal testing. SPILLO-PBSS has been successfully used in several research projects, such as a study in which a compound (MV1035) was found to reduce migration and invasiveness in U87 glioblastoma (GBM) cell lines: the human structural proteome was analyzed and the RNA demethylase ALKBH5 has been identified as a target responsible for the observed effects (target experimentally validated). Another top-ranked target identified by SPILLO-PBSS, the DNA repair protein AlkB homolog 2 (ALKBH2), abundantly expressed in GBM cell lines, resulted particularly interesting for its pivotal role in the onset of resistance to Temozolomide (TMZ), the standard firstline treatment for GBM.
Neurodegeneration: can metabolites from Eremurus persicus help?
The number of patients affected by neurodegenerative diseases is increasing worldwide, and no effective treatments have been developed yet. Although precision medicine could represent a powerful tool, it remains a challenge due to the high variability among patients. To identify molecules acting with innovative mechanisms of action, we performed a computational investigation using SAFAN technology, focusing specifically on HuD. This target belongs to the human embryonic lethal abnormal visual-like (ELAV) proteins and plays a key role in neuronal plasticity and differentiation. The results highlighted that the molecule able to bind the selected target was (R)-aloesaponol-III-8-methyl ether [(R)-ASME], a metabolite extracted from Eremurus persicus. Notably, this molecule is a TNF-alpha inhibitor, a cytokine involved in neuroinflammation. To obtain a suitable amount of (R)-ASME to confirm its activity on HuD, we optimized the extraction procedure. Together with ASME, another related metabolite, germichrysone, was isolated. Both ASME and germichrysone underwent biological investigation, but only ASME confirmed its ability to bind HuD. Given the multifactorial nature of neurodegenerative diseases, we decided to investigate ASME as a proteasome activator, being molecules endowed with this kind of activity potentially able to counteract aggregations of dysregulated proteins. ASME was able to activate the considered target both in enzymatic and cellular assays. Therefore, ASME may be considered a promising hit in the fight against neurodegenerative diseases
Anti-tumor Efficacy Assessment of the Sigma Receptor Pan Modulator RC-106. A Promising Therapeutic Tool for Pancreatic Cancer
Introduction: Pancreatic cancer (PC) is one of the most lethal tumor worldwide, with no prognosis improvement over the past 20-years. The silent progressive nature of this neoplasia hampers the early diagnosis, and the surgical resection of the tumor, thus chemotherapy remains the only available therapeutic option. Sigma receptors (SRs) are a class of receptors proposed as new cancer therapeutic targets due to their over-expression in tumor cells and their involvement in cancer biology. The main localization of these receptors strongly suggests their potential role in ER unfolded protein response (ER-UPR), a condition frequently occurring in several pathological settings, including cancer. Our group has recently identified RC-106, a novel pan-SR modulator with good in vitro antiproliferative activities toward a panel of different cancer cell lines. In the present study, we investigated the in vitro properties and pharmacological profile of RC-106 in PC cell lines with the aim to identify a potential lead candidate for the treatment of this tumor.Methods: Pancreatic cancer cell lines Panc-1, Capan-1, and Capan-2 have been used in all experiments. S1R and TMEM97/S2R expression in PC cell lines was quantified by Real-Time qRT-PCR and Western Blot experiments. MTS assay was used to assess the antiproliferative effect of RC-106. The apoptotic properties of RC-106 was evaluated by TUNEL and caspase activation assays. GRP78/BiP, ATF4, and CHOP was quantified to evaluate ER-UPR. Proteasome activity was investigated by a specific fluorescent-based assay. Scratch wound healing assay was used to asses RC-106 effect on cell migration. In addition, we delineated the in vivo pharmacokinetic profile and pancreas distribution of RC-106 in male CD-1 mice.Results: Panc-1, Capan-1, and Capan-2 express both SRs. RC-106 exerts an antiproliferative and pro-apoptotic effect in all examined cell lines. Cells exposure to RC-106 induces the increase of the expression of ER-UPR related proteins, and the inhibition of proteasome activity. Moreover, RC-106 is able to decrease PC cell lines motility. The in vivo results show that RC-106 is more concentrated in pancreas than plasma.Conclusion: Overall, our data evidenced that the pan-SR modulator RC-106 is an optimal candidate for in vivo studies in animal models of PC
Anti-Multiple Myeloma Potential of Secondary Metabolites from Hibiscus sabdariffa
Multiple myeloma (MM) belongs to hematological cancers and its incidence is increasing worldwide. Despite recent advances in its therapy, MM still causes many deaths every year. In fact, current therapies sometimes fail and are associated with severe adverse effects, including neurotoxicity. As a part of our ongoing efforts to discover new potential therapies against MM, we prepared Hibiscus sabdariffa extracts obtained by a microwave-assisted solvent extraction and investigate their activity by in vitro assays on the RPMI-8226 cell line. The bioguided fractionation of the crude ethanolic extract allowed the identification of HsFC as the most effective extract. We assessed cell viability (MTT and Tripan blue test), cell migration (Boyden chamber assay), and neurotoxicity (DRG neurotoxicity assay). The promising results prompted us to further fractionate HsFC and we obtained two molecules effective against RPMI-8226 cells without neurotoxic effects at their active concentrations. Moreover, both compounds are able to significantly reduce cell migration
Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair
Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viabilit
Chemotherapy-Induced Peripheral Neuropathy and Changes in Cytoskeleton
Despite the different antineoplastic mechanisms of action, peripheral neurotoxicity induced by all chemotherapy drugs (anti-tubulin agents, platinum compounds, proteasome inhibitors, thalidomide) is associated with neuron morphological changes ascribable to cytoskeleton modifications. The “dying back” degeneration of distal terminals (sensory nerves) of dorsal root ganglia sensory neurons, observed in animal models, in in vitro cultures and biopsies of patients is the most evident hallmark of the perturbation of the cytoskeleton. On the other hand, in highly polarized cells like neurons, the cytoskeleton carries out its role not only in axons but also has a fundamental role in dendrite plasticity and in the organization of soma. In the literature, there are many studies focused on the antineoplastic-induced alteration of microtubule organization (and consequently, fast axonal transport defects) while very few studies have investigated the effect of the different classes of drugs on microfilaments, intermediate filaments and associated proteins. Therefore, in this review, we will focus on: (1) Highlighting the fundamental role of the crosstalk among the three filamentous subsystems and (2) investigating pivotal cytoskeleton-associated proteins