Anti-Tumoral Effects of A Benzimidazolyl Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines

Anti-Tumoral Effects of A Benzimidazolyl Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-Tumoral Effects of A Benzimidazolyl Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines Alice Nicolai, Valentina Noemi Madia, Antonella Messore, Alessandro De Leo, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-233855/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nocodazole is a well-known anti-proliferative agent, thanks to its anti-microtubule activity. Herein we report a benzimidazolyl carbamate ester derivative, namely RDS 60, as structural analog of this compound. We evaluated the anti-neoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines. We found that RDS 60 significantly inhibited replication of both HNSCC without inducing any significant cytotoxic effect on non-transformed human dermal fibroblasts. The treatment of the two cell lines with 1 µM RDS 60 for 24 hours determined the incapacity of cells to develop normal bipolar mitotic spindles contemporaneously to a block of the cell cycle in G2/M and to cytoplasmic accumulation of cyclin B1. Moreover, doubling the dosage of RDS 60, an activation of apoptosis in both HNSCC was observed. Additionally, RDS 60 treatment reversed the epithelial-mesenchymal transition and inhibited cell migration and extracellular matrix infiltration of both HNSCC. These results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and invasion of the two HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignant phenotype of HNSCC suggests its potential role as an interesting and powerful tool for new approaches in treating these tumors. Internal Medicine General Cell Biology & Physiology benzimidazole head and neck squamous cell carcinoma apoptosis epithelial-mesenchymal transition tubulin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nocodazole is a benzimidazol-2-yl carbamate ester derivative that interacts with microtubules by targeting tubulin. In fact, nocodazole exerts a high affinity for beta tubulin, developing hydrogen bonds between the single beta subunits and consequently inhibiting first the aggregation of alpha and beta subunits and then the interaction among tubulin heterodimers; this altered state finally induces the depolymerization of microtubules [1, 2]. The impairment of microtubules polymerization determines an alteration of mitotic spindle organization with the consequent incapacity to carry out mitosis [3, 4]. Whenever cells with high replication rate are treated with nocodazole, they are not able to organize a normal bipolar mitotic spindle, so that they do not complete mitosis and are blocked in G2 or M phase of cell cycle. Yet, the prolonged arrest in pro-metaphases is a starting point for apoptosis [5]. Despite its antitumoral activity, nocodazole did not reach the clinical phase so far [6]. On the other hand, several previous reports have suggested that benzimidazole is the most prominent heterocycle having good cytotoxic properties against different types of cancer cell lines [7-9]. Therefore, we decided to deepen our knowledge on the anti-tumor activity of a benzimidazole derivative structurally related with nocodazole that we previously synthesized and reported [10], namely RDS 60 (Fig. 1). To this aim, we evaluated the activity of RDS 60 in terms of anti-mitotic and apoptosis inducer agent and we tested its potential anti-tumoral efficiency directed to human head and neck squamous cell carcinomas (HNSCC), a group of tumors detectable in various regions of the oral cavity, oropharynx, hypopharynx and larynx. We chose these types of neoplasms because they are extremely aggressive; in fact, almost 50% of the newly diagnosed HNSCC has a survival rate below 5 years [11]. HNSCC are collocated at the sixth position among malign tumors more diffuse in the world [12] and they are all characterized by a poor prognosis, mainly due to metastases, to the development of multiple primary tumors and to local and regional often inoperable relapses [13, 14]. The presence of distant metastases at the moment of diagnosis is associated to the high mortality rate of HNSCC [15] since the chemotherapy used for metastatic disease gives often low responses [16, 17]. Herein, we demonstrated that our benzimidazol-2-yl carbamate ester RDS 60 was capable to impair tubulin assembling in the mitotic spindle, to block the cell cycle in G2 phase, to induce apoptosis and to reverse the invasive phenotype of two HNSCC cell lines. Materials And Methods RDS 60 synthesis RDS 60 (ethyl (5-((1H-pyrrol-1-yl)methyl)-1H-benzo[d]imidazol-2-yl)carbamate) (Fig. 1a) was synthesized as previously described [10]. However, we modified the first two reaction steps in order to improve the overall reaction yields. Indeed, we applied a reduction reaction according to Moore et al. [18] of the commercially available carboxylic acid into alcohol that was subsequently converted in the corresponding benzyl chloride (Fig. 1b) via an aliphatic nucleophilic substitution. The subsequent synthetic steps resemble the ones previously reported [10]. Therefore, we report the details of the first two synthetic steps since the other ones can be found in ref. 10. Melting point (°C), recrystallization solvent, yield (%), chromatographic system, IR, 1 H NMR, formula, Mr and analyzed elements for derivative RDS 60 agreed with the ones previously described [10]. RDS 60 samples used for biological evaluation were 99% pure as determined by elemental analysis. Synthesis of 1 A solution of AlCl 3 (3.77 g; 28.28 mmol) in THF dry (4.6 mL) was added dropwise into a solution of 3,4-dinitrobenzoic acid (5 g; 23.57 mM) and NaBH 4 (3.21 g; 84.85 mmol) in THF dry (46 mL) at 0 °C within 20 min. The reaction was stirred at 25 °C for 1 hour (h) and for 2 h to reflux and then poured into ice-cold water (115 mL). The pH was adjusted to 7 with 1 N HCl and the mixture was extracted with ethyl acetate (3 x 50 mL). The collected extracts were washed with brine (3 x 100 mL) and dried. Removal of the solvent furnished crude residue that was washed with ethyl acetate in order to discard the boron salt, yielding intermediate 1 (3.37 g, 72.1 %) as a yellow solid. Synthesis of 2 To a solution of compound 1 (16.27 g, 81.85 mM) in CHCl 3 (126 mL) was added PCl 5 (6.63 g, 31.85 mM) portionwise at 0 °C within 15 min. The reaction mixture was stirred at 0 °C for 15 min and at 25 °C for 2.5 h and then poured into ice-cold water. The organic layer was washed with 5% w/v Na 2 CO 3 (3 x 100 mL) and with NaCl ss (3 x 100 mL) and dried. Removal of the solvent furnished the crude compound 2 that underwent to the following step without further purification. Cell cultures and treatment Two HNSCC cell lines were utilized: tongue carcinoma CAL27 and pharynx carcinoma FaDu (ATCC, USA), and one human dermal fibroblast primary culture HF [19, 20]. Cells were grown in RPMI 1640 medium with 10% fetal calf serum (FCS), 2 mM glutamine and 50 U/mL penicillin-streptomycin (Sigma-Aldrich). The compound RDS 60 was solubilized in dimethylsulfoxide (DMSO) (Sigma) for a 10 mM stock solution and utilized to final concentrations from 100 nM to 10 μM for 24 and 48 h. Control cells were treated with equivalent amounts of DMSO in every experiment. Cytotoxicity assay To determine cytotoxicity, a sulforhodamine-B colorimetric assay was performed: 5x10 3 cells were plated in 96-well plates, grown for 24 h and then treated with 100 nM, 1, 2.5, 5, 10 μM RDS 60 for 24 and 48 h. Cells were then fixed with 50% trichloroacetic acid for 1 h to 4 °C and stained for 30 min to room temperature (RT) with 0.4% sulforhodamine-B in 1% acetic acid. Excess dye was removed by washing four times with 1% acetic acid. Protein-bound dye was dissolved in 10 mM Tris pH 10 and optical density was determined at 510 nm using a microplate reader. Flow cytometry analysis Cells were seeded in 60-mm plates, grown for 24 h and then treated with 1 μM RDS 60 or equivalent amounts of DMSO for 24 h. The cells were harvested by trypsinization, washed twice with cold PBS, fixed in 70% ethanol at 4°C overnight. Cells were rinsed twice with PBS and incubated with 50 µL of RNAse (100 µg/mL, Sigma) to ensure that only DNA was stained, and 200 µL of Propidium Iodide (PI) (50 µg/mL, Sigma). Cell cycle analysis was performed using a FACS CantoII equipped with 488nm laser and DIVA Software (BD Biosciences). The cells were first gated using a forward vs. side scatter (FSC vs. SSC) strategy, and upon 488 nm laser excitation, PI fluorescence was then detected above 580 nm. Data were analyzed using Flow Jo software (Flow Jo LLC, OR, USA). Immunofluorescence Cells were grown on Labteck chamber slides (Nunc) for 24 h and then treated with 1μM RDS 60 or with DMSO for 24 h. Cells were washed with PBS with Ca/Mg (washing buffer) and fixed with 4% buffered paraformaldehyde (Sigma Aldrich) for 20 min at 4 °C, then permeabilized with PBS, 5% FCS, 0.5% TritonX100 for 30 min at RT and incubated for 1 h to RT with the primary monoclonal antibody to beta-tubulin (1:100 diluted; Immunological Sciences). Alternatively, cells were permeabilized with 0.1 % TritonX100 for 10 min to RT, incubated with 3% BSA for 1 h to RT and incubated with the primary rabbit polyclonal antibody to cyclin B1 in 0.1% BSA (1:200 diluted; Elabscience, USA) overnight at 4°C. Cells were washed twice with washing buffer and incubated with the secondary anti-mouse or anti-rabbit antibody FITC conjugated (1:400 diluted; Molecular Probes) for 1 h to RT. Cells were washed twice with washing buffer and DNA was stained with Hoechst for 15 min to RT. The slide was mounted with ProLong-Antifade (Life Technologies) and analyzed by a fluorescence microscope (Olympus BX52). Image acquisition and processing were conducted by IAS 2000 software. Western blot analysis Cells were seeded in 100-mm plates, grown for 24 h and treated with 2.5 μM RDS 60 or with equivalent amounts of DMSO for 24 h. Cells were then scraped in lysis buffer (1% Triton, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) with protease inhibitor cocktail (Roche Applied Science, Germany) for 30 min at 4 °C. Lysates were centrifuged at 16,000xg for 15 min at 4 °C and the supernatant was collected. Protein concentration was evaluated using Protein Concentration Assay (Bio-Rad Laboratories, CA, USA). Protein lysates (50-100 μg) were separated by molecular weight with 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h to RT in 5% nonfat dry milk and incubated with primary antibodies opportunely diluted in PBS-Tween overnight at 4°C, washed in Tris-buffered saline with 0.1% Tween-20 and incubated with horseradish-peroxidase-conjugated-anti-mouse/rabbit-IgG (1:5000; Sigma-Aldrich, MO, USA) for 1 h to RT. Filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescence Substrate; Thermo Fisher Scientific, USA) using Kodak X-Omat films (Kodak, USA). Primary antibodies were: mouse anti-poly (ADP-ribose)-polymerase (PARP-1) (diluted 1:500; Santa Cruz Biotechnology, TX, USA); mouse anti-cleaved-caspase 8 (diluted 1:500; Cell Signaling Technology, MA, USA); mouse anti-caspase 9 (diluted 1:500; Cell Signaling Technology); mouse anti-B-cell-lymphoma-2 (Bcl-2) (diluted 1:200; Santa Cruz Biotechnology); rabbit anti-Bcl-2-associated-X-protein (Bax) (diluted 1:250; Santa Cruz Biotechnology); rabbit anti E-cadherin (1:1000 diluted; GeneTex); rabbit anti N-cadherin (1:1000 diluted; GeneTex); rabbit anti-tubulin (diluted 1:4000; Immunological Sciences). Experiments were performed in triplicate, the bands from the blots were quantified using ImageJ v.1.48 software (National Institutes of Health, Bethesda, MD, USA), the mean values were calculated and expressed as densitometric units (DU). Invasion assay Invasion assay was performed with Matrigel Invasion Chambers (Corning) consisting of inserts with 8 μm pore membrane pretreated with Matrigel: 5x10 5 /mL cells were plated in serum-free medium plus DMSO or in serum-free medium plus 1 μM RDS 60 in the insert chamber, the lower chamber contained complete medium with FCS. After 18 h of culture the inserts were washed with PBS with Ca/Mg and fixed by 100% methanol for 20 min at 4 °C, washed twice with PBS with Ca/Mg and stained for 20 min at RT with haematoxylin. The inserts were then mounted on slides and the cells that migrated through the filter pores to the lower side of the membrane were counted by an optical microscope (Olympus BX52). Imagine acquisition and processing were conducted by IAS 2000 software. Statistical analysis and graphic programs All results were analyzed using one-way analysis of variance, and significance was evaluated using Tukey’s honest significant difference post hoc test. All figures were created using Adobe Photoshop CS5 and all graphs were produced and statistical analyses conducted using Graph Pad Prism 5.0. Results Given the antitumoral activity of the benzimidazole-based small molecule nocodazole, we decided to deepen the activity of its analog belonging to our in-house library, namely RDS 60. Indeed, this compound shares the same benzimidazol-2-yl carbamate ester structure of nocodazole and differs for the replacement of the 2-thienyl substituent with its isostere1-pyrrolyl ring and for the reduction of the carbonyl group with a methylene one (Fig. 1). To evaluate the effects of this new compound on cell viability and replication, two HNSCC cell lines were treated for 24 and 48 h with 100 nM, 1 μM, 2.5 μM, 5 μM, 10 μM RDS 60. A significant decrease in cell viability was evidenced at 24 h of RDS 60 treatment at 2.5 μM in CAL 27 and at 10 μM in FaDu; the decrease of proliferation became significant at 1 μM RDS 60 for both tumor cell lines at 48 h of treatment (Fig. 2). We repeated the time points of RDS 60 concentrations at 1, 10, 20, 30, 40 μM to calculate the EC 50 that resulted 10.8 μM for CAL27 and 12.4 μM for FaDu at 24 h; 2.5 μM for CAL27 and 2.9 μM for FaDu at 48 h. To evaluate the cytotoxicity of the compound on normal somatic cells, human dermal fibroblasts HF were treated for 24 and 48 h with 100 nM, 1 μM, 2.5 μM and 10 μM RDS 60. This treatment led to decreased viability of HF only at the highest concentration of RDS 60 (Fig. 2). Therefore, we decided to perform the following experiments on tumor cells utilizing the compound at low concentrations around 1-2 μM, correspondent to not cytotoxic doses for HF. Since the molecular target of nocodazole, analog of RDS 60, is tubulin, with the impairment of regular organization of microtubules, we analyzed whether RDS 60 could interfere in some way with tubulin and with its assembly into the mitotic spindle. To understand this aspect, we performed an immunofluorescent staining of beta-tubulin in both HNSCC untreated and treated with 100 nM and 1 μM RDS 60 for 24 h and then we observed the cells undergoing mitosis using a fluorescent microscope. We found that, while all untreated cells developed normal typical bipolar mitotic spindles, in the cells treated with both concentrations of RDS 60 it was impossible to find a normal mitotic spindle, because every mitotic spindle had abnormal shape and size, with tripolar or multipolar structure, certainly determining abortive mitosis (Fig. 2). At this point we investigated whether RDS 60 could interfere with the cell cycle, so we analyzed both cell lines by flow cytometry at 24 h of treatment with 1 μM RDS 60 after staining with PI. We found that the treatment with 1 μM RDS 60 induced a notable increase of cells in G2/M phase and a parallel decrease of cells in S and G0/G1 phases. Specifically, RDS 60 increased the percentage of FaDu in G2/M from 27% to 69% and of CAL27 from 32% to 56% (Fig. 3). Consequent to this result, we evaluated cyclin B1 expression, since an increase of this protein is essential for the regular control of cell cycle transition from S to G2 and then to M phases [21]. So, we observed cyclin B1 by immunofluorescence and found that its staining was significantly enhanced upon 100 nM and 1 μM RDS 60 treatment for 24 h (Fig. 3). This result was in accordance with the increase of cells blocked in G2/M phase. Furthermore, we also showed that cyclin B1 localization was exclusively cytoplasmic in both treated and untreated cells and that the protein didn’t translocate into the nucleus (Fig. 3). Since the block of cell cycle can represent a starting point for apoptosis, we investigated whether RDS 60 could induce apoptosis. Therefore, the expression of PARP-1 was evaluated, because its cleavage by caspase 3 occurs during the late phases of apoptosis so that cleaved PARP-1 is considered a marker of apoptosis. The two HNSCC were treated with 2 μM RDS 60 for 24 h and the decrease of uncleaved PARP-1 and the appearance of cleaved PARP-1 was observed in both (Fig. 4). In addition, other two protein correlated to apoptosis were evaluated: Bcl-2, an anti-apoptotic protein, and Bax, a pro-apoptotic Bcl-2-associated protein; because an increased Bax/Bcl-2 ratio is usually observed during apoptosis. We found that the pro-apoptotic protein Bax was up-regulated in both cell lines treated with 2 μM RDS 60 for 24 h, while the anti-apoptotic protein Bcl-2 was down-regulated exclusively in FaDu. The resulting Bax/Bcl2 ratio increased in both HNSCC upon treatment (Fig. 4), confirming the apoptotic response. Finally, the activation of initiator caspases 8 and 9 was analyzed, since the presence of proteolytic fragments of caspases is indicative of their activation and is an additional marker of apoptosis. Cleaved caspase 8 appeared in both cell lines after RDS 60 treatment, while caspase 9 was not modified by the treatment (Fig. 4), suggesting that RDS 60 activated apoptosis through the extrinsic pathway conducted by caspase 8. We then investigated the epithelial-mesenchymal transition (EMT), that is a fundamental feature of malignant cells. Therefore, the modulation of two important markers of EMT, E-cadherin and N-cadherin, was analyzed and found that 24 h of 1 μM RDS 60 treatment was able to reverse EMT phenotype in both HNCSS. In fact, Western blot analysis showed that E-cadherin was low in basal conditions and increased after RDS 60 treatment, while N-cadherin from highly expressed in untreated cells became down-regulated after treatment (Fig. 5). In order to quantify the degree of malignity of these tumor cells, a Matrigel invasion assay was performed and both HNSCC basally showed a high rate of motility, while 16 h of 1 μM RDS 60 treatment was able to completely abolish this malignant attitude, since the number of cells able to migrate dramatically diminished when treated: from 71 to 17 for FaDu and from 92 to 23 for CAL27 (Fig. 5). The invasion capacity, represented by the number of cells capable to cross through the porous membrane, decreased significantly of 76 % in FaDu and 75 % in CAL27 after treatment (Fig. 5). Taken together, these data demonstrated that RDS 60 played an important role not only in blocking the cell cycle and entering apoptosis, but also in reducing the invasive phenotype of HNSCC cells. Discussion During the last decades microtubules have constituted an interesting target in cancer therapy and the research still now is continuing to develop new targeted approaches to improve the existing compounds in order to increase their selectivity for tumor cells and decrease their unwanted side effects [22, 23]. Given our interest in developing new anti-cancer agents, we chose to design and synthesize a derivative of the microtubule targeting compound nocodazole and then to evaluate its anti-tumoral activity on two HNSCC cell lines. We demonstrated that this compound named RDS 60 was a potent inhibitor of proliferation in both tested HNSCC, while it did not exert any important cytotoxic effects on human dermal fibroblasts. The negative regulation performed by RDS 60 on cell replication was obtained by targeting beta tubulin and hampering the assembly of bipolar mitotic spindles with the following abortion of mitosis, as we illustrated. In accordance with these events, RDS 60 arrested the cell cycle at the G2 phase, supported also by an impressive accumulation of cyclin B1 into the cytoplasm. It is known that cyclin B1 increases when the cell is approaching G2 phase and, before M phase, it is phosphorylated and must translocate from the cytoplasm into the nucleus in order to initiate mitosis [21]. We saw that cyclin B1 increased after RDS 60 treatment, but it remained totally localized into the cytoplasm; this information was first of all confirming that the cells were blocked in G2 phase and it was secondly suggesting that cyclin B1 was not activated and, for this reason, the cell cycle was interrupted between G2 and M phase. In a similar way, the microtubule depolymerizing drug nocodazole inhibits proliferation through a block of cell cycle in G2/M [24]. Usually, a cell undergoing a prolonged arrest in pro-metaphase starts towards apoptosis [5]; in fact, RDS 60, subsequently to its anti-proliferative effect, acted as an apoptosis inducer for both HNSCC, as demonstrated by the cleavage of PARP-1 and by the increase of the Bax/Bcl-2 ratio. This apoptosis followed the extrinsic pathway, as demonstrated by the specific cleavage for caspase 8 and not for caspase 9. We then studied some features of HNSCC malignant and aggressive phenotype and found that both cell lines basally expressed low rates of E-cadherin and high rates of N-cadherin, confirming the typical EMT pattern of an invasive tumor [25]. We demonstrated that RDS 60 was able to reverse this EMT phenotype, by up-regulating E-cadherin and down-regulating N-cadherin in both HNSCC. The attitude to amplify cell motility and stroma infiltration reflects the clinical aggressive behavior usually represented by these tumors [26, 27] and, as expected, the two HNSCC showed a high basal rate of invasiveness into the extracellular matrix Matrigel. Interestingly, the treatment with RDS 60 markedly impaired this motility, reducing the invasion capacity up to 4 folds respect untreated cells. This ability to reduce the motility of HNSCC can be related to the target of RDS 60 represented by tubulin, which is the constituent not only of stable but also of dynamic microtubules; consequently, any kind of tubulin modification can reduce microtubules stability [28]. For example, it has been shown that post-translational deacetylation of tubulin alters the microtubule structure and promotes cell motility and the deacetylation of tubulin obtained with the overexpression of histone deacetylase 6 (HDAC6) is sufficient to enhance fibroblast motility [29]. We can hypothesize that RDS 60, through the destabilization of tubulin, can interfere with dynamic microtubules organization and consequently slow HNSCC cell mobility. Taken together these data suggest that RDS 60 could represent a potential innovative anticancer agent for HNSCC, since this molecule is able to block tumor cells at G2 phase, to induce apoptosis and to direct the invasive cells toward a less malignant phenotype. Totally, we can present RDS 60 as a valuable additional tool capable to control and attenuate HNSCC malignity and aggressiveness. Declarations Compliance with Ethical Standards Disclosure of potential conflicts of interest The authors declare that there is no conflict of interest. Research involving Human Participants and/or Animals This research doesn’t involve human participants and/or animals Informed Consent This research didn’t use human participants or biological materials or any clinical data. Ethics approval and consent to participate This article does not contain any study with human participants or animals. Availability of data and materials All data and materials have been described and included in the manuscript. Competing interests All individual authors declare that they have no conflict of interest. Funding This research was supported by Sapienza University through the grant Ateneo “Sapienza”. Authors’contributions Conception and design: Costi R, Di Salvo R, Artico M, S Scarpa Development of chemical methodology: Messore A, De Leo A, Ialongo D, Tudino V, Scipione L Development of biological methodology: Nicolai A, Tortorella E, Taurone S, Pergolizzi T Writing: Scarpa S, Madia VN, Costi R References Florian S, Mitchison TJ (2016) Anti-microtubule drugs. Methods Mol Biol 1413:403-421. Vasquez RJ, Howell B, Yvon AM, Wadsworth P, Cassimeris L (1997) Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. 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Madia","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"Noemi","lastName":"Madia","suffix":""},{"id":11464157,"identity":"7f3cabe2-4b29-4d84-a81f-8eaab5e4520f","order_by":2,"name":"Antonella Messore","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Antonella","middleName":"","lastName":"Messore","suffix":""},{"id":11464158,"identity":"bc16208d-34b4-44df-9989-dde502e121a0","order_by":3,"name":"Alessandro De Leo","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"De Leo","suffix":""},{"id":11464159,"identity":"89b081e3-a061-40e2-abdc-ae38b7293452","order_by":4,"name":"Davide Ialongo","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Davide","middleName":"","lastName":"Ialongo","suffix":""},{"id":11464160,"identity":"29dc86e3-0ed2-4b0e-988f-431da5d9739d","order_by":5,"name":"Valeria Tudino","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Tudino","suffix":""},{"id":11464161,"identity":"2f838576-8424-457f-901d-1ed8e4564d00","order_by":6,"name":"Elisabetta Tortorella","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elisabetta","middleName":"","lastName":"Tortorella","suffix":""},{"id":11464162,"identity":"f528ed29-04c6-4ec1-8f9b-3f08df49fd46","order_by":7,"name":"Luigi Scipione","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luigi","middleName":"","lastName":"Scipione","suffix":""},{"id":11464163,"identity":"cc4ca621-5916-46cd-a5d6-07fbc2988863","order_by":8,"name":"Samanta Taurone","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Samanta","middleName":"","lastName":"Taurone","suffix":""},{"id":11464164,"identity":"93a13d0a-648c-4e3b-a8d2-08d3884fd348","order_by":9,"name":"Tiziano Pergolizzi","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tiziano","middleName":"","lastName":"Pergolizzi","suffix":""},{"id":11464165,"identity":"5fce26da-fb4a-486d-9adc-8bb6c0b4c5ce","order_by":10,"name":"Marco Artico","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Artico","suffix":""},{"id":11464166,"identity":"53c773c1-bee2-485a-87d0-923d641f2bba","order_by":11,"name":"Roberto Di Santo","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roberto","middleName":"Di","lastName":"Santo","suffix":""},{"id":11464167,"identity":"97596b7d-da3d-4ba8-9e15-9e0cb5a86fce","order_by":12,"name":"Roberta Costi","email":"","orcid":"","institution":"Sapienza University of Rome: Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Costi","suffix":""},{"id":11464168,"identity":"e6812105-89a4-4ca2-ac1f-6afb8ee765f0","order_by":13,"name":"Susanna Scarpa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYAgAGwYGZjBDgoGNSC1pMC0WDGxE6jkMY1Qw4LJGfkb6w8+FO+wY5NsPP/tcUHM+cTs778NHN2okGPjkG7BqMbiRYyw980wyg8GZNOPZM47dTtzZzG5snHNMAqfDDCRyGKR525iBDAZjZh6224kbDrOxSeew4dYCdNjj37xt9UAG+2dmnn/noFr+4dbCcCPBDGgL0Nc3eIyZedsOQLTktuFx2Jk3Zta8bcd5DM7kFDPP7Es2BmphNs7tk+BhY0vA7rD29Me3eduq5eTbj29mLvhmJ7vh/DHGxznf6uTkmw/gcBkE8IAIZgwRgoCZsJJRMApGwSgYiQAAlqVN9WEGo90AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3169-3968","institution":"Universita degli Studi di Roma La Sapienza Dipartimento di Medicina Sperimentale","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Susanna","middleName":"","lastName":"Scarpa","suffix":""}],"badges":[],"createdAt":"2021-02-11 12:15:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-233855/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-233855/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6020583,"identity":"e703224c-9249-4a18-8cfc-561d7c671c5b","added_by":"auto","created_at":"2021-02-16 20:21:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":307799,"visible":true,"origin":"","legend":"a) Chemical structure of RDS 60; b) Synthesis of RDS 60: i) AlCl3, NaBH4, THF dry, 0 °C to reflux, 3 h and 20 min; ii) PCl5, CHCl3, 0 °C to RT, 2 h and 45 min; iii) 1. (1s,3s)-1,3,5,7-tetraazaadamantane, NaI, EtOH abs., CHCl3, RT, 24 h, 2. HCl (37%), EtOH, 55 °C, 4 h, 3. Na2CO3(ss), RT; iv) 2,5-dimethoxytetrahydrofuran, AcOH, reflux, 5 min; v) H2/Pd, AcOEt, 45 °C, 4 h; vi) S-methylisothiourea, PTSA, EtOH, reflux, 45 min.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/7f7036fc2162e4e595cadeb6.png"},{"id":6020585,"identity":"1a704f87-b356-4f6b-8f83-ed24a84eaa52","added_by":"auto","created_at":"2021-02-16 20:21:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1558320,"visible":true,"origin":"","legend":"Cell viability of untreated (CTR) and RDS 60 treated FaDu, CAL27 and human normal fibroblasts (HF) expressed as percentages of alive cells ±SD. Treatments were performed for 24 h (dark gray) and 48 h (light gray). * p \u003c0.01; ** p \u003c0.001. Lower right panel - Immunofluorescence staining of beta tubulin on CAL27 and FaDu untreated (C) and treated with 1 μM RDS 60 (T) for 24 h. Magnification 40X. Smaller panels show mitotic spindles at higher magnification (100X) from untreated (C) and RDS 60 (T) treated cells.","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/c5630abb2610d1973952bacc.png"},{"id":6020342,"identity":"04b9b8c1-8590-4e79-b8b4-aebcabc4ab90","added_by":"auto","created_at":"2021-02-16 20:18:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2168208,"visible":true,"origin":"","legend":"Upper panel - Cell cycle was investigated by flow cytometry analysis of DNA content after PI staining of FaDu and CAL27 cells untreated (CTR) and treated with 1 μM RDS 60 for 24 h. The histogram shows the media ±SD representative of results obtained from three independent experiments. Lower panel - Immunofluorescence staining of cyclin B1 on FaDu (a, b, c) and CAL27 (d, e, f) untreated (a, d) and treated with 1 μM RDS 60 for 24 h shown with (b, e) and without (c, f) nuclear staining. Magnification 40X.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/a2f5ec6964d1c0272edfeef1.png"},{"id":6020584,"identity":"883c8c84-6708-4ac3-949e-67472f2ba3c4","added_by":"auto","created_at":"2021-02-16 20:21:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141944,"visible":true,"origin":"","legend":"Western blot of PARP-1, Bax, Bcl-2, cleaved caspase 8, caspase 9 and tubulin from FaDu and CAL27 cells untreated (CT) and treated (TR) with 2 μM RDS 60 for 24 h. The means from densitometry quantifications of three different experiments of the Bax to Bcl-2 ratio normalized to tubulin are indicated.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/28ff5fc23ac707ad911092ba.png"},{"id":6020343,"identity":"d2fa1ed3-611b-4808-9f63-4c4e0d290802","added_by":"auto","created_at":"2021-02-16 20:18:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1335743,"visible":true,"origin":"","legend":"Left upper panel - Western blot of EMT related proteins E-cadherin and N-cadherin for untreated (CT) and 1 μM RDS 60 treated (TR) for 24 h FaDu and CAL27. Lower panel - Matrigel invasion assay of FaDu (a, c) and CAL27 (b, d) cells untreated (a, b) and treated with 1 μM RDS 60 for 16 h (c, d). Right upper panel - Migrated cells in five random fields from three independent experiments were counted and the means±SD are reported in the graphic with **P\u003c0.001.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/59f929de2b11ee0b19723e06.png"},{"id":13660687,"identity":"06173892-3f97-4121-9024-563aba3c8034","added_by":"auto","created_at":"2021-09-17 10:25:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2832783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-233855/v1/5ba7e0ec-7b8d-48a3-8fbc-52584a2b718e.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAnti-Tumoral Effects of A Benzimidazolyl Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNocodazole is a benzimidazol-2-yl carbamate ester derivative that interacts with microtubules by targeting tubulin. In fact, nocodazole exerts a high affinity for beta tubulin, developing hydrogen bonds between the single beta subunits and consequently inhibiting first the aggregation of alpha and beta subunits and then the interaction among tubulin heterodimers; this altered state finally induces the depolymerization of microtubules [1, 2]. The impairment of microtubules polymerization determines an alteration of mitotic spindle organization with the consequent incapacity to carry out mitosis [3, 4]. Whenever cells with high replication rate are treated with nocodazole, they are not able to organize a normal bipolar mitotic spindle, so that they do not complete mitosis and are blocked in G2 or M phase of cell cycle. Yet, the prolonged arrest in pro-metaphases is a starting point for apoptosis [5]. Despite its antitumoral activity, nocodazole did not reach the clinical phase so far [6]. On the other hand, several previous reports have suggested that benzimidazole is the most prominent heterocycle having good cytotoxic properties against different types of cancer cell lines [7-9]. Therefore, we decided to deepen our knowledge on the anti-tumor activity of a benzimidazole derivative structurally related with nocodazole that we previously synthesized and reported [10], namely RDS 60 (Fig. 1). To this aim, we evaluated the activity of RDS 60 in terms of anti-mitotic and apoptosis inducer agent and we tested its potential anti-tumoral efficiency directed to human head and neck squamous cell carcinomas (HNSCC), a group of tumors detectable in various regions of the oral cavity, oropharynx, hypopharynx and larynx. We chose these types of neoplasms because they are extremely aggressive; in fact, almost 50% of the newly diagnosed HNSCC has a survival rate below 5 years [11]. HNSCC are collocated at the sixth position among malign tumors more diffuse in the world [12] and they are all characterized by a poor prognosis, mainly due to metastases, to the development of multiple primary tumors and to local and regional often inoperable relapses [13, 14]. The presence of distant metastases at the moment of diagnosis is associated to the high mortality rate of HNSCC [15] since the chemotherapy used for metastatic disease gives often low responses [16, 17].\u003c/p\u003e\n\u003cp\u003eHerein, we demonstrated that our benzimidazol-2-yl carbamate ester RDS 60 was capable to impair tubulin assembling in the mitotic spindle, to block the cell cycle in G2 phase, to induce apoptosis and to reverse the invasive phenotype of two HNSCC cell lines.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eRDS 60 synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRDS 60 (ethyl (5-((1H-pyrrol-1-yl)methyl)-1H-benzo[d]imidazol-2-yl)carbamate) (Fig. 1a) was synthesized as previously described [10]. However, we modified the first two reaction steps in order to improve the overall reaction yields. Indeed, we applied a reduction reaction according to Moore et al. [18] of the commercially available carboxylic acid into alcohol that was subsequently converted in the corresponding benzyl chloride (Fig. 1b) via an aliphatic nucleophilic substitution. The subsequent synthetic steps resemble the ones previously reported [10]. Therefore, we report the details of the first two synthetic steps since the other ones can be found in ref. 10. Melting point (\u0026deg;C), recrystallization solvent, yield (%), chromatographic system, IR, \u003csup\u003e1\u003c/sup\u003eH NMR, formula, Mr and analyzed elements for derivative RDS 60 agreed with the ones previously described [10]. RDS 60 samples used for biological evaluation were 99% pure as determined by elemental analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA solution of AlCl\u003csub\u003e3 \u003c/sub\u003e(3.77 g; 28.28 mmol) in THF dry (4.6 mL) was added dropwise into a solution of 3,4-dinitrobenzoic acid (5 g; 23.57 mM) and NaBH\u003csub\u003e4\u003c/sub\u003e (3.21 g; 84.85 mmol) in THF dry (46 mL) at 0 \u0026deg;C within 20 min. The reaction was stirred at 25 \u0026deg;C for 1 hour (h) and for 2 h to reflux and then poured into ice-cold water (115 mL). The pH was adjusted to 7 with 1 N HCl and the mixture was extracted with ethyl acetate (3 x 50 mL). The collected extracts were washed with brine (3 x 100 mL) and dried. Removal of the solvent furnished crude residue that was washed with ethyl acetate in order to discard the boron salt, yielding intermediate 1 (3.37 g, 72.1 %) as a yellow solid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo a solution of compound 1 (16.27 g, 81.85 mM) in CHCl\u003csub\u003e3\u003c/sub\u003e (126 mL) was added PCl\u003csub\u003e5\u003c/sub\u003e (6.63 g, 31.85 mM) portionwise at 0 \u0026deg;C within 15 min. The reaction mixture was stirred at 0 \u0026deg;C for 15 min and at 25 \u0026deg;C for 2.5 h and then poured into ice-cold water. The organic layer was washed with 5% w/v Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (3 x 100 mL) and with NaCl\u003csub\u003ess\u003c/sub\u003e (3 x 100 mL) and dried. Removal of the solvent furnished the crude compound 2 that underwent to the following step without further purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cultures and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo HNSCC cell lines were utilized: tongue carcinoma CAL27 and pharynx carcinoma FaDu (ATCC, USA), and one human dermal fibroblast primary culture HF [19, 20]. Cells were grown in RPMI 1640 medium with 10% fetal calf serum (FCS), 2 mM glutamine and 50 U/mL penicillin-streptomycin (Sigma-Aldrich). The compound RDS 60 was solubilized in dimethylsulfoxide (DMSO) (Sigma) for a 10 mM stock solution and utilized to final concentrations from 100 nM to 10 \u0026mu;M for 24 and 48 h. Control cells were treated with equivalent amounts of DMSO in every experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine cytotoxicity, a sulforhodamine-B colorimetric assay was performed: 5x10\u003csup\u003e3\u003c/sup\u003e cells were plated in 96-well plates, grown for 24 h and then treated with 100 nM, 1, 2.5, 5, 10 \u0026mu;M RDS 60 for 24 and 48 h. Cells were then fixed with 50% trichloroacetic acid for 1 h to 4 \u0026deg;C and stained for 30 min to room temperature (RT) with 0.4% sulforhodamine-B in 1% acetic acid. Excess dye was removed by washing four times with 1% acetic acid. Protein-bound dye was dissolved in 10 mM Tris pH 10 and optical density was determined at 510 nm using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 60-mm plates, grown for 24 h and then treated with 1 \u0026mu;M RDS 60 or equivalent amounts of DMSO for 24 h. The cells were harvested by trypsinization, washed twice with cold PBS, fixed in 70% ethanol at 4\u0026deg;C overnight. Cells were rinsed twice with PBS and incubated with 50 \u0026micro;L of RNAse (100 \u0026micro;g/mL, Sigma) to ensure that only DNA was stained, and 200 \u0026micro;L of Propidium Iodide (PI) (50 \u0026micro;g/mL, Sigma). Cell cycle analysis was performed using a FACS CantoII equipped with 488nm laser and DIVA Software (BD Biosciences). The cells were first gated using a forward vs. side scatter (FSC vs. SSC) strategy, and upon 488 nm laser excitation, PI fluorescence was then detected above 580 nm. Data were analyzed using Flow Jo software (Flow Jo LLC, OR, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were grown on Labteck chamber slides (Nunc) for 24 h and then treated with 1\u0026mu;M RDS 60 or with DMSO for 24 h. Cells were washed with PBS with Ca/Mg (washing buffer) and fixed with 4% buffered paraformaldehyde (Sigma Aldrich) for 20 min at 4 \u0026deg;C, then permeabilized with PBS, 5% FCS, 0.5% TritonX100 for 30 min at RT and incubated for 1 h to RT with the primary monoclonal antibody to beta-tubulin (1:100 diluted; Immunological Sciences). Alternatively, cells were permeabilized with 0.1 % TritonX100 for 10 min to RT, incubated with 3% BSA for 1 h to RT and incubated with the primary rabbit polyclonal antibody to cyclin B1 in 0.1% BSA (1:200 diluted; Elabscience, USA) overnight at 4\u0026deg;C. Cells were washed twice with washing buffer and incubated with the secondary anti-mouse or anti-rabbit antibody FITC conjugated (1:400 diluted; Molecular Probes) for 1 h to RT. Cells were washed twice with washing buffer and DNA was stained with Hoechst for 15 min to RT. The slide was mounted with ProLong-Antifade (Life Technologies) and analyzed by a fluorescence microscope (Olympus BX52). Image acquisition and processing were conducted by IAS 2000 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 100-mm plates, grown for 24 h and treated with 2.5 \u0026mu;M RDS 60 or with equivalent amounts of DMSO for 24 h. Cells were then scraped in lysis buffer (1% Triton, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) with protease inhibitor cocktail (Roche Applied Science, Germany) for 30 min at 4 \u0026deg;C. Lysates were centrifuged at 16,000xg for 15 min at 4 \u0026deg;C and the supernatant was collected. Protein concentration was evaluated using Protein Concentration Assay (Bio-Rad Laboratories, CA, USA). Protein lysates (50-100 \u0026mu;g) were separated by molecular weight with 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h to RT in 5% nonfat dry milk and incubated with primary antibodies opportunely diluted in PBS-Tween overnight at 4\u0026deg;C, washed in Tris-buffered saline with 0.1% Tween-20 and incubated with horseradish-peroxidase-conjugated-anti-mouse/rabbit-IgG (1:5000; Sigma-Aldrich, MO, USA) for 1 h to RT. Filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescence Substrate; Thermo Fisher Scientific, USA) using Kodak X-Omat films (Kodak, USA). Primary antibodies were: mouse anti-poly (ADP-ribose)-polymerase (PARP-1) (diluted 1:500; Santa Cruz Biotechnology, TX, USA); mouse anti-cleaved-caspase 8 (diluted 1:500; Cell Signaling Technology, MA, USA); mouse anti-caspase 9 (diluted 1:500; Cell Signaling Technology); mouse anti-B-cell-lymphoma-2 (Bcl-2) (diluted 1:200; Santa Cruz Biotechnology); rabbit anti-Bcl-2-associated-X-protein (Bax) (diluted 1:250; Santa Cruz Biotechnology); rabbit anti E-cadherin (1:1000 diluted; GeneTex); rabbit anti N-cadherin (1:1000 diluted; GeneTex); rabbit anti-tubulin (diluted 1:4000; Immunological Sciences). Experiments were performed in triplicate, the bands from the blots were quantified using ImageJ v.1.48 software (National Institutes of Health, Bethesda, MD, USA), the mean values were calculated and expressed as densitometric units (DU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvasion assay was performed with Matrigel Invasion Chambers (Corning) consisting of inserts with 8 \u0026mu;m pore membrane pretreated with Matrigel: 5x10\u003csup\u003e5\u003c/sup\u003e/mL cells were plated in serum-free medium plus DMSO or in serum-free medium plus 1 \u0026mu;M RDS 60 in the insert chamber, the lower chamber contained complete medium with FCS. After 18 h of culture the inserts were washed with PBS with Ca/Mg and fixed by 100% methanol for 20 min at 4 \u0026deg;C, washed twice with PBS with Ca/Mg and stained for 20 min at RT with haematoxylin. The inserts were then mounted on slides and the cells that migrated through the filter pores to the lower side of the membrane were counted by an optical microscope (Olympus BX52). Imagine acquisition and processing were conducted by IAS 2000 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis and graphic programs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll results were analyzed using one-way analysis of variance, and significance was evaluated using Tukey\u0026rsquo;s honest significant difference post hoc test. All figures were created using Adobe Photoshop CS5 and all graphs were produced and statistical analyses conducted using Graph Pad Prism 5.0.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGiven the antitumoral activity of the benzimidazole-based small molecule nocodazole, we decided to deepen the activity of its analog belonging to our in-house library, namely RDS 60. Indeed, this compound shares the same benzimidazol-2-yl carbamate ester structure of nocodazole and differs for the replacement of the 2-thienyl substituent with its isostere1-pyrrolyl ring and for the reduction of the carbonyl group with a methylene one (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the effects of this new compound on cell viability and replication, two HNSCC cell lines were treated for 24 and 48 h with 100 nM, 1 \u0026mu;M, 2.5 \u0026mu;M, 5 \u0026mu;M, 10 \u0026mu;M RDS 60. A significant decrease in cell viability was evidenced at 24 h of RDS 60 treatment at 2.5 \u0026mu;M in CAL 27 and at 10 \u0026mu;M in FaDu; the decrease of proliferation became significant at 1 \u0026mu;M RDS 60 for both tumor cell lines at 48 h of treatment (Fig. 2). We repeated the time points of RDS 60 concentrations at 1, 10, 20, 30, 40 \u0026mu;M to calculate the EC\u003csub\u003e50\u003c/sub\u003e that resulted 10.8 \u0026mu;M for CAL27 and 12.4 \u0026mu;M for FaDu at 24 h; 2.5 \u0026mu;M for CAL27 and 2.9 \u0026mu;M for FaDu at 48 h. To evaluate the cytotoxicity of the compound on normal somatic cells, human dermal fibroblasts HF were treated for 24 and 48 h with 100 nM, 1 \u0026mu;M, 2.5 \u0026mu;M and 10 \u0026mu;M RDS 60. This treatment led to decreased viability of HF only at the highest concentration of RDS 60 (Fig. 2). Therefore, we decided to perform the following experiments on tumor cells utilizing the compound at low concentrations around 1-2 \u0026mu;M, correspondent to not cytotoxic doses for HF.\u003c/p\u003e\n\u003cp\u003eSince the molecular target of nocodazole, analog of RDS 60, is tubulin, with the impairment of regular organization of microtubules, we analyzed whether RDS 60 could interfere in some way with tubulin and with its assembly into the mitotic spindle. To understand this aspect, we performed an immunofluorescent staining of beta-tubulin in both HNSCC untreated and treated with 100 nM and 1 \u0026mu;M RDS 60 for 24 h and then we observed the cells undergoing mitosis using a fluorescent microscope. We found that, while all untreated cells developed normal typical bipolar mitotic spindles, in the cells treated with both concentrations of RDS 60 it was impossible to find a normal mitotic spindle, because every mitotic spindle had abnormal shape and size, with tripolar or multipolar structure, certainly determining abortive mitosis (Fig. 2).\u003c/p\u003e\n\u003cp\u003eAt this point we investigated whether RDS 60 could interfere with the cell cycle, so we analyzed both cell lines by flow cytometry at 24 h of treatment with 1 \u0026mu;M RDS 60 after staining with PI. We found that the treatment with 1 \u0026mu;M RDS 60 induced a notable increase of cells in G2/M phase and a parallel decrease of cells in S and G0/G1 phases. Specifically, RDS 60 increased the percentage of FaDu in G2/M from 27% to 69% and of CAL27 from 32% to 56% (Fig. 3). Consequent to this result, we evaluated cyclin B1 expression, since an increase of this protein is essential for the regular control of cell cycle transition from S to G2 and then to M phases [21]. So, we observed cyclin B1 by immunofluorescence and found that its staining was significantly enhanced upon 100 nM and 1 \u0026mu;M RDS 60 treatment for 24 h (Fig. 3). This result was in accordance with the increase of cells blocked in G2/M phase. Furthermore, we also showed that cyclin B1 localization was exclusively cytoplasmic in both treated and untreated cells and that the protein didn\u0026rsquo;t translocate into the nucleus (Fig. 3).\u003c/p\u003e\n\u003cp\u003eSince the block of cell cycle can represent a starting point for apoptosis, we investigated whether RDS 60 could induce apoptosis. Therefore, the expression of PARP-1 was evaluated, because its cleavage by caspase 3 occurs during the late phases of apoptosis so that cleaved PARP-1 is considered a marker of apoptosis. The two HNSCC were treated with 2 \u0026mu;M RDS 60 for 24 h and the decrease of uncleaved PARP-1 and the appearance of cleaved PARP-1 was observed in both (Fig. 4). In addition, other two protein correlated to apoptosis were evaluated: Bcl-2, an anti-apoptotic protein, and Bax, a pro-apoptotic Bcl-2-associated protein; because an increased Bax/Bcl-2 ratio is usually observed during apoptosis. We found that the pro-apoptotic protein Bax was up-regulated in both cell lines treated with 2 \u0026mu;M RDS 60 for 24 h, while the anti-apoptotic protein Bcl-2 was down-regulated exclusively in FaDu. The resulting Bax/Bcl2 ratio increased in both HNSCC upon treatment (Fig. 4), confirming the apoptotic response. Finally, the activation of initiator caspases 8 and 9 was analyzed, since the presence of proteolytic fragments of caspases is indicative of their activation and is an additional marker of apoptosis. Cleaved caspase 8 appeared in both cell lines after RDS 60 treatment, while caspase 9 was not modified by the treatment (Fig. 4), suggesting that RDS 60 activated apoptosis through the extrinsic pathway conducted by caspase 8.\u003c/p\u003e\n\u003cp\u003eWe then investigated the epithelial-mesenchymal transition (EMT), that is a fundamental feature of malignant cells. Therefore, the modulation of two important markers of EMT, E-cadherin and N-cadherin, was analyzed and found that 24 h of 1 \u0026mu;M RDS 60 treatment was able to reverse EMT phenotype in both HNCSS. In fact, Western blot analysis showed that E-cadherin was low in basal conditions and increased after RDS 60 treatment, while N-cadherin from highly expressed in untreated cells became down-regulated after treatment (Fig. 5). In order to quantify the degree of malignity of these tumor cells, a Matrigel invasion assay was performed and both HNSCC basally showed a high rate of motility, while 16 h of 1 \u0026mu;M RDS 60 treatment was able to completely abolish this malignant attitude, since the number of cells able to migrate dramatically diminished when treated: from 71 to 17 for FaDu and from 92 to 23 for CAL27 (Fig. 5). The invasion capacity, represented by the number of cells capable to cross through the porous membrane, decreased significantly of 76 % in FaDu and 75 % in CAL27 after treatment (Fig. 5).\u003c/p\u003e\n\u003cp\u003eTaken together, these data demonstrated that RDS 60 played an important role not only in blocking the cell cycle and entering apoptosis, but also in reducing the invasive phenotype of HNSCC cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring the last decades microtubules have constituted an interesting target in cancer therapy and the research still now is continuing to develop new targeted approaches to improve the existing compounds in order to increase their selectivity for tumor cells and decrease their unwanted side effects [22, 23]. Given our interest in developing new anti-cancer agents, we chose to design and synthesize a derivative of the microtubule targeting compound nocodazole and then to evaluate its anti-tumoral activity on two HNSCC cell lines. We demonstrated that this compound named RDS 60 was a potent inhibitor of proliferation in both tested HNSCC, while it did not exert any important cytotoxic effects on human dermal fibroblasts. The negative regulation performed by RDS 60 on cell replication was obtained by targeting beta tubulin and hampering the assembly of bipolar mitotic spindles with the following abortion of mitosis, as we illustrated. In accordance with these events, RDS 60 arrested the cell cycle\u0026nbsp;at the G2 phase, supported also by an impressive accumulation of cyclin B1 into the cytoplasm. It is known that cyclin B1 increases when the cell is approaching G2 phase and, before M phase, it is phosphorylated and must translocate from the cytoplasm into the nucleus in order to initiate mitosis [21]. We saw that cyclin B1 increased after RDS 60 treatment, but it remained totally localized into the cytoplasm; this information was first of all confirming that the cells were blocked in G2 phase and it was secondly suggesting that cyclin B1 was not activated and, for this reason, the cell cycle was interrupted between G2 and M phase. In a similar way, the microtubule depolymerizing drug nocodazole inhibits proliferation through a block of cell cycle in G2/M\u0026nbsp;[24].\u0026nbsp;Usually, a cell undergoing a prolonged arrest in pro-metaphase starts towards apoptosis [5]; in fact, RDS 60, subsequently to its anti-proliferative effect, acted as an apoptosis inducer for both HNSCC, as demonstrated by the cleavage of PARP-1 and by the increase of the Bax/Bcl-2 ratio. This apoptosis followed the extrinsic pathway, as demonstrated by the specific cleavage for caspase 8 and not for caspase 9.\u003c/p\u003e\n\u003cp\u003eWe then studied some features of HNSCC malignant and aggressive phenotype and found that both cell lines basally expressed low rates of E-cadherin and high rates of N-cadherin, confirming the typical EMT pattern of an invasive tumor [25]. We demonstrated that RDS 60 was able to reverse this EMT phenotype, by\u0026nbsp;up-regulating E-cadherin and down-regulating N-cadherin in both HNSCC. The attitude to amplify cell motility and stroma infiltration reflects the clinical aggressive behavior usually represented by these tumors [26, 27] and, as expected, the two HNSCC showed a high basal rate of invasiveness into the extracellular matrix Matrigel. Interestingly, the treatment with RDS 60 markedly impaired this motility, reducing the invasion capacity up to 4 folds respect untreated cells. This ability to reduce the motility of HNSCC can be related to the target of RDS 60 represented by tubulin, which is the constituent not only of stable but also of dynamic microtubules; consequently, any kind of tubulin modification can reduce microtubules stability [28]. For example, it has been shown that post-translational deacetylation of tubulin alters the microtubule structure and promotes cell motility and the deacetylation of tubulin obtained with the overexpression of histone deacetylase 6 (HDAC6) is sufficient to enhance fibroblast motility [29]. We can hypothesize that RDS 60, through the destabilization of tubulin, can interfere with dynamic microtubules organization and consequently slow HNSCC cell mobility.\u003c/p\u003e\n\u003cp\u003eTaken together these data suggest that RDS 60 could represent a potential innovative anticancer agent for HNSCC, since this molecule is able to block tumor cells at G2 phase, to induce apoptosis and to direct the invasive cells toward a less malignant phenotype. Totally, we can present RDS 60 as a valuable additional tool capable to control and attenuate HNSCC malignity and aggressiveness.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving Human Participants and/or Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research doesn\u0026rsquo;t involve human participants and/or animals\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research didn\u0026rsquo;t use human participants or biological materials or any clinical data.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any study with human participants or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials have been described and included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll individual authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Sapienza University through the grant Ateneo \u0026ldquo;Sapienza\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: Costi R, Di Salvo R, Artico M, S Scarpa\u003c/p\u003e\n\u003cp\u003eDevelopment of chemical methodology: Messore A, De Leo A, Ialongo D, Tudino V, Scipione L\u003c/p\u003e\n\u003cp\u003eDevelopment of biological methodology: Nicolai A, Tortorella E, Taurone S, Pergolizzi T\u003c/p\u003e\n\u003cp\u003eWriting: Scarpa S, Madia VN, Costi R\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFlorian S, Mitchison TJ (2016) Anti-microtubule drugs. 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Nature 421:230. \u003ca href=\"https://doi.org/10.1038/421230a\"\u003ehttps://doi.org/10.1038/421230a\u003c/a\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"benzimidazole, head and neck squamous cell carcinoma, apoptosis, epithelial-mesenchymal transition, tubulin","lastPublishedDoi":"10.21203/rs.3.rs-233855/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-233855/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNocodazole is a well-known anti-proliferative agent, thanks to its anti-microtubule activity. Herein we report a benzimidazolyl carbamate ester derivative, namely RDS 60, as structural analog of this compound. We evaluated the anti-neoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines. We found that RDS 60 significantly inhibited replication of both HNSCC without inducing any significant cytotoxic effect on non-transformed human dermal fibroblasts. The treatment of the two cell lines with 1\u0026nbsp;\u0026micro;M RDS 60 for 24 hours determined the incapacity of cells to develop normal bipolar mitotic spindles contemporaneously to a block of the cell cycle in G2/M and to cytoplasmic accumulation of cyclin B1. Moreover, doubling the dosage of RDS 60, an activation of apoptosis in both HNSCC was observed. Additionally, RDS 60 treatment reversed the epithelial-mesenchymal transition and inhibited cell migration and extracellular matrix infiltration of both HNSCC. These results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and invasion of the two HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignant phenotype of HNSCC suggests its potential role as an interesting and powerful tool for new approaches in treating these tumors.\u003c/p\u003e","manuscriptTitle":"Anti-Tumoral Effects of A Benzimidazolyl Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-16 20:18:08","doi":"10.21203/rs.3.rs-233855/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ac17fe9-9af9-4e26-b026-f5394977b31e","owner":[],"postedDate":"February 16th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2406824,"name":"Internal Medicine"},{"id":2406825,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2021-02-16T20:18:09+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-16 20:18:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-233855","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-233855","identity":"rs-233855","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}