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The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

Rigosertib is a small molecule in preclinical development that, due to its characteristics as a dual PLK1 and PI3K inhibitor, is particularly effective in counteracting the advance of different types of tumors. In this work, we evaluated the efficacy of Rigosertib and the expression of p53 in five different human tumor cell lines in vitro, A549 (lung adenocarcinoma), MCF-7 and MDA-MB231 (breast cancer cells), RPMI 8226 (multiple myeloma), and U87-MG (glioblastoma). We demonstrated that in all cell lines, the effect was dose- and time-dependent, but A549 cells were the most sensible to the treatment while higher concentrations were required for the most resistant cell line U87-MG. Moreover, the highest and lowest p53 levels have been observed, respectively, in A459 and U87-MG cells. The alterations in the cell cycle and in cell-cycle-related proteins were observed in A549 at lower concentrations than U87-MG. In conclusion, with this article we have demonstrated that Rigosertib has different efficacy depending on the cell line considered and that it could be a potential antineoplastic agent against lung cancer in humans.

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.2

Proteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.

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 viability

Effective therapies for multiple sclerosis (MS) are still missing. This neurological disease affects more than 2.5 million people worldwide. To date, biological immunomodulatory drugs are effective and safe during short-term treatment, but they are suitable only for parenteral administration and they are expensive. Accordingly, academic and industrial environments are still focusing their efforts toward the development of new MS drugs. Considering that neurodegeneration is a contributory factor in the onset of MS, herein we will focus on the crucial role played by sigma 1 receptors (S1Rs) in MS. A pilot study was performed, evaluating the effect of the S1R agonist (R)-RC33 on rat dorsal root ganglia experimental model. The encouraging results support the potential of S1R agonists for MS treatment.

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.

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.

Cholangiocarcinoma is the first most common cancer of the biliary tract. To date, surgical resection is the only potentially curative option, but it is possible only for a limited percentage of patients, and in any case survival rate is quite low. Moreover, cholangiocarcinoma is often chemotherapy-resistant, and the only drug with a significant benefit for patient’s survival is Gemcitabine. It is necessary to find new drugs or combination therapies to treat nonresectable cholangiocarcinoma and improve the overall survival rate of patients. In this work, we evaluate in vitro the antitumoral effects of Rigosertib, a multi-kinase inhibitor in clinical development, against cholangiocarcinoma EGI-1 cell lines. Rigosertib impairs EGI-1 cell viability in a dose- and time-dependent manner, reversibility is dose-dependent, and significant morphological and nuclear alterations occur. Moreover, Rigosertib induces the arrest of the cell cycle in the G2/M phase, increases autophagy, and inhibits proteasome, cell migration, and invasion. Lastly, Rigosertib shows to be a stronger radiosensitizer than Gemcitabine and 5-Fluorouracil. In conclusion, Rigosertib could be a potential therapeutic option, alone or in combination with radiations, for nonresectable patients with cholangiocarcinoma.

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.

In this manuscript we report on the design, synthesis and evaluation of dual Sigma 1 Receptor (S1R) modulators/Acetylcholinesterase (AChE) inhibitors endowed with antioxidant and neurotrophic properties, potentially able to counteract neurodegeneration. The compounds based on arylalkylaminoketone scaffold integrate the pharmacophoric elements of RRC-33, a S1R modulator developed by us, donepezil, a well-known AChE inhibitor, and curcumin, a natural antioxidant compound with neuroprotective properties. A small library of compounds was synthesized and preliminary in vitro screening performed. Some compounds showed good S1R binding affinity, selectivity towards S2R and N-Methyl- d -Aspartate (NMDA) receptor, AChE relevant inhibiting activity and are potentially able to bypass the BBB, as predicted by the in silico study. For the hits 10 and 20, the antioxidant profile was assessed in SH-SY5Y human neuroblastoma cell lines by evaluating their protective effect against H2O2 cytotoxicity and reactive oxygen species (ROS) production. Tested compounds resulted effective in decreasing ROS production, thus ameliorating the cellular survival. Moreover, compounds 10 and 20 showed to be effective in promoting the neurite elongation of Dorsal Root Ganglia (DRG), thus demonstrating a promising neurotrophic activity. Of note, the tested compounds did not show any cytotoxic effect at the concentration assayed. Relying on these encouraging results, both compounds will undergo a structure optimization program for the development of therapeutic candidates for neurodegenerative diseases treatment.

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.

Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

Aim: Nowadays, there is a great interest in the therapeutic potential of sigma1 receptor ligands for treating different CNS pathologies. Our previous investigations led to identify (R)-RC-33 as a potent and selective S1R agonist. Results: Herein, we report the gram-scale synthesis, pharmacokinetic profile and CNS distribution of (R)-RC-33 in the mouse to determine the most suitable dosage schedule for in vivo administration. For comparative purposes, the same experiments were also performed with PRE-084, the most widely used S1R agonist commonly in pharmacological experiments. Discussion: (R)-RC-33 shows a similar pharmacokinetic profile and a better CNS distribution when compared with PRE-084. Conclusion: (R)-RC-33 may be a promising candidate for in vivo studies in animal models of neurodegenerative diseases.