A cytotoxicity study proceeded using A431 and BJ1 cell lines. The expression alteration of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and DNA damage and fragmentation using qRT-PCR and comet assays were also investigated. Optimized CXB-TES formulation was spherically shaped and displayed a vesicle size of 75.9 ± 11.4 nm, a surface charge of −44.7 ± 1.52 mV, and an entrapment efficiency of 88.8 ± 7.2%. The formulated TES-based hydrogel displayed a sustained in vitro CXB release pattern for 24 h with an enhanced flux and permeation across rat skin compared with the control (free drug-loaded hydrogel). Interestingly, CXB-TES hydrogel has a lower cytotoxic effect on normal skin cells compared with TES suspension and CXB powder. Moreover, the level of expression of the CDKN2A gene was significantly  decreased in skin tumor cell lines compared with normal skin cell lines, indicating that TES are the suitable carrier for topical delivery of CXB to the cancer cells suppressing their progression. In addition, apoptosis demonstrated by comet and DNA fragmentation assays was evident in skin cancer cells exposed to CXB-loaded TES hydrogel formulation. In conclusion, our results illustrate that CXB-TES-loaded hydrogel could be considered a promising carrier and effective chemotherapeutic agent for the management of skin carcinoma.The topical delivery of therapeutics is a promising strategy for managing skin conditions. Cyclooxygenase-2 (COX-2) inhibitors showed a possible target for chemoprevention and cancer management. Celecoxib (CXB) is a selective COX-2 inhibitor that impedes cell growth and generates apoptosis in different cell tumors. Herein, an investigation proceeded to explore the usefulness of nano lipid vesicles (transethosomes) (TES) of CXB to permit penetration of considerable quantities of the drug for curing skin cancer. The prepared nanovesicles were distinguished for drug encapsulation efficiency, vesicle size, PDI, surface charge, and morphology. In addition, FT-IR and DSC analyses were also conducted to examine the influence of vesicle components. The optimized formulation was dispersed in various hydrogel bases. Furthermore,  One of the most prevalent types of skin cancer is squamous cell carcinoma (SCC), which accounts for 20% of non-melanocytic skin cancers . This tyCelecoxib (CXB) belongs to cyclooxygenase-selective COX-2 inhibitors with pronounced analgesic and anti-inflammatory activities . Such peThe stratum corneum (SC), the skin’s top layer, is a significant barrier and rate-limiting step for drug diffusion through the skin . Lipid vIn vitro performance of the CXB-TES gel was examined using a skin cancer cell line (A431). Additionally, the optimized preparation was assessed for its safety, DNA damage and fragmentation, and the expression alteration of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene related to skin cancer progression.The objective of this research is to develop CXB -loaded TES within a topical hydrogel system to improve the drug’s skin permeability, allowing for enhanced delivery of CXB to tumor microenvironment to impede cell growth in the tumor cell, as well as minimizing the non-specific distribution in normal cells associated with chemotherapeutics and avoiding the side effect associated with oral administration of CXB. ®, 12,000–14,000 Da), Ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO), MTT Salt: 3--2,5-diphenyl tetrazolium bromide and trypsin were obtained from Sigma-Aldrich Co. . Carbopol® 934 (CP) was obtained from BDH Chemicals Ltd. . A human skin carcinoma (A431), and a normal human skin fibroblast cell (BJ1) were supplied from American Type Culture Collection (ATCC) . Cell supplements, such as Dulbecco modified Eagle-medium (DMEM) and DMEM F12 media , Foetal bovine serum (FBS), and antibiotic-antimycotic  were obtained from Lonza Walkersville, Inc. . All compounds were analytical grade and utilized without additional purification.Celecoxib was kindly obtained from Sedico Pharmaceuticals Co. . Span 60, Tween 80, sodium deoxycholate, hydroxypropyl methylcellulose (HPMC), dialysis membrane,  at room temperature (25 ± 0.1 °C) for 10 min utilizing a magnetic stirrer, followed by addition of deionized water (Qs to 100%) dropwise for a further 5 min. Similarly, TES were prepared using different edge activators, either sodium deoxycholate (SDC), Tween 80 (T80) or span 60 (S60), which were added along with the drug and lipid components. The constituents of the various formulations are summarized in CXB-entrapped ES and TES were formulated using the previously described cold method ,28. BrieA dynamic laser light scattering (DLS) technique was applied to estimate the particle size distribution of the fabricated lipid vesicles at 25.5 ± 0.5 °C, using the Malvern Zeta-sizer Nano-ZS . Before each measurement, the lipid vesicle suspension dispersed in deionized water at 1 mg/mL and was vortexed for at least one minute. The surface charge of the developed CXB-lipid vesicles was determined using electrophoretic mobility data acquired in deionized water. Measurements were estimated in triplicates.max of 255 nm, after cooling centrifugation, of CXB-nano suspension at 18,000 rpm and 4.0 °C for 30 min, utilizing a large-capacity bench-top refrigerated centrifuge . The average value of EE for triplicate samples was calculated using Equation (1):WT = weight of the total CXB in the supernatant and sediment, and WF = weight of the free un-incorporated CXB quantified in the supernatant, respectively [The entrapment efficiency (EE%) of CXB within nano ES and TES was determined indirectly by measuring free un-entrapped CXB spectrophotometrically using a Shimadzu UV-1601 PC , with λectively .To evaluate CXB-lipid interactions, the DSC-thermograms were acquired using a Themys One+ differential scanning calorimeter . Samplings measuring 1–2 mg of CXB powder, Phospholipone-90G (PL-90G), sodium deoxycholate (SDC), blank, dried TES powder, and optimized CXB-TES dried powder (F4) were placed in aluminum pans and heated at a scanning rate of  in the presence of nitrogen, with the flow rate set to 40 mL/min. By using CALISTO-thermal analysis software 2.0 , the melting point and enthalpy  can be estimated.−1.The chemical attributes of CXB powder, Phospholipone-90G (PL-90G), sodium deoxycholate (SDC), blank, dried TES powder, and optimized CXB-TES dried powder were evaluated using FT-IR spectrophotometry . Samples (4–5 mg) were mixed with potassium bromide and were compressed into discs. The FT-IR spectrum was recorded from 4000 to 400 cmw/v) was used to negatively stain the vesicles by adding 20 µL for a few seconds, followed by drying overnight at room temperature. Images were captured using the AMT-700 camera .Optimized CXB-TES were imaged employing a transmission electron microscope (TEM)  at 80 kV accelerating voltage. The sample was treated by adding 20 µL of the TES suspension onto a Formvar-coated 300 mesh grid stabilized with evaporated carbon film for 1 min. A filter paper was used to remove the excess sample. The aqueous solution of uranyl acetate  0.5% w/w. To form hydrogels, the polymer was dissolved in deionized water. The aqueous polymer dispersion was hydrated for 5 h until a transparent gel formed. Gel viscosity was determined using a Brookfield DV+ II model LV viscometer at 1.5 rpm and 25 ± 0.1 °C. CXB-loaded TES were centrifuged at 36.670× g, 4.0 ± 0.5 °C for 30 min. The acquired pellets were blended with particular amounts of the gels so that the ultimate CXB concentration in the hydrogel was set to 1.0% w/w. The obtained gels were vortexed until homogeneous [A CXB-loaded TES formulation (Formula F4) was dispersed in various hydrogel bases, namely hydroxypropyl methylcellulose (HPMC) 2.5% ogeneous .The clarity of the formed gels was examined visually, via inspection of any signs of turbidity or phase separation . In addimax of 255 nm using a spectrophotometer . EE% was assessed using the linear regression equation attained from ethanol’s CXB standard calibration curve. The mean percent of EE% was presented as an average of 3 readings.A measure of 0.5 g of CXB-loaded TES gel formulation was dissolved in ethanol to dissolve vesicles and extract the CXB. Absorbance was measured spectrophotometrically at λIn vitro drug release from the optimized CXB-loaded TES formulation (Formula F4) compared to CXB-TES-loaded HPMC-based hydrogel, and CXB-TES-loaded-Carbopol-934 based hydrogel was studied as described previously [®, MWT cutoff 12,000–14,000) was extended over the open bottom end of a glass tube and tightened by a rubber band. A measure of 1 mL of TES suspension (Formula F4) containing 5 mg CXB, or TES gel , was placed over the cellulose membrane. The glass tube was submerged in phosphate buffer  with 1% w/v SLS at 37 ± 0.5 °C and shaken at 75 rpm employing a thermostatically controlled water bath shaker . Aliquots (5 mL) were replaced with release media. The CXB concentration was estimated spectrophotometrically utilizing a Shimadzu UV-1601 PC , at λmax of 255 nm, at predetermined time points . In vitro release assay was performed in triplicate. Various mathematical models  were employed to specify the kinetics and mechanism of CXB release from the developed formulations [eviously ,31. A prulations ,34,35,36in vitro drug release test was used. At predefined time intervals (up to 6 h), 3 mL of the release medium was replaced with new media. Analysis of samples was done at λmax of 255 nm. All experiments were done in triplicates. The permeability studies were conducted using rat abdominal skin as per earlier published methods ,37. The n, mg/cm2) was plotted versus time (h) to construct the permeation profiles [appP value) was calculated in accordance with the following equation:Q/Δt is the linear mass appearance rate of the drug of interest in the acceptor chamber, A is the skin’s surface area . The steady-state flux  was estimated according to the below equation:The cumulative amount of CXB permeated per unit of skin membrane area . Next, the tested preparations or doxorubicin (positive control) were added to cells, followed by incubation for 48 h. Afterward, the medium was substituted with 40 µL MTT salt (2.5 μg/mL), and cells were incubated for another 4 h. Next, a 200 μL of 10% sodium dodecyl sulfate (SDS) in deionized water was put in each well, and then cells were incubated overnight at 37 ± 0.5 °C to pause the reaction and solubilize the resulting crystals. The absorbance was recorded at λmax of 540 nm. Probit analysis was done for IC50 and IC90 calculation using SPSS 11 program.A cytotoxicity test was performed based on the mitochondrial-dependent reduction of yellow MTT -2,5-diphenyltetrazolium bromide) to insoluble formazan crystals . The assmax of 260 nm. The purity of the total RNA was assessed by the absorbance ratio (260/280 nm), which is between 1.8 and 2.1, indicating a pure RNA sample [+ RNA separated from either normal or cancer skin cell lines was reverse transcribed into cDNA, employing a RevertAid TM First Strand cDNA Synthesis Kit , 20 µL. A master mix containing 5 μg of total RNA was used [2, 10× RT buffer , 10 mM each of dNTP, 50 µM oligo-dT primer, 20 IU ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 IU MuLV reverse transcriptase. Centrifugation of each mixture was done at 1000× g for 30 sec. Afterward, each mixture was transferred to the thermocycler at 25 ± 0.5 °C for 10 min, followed by 1 h at 42 ± 0.5 °C. Finally, a denaturation step at 99 ± 0.5 °C for 5 min was performed. The reaction tubes containing RT preparations were ice-cooled until utilized for cDNA amplification.The total RNA genome was isolated from normal and cancer skin cell cultures using RNeasy Mini Kit  supplemented with DNaseI (Qiagen). The procedures were performed following the company’s protocol. To degrade DNA residues in the isolated total RNA, one unit of RQ1 RNAse-free DNAse  resuspended in DEPC-treated water was used. Analysis was done at λA sample . In addiA sample . Aliquotwas used . The mas® Premix Ex TaqTM (12.5 mL), 0.2 mM sense primer (0.5 mL), 0.2 mM antisense primer (0.5 mL), distilled water (6.5 mL), plus 5 mL of cDNA template [−ΔΔCT method [The qRT-PCR was performed using normal and cancer skin cell lines to get cDNA copy numbers. The StepOne™ Real-Time PCR System was used from Applied Biosystems . The reaction mixture (25 mL) comprised of 1× SYBRtemplate . In thretemplate . Each roT method ,47.4 cells) were suspended in 0.75% low-gelling-temperature agarose. The cells suspended in agarose were pipetted onto dry pre-coated microscope slides. Then, the slides were immersed in lysing solution at 50 ± 0.5 °C  for at least 4 h, followed by washing overnight using Tris/borate/EDTA buffer, pH 8.0, at room temperature. Afterward, samples were transferred for electrophoresis at 0.6 V/cm for 25 min, then stained with propidium iodide. The slides were analyzed using a fluorescence microscope equipped with a CCD camera. For slide scoring, Comet images (150 separate images) were analyzed for the percentage of DNA in the comet tail, the tail moment, and the content of DNA. The percent of DNA in the comet tail (100 tested cells/sample) was used to indicate the quantity of DNA damage. A visual score was assigned to each cell based on comet tail length migration and the relative proportion of DNA in the nucleus: class 0 , class 1 , class 2 , and class 3  [The comet assay was performed using healthy and tumor skin cell lines. The test was conducted as described previously . Brieflynucleus) .6 cells) were seeded in different Petri dishes  and then exposed to the tested formulations for 24 h. Afterward, all cells (including floating cells) were trypsinized, washed, and lysed. The lysis buffer used to dissolve the cell membranes comprised 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% Triton X-100 for 30 min on ice. Liquidates were cleaned using a vortex mixer and centrifuged at 10,000× g for 20 min. The extraction of fragmented DNA in the supernatant was done using an equal volume of neutral phenol: chloroform: isoamyl alcohol mixture (25:24:1). Quantitative analysis of DNA content was done electrophoretically on 2% agarose gels containing 0.1 μg/mL ethidium bromide.The DNA fragmentation test was conducted in normal and cancer skin cell lines accordingly, with the procedure specified by Yawata  with somt-test was used to examine the groups’ statistically significant differences .At least three duplicates of each experiment were conducted, and means±SD were recorded. One-way ANOVA with Tukey Kramer multiple estimates or a two-sided Student’s Particle size (mean diameter), polydispersity index (PDI), and zeta potential of the developed lipid vesicles are displayed in The zeta potential is a crucial physical characteristic for exploring the stability of vesicles. At the same time, particle aggregation decreases as zeta potential increases more than 30 mV due to the electrostatic repulsion of the charged particles . All prep-value of 3.99, hence S60 increases EE%. Additionally, the interaction between the highly hydrophobic alkyl domains of S60 and the hydrophobic parts of the vesicles prevents drug escape due to condensed layers of vesicles [EE% of the prepared TES varied from 77.9 ± 1.5% to 89.4 ± 1.9%. The EE% of formulations containing S60 was higher than the EE% of the formulation prepared using T80. This result could be ascribed to the difference in the used EA’s hydrophilic-lipophilic balance (HLB) values. The higher the lipophilicity of EA , the better the entrapment of lipophilic drugs. CXB is highly lipophilic with a log vesicles .EA’s lipid phase transition temperature (Tc) impacts EE%; the higher the Tc, the greater its capacity to build a more ordered structure and a less leaky bilayer, which may also increase EE%. Using EAs with a lower Tc may cause abnormal structure formation and increase vesicle bilayer fluidity, limiting drug EE%. This effect appears with a formulation prepared using S60 (F8–F10), which has a high Tc value of 53 °C, showing higher EE% . As the −1 for the drug alone, PL-90G alone, SDC alone, their corresponding physical mixture, and the selected blank and loaded TES  are shown in w/w loaded TES hydrogel containing CXB was 6400 ±150 cPs, while the viscosity of carbopol 934 0.5% w/w loaded TES hydrogel containing CXB was 15,500 ± 100 cPs.The prepared TES hydrogels were homogenous, clear with no inspection of phase separation without appreciable lumps, and uniform in consistency. The pH of the prepared TES gel was 6.5, and 5.8 for HPMC-loaded and CP-934 loaded TES hydrogels. Furthermore, high CXB contents were found in the prepared TES hydrogels: 99.5 ± 2.5 and 98.99 ± 3.7% for HPMC and CP-934 TES hydrogels, respectively. This high EE% was acceptable and approved the formulation technique. The viscosity of HPMC 2.5% in vitro release study of CXB from the optimized TES formulation (F4) was carried out in PBS  at 37 ± 0.5 °C, employing a modified dialysis membrane diffusion technique [in vitro release profile of CXB-TES was compared with that of CXB-TES-loaded HPMC-based gel and CXB-TES-loaded Carbopol-934-based gel . After 24 h, the cumulative drug release followed this order: TES suspension > TES HPMC hydrogel > TES Carbopol-934 hydrogel. The differences were statistically significant . Similar conclusions were observed previously, where the vesicle dispersion within different gel bases led to the subsequent reduction in drug release rates [After 24 h, the cumulative release from CXB-TES suspension was 36.81 ± 0.79%, which was significantly higher than that released from HPMC and CP 934-based TES hydrogel  calculated by various mathematical models are represented in To actually investigate the mechanisms promoting CXB release from various preparations, kinetic analysis was done by fitting data to either a zero-order, first-order, Higuchi diffusion, or Korsmeyer–Peppas model ,83. The n values were 0.5 < n < 1.0, indicating anomalous non-Fickian drug diffusion. The drug transport mechanism is controlled by gel erosion, and diffusion is merged with the distention of lipid bilayers [It was found that the best fit model for CXB release from the developed hydrogels was the Higuchi diffusion model. The estimated bilayers . These fbilayers ,85. The in vitro model for comparing transdermal permeation properties of CXB-TES-loaded HPMC hydrogel and free CXB-loaded HPMC hydrogel to understand the in vivo performance of the optimized TES-based hydrogel preparation (2 after 6 h) compared to 111.81 ± 3.31 μg/cm2 drug permeation from the free CXB-loaded gel formulation at the same time . The in vitro permeation parameters also revealed that the TES-based gel formulation showed significantly  higher transdermal flux (Jss) compared to the free drug-loaded gel formulation, as presented in Papp) calculated for the transport of drug-containing TES gel across the skin is 2.394 × 10−2 ± 0.102 cm/h. The Papp value calculated from the linear flux of free drug-containing gel across the skin membrane is 1.702 ± 0.12 × 10−2 cm/h, equivalent to a 1.4-fold increased transfer rate of CXB when encapsulated as TES hydrogel.Freshly excised rat skin was used as an paration . The cumThe characteristics of the gel, accompanied by the size and vesicular nature of TES, are considered the primary reason for this observation. The greater penetration of the sodium deoxycholate TES gel may result from its decreased size, negative zeta potential, and improved fluidity of the lipid bilayers. Various methods, including adsorption and diffusion of vesicles onto the skin’s surface and that the vesicles operate as penetration enhancers that lower the barrier characteristics of the stratum corneum, might demonstrate the capacity of vesicles to facilitate drug transfer through the skin . The ves50 1.4, 17.5, and 1.8, respectively. However, in the case of blank gel and CXB gel, the mortality on A431 cell lines were 10.3% and 18.4% at 100 µg/mL, as depicted in The cytotoxicity of various formulations was tested on the human skin cancer cell line (A431) at 100 µg/mL to prove the activity of CXB-TES against cancer cells. The MTT test was used to compare their findings to those of a normal skin fibroblast cell line (BJ1). The results revealed that CXB-TES suspension, CXB-TES hydrogel, and CXB powder exhibited 100% mortality with IC50 of TES-based gel might be attributed to the inclusion of CXB-TES within the network structure of HPMC gel, leading to a slower flux and permeability with a more sustained release compared to CXB-TES dispersion and free CXB powder that could permeate more freely through the skin [Moreover, ethanol might destabilize cell membranes, facilitating their entry . The higthe skin .50: 1.6 and 1.8 µg/mL, respectively. On the other hand, TES hydrogel has a lower cytotoxic effect on the normal cell with IC50 equal to 101.1 µg/mL, and TES suspension has IC50 equal to 82.7 µg/mL. This elucidated that the TES gel is safer than CXB powder by 100-fold, indicating an important advantage of the developed formulation for anti-cancer therapeutic. Remarkably, the possibility of applying CXB as TES gel can represent an innovative approach to hamper the toxicity issues related to free drug application.Comparing the effect of TES suspension, TES hydrogel, and CXB powder on normal cell lines (Bj1), the results indicated that CXB powder has the same cytotoxicity on normal and cancer cell lines with relatively similar ICp ≤ 0.05; ANOVA/Tukey) in normal skin cell lines treated with TES suspension and CXB powder, respectively, compared with untreated normal skin cell lines  in normal skin cell lines treated with doxorubicin compared to negative control normal cell lines. In addition, treatment of normal skin cell lines with TES hydrogel increased the expression levels of the CDKN2A gene compared with negative control normal cell lines. Still, the expression levels of the TES gel-treated group were lower than those in the TES suspension and CXB powder groups.Gene expression analysis was performed on normal and cancerous skin cell lines using skin cancer-related genes, namely the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene a,b. The ll lines a. Moreovp ≤ 0.01; ANOVA/Tukey) in skin cancer cell lines treated with TES suspension, TES gel, and doxorubicin (+ve control), respectively, compared with negative control skin cancer cell lines. Furthermore, the expression levels of the CDKN2A gene declined considerably  in skin cancer cell lines treated with CXB powder compared to negative control skin cancer cell lines. Treatment of skin cancer cell lines with CXB gel slightly decreased the expression levels of the CDKN2A gene compared with negative control cancer cell lines. In contrast, the expression levels of the CDKN2A gene in a group of skin cancer cell lines treated with blank HPMC gel were relatively similar to the CDKN2A expression levels in negative control skin cancer cell lines.On the other hand, the expression levels of the CDKN2A gene in groups of normal skin cell lines treated with blank HPMC gel and CXB gel were relatively near to the CDKN2A expression levels in negative control normal cell lines. Expression levels of the CDKN2A gene in skin cancer cell lines are summarized in Melanoma is a complicated disorder, comprising environmental, phenotypic, and hereditary risk factors . ApproxiSwitching off the CDKN2A tumor suppressor gene’s expression and encoding the p16INK4a protein is linked with the emergence of different cancers. The p16INK4a protein contributes a vital role in the cell cycle and senescence by regulating the cyclin-dependent kinase (CDK) 4/6 and cyclin D complexes. Genetic and epigenetic deviations of the CDKN2A gene result in increased tumorigenesis and metastasis with a poor prognosis. In this scenario, restoring genetic and epigenetic reactivation of CDKN2A is a practical strategy for the inhibition and cure of cancer. Previously, Chin-Cheng Su et al.  investigp ≤ 0.01; ANOVA/Tukey) the highest DNA damage values (23.63 ± 0.93%) compared with the control skin cell lines (6.82 ± 0.86%)  in normal skin cell lines treated with doxorubicin compared with the control skin cell lines, but these DNA damage values were lower than those in the group treated with CXB powder. Treatment of normal skin cell lines with TES suspension and TES gel exhibited a similar rise  in the DNA damage values compared to those in the control group. However, treatment of normal skin cell lines with blank gel and CXB gel showed DNA damage values similar to those in the control group. The damage to the DNA in skin cancer cell lines is summarized in p ≤ 0.01; ANOVA/Tukey) the highest DNA damage values (27.63 ± 1.21%) compared with negative skin cancer cell lines (11.21 ± 0.87%)  in skin cancer cell lines treated with doxorubicin (24.82 ± 0.80%) and TES suspension (23.43 ± 0.93%) compared with negative skin cancer cell lines, but these DNA damage values were lower than those in the group treated with CXB powder. Treatment of skin cancer cell lines with TES gel exhibited a considerable rise  in the DNA damage values (21.41 ± 0.51%) in comparison to those in the control group (11.21 ± 0.87). However, treatment of skin cancer cell lines with blank gel and CXB gel induced DNA damage values  lower than other compound treatments with those in negative cancer cell lines  . In addi± 0.87%) . Furtherll lines .1 cell cycle arrest, followed by p53-dependent autophagy.In the same line with our observations, Liu et al.  proved tCXB-loaded liposomes have been shown to effectively induce apoptosis in rat hepatocytes compared to CXP powder . FurtherPrior studies indicated that different targets of NSAIDs, besides COX-2, may exist, such as 15-lipoxygenase-1, AKT/PKB Kinase, PPAR delta, and P21 Ras. In addition, calcium ion homeostasis, nuclear factor kB (NFkB), and inducible nitric oxide synthase may play an essential role in apoptosis induction by CXB ,105,106.p ≤ 0.01; ANOVA/Tukey) increase (27.6 ± 0.54%) in DNA fragmentation rates compared with those in untreated normal skin cell lines (8.1 ± 0.24%). Additionally, the DNA fragmentation values were increased significantly in normal skin cell lines treated with doxorubicin (21.7 ± 0.66%), TES suspension (20.6 ± 0.56%), TES hydrogel (19.4 ± 0.48%), CXB hydrogel (17.1 ± 0.23%), and blank HPMC gel (15.2 ± 0.30%) compared with those in untreated normal skin cell lines (8.1 ± 0.24%). Moreover, the agarose gel in p ≤ 0.01; ANOVA/Tukey) rise (43.7 ± 0.53%) in the DNA fragmentation rates compared with those in untreated skin cancer cell lines (13.7 ± 0.45%). Furthermore, the DNA fragmentation rates were increased significantly in skin cancer cell lines treated with TES suspension (35.5 ± 0.58%), doxorubicin (33.8 ± 0.48%), TES hydrogel (31.6 ± 0.61%), CXB gel (21.2 ± 0.66%), and blank HPMC hydrogel (17.9 ± 0.15%) compared with those in untreated skin cancer cell lines (13.7 ± 0.45%). Additionally, the agarose gel in The rates of DNA fragmentation in normal and cancer skin cell lines are summarized in Similarly, Ramer et al.  indicate50 value of 17.5 μg/mL) while expressing a pronounced safety against normal skin cell lines compared to free CXB. Moreover, the level of CDKN2A gene expression was significantly  decreased in skin cancer cell lines relative to normal skin cell lines, indicating that TES are a good carrier for topical delivery of CXB to the cancer cells, suppressing their progression. CXB-TES hydrogel induced DNA fragmentation and apoptosis in skin cancer cells. The superior physicochemical performance of the hydrogel, combined with the elasticity and sustaining drug release effect of the TES, could improve skin retention capacity and penetration. Consequently, using TES hydrogel as a potential carrier for the topical delivery of CXB is considered a promising strategy for localized management of skin cancer with the ability to overcome reported adverse effects. The future perspectives will focus on clinical investigations into the therapeutic effect of CXB-TES hydrogel in humans.This study assessed a new therapeutic approach for skin cancer management. TES were prepared by the established cold method with different edge activators. The optimized vesicles were spherical and displayed a high entrapment efficiency (88.8 ± 7.2%), nanometric size, and negative zeta potential. Loading the developed CXB-TES within hydrogel formulation showed more efficiency in controlling drug release compared to TES dispersion. Additionally, the application of TES hydrogel strongly augmented CXB penetration and flux into rat skin approximately 1.4-fold relative to free CXB hydrogel. The developed TES hydrogel significantly reduced A431 cancer cell viability (IC


Rhabdomyosarcoma (RMS) in adults is a rare and aggressive disease, which lacks standard therapies for relapsed or advanced disease. This retrospective study aimed to describe the activity of BOMP-EPI , an alternative platinum-based regimen, in adult patients with relapsed/metastatic RMS. In the study, 10 patients with RMS with a median age at diagnosis of 20.8 years and a female/male distribution of 6/4 received a mean of 2.5 cycles of BOMP-EPI. The best RECIST response was a complete response in 1/10 (10%) patients, a partial response in 5/10 (50%), stable disease in 3/10 (30%) and progression in 1/10 (10%). With a median follow-up in the alive patients from the start of therapy of 30.5 months (15.7–258), all patients progressed with a median progression-free survival of 8.47 months (95% CI 8.1–8.8), and 7/10 patients died with a median overall survival of 24.7 months (95% CI 13.7–35.6). BOMP-EPI was an active chemotherapy regimen in adults with pediatric-type metastatic RMS, with outcomes in terms of survival that seem superior to what was expected for this poor-prognosis population. Low HMGB1 expression level was identified as a predictive factor of better response to this treatment. PAX-FOXO1 fusion (PAX3 or PAX7). Sclerosing RMS (10% of cases) is associated with an abundant hyaline matrix, affecting children and adults. Pleomorphic RMS is a highly aggressive subtype (10% of cases), affecting adults. Rhabdomyosarcoma (RMS) is a high-grade malignant neoplasm derived from the primitive mesenchymal cells with a propensity for myogenic differentiation, which can be developed in any part of the body. RMS is the most common soft tissue sarcoma (STS) in children, constituting 50% of sarcoma cases in childhood ,2,3. RMSStage , location, size, disease volume after surgery (classified in the intergroup rhabdomyosarcoma study (IRS) groups), histologic subtype, age and the presence of specific molecular rearrangements are the main prognostic factors. Currently, the upfront treatment of RMS consists in a multimodal therapy, which includes surgical resection, systemic therapy based on intensive multidrug regimens and radiotherapy. According to the last 2005 EpSSG RMS stratification, first-line chemotherapy includes vincristine and actinomycin (VA) for the low-risk group, adding ifosfamide (IVA) for standard and high-risk groups and doxorubicin for the very high-risk group (IVADo). This multimodal therapy has shown overall survival rates over 90% in patients with a low-risk localized disease, but only of 21% or 30% in patients with a metastatic or recurrent disease, respectively ,5. Age iPlatinum-based regimens have shown activity in pediatric patients with RMS enrolled in small clinical trials, with overall response rates between 28% and 39% ,11,12,13DNA-platinum adducts can be recognized by the nonhistone chromosomal high-mobility group (HMGs) proteins, which are involved in the maintenance and functional regulation of DNA, such as replication, recombination, transcription and DNA repair ,19. HMGsThis study aims to describe the activity of alternative platinum-based regimes  in AYA and adult patients with metastatic/relapsed RMS and identify low HMGB1 expression level as a predictive factor of better response to this treatment.In this study, 10 patients with RMS with a median age at diagnosis of 20.8 years (17–44) were treated with BOMP-EPI at a median age of 22.6 years (18–46). There was a female predominance (60%/40%), and the most frequent subtype was alveolar RMS (6/10). In 60% of cases, primary tumors arose in the head and neck. All patients had a high- or very high-risk disease based on EpSSG classification and five patients had metastatic disease at diagnosis. All except for one patient underwent surgery for the primary tumor .Three patients with metastatic disease at diagnosis received BOMP-EPI as their upfront therapy and seven patients with relapse ) received BOMP-EPI after pretreatment with alkylating agents  . Three pp = 0.61) or OS (26.2 months (95% CI 13.7–35.6) in alveolar RMS vs. 10.8 months (95% CI 1–35.8) in embryonal/spindle cell RMS, p = 0.34). Three patients received a rechallenge of BOMP-EPI at progression after stopping therapy while in response . At rechallenge, two of three obtained a new PR, with disease control for 15.8 months in one patient and without evidence of disease after 229 months of follow-up in another patient. More in detail, this last patient had been diagnosed from a prostatic embryonal rhabdomyosarcoma with pulmonary metastasis when he was 42 years old. After diagnostic biopsy, he started induction therapy with BOMP-EPI, receiving six cycles, achieving a complete radiological response. Surgery (prostatectomy) was performed. After 29 months of follow-up, a disease relapse on the proximal femur was diagnosed. Induction chemotherapy with BOMP-EPI was restarted, receiving five cycles, achieving again a radiological complete response. The patient then underwent radical surgery, with a hip replacement. After surgery, he received high-dose chemotherapy with autologous stem-cell rescue. He is free of disease after 19 years of follow-up.One patient (10%) showed a complete response (CR), five patients (50%) showed a partial response (PR), three patients (30%) showed a stable disease (SD) and one patient (10%) showed progression, according to the RECIST response . At a mep < 0.05) and a similar tendency with PFS . HMGB1 mRNA levels in the patient with a complete response (RMS-05) were 3–10-fold lower than in the other patients , after relapse (RMS-04 andRMS-08) with other therapies (IVA/V or EPI-IFOS) or before relapse  with other therapies (IVA/V or IVA). Mean HMGB1 and HMGB2 expression levels were approximately 10-fold higher than HMGA2 and HMGB3 or two times higher than HMGA1 . HMGB1 mpatients a. No sigpatients b,c.HMGB1, HMGB2 and HMGA2 expression levels were analyzed in six RMS cell lines: three embryonal  and three alveolar . HMGB1 mRNA levels were 4–15-fold lower in A204 and Hs729T cell lines than in RH30, RH41, RH28 and RD cell lines. A204 and Hs729T cell lines also showed approximately two times less of HMGB1 protein than in RH30, RH41, RH28 and RD cell lines a,d. MoreThus, low HMGB1 expression levels observed in patients with RMS with a better response to BOMP-EPI and in RMS cell lines with lower cisplatin IC50 could indicate that HMGB1 is a predictive biomarker of cisplatin response.HMGB1 was downregulated in the six RMS cell lines by transduction with lentivirus harboring shRNA against HMGB1 (shHMGB1) or nontargeting shRNA for control (shControl). HMGB1 mRNA levels were decreased by 40%–50% in RH30, RH28, RD and A204 cell lines and 25% in RH41 and Hs729T cell lines transduced with shHMGB1 lentivirus versus transduction with shControl lentivirus . All theRH30 cell lines with stable downregulation of HMGB1 (RH30 shHMGB1) after transduction with shHMGB1 lentivirus were selected by puromycin treatment for 2 weeks as well as for transduction with shControl lentivirus (RH30 shControl). RH30 shHMGB1 cell lines were treated with cisplatin at 5, 10 and 20 µM over 48 h, showing additional sensitivity to a cisplatin dose of 20% versus RH30 shControl with the different cisplatin doses .Treatment with platinum-based chemotherapy in this retrospective series of adult patients with advanced rhabdomyosarcoma suggests activity from this regimen, with an overall response rate (ORR) of 60% and a median PFS exceeding 8 months. Toxicity was mainly hematologic, as expected, with two patients withholding therapy because of side effects, but there were no toxic deaths. Our series has some limitations, its retrospective nature and its limited size being the major issues. Consequently, our findings and conclusions are hypothesis generating and should be validated in larger prospective series. However, data regarding the systemic therapy of adult patients with advanced or relapsed rhabdomyosarcoma is scarce in the literature. The management of adult patients with RMS remains controversial owing to their bare representation in clinical trials, but it seems that they do benefit from the same pediatric regimes . There iHMGB1, HMGB2 and HMGA2 genes have been analyzed in tumors of these patients with RMS because they could affect DNA-platinum adduct repair and, in consequence, their sensitivity to this therapy. HMGB1 mRNA levels showed a significant negative correlation with OS and the same tendency for PFS, which is especially interesting in the patient with complete response who showed the lowest HMGB1 mRNA levels and the highest PFS and OS of this RMS series. The A204 and H729T cell lines, which are two of the most sensitive RMS cell lines to cisplatin treatment also showed the lowest levels of HMGB1 mRNA and protein levels. HMGB1 knockdown in the six RMS cell lines assayed showed a strong reduction of the proliferation, indicating HMGB1 could be a new target for the treatment of RMS. On the other hand, the overexpression of HMGB1 has been associated with proliferation, angiogenesis, the evasion of programmed cell death, invasion and metastasis. Moreover, HMGB1 can be released to the extracellular milieu and acts as a damage-associated molecular pattern (DAMP) molecule with function in the inflammatory response ,39. Our HMGB2 and HMGA2 mRNA levels did not correlate with OS and PFS in these patients with RMS. However, the lowest levels of HMGB2 mRNA and protein were also observed in A204 and H729T RMS cell lines, and the lowest levels of HMGA2 mRNA and protein were observed in RH28 and H729T RMS cells. Hs729T, RH28 and A204 cell lines are the most sensitive RMS cell lines to cisplatin among the six RMS cell lines analyzed. HMGB2 overexpression has been associated with resistance to cisplatin in head and neck squamous cell carcinoma , as wellIn conclusion, in our experience, platinum-based regimens are active in adult patients with relapsed and advanced rhabdomyosarcoma, with manageable expected toxicity. These observations deserve their own explorations in a prospective study. Moreover, low HMGB1 expression levels could be used as predictive factors of good responses to BOMP-EPI treatment.Embryonal RMS cell line RD was purchased from Merck , embryonal RMS cell line A204 (ATCC HTB-82) and Hs729T (ATCC HTB-153) were purchased from ATCC  and alveolar RMS cell line RH41 was purchased from DSMZ . Alveolar RMS cell lines RH30 and RH28 were kindly provided by Dr. Amancio Carnero  and Dra. Soledad Gallego , respectively.RD, RH30 and Hs729T were cultured in DMEM medium . RH41 and RH28 were cultured in RPMI medium . A204 was cultured in McCoy’s 5A medium . All cell culture mediums were supplemented with 10% FBS , 100 units/mL penicillin and 100 µg/mL streptomycin . Additionally, DMEM medium was supplemented with 100 µM sodium pyruvate  and 1 mM MEM NEAA .2 atmosphere and at 37 °C, and they were regularly tested for mycoplasma contamination.All cell lines were incubated in a 5% COEach RMS cell line was seeded in MW24 at 25,000 cells/well and was incubated with lentivirus, which expresses HMGB1 (shHMGB1) or control (shControl) short hairpin RNAs at a MOI = 1  plus polybrene  at 5 µg/mL overnight. The number of cells was counted with trypan blue in a Neubauer chamber after 4 and 8 days of the transduction. Cells with stable expression of shHMGB1 or shControl were selected by treatment with puromycin at 0.25 µg/mL.Patients diagnosed with RMS (excluding pleomorphic histologic subtype) from August 1994 to November 2015 in Hospital Universitario Virgen del Rocío (Seville) and Hospital Universitario Son Espases  were retrospectively reviewed. Patients were eligible for this analysis if they had more than 16 years at diagnosis and they had been treated with platinum-based regimes, specifically BOMP-EPI   Table 6Table 6. https://www.htgmolecular.com/assays/obp, accessed on 12 September 2020) related to tumor biology. RNA was extracted from paraffined tumor samples with at least 70% of tumor area or from selected tumor area by macrodissection in cases with less than 70% of tumor area or more than 20% of necrosis. RNA-Seq libraries were synthesized in the HTG EdgeSeq system by using the HTG EdgeSeq chemistry and following the specific instructions and recommendations for sequencing with the Illumina technology. RNA-Seq libraries were cleaned with Agencourt AMPure XP  and quantified with the KAPA Library Quantification kit  by qPCR, according to the manufacturer’s instructions. Library denaturation was performed by adding the first 2 N NaOH to the library, followed by the addition of 2 N HCl. The PhiX was spiked in at a 5% (concentration of 12.5 pM).Paraffined tumor samples were obtained from Hospital Universitario Virgen del Rocío and Hospital Universitari Son Espases, after receiving the patient’s informed consent for the study. FOXO1 rearrangement was confirmed by FISH in all RMS cell lines and BOMP-EPI-treated tumor samples. Gene expression analysis was performed with HTG EdgeSeq technology , using the EdgeSeq Oncology Biomarker Panel (OBP) for quantification of 2549 human RNA transcripts .A total of 2500 or 5000 cells/well were seeded in 96 well plates and were treated 24 h later with 0.1, 1, 10 and 100 µM of cisplatin (Sigma-Aldrich) or drug vehicle. After 72 h, 20 µL of Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS)  was added to each well, and absorbance was measured at 490 nm in iMark microplate absorbance reader . The IC50 of each RMS cell line was calculated with a nonlinear regression. A total of 5000 cells/well of RH30 shControl and shHMGB1 were seeded in 96 well plates and treated 8 h later with 5, 10 and 20 µM of cisplatin (Sigma-Aldrich) or drug vehicle. After 48 h, 20 µL of Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS)  was added to each well, and absorbance was measured at 490 nm in iMark microplate absorbance reader .RNA was extracted from RMS cell lines with the RNA PureLink RNA Mini kit , quantified with the NanoDrop One C spectrophotometer  and reverse transcribed to cDNA using the High-Capacity Reverse cDNA Transcription kit . Expression levels of each gene were quantified by RT-qPCR in an ABI Prism 7900HT real-time PCR system by using the TaqMan Universal PCR Master Mix  and the specific TaqMan Gene Expression Assays (Applied Biosystems) HMGA2 (Hs00171569_m1), HMGB1 (Hs01923466_g1), HMGB2 (Hs01127828_g1) and GAPDH (Hs03929097_g1) as a housekeeping gene for data normalization.Cell lysis and protein extraction were carried out using the RIPA buffer , 0.5 M EDTA (Thermo Fisher Scientific), Triton X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)), supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Further, 20 µg of protein from each sample were separated by SDS-PAGE and transferred to a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h with 5% BSA  in 1x TBS-T  and incubated with specific antibodies HMGA2 , HMGB1 , HMGB2  and ɑ-tubulin  overnight at 4 °C. Then, membranes were washed with 1x TBS-T and incubated with rabbit antimouse IgG (Sigma-Aldrich) or goat antirabbit IgG , both conjugated with peroxidase. HRP substrate  was used for chemiluminescent detection, and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad).


Although the overall survival of advanced soft-tissue sarcoma (STS) patients has increased in recent years, the median progression-free survival is lower than 5 months, meaning that there is an unmet need in this population. Among second-line treatments for advanced STS, eribulin is an anti-microtubule agent that has been approved for liposarcoma. Here, we tested the combination of eribulin with gemcitabine in preclinical models of L-sarcoma. The effect in cell viability was measured by MTS and clonogenic assay. Cell cycle profiling was studied by flow cytometry, while apoptosis was measured by flow cytometry and Western blotting. The activity of eribulin plus gemcitabine was evaluated in in vivo patient-derived xenograft (PDX) models. In L-sarcoma cell lines, eribulin plus gemcitabine showed to be synergistic, increasing the number of hypodiploid events (increased subG1 population) and the accumulation of DNA damage. In in vivo PDX models of L-sarcomas, eribulin combined with gemcitabine was a viable scheme, delaying tumour growth after one cycle of treatment, being more effective in leiomyosarcoma. The combination of eribulin and gemcitabine was synergistic in L-sarcoma cultures and it showed to be active in in vivo studies. This combination deserves further exploration in the clinical context. Halichondria okadai, and it has been approved for the treatment of metastatic breast cancer, and recently also for unresectable or metastatic liposarcoma (mLPS) patients who have received a prior anthracycline regimen  is a deoxycytidine analogue used in the treatment of a large spectrum of tumours, including STS . GemcitaThis study aims to investigate the potential synergism of eribulin plus gemcitabine in L-sarcomas in vitro, assessing the mechanisms underlying this synergism and the translation to in vivo studies to determine the effectiveness and safety of this drug combination, as well as to analyse the potential benefit in the clinical setting.50) concerning cell viability in each cell line. MTS experiments analysed 72 h after adding the drug revealed IC50 viability values at nanomolar (nM) concentration in all the cell lines, confirming the cytotoxic effect previously described for both drugs   and LMS (SK-UT-1 and CP0024). First, we identified the optimal drug concentration for each compound calculating the half-maximal inhibitory concentration (ICth drugs . Our resth drugs C. Drops th drugs D. This il lines) . Statistl lines) .p = 0.002 and p = 0.033 for 12-6- and 24-12 h combination, respectively) .  . In the ctively) . The samtively). .To understand the molecular mechanisms responsible for the synergy observed in cell viability experiments with the sequential combination, we decided to check whether the reduction in cell viability could be a consequence of an increase in apoptotic events. Since, so far, all experiments have revealed similar results in the four cell lines tested, we decided to perform the subsequent analysis only in one LPS cell line (93T449) and two LMS cell lines (CP0024 and SK-UT-1); that became our focus of interest. Using flow cytometry to analyse DNA content, we quantified cell cycle profiles after the incubation with the drugs of interest. Consistent with the role of eribulin in microtubule polymerization, an arrest in G2/M is observed in long treatments and it seems to be reversible since the percentage of G2 cells diminished from 12 to 24h, except in the SK-UT-1 cell line A,B. Howep = 0.046) (p = 0.152) but in the 24-12H experiment we also observe an γ-H2AX foci accumulation when treating with gemcitabine that we did not observe in other cell lines, with a significant difference between G12H and E24H    A. The sa= 0.046) D. For th= 0.049) B. In the= 0.088) C. Γ-H2AX= 0.088) D. Intere= 0.088) D.p = 0.0002; Wilcoxon test   and one LPS PDX model (LPS-IBIS-015). The original patient localization of tumour was uterine (LMS-IBIS-002) and deltoid (LMS-IBIS-010) LMS and retroperitoneal dedifferentiated LPS (LPS-IBIS-015) . We obsexon test B. We couon test) B. Bodyweon test) C. Both mIBIS-002 D and LMSIBIS-002 D models,p-value: 0.0033)  . Bodywei 0.0033) .Based on the angiogenic capabilities of eribulin, we decided to study vessel formation in the tumours of our three PDX models in response to treatment . In the 50 of eribulin for our cell lines is in the nanomolar range, as demonstrated by Hayasaka et al. [To identify a new strategy in the treatment of L-sarcomas in second or successive lines, we have focused on the combination of eribulin with gemcitabine. The combination is synergistic in in vitro experiments with LMS and LPS cell lines and it is particularly effective in PDX models of LMS. Eribulin is a microtubule-targeting agent that has been approved for the treatment of patients with unresectable or metastatic LPS previously treated with anthracyclines ,26. The a et al. , Stehle a et al. , and by a et al. . Our LMSa et al. ,30. Sinca et al. . Additioa et al. . For thea et al. . A Phasea et al. .50 is lower when eribulin is applied before gemcitabine, and in the case of LPS, for 94T778 this is also the case but not for 93T449, where concomitance or gemcitabine before eribulin would work better. Regarding the combination indexes, there is a strong synergy in all cell lines when we treat with eribulin before gemcitabine at low concentrations (0.1 and 1 nM) of both drugs. This low concentration-synergy has been reported in other studies [The combination of eribulin with gemcitabine was tested in our L-sarcoma lines with three different sequences: eribulin plus gemcitabine, eribulin before gemcitabine, and vice versa to find the best combination scheme to obtain a strong synergism. Eribulin before gemcitabine proves to be the best approach, as it is the approach with the lowest combination index (CI) in our cell lines. In the two LMS cell lines, CP0024 and SK-UT-1, the ED studies ,36, probPrevious preclinical studies described the G2 m arrest caused by eribulin, which becomes more pronounced over time, becoming irreversible . KuznetsCell death occurs after mitotic arrest and is characterized by the inactivation of anti-apoptotic Bcl-2 proteins and by the activation of Bax in Ewing sarcoma cell lines, where caspases contribute only partially . In rhabGemcitabine, being a deoxycytidine triphosphate analogue, is incorporated into the DNA of cells during replication, causing DNA damage that the cell cannot repair, leading to G1-cell arrest. There is wide evidence of gemcitabine combinations with other drugs such as inhibitors of microtubules, PARP, or proteins such as p330. These induce a synergy caused by an increase in apoptosis, linked to an increase in DNA damage, which is measured, in most cases, by an increase in γ-H2AX ,43,44. TTP53 is mutated in this LPS line, which is more sensitive than the other line of this type of sarcoma (93T449) to eribulin, or to determine whether the expression/amplification levels of MDM2, a negative regulator of p53, are higher in 94T778. Both lines come from the same patient, but 94T778 comes from the second metastasis of that patient. In this case, the mutational/inactivation status of p53 could account for the different sensitivities observed in our preclinical setting. In line with this, it was recently reported that mutations in TP53 are associated with a higher PFS in patients with LMS treated with eribulin [Previous studies from our laboratory looked at the p53 status of our cells: 93T449 has no mutations, SK-UT-1 has two missense mutations at residues 524 and 743, and line CP0024 has a heterozygous mutation at residue 52. Recently, it has been shown that a complete loss of p53 function sensitizes lung cancer cells to eribulin [eribulin .In a phase-II trial measuring the efficacy of eribulin and gemcitabine combination in L-sarcomas, with PFS as the primary endpoint, treatment is carried out following the same scheme as in our in vivo PDX approach . We wereIt has been described that the antitumour activity of eribulin is also based on the changes it produces in the microenvironment, mainly through the associated immune response and vascular remodelling. In breast cancer xenografts, eribulin improves tumour perfusion through vascular remodelling based on an increase in the number of small functional microvessels . In our 2. Tissue culture supplements were all purchased from Sigma-Aldrich . Cells were checked routinely and found to be free of contamination by mycoplasma or fungi, and their authenticity was checked before experiments. All the cell lines were discarded after 7–8 passes and new lines were obtained from frozen stocks.The LPS 93T449 (CRL-3043™), 94T778 (CRL-3044™), and LMS SK-UT-1(HTB-114™) human cell lines were obtained from the American Type Culture Collection . The CP0024 LMS human primary cell line was established from fresh tumour samples in our laboratory. The SK-UT-1 cell line was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin/ampicillin (P/S), 1% sodium pyruvate, 0.1% MEM-non-essential amino acid solution, and 0.1% HEPES buffer. Both CP0024, 93T449, and 94T778 were cultured in RPMI medium supplemented with 10% FBS, 1% P/S, and 1% Fungizone. All cell lines were maintained at 37 °C with 5% CO50 concentrations, which induced 50% cell death, were calculated for eribulin ) and gemcitabine hydrochloride (Sigma Aldrich) in all cell lines. DMSO was used as a drug vehicle and negative control. SK-UT-1 (2 × 103), CP0024 (2.5 × 103), 93T449, and 94T778 (3 × 103) cells were seeded in 96-well plates and treated for 72 h (h) with drug concentrations ranging from 10−7 to 10−11M for eribulin and from 10−6 to 10−10M for gemcitabine. Cell viability was measured using the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method , recording absorbance at 490 nm in an iMarkTM microplate absorbance reader  spectrophotometer. Data were analysed using Prism 6.0 .IC3), CP0024 (2.5 × 103), 93T449, and 94T778 (3 × 103) cells were seeded in 96-well plates, 24 h before treatment with eribulin plus gemcitabine (concomitant treatment), eribulin 24 h prior to gemcitabine and gemcitabine 24 h prior to eribulin in concentrations ranging 10−8 and 10−10 M for both drugs, always in a 1:1 ratio of both drugs. After 72 h, cell viability was measured using the MTS method , recording absorbance at 490 nm. The combination index ED50, ED75, and ED90 were then calculated with Calcusyn Version 2.0 , following Chou-Talalay’s combination index theorem.SK-UT-1  and 1 nM (CP0024 and SK-UT-1), and gemcitabine was used at 30 nM (93T449) and 3 nM (CP0024 and SK-UT-1) in the following conditions: eribulin monotherapy (24 or 12 h), gemcitabine monotherapy (24 or 12 h), and combination treatment (24 h eribulin plus 12 h gemcitabine or 12 h eribulin plus 6 h gemcitabine). DMSO was used as a drug vehicle and negative control.4) were seeded in 10 cm dishes and treated with the same scheme used in cell viability assays. Each condition was tested in triplicate. After 10 days, colonies were fixed and stained with crystal violet assay. After extensive washing, colonies were counted manually, and the relative number of observed colonies was represented in a graph.Cells  in PBS, incubating for 1 h at RT in the dark with horizontal shaking. The cell cycle was measured by flow cytometry ) and data were analysed with FlowJo software .After treatment, cells were trypsinized, centrifuged, and washed with 1X PBS, followed by a 30 min (min) incubation at 4 °C in 70% ethanol. After centrifugation at 1800 rpm for 10 min, cells were resuspended in a mixture of 1 mg/mL propidium iodide (Sigma Aldrich) and 50 mg/mL RNaseA  following the manufacturer’s instructions. Apoptosis levels were determined by flow cytometry (Canto II flow cytometer) and data were analysed with both BD FACS Diva and FlowJo software.® 20 (Bio-Rad)) solution (blocking solution) and incubated with primary antibody in blocking solution overnight at 4 °C. The primary antibodies used were: β-actin , γ-H2AX , cleaved-caspase-3 , cleaved PARP-1 , and p21 . Blots were then washed in 1X TBS-T and incubated with either rabbit anti-mouse IgG  or goat anti-rabbit IgG  peroxidase-labelled antibodies in 1X TBS-T for 1 h. HRP substrate was used for chemiluminescent detection ) and image acquiring was performed using Chemidoc Imaging System (Bio-Rad). Blots were analysed using Image Lab from Bio-Rad. The experiments were performed in triplicate.Cells were lysed in RIPA buffer , 0.5M EDTA , Triton™ X-100 (Sigma-Aldrich), 10% Sodium Deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich), and 3M NaCl (Thermo Fisher Scientific)). Equal amounts of total protein, determined by the Bradford method  with QuickStart Bovine Serum Albumin Standard Set (Bio-Rad) for calibration curve, were separated by SDS-PAGE on 10–15% polyacrylamide gels and transferred to nitrocellulose membranes . For immunodetection, blots were soaked in 5% BSA (Albumin (BSA) Fraction V (pH 7.0) (PanReac AppliChem)) in TBS-T . Then, cells were washed with 200 mM glycine solution for 15 min at RT and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 30 min at RT. Cells were blocked in 1% BSA 2X PBS solution for 30 min at RT. Subsequently, they were incubated with the γ-H2AX antibody 1:100 diluted in blocking solution overnight at 4 °C. Washes were made with 1X PBS for 5 min and incubated with the goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (ThermoFisher Scientific) diluted in blocking solution for 1 h at RT in the dark. After washing with 1X PBS, nuclei were stained with DAPI  diluted in PBS (1:1000 dilution) for 15 min RT in the dark. Crystals were mounted on the slides with ProLong Gold solution (Life Technologies). The slides were stored at 4 °C until analysis. Images were obtained with the Leica TCS-SP2-AOBS confocal microscope and analysed with LCS Lite and Fiji Software v1.8.0.Cells were seeded in 10 mmThree PDX models were used for the in vivo studies: LMS-IBiS-002, LMS-IBiS-010, and LPS-IBiS-015. For LMS-IBiS-002, pathologic evaluation confirmed the original diagnostic of a leiomyosarcoma spindle cell type. Microscopically, it is a well-demarcated, unencapsulated tumour with an expansive growth front. It consists of interlacing bundles of spindle cells with eosinophilic cytoplasm. There were areas of intense nuclear pleomorphism, as well as frequent mitotic figures. No areas of coagulative necrosis, lymphatic vascular invasion were identified. Although, no immunohistochemical markers were stained for the original diagnosis, we were able to detect diffuse expression of smooth muscle actin (SMA) and h-caldesmon in our PDX. For LMS-IBiS-010, the patient tumour expressed SMA and h-caldesmon, while desmin protein expression was negative. Our PDX model maintained the positive expression of h-caldesmon, but had loss at least in the tumour block tested the expression of SMA. For LPS-IBiS-015, pathologist described the original tumour as a Grade 3 dedifferentiated liposarcoma with muscular differentiation, and this tumour was positive for MDM2, SMA, and Desmin and negative for myogenin, CD117, DOG1, and S100 protein. In our PDX samples, tumours maintained the positivity for SMA, MDM2, h-caldesmon, and DOG-1.3 volume) were implanted subcutaneously on the right flank and developed in 3–7 weeks until reaching a minimum volume of 150 mm3 to start treatments. Tumours were measured using callipers.Six- to eight-week-old female nude mice ) were used. The mice were anesthetized with 100 µL of a mixture of the anaesthetics diazepam  and ketamine  in a 1:3 ratio administered by intraperitoneal injection. Tumour samples  in a CO2 chamber, following the ethical standards of animal treatment. This project has the approval of Consejería de Agriculutra, Pesca y Desarrollo rural under the code 16/05/2017/061.The mice were randomized according to their tumour size to the following 4 treatment groups: control group ; eribulin group (1.6 mg/kg dose intravenously); gemcitabine group ; and eribulin and gemcitabine combination group, receiving doses of 1.6 mg/kg of intravenous eribulin and 120 mg/kg of intraperitoneal gemcitabine, eribulin 3 h prior to gemcitabine. For intraperitoneal treatments, we used an insulin needle (0.3 mm (29 G) × 12.7 mm)  and for intravenous treatment, we treated by either of the two lateral veins of the tail with a needle (0.40 mm (27G) × 10 mm) . Mice were immobilized in a restrainer for mice of 35 g (90 × 30 mm) during tail treatment. The mice received the appropriate treatment for 2 weeks . Mice were monitored daily for signs of distress and weighed three times a week. The tumour size was measured, and size was estimated according to the following equation: tumour volume  =  [length × width2O  for 1 day and then paraffin-embedded. Cross-sections were prepared and stained with rabbit anti-mouse CD31, (PECAM-1) (D8V9E) XP®, Cell Signalling, 1:90 dilution) using Histofine Simple Stain Mouse MAX PO and DAB substrate kits . Tissue morphology was visualized by haematoxylin and eosin (H&E) staining. Quantification of vascular morphology and microvessel density (MVD) was conducted by an expert pathologist (RR) with an Olympus BX61 optical microscope. To calculate the proportion (%) of small and large vessels, a piecewise threshold of the vascular area was selected. Vascular “hot spot” regions, defined as regions of high vascular density in the tumour, were identified at 100× magnification. Individual mature and immature microvessel counts were made on at least 5 different fields (0.95 mm2/field) and MVD was expressed as the average microvessel count per high-power field (HPF).Fragments of resected tumour PDX from nude mice were fixed in a 1:4 formol solution in HParaffin blocks corresponding to tumour samples from LMS-IBiS-002, LMS-IBiS-010, and LPS-IBiS-015 mouse models were cut into 10 µm slices. RNA, including small non-coding RNA, was extracted using the RecoverAll Total Nucleic Acid Isolation kit , following the manufacturer’s instructions. RNA samples were retrotranscribed to cDNA using the high-capacity reverse transcription kit (Invitrogen), following instructions from the cDNA TaqManTM Small RNA Assays user guide. TaqMan probes (Thermo-Fisher) for apoptosis miRNA MIR184 (Hs06637236_s1) and necrosis miRNA MIR21 (Hs04231424_s1) were used as targets, U6 snRNA (001973) was used as an endogenous control.t-test. Differences were considered significant when p < 0.05. Statistical analysis was performed using Prism 6.0 . In addition, the log rank (Mantel-Cox) test was used for survival analysis in the PDX experiment, and the Wilcoxon signed-rank test was used for the assessment of tumour growth inhibition.Data of biological replicates were grouped and presented as mean ± standard error of the mean. Differences between two treatment conditions were statistically analysed when indicated, using the unpaired Student’s In conclusion, we have demonstrated that the eribulin and gemcitabine combination is synergistic in LMS and LPS cell lines. This synergism could be explained in part by the accumulation of DNA damage and the sub-G1 population. Eribulin plus gemcitabine combination is feasible in L-sarcoma PDX models, and survival in mice treated with the combination augments in comparison with eribulin monotherapy. Further analysis is needed to explain the mechanics underlying this synergism and its translation to the clinic onset.


MS47 and MS49, developed previously as G-quadruplex stabilizers, focusing specifically upon the human melanoma MDA-MB-435 cell line. At the National Cancer Institute (NCI), USA, bis-triazole MS47 (NCS 778438) was evaluated against a panel of sixty human cancer cell lines, and showed selective, distinct multi-log differential patterns of activity, with GI50 and LC50 values in the sub-micromolar range against human cancer cells. MS47 showed highly selective cytotoxicity towards human melanoma, ovarian, CNS and colon cancer cell lines; in contrast, the leukemia cell lines interestingly showed resistance to MS47 cytotoxic activity. Further studies revealed the potent cell growth inhibiting properties of MS47 and MS49 against the human melanoma MDA-MB-435 cell line, as verified by MTT assays; both ligands were more potent against cancer cells than MRC-5 fetal lung fibroblasts (SI > 9). Melanoma colony formation was significantly suppressed by MS47 and MS49, and time- and dose-dependent apoptosis induction was also observed. Furthermore, MS47 significantly arrested melanoma cells at the G0/G1 cell cycle phase. While the expression levels of Hsp90 protein in melanoma cells were significantly decreased by MS49, corroborating its binding to the G4-DNA promoter of the Hsp90 gene. Both ligands failed to induce senescence in the human melanoma cells after 72 h of treatment, corroborating their weak stabilization of the telomeric G4-DNA.Here, we describe the anticancer activity of our novel bis-triazoles  BrieflyTM mycoplasma detection kits (in accordance with the manufacturer’s instructions) ensured that cultures were free of bacterial infection (contamination). Sub-cultured cells were grown in RPMI-1640 media  supplemented with 10% (v/v) heat-inactivated FBS . Cells were kept at 37 °C and 100% humidity in a 5% CO2 incubator .The MDA-MB-435 human melanoma cell line was obtained from the American Type Culture Collection (ATCC)  and kept in liquid nitrogen. Upon achieving 70–80% confluence, cells were passaged twice weekly, and modest passage numbers (≤20) were utilized in all tests. The use of Lonza MycoAlertMS47 and MS49 on human melanoma MDA-MB-435 cell line. Cells were seeded at a density of 3 × 103 per well (180 µL per well) into 96-well plates  and allowed to adhere at 37 °C/5% CO2 for 24 h. Fresh top stock solutions of the test compounds (10 mM in DMSO) were prepared, followed by the preparation of serial dilutions in RPMI-1640 media for addition to cancer cells. The control wells only received the vehicle (20 µL media per well). Final concentrations of the test compound in the wells were: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 µM. While the final DMSO concentration in the wells never surpassed 1%. Vehicle control experiments were conducted (0.0001–1% DMSO). Test plates were incubated for further 72 h at 37 °C and 5% CO2. At the time of test compound addition (T0) and after 72 h exposure, cell viability was determined by the addition of MTT reagent . To allow the reduction in MTT by live cells to insoluble crystals of dark purple formazan, test plates were incubated for 3 h. Then, the supernatant was removed from each well by means of aspirator, and DMSO (150 µL per well) was added to solubilize the cellular formazan. The absorbance was measured at 550 nm using a plate reader from Anthos Labtec systems. The intensity recorded (OD) is proportional to metabolic activity, which coincides with the number of live cells. By using Microsoft Excel 2010 software, GI50 values (test compound concentrations that inhibit cell growth by 50%) were estimated. Results are presented as mean of three separate experiments .The chosen method used in this study was originally described by Saleh et al. ,26 and aMS47 and MS49 , medium containing MS47 and MS49 was aspirated, cells were gently washed twice with 1.0 mL medium, and 2.0 mL of fresh medium were added prior to incubation for ~10–14 days. When colonies of more than 50 cells were visible in the control wells, experiments were halted. The colonies were washed twice with 1.0 mL of cold PBS, fixed with 100% methanol (1.0 mL/well) for 10 min, stained with 0.5 mL of 0.05% methylene blue , washed (3× dH2O), air dried and counted. Prism software was used to calculate the mean survival fraction (%) of untreated and treated cell colonies.The clonogenic cell survival test examines the capacity of a single cell to withstand a brief exposure to test chemicals and maintain its proliferative potential to generate offspring colonies ,28. The 2 culture dishes, cells were sown at a density of 1–2 × 106 per dish, allowed to adhere for 24 h, and exposed to 1 × GI50 and 2 × GI50 of MS47 and MS49 for 72 h. After exposure to the treatment, protein lysates were prepared, and protein concentrations were determined using the Bradford assay [Western blotting was performed according to Chen et al.’s instructions . In 10 crd assay . Using Srd assay . The denv/v) Triton X-100, 0.1 mg/mL of ribonuclease A and 0.1% (w/v) sodium citrate in d-H2O according to Nicoletti et al.’s technique [5 cells/well were seeded in 6-well plates and incubated overnight before being treated with MS47 and MS49 at 0.5 ×, 1 × and 2 × GI50 doses for 24, 48 and 72 h. At the end of the specified time period, cells were extracted, pelleted by centrifugation, then resuspended in 0.3–0.5 mL of fluorochrome solution, and kept in the dark at 4 °C overnight. Beckman Coulter FC500 flow cytometer was utilized for cell cycle analyses . FCS express analysis software version 7  was used to analyze the data, and model 1 of multivariate analysis was employed as the mathematical model. The analysis was performed automatically by selecting the best-fitting cycle, one cycle at a time, and displaying only the major phases of the cell cycle, G0/G1, S, and G2/M.Cell cycle analysis was performed using a fluorochrome solution comprising 50 mg/mL propidium iodide (PI), 0.1%  and 1× annexin-V buffer (100 µL) were applied to cells; after 15 min of incubation at room temperature in the dark, PI  and 400 µL annexin-V buffer were added. Prior to analysis with Beckman Coulter FC500 flow cytometer, cells were maintained on ice and in the dark for 10 min . Using version 7 of FCS express analysis software, data were analyzed .In 6-well plates, cells were sown at a density of 1.0 × 104 cells/well in 6-well plates, incubated overnight, and then treated with MS47 and MS49 at 0.5 × and 1 × GI50 concentrations for 72 h. After 72 h, the medium containing treating ligands was aspirated, cells were washed twice with 1× PBS (2 mL per well), 1× fixation solution  was added (2 mL per well), and then the cells were left to fix at room temperature for 10–15 min. The fixation solution was eliminated, cells were washed twice with 1× PBS, then 1.0 mL of the freshly prepared staining solution (SA) β-galactosidase was added to each well, and cells were incubated at 37 °C for 12–16 h in a dry incubator (no CO2). While the staining solution (SA) β-galactosidase was still on the plate, the cells were examined using a phase contrast microscope (10× magnification) for the appearance of a blue hue. Using Prism software or Microsoft Excel, the percentage of stained cells was determined.Investigation of senescence-associated (SA) β-galactosidase  followed the chromogenic experiment which was published by Debacq-Chainiaux et al.  using lip < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.00005 compared to untreated control) were identified. The significance level for clonogenic assay was determined using Dunnett’s multiple comparison test (**** p < 0.0001 compared to untreated control).Experiments were performed a minimum of three times, with sample experiments depicted in the Figures. The statistical significance was determined using one-way and two-way analyses of variance (ANOVAs). Using Tukey’s multiple comparison test, levels of significance (* in vitro screen comprises a panel of sixty distinct human tumor cell lines (from nine different organ sites), against which compounds are tested over a specific concentration range (10 nM–100 µM) to evaluate the relative degree of growth inhibition or cytotoxicity against each cell line. For each studied drug, a characteristic profile or “fingerprint” of cellular response is established [50), total growth inhibition (TGI), and 50% lethal concentration (LC50) “net cell killing” or “cytotoxicity parameter”, which are the concentrations of the test drug at which the percentages of the cell growth (PG) are +50%, 0%, and −50%, respectively [50, TGI or LC50 data for a given chemical evaluated against each cell line in the NCI in vitro screen. Deltas are represented as horizontal bars in relation to a vertical line representing the mean panel [50, TGI and LC50. Relatively sensitive cell lines have negative deltas and right-extending bars. While relatively resistant cell lines have positive deltas and bars that extend to the left [The NCI ablished . The “doablished  that mosectively . The meaan panel . Each bathe left .in vitro screening consists of two consecutive stages: the NCI 60 cell one-dose screen and the NCI 60 cell five-dose screen [−5 M) against a panel of sixty distinct human cancer cell lines. The data from a single dose are presented as a mean graph of the percent growth (PG) of the treated cells, allowing for the detection of both growth inhibition  and fatality . For instance, a score of 100 indicates no growth inhibition, 40 indicates 60% growth inhibition, 0 indicates no net growth throughout the period of the experiment, −40 indicates 40% lethality (cytotoxicity), and −100 indicates all cells are dead [50, TGI and LC50 [The methodology of NCI 60 panel e screen . In the are dead . Only coand LC50 ,38,40.MS47 was selected by the National Cancer Institute for testing against the NCI 60 panel of human tumor cell lines. The mean graph of the initial single-dose (10 μM) screen of MS47 (named as NSC778438) is presented in MS47. The highest cytotoxic activity for MS47 (yellow highlight) is 96.85% against the melanoma cell line SK-MEL-5, followed by 94.25% against the breast cancer BT-549 cell line, followed by 92.76% against the CNS cancer SNB-75 cell line and 92.63% against the prostate cancer DU-145 cell line. Although leukemic cell lines showed resistance to the lethality of MS47 (LC50 > 100 µM)  , MS47 deMS47. The renal cancer TK-10 and UO-31 cell lines, ovarian cancer NCI/ADR-RES cell line, and colon cancer HCT-15 cell line have the lowest growth inhibition and highest relative resistance.Renal cancer cell lines demonstrate an exceptional resistance to MS47 (NSC778438) satisfied the pre-determined criteria for the threshold inhibition in a minimum number of cells in the NCI 60 cell one-dose screening, so it was tested against the panel of sixty tumor cell lines of NCI at five small doses. 50, TGI and LC50 values (µM) and dose–response curves of the MS47 (NSC778438) against the panel of NCI 60 human cancer cell lines that resulted from the NCI 60 cell five-dose screening. While the mean graphs of the log10 values (Molar) of GI50, TGI and LC50 of MS47 (NSC778438) obtained from the NCI 60 cell line experiments can be found in the Ligand MS47 (NSC778438) against most of the NCI 60 human cancer cell lines. The NCI 60 GI50 values range from 0.111 µM to 24.1 µM with the exception of the ovarian cancer NCI/ADR-RES and renal cancer CAKI-1 cell lines, which showed significant resistance to MS47 with GI50 values > 100 µM. The most sensitive cell line to MS47 was the melanoma MALME-3M cell line, with the lowest sub-micromolar GI50 value being 0.111 µM, followed by the ovarian cancer OVCAR-4, breast cancer MCF7 and MDA-MB-468, colon cancer COLO 205, CNS cancer U251, SNB-75 and SNB-19, melanoma MDA-MB-435 and LOX IMVI, renal cancer SN12C, and non-small cell lung cancer NCI-H522 cell lines, showing sub-micromolar GI50 values < 0.2 µM. The least growth inhibitory activity of MS47 was for the renal cancer 786-0 cell line (GI50 = 24.1 µM).The results in MS47 (NCS778438) against the NCI 60 human cancer cell lines can be described by the NCI 60 TGI values, which range from 0.26 µM to 38.1 µM. Resistance to MS47  can be found in the leukemia CCRF-CEM, colon cancer HCT-15, ovarian cancer NCI/ADR-RES, and renal cancer 786-0 and CAKI-1 cell lines. MS47 showed the highest potency in total growth inhibition against the ovarian cancer OVCAR-4 cell line (TGI = 0.26 µM), followed by the melanoma MALME-3M cell line (TGI = 0.265 µM), and other cell lines showing sub-micromolar TGI values less than 0.4 µM. While the lowest potency in the total growth inhibitory activity was against the renal cancer UO-31 cell line.The total growth inhibition activity of 50 values show the lethality and cytotoxicity of MS47 (NCS778438) against the NCI 60 human cancer cell lines. The values range from 0.515 µM to 86.1 µM. The ovarian cancer OVCAR-4 cell line showed the highest sensitivity to MS47 (LC50 = 0.515 µM), followed by the melanoma MDA-MB-435 and MALME-3M, colon cancer COLO 205, and CNS cancer U251 cell lines, with sub-micromolar LC50 values of 0.602, 0.636, 0.644 and 0.694 µM, respectively, and other cell lines with LC50 values of <1 µM. The least lethal activity shown by MS47 was for the renal cancer UO-31 cell line (LC50 = 86.1 µM). All of the leukemia cell lines showed interesting resistance to MS47 cytotoxic activity, in addition to the colon cancer HCT-15, ovarian cancer NCI/ADR-RES, renal cancer 786-0 and CAKI-1, prostate cancer PC-3 and breast cancer HS 578T cell lines, with LC50 values of >100 µM. The resistance and sensitivity shown by the panel of sixty human cancer cell lines reflected a significant selective cytotoxic  activity for MS47.The NCI 60 LCMS47 (NSC778438) has potent and selective cell growth inhibition and cytotoxic activities against tumor cell lines isolated from distinct organs, making it a promising anticancer drug candidate for further development for the treatment of multiple carcinomas, such as renal, melanoma, ovarian, colon, breast, and CNS cancers.The NCI 60 cell five-dose screening results indicated that MS47 indicated by the NCI 60 panel in vitro screen results and the higher selective potent growth inhibitory shown by MS49 indicate that further preclinical evaluations of both ligands are warranted. Specifically notable is the sensitivity of the melanoma MDA-MB-435 cell line (LC50 = 0.602 µM); thus, this cell line was selected for more thorough interrogation of anticancer activity.The cytotoxicity of MS47 and MS49 were tested further in vitro using an MTT assay [in vitro anticancer evaluation of both ligands as it is one of the most sensitive cell lines to the lethal effect of MS47, as shown by the NCI 60 panel data.The growth inhibitory activities of TT assay ,41,42 ag50) were determined from the dose–response curves  to evaluate their putative selective cytotoxic activities are also presented in MS47 and MS49 inhibit the growth of the human melanoma MDA-MB-435 cell line potently. However, ligand MS49  shows more potent growth inhibitory effect than MS47 . Comparing the GI50 values with those of human normal lung MRC-5 fibroblasts, both ligands demonstrate greater potency in the melanoma cell line than in non-tumorigenic lung fibroblasts, with indicated cancer selectivity indices (SI) of 9.8 for MS47 and 17.7 for MS49, revealing their good selective growth inhibitory effects for cancer cells over normal cells.The estimated concentrations at 50% cell growth inhibition  were determined in vitro using the clonogenic cell survival assay [MS47 and MS49 significantly inhibit MDA-MB-435 colony formation after 24 h exposure . This might indicate that for any circulating cells, brief exposure to a test agent may inhibit their ability to form metastatic tumor colonies. The two-way ANOVA found a significant effect of the ligands’ concentration F = 87.77, (p < 0.0001), a non-significant effect of the treatment F = 4.053 × 10−30, (p > 0.99), and non-significant effect of interaction F = 1.53 × 10−30, (p > 0.999). Dunnett’s multiple comparison test determined the significance between control and 1 × GI50 and 2 × GI50 for both of the ligands. The clonogenic assay emphasized the anticancer cytotoxicity and potency of MS47 and MS49, as the formation of colonies was significantly inhibited (**** p < 0.0001).The effects of al assay ,28. MS47exposure A,B. At vMS47 and MS49 and help establish the cell death modality, both a cell cycle analysis and an apoptosis assay were performed [To determine whether the MDA-MB-435 cell cycle was perturbed by treatment with erformed .MS47 and MS49 on the cell cycle was investigated by flow cytometry. Cells were exposed to MS47 and MS49  for 24, 48 and 72 h  caused significant arrest in the G0/G1 and G2/M phases of the cell cycle in the human melanoma MDA-MB-435 cells (** p < 0.005) and (* p < 0.05), respectively (MS49 (2 × GI50) did not show any significant difference compared to the control untreated group and other treatments after 72 h of exposure. Thus, in terms of cell cycle arrest, MS47 (2 × GI50) exerts a more significant effect on treated cells in comparison to MS49 (2 × GI50) (* p < 0.05). Consequently, we conclude that MS47 may trigger cell death following the arrest of cells at G0/G1.The effect of treating the human melanoma MDA-MB-435 cells with and 72 h . Two-wayreatment A,B. In cectively C,D. WhilMS47 (2 × GI50) and MS49 (2 × GI50), we observed the presence of sub-G0/G1 events after 72 h, indicating the apoptotic death of the cancer cells. Additionally, the G2/M phase was depleted in both ligands in comparison to other treated groups in an observable manner. However, this depletion was significant only after treating the cells with MS47 (2 × GI50) (* p < 0.05). Interestingly, the depletion of G2/M phase and the enhancement of the S, G0/G1, and sub-G0/G1 phases may indicate that our compounds triggered the apoptotic cell death. Cell cycle profiles are shown in the For both MS47 and MS49 may initiate apoptosis, human melanoma MDA-MB-435 cells were treated with both ligands  for 24, 48 and 72 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry (To investigate the cell death mechanism and test the hypothesis that ytometry . CorrespMS47 (0.5 × GI50) and (2 × GI50) have a direct effect on the cell viability as the number of healthy cells decreased significantly compared to the control untreated cells (* p < 0.05). Tukey’s multiple comparison test shows that MS47 (0.5 × GI50) and (2 × GI50) decreased the percentage of healthy cells compared to that recorded in the control untreated cells (* p < 0.05). Additionally, the percentage of late apoptotic cells was significantly increased in the treated group MS47 (2 × GI50), as the number of apoptotic cells was ten-fold higher in the MS47-treated (2 × GI50) cells compared to the control cells (** p < 0.005). Whereas for MS49, the number of viable cells decreased significantly when the cells were treated with (1 × GI50) and (2 × GI50) MS49 compared to the control untreated group at (* p < 0.05) and (** p < 0.005), respectively. Similarly to MS47 (2 × GI50), MS49 (2 × GI50) showed a 10-fold significant increase in the percentage of late apoptotic cells compared to that of the control untreated group (** p < 0.005), as shown in After 24 h of the treatment, the two-way ANOVA test found that MS47 caused significant decrease in the percentage of viable cells (* p < 0.05), and a significant increase in the percentage of apoptotic cells compared to the control cells (** p < 0.005). Tukey’s multiple comparison test found that the percentage of late apoptotic cells increased up to 77% (by two-fold) compared to the 24 h treatment (42%) (** p < 0.005) following exposure to MS47 (2 × GI50). Similarly, MS49 caused significant programmed cell death; the late apoptotic population increased, while the viable cell population decreased in a significant manner when the cells were treated with MS49 (2 × GI50) for 48 h compared to the other treatments and the control untreated cells at (*** p < 0.0005) and (* p < 0.05), respectively (The same cytotoxic (apoptotic) effect was observed after 48 h of treatment. The two-way ANOVA found that ectively B.MS47 and MS49, the same cytotoxic (apoptotic) effect persisted. Tukey’s multiple comparison test found that for the MS47-treated cells (2 × GI50), the percentage of viable cells decreased while the percentage of late apoptotic cells increased significantly compared to that of the control group (*** p < 0.0005). For MS49, 1 × GI50 exposure caused a significant decrease in the viable cell population (* p < 0.05), accompanied by a significant increase the late apoptotic cells (*** p < 0.0005). MS49 at 2 × GI50 showed a strong significant decrease in the number of viable cells compared to all the other treated groups (**** p < 0.00005), and a significant increase in the percentage of late apoptotic cells (*** p < 0.0005) (After 72 h exposure to  0.0005) C,D.Heat-shock proteins (Hsps) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation while preventing protein aggregation. Many solid tumors and haematological malignancies have an increase in heat-shock proteins . Client MS47 and MS49 on the expression levels of Hsp90 protein chaperone in human melanoma MDA-MB-435 cells. Protein lysates of melanoma cells following 72 h of exposure to MS47 and MS49 (1 × GI50 or 2 × GI50 μM) were prepared. One-way ANOVA showed no significant effect of MS47 treatment on the levels of Hsp90 protein in the human melanoma MDA-MB-435 cells  F = 5.841, p = 0.0391 (MS49 (with piperidinyl group) to induce stabilization of the G-quadruplex formed by the Hsp90a promoter, with a binding affinity higher than that of MS47 (with diethylamino group), as reported previously [Western blots  were per35 cells A. Howeve= 0.0391 B. This seviously .Blot images are splices to present the intended groups. The original image files for the blots are found in the MS47 and MS49 induce senescence in the human melanoma MDA-MB-435 cells. MS47 nor MS49 at the concentrations tested (0.5 × GI50 and 1 × GI50) induced senescence in the treated cells after 72 h of exposure as visualized by the absence of blue color. No statistical analyses were performed since the stained cells did not show any color. We speculate that the weak binding of both of the ligands to G4-DNA of the telomeric region may not be strong enough to stabilize the G-quadruplex; alternatively, the exposure period may not be long enough to impact the length of telomeres sufficiently to trigger senescence.Due to cellular replication, telomeres shorten, resulting in cell cycle arrest (growth arrest), also known as replicative senescence . TelomerMS47 (NSC778438), we demonstrate a promising novel compound with wide-spectrum selective cytotoxic activity against different types of cancer. Several cell lines showed sensitivity toward our drug. The NCI 60 GI50, TGI and LC50 values of MS47 range from 0.111 µM to 24.1 µM, 0.26 µM to 38.1 µM and 0.515 µM to 86.1 µM, respectively. MS47 displays the most selective lethal activity towards the ovarian cancer OVCAR-4 (LC50 = 0.515 µM) cell line, followed by the melanoma MDA-MB-435 and MALME-3M, colon cancer COLO 205, and CNS cancer U251 cell lines, with sub-micromolar LC50 values of 0.602, 0.636, 0.644 and 0.694 µM, respectively, and other cell lines with LC50 values less than 1 µM. In MTT assays, MS47 and MS49 demonstrated a potent cell growth inhibitory effect on the human melanoma MDA-MB-435, revealing selectivity (enhanced potency) over embryonic lung fibroblasts. Moreover, at 1 × GI50 and 2 × GI50 values, both ligands inhibited clonogenic cell survival  and induced significant apoptotic cell death. Western blot analyses showed that the G4 stabilizer MS49 (at 2 × GI50) significantly decreased the levels of Hsp90 in human melanoma MDA-MB-435 cells. MS47 and MS49 represent potent experimental antitumor agents, and can be used to treat cancer by inducing apoptotic cell death. Additionally, high concentrations of both ligands can be used as potent inhibitors for the progression of the cell cycle, as they have a role in the perturbation of the cell cycle after 72 h of treatment. To our knowledge, this is the first report on our prepared compounds MS47 and MS49, which are novel and have not been synthesized by others. For our future work, the same screening will be repeated on other human melanoma cell lines, such as LOX IMVI and MALME-3M, to confirm if the observations/mechanisms of action are the same for multiple human melanoma cell lines. Furthermore, the molecular targets and mechanisms of action of anticancer bis-triazoles MS47 and MS49 in human melanoma MDA-MB-435 and other cancer cell lines will be interrogated. Our in vitro evaluation will include RNA sequencing (RNA-Seq) technology which quantitatively analyzes the whole-transcriptome sequencing  of the RNA extracts of the treated cells to study the gene expression of cancer drug targets pertinent to tumorigenesis.Based on the observed activity of


Multi recurrent or high-grade meningiomas remain an unmet medical need in neuro-oncology. Several studies have highlighted the potential therapeutic efficacy of mTor inhibitors to control tumoral growth of meningiomas. However, a positive feedback on AKT oncogenic pathway from these drugs may explain the modest success. Our aim was to target Pi3kinase upstream mTor, and MAP kinase pathway, overactivated in meningiomas, alone or in combined targeting in comparison to mTor targeting. Our in vitro results obtained on three meningioma cell lines and on a large series of fresh human meningiomas, including 35 WHO grade 1, 23 grade 2, and five grade 3, showed that co-targeting Pi3kinase and MAP kinase seemed promising, opening new therapeutic strategies in these tumors.NF2 genomic alteration. Alpelisib induced a higher inhibitory effect on cell viability and proliferation than everolimus in all cell lines and 32 randomly selected tumors no matter the genomic status, the histological subtype or grade. Trametinib also strongly inhibited cell proliferation and induced AKT activation. Combined treatment with alpelisib plus trametinib reversed the AKT activation induced by trametinib and induced an additive inhibitory effect irrespective of the cell lines or tumor features. Co-targeting pathways seems promising and may be considered particularly for aggressive meningioma.Recurrent or high-grade meningiomas are an unmet medical need. Recently, we demonstrated that targeting mTOR by everolimus was relevant both in vitro and in humans. However, everolimus induces an AKT activation that may impact the anti-proliferative effect of the drug. Moreover, the MAP kinase pathway was shown to be involved in meningioma tumorigenesis. We therefore targeted both the Pi3k-AKT-mTOR and MAP kinase pathways by using combinations of the Pi3k inhibitor alpelisib and the MEK inhibitor trametinib. Our study was performed in vitro on the human meningioma cell lines and on a large series of primary cultures providing from 63 freshly operated meningiomas including 35 WHO grade 1, 23 grade 2, and five grade 3, half of which presented a  Recurrent or high-grade meningiomas are an unmet medical need in neuro-oncology . The sixAlthough mutations in components of the phosphoinositide-3-kinase/AKT/mammalian target of rapamycin (Pi3K-AKT-mTOR) signaling pathway are rare and mostly observed in non-aggressive meningioma , we haveWe have never observed apoptosis under everolimus or octreotide in our in vitro studies on meningiomas suggesting that only an antiproliferative effect was induced by these drugs in vivo ,12. We hAlpelisib, everolimus, and trametinib were obtained from Novartis International AG . Drugs were dissolved in 100% dimethylsulfoxide (DMSO) to a 10 mM concentration and stored at −80 °C before dilution to intermediate concentrations in cell culture medium. In all the pharmacological experiments, control cells were treated with an equivalent 0.1% vehicle DMSO concentration, equivalent to the 0.1% final DMSO concentration in the drug dilution.2.This study was carried out on three human meningioma cell lines, Ben-Men1, CH-157MN and IOMM-Lee. The Ben-Men-1 comes from WHO grade 1 meningothelial meningioma , CH-157MN comes from meningioma with unknown WHO grade (accession number CVCL_5723kindly given by CERIMED), IOMM-Lee comes from WHO grade 3 bone tumor invasion of a meningioma . The three cell lines were cultured in DMEM-F12 (Dulbecco’s Modified Eagle Medium) 10% FCS , 1% streptomycin/penicillin, 1% fungizone , at 37 °C with 7% CO3 and disaggregated into single cells by exposure to 0.37% type I collagenase  for 2 h at 37 °C in culture medium (the same than that of cell lines). The cells were cultured in complete medium (10% of FCS) and the experiments were performed in the first 3 weeks after surgery to preserve differentiated features of tumoral cells as previously described  (−9 M) 30 ± 6%, trametinib (10−7 M) 30 ± 8%, vs. combined treatment 52 ± 5%  and one without (IOMM-Lee). Among the 3 cell lines, the behavior of Ben-Men1- is closest to those of human fresh tumors, probably due to the accumulation of oncogenic events as BRAF or NRAS mutation in the 2 others CH-157MN and IOMM-Lee. We have previously demonstrated that meningioma primary cell cultures are a highly relevant model to study targeted therapies, decipher molecular mechanisms and establish proof-of-concept to set up clinical trials [In a first step, the drugs were assessed on 3 human meningioma cell lines, 2 with l trials ,12,13,14l trials ,12 that l trials ,13,14. TPi3K or AKT components but also NF2 status. Moreover, alpelisib induced a stronger inhibitory effect in comparison to everolimus with a proapoptotic effect in 50% of tumors. We demonstrated that MEK inhibition decreases cell proliferation, slightly stronger in WHO grade 1 than WHO 2 and 3. Other pathways, such as the Hippo signaling cascade, may be involved in such variable responses [We have demonstrated that targeting the Pi3K-AKT-mTOR pathway is relevant in all meningiomas; regardless of any mutations of esponses . Alpelisesponses . In that