Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied . Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.  Human noroviruses (HuNoVs) are recognised as a leading global cause of sporadic and epidemic viral gastroenteritis  and accoVarious HuNoV infection models have yielded valuable insights into the NoV life cycle in recent years ,10,11,12Norovirus genus within the Caliciviridae family of small, non-enveloped, positive sense, single-stranded RNA viruses [Human noroviruses and MuNoVs belong to the  viruses ,19. The  viruses ,21. The  viruses . ORF2 an viruses .Norovirus genus [Viral recombination is a key mechanism in the evolution and diversity of NoVs; increasing evidence indicates that recombination shapes NoV pathogenesis and fitness and drives the evolution of emerging strains . Numerouus genus ,27. In cus genus ,28,29,30We recently identified a set of checkpoints, including their respective drivers and constraints, that must be successfully bypassed for the generation of a viable recombinant NoV ,31. FollIn the event of an asynchronous infection, the uptake of multiple viruses into a single cell is dependent on factors that may limit consecutive entry of more than one virus particle per cell in a process known as superinfection exclusion. Superinfection exclusion is defined as the ability of an established virus to prevent a secondary infection by the same or a closely related virus . The priHere, we determine the effect of a temporal separation of in vitro infections with the two homologous parental MuNoV strains MNV-1 WU20 and CW1 on the composition of MuNoV populations. A clear advantage of in vitro systems to study viral population dynamics is that they present a well-defined entity containing only viruses and cells. Effects of other factors interfering with cell coinfection (such as the host immune response or microbiome) may thus be discounted.Our results demonstrate that a time interval from one to two hours onwards between two consecutive NoV infections allows establishment of a barrier that reduces or prevents superinfection; this first demonstration of time-dependent viral interference for NoVs has clear implications for NoV epidemiology, risk assessment, and potentially treatment. A graphical overview of all assays is provided in −1) and streptomycin (5 mg ml−1) , and 1% 1 M HEPES buffer (pH 7.6) (Invitrogen) at 37 °C with 5% CO2.The murine macrophage cell line RAW264.7 (ATCC TIB-71) was maintained in Dulbecco’s modified Eagle’s medium (DMEMc)  containing 10% heat inactivated foetal calf serum (FCS) (BioWhittaker), 2% of an association of penicillin . Supernatants were purified by ultracentrifugation on a 30% sucrose cushion in a SW28 rotor  at 23,000 rounds per min for 2 h at 4 °C. Pellets were suspended in 500 µL phosphate-buffered saline (PBS), aliquoted, and frozen at −80 °C. Viral titres were determined via plaque assay for the seventh passage of WU20 and the eighth of CW1 (WU20 P7 and CW1 P8), as described by Hyde et al., 2009 [Murine NoV isolates MNV-1 CW1 and WU20 . After 1 h, the WU20 inoculums (300 µL) were removed and stored at −80 °C. The cells were washed twice with PBS and were infected with CW1 at various MOIs  at delays of 0 min (coinfection), 30 min, and 1, 2, 4, 8, 12, and 24 h (superinfections). For coinfections, CW1 and WU20 inoculums in a final volume of 300 µL were simultaneously added to cells. Cells and virus then remained on ice for 1 h, whereupon the inoculum was removed. For superinfections, CW1 inoculums were asynchronously dispensed onto cells at the appropriate delays, whereupon cells and virus remained on ice for 1 h until removal of the inoculums; the cells were then washed twice with PBS and 300 µL DMEMc were added. Twenty-four hours post co-or superinfection, both cells and supernatants were frozen and stored at −80 °C until further analysis.Monolayers of RAW 264.7 cells were prepared in 24-well plates at a density of 5 × 10RNA extractions were performed with Tri Reagent solution  on 120 µL of co-and superinfection supernatants. Extracted RNA was reverse-transcribed into complementary DNA (cDNA) using an iScript cDNA Synthesis kit . The extracted and reverse-transcribed cDNA was quantitatively analysed via real time quantitative PCR (qPCR), employing primers to allow discrimination between CW1 and WU20 based upon single nucleotide polymorphisms (SNPs) at the 5′ genomic extremity , as described by Mathijs et al. 2010. Primers and probes used in the quantification of genomic copies correspond to those listed in SpeI-linearised and purified plasmids were transcribed with T7 RNA polymerase, treated with DNAse, and quantified via spectrophotometer. Genomic copy numbers of transcribed RNA were deduced and serial ten-fold dilutions were prepared with ultrapure RNAse free water (Invitrogen). Aliquots of the master stock were stored at −80 °C and measured before dilution and use. Final results were normalised using transcripts of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . A 5 µL qPCR mix  was set up by adding 1 µL of cDNA to 2.50 µL of iQ supermix, 0.1 µL of both GAPDH-forward and -reverse primers , 0.2 µL of the GAPDH-probe  and 1.1 µL of nuclease free water. Cycling conditions included an initial 5-min denaturation at 95 °C followed by 38 cycles of 10 s at 95 °C and 40 s at 60 °C.Quantifications were performed as previously described by Mauroy et al. (2012) ; for gen6 RAW264.7 cells/well) were inoculated at room temperature with 1 mL of serial dilutions of virus-containing culture fluids of the co- and superinfection assays. After 1 h, inoculums were removed and cells were overlaid with 2 mL of medium containing 70% DMEM-Glutamax (4.5 g glucose l-1 and 15 mM sodium hydrogen carbonate), 2.5% FCS, 2% PS, 1% HEPES and 0.7% SeaPlaque agarose  per well. After 48 h of incubation , 36 individual plaques were randomly selected per condition. Infected cells from the plaque margins were picked with a needle under a microscope and were diluted into fresh DMEMc before propagation by inoculation onto RAW 264.7 cells grown in 24-well plates. After 72 h, supernatants were collected and frozen at −80 °C until further analysis.Cells and supernatants from the co- and superinfection step were frozen and thawed once and then utilised as inoculums in a plaque assay for purification of infectious progeny viruses following the method described by Hyde et al. 2009 with slight modifications . BrieflyFollowing RNA extraction and reverse transcription, cDNAs of individual plaque-purified virus progenies were analysed via two parallel real time PCR runs employing two pairs of primers to allow discrimination between CW1, WU20 (and recombinant) signals based upon single nucleotide polymorphisms (SNPs) at both genomic extremities  as described by Mathijs et al. 2010 . Five µlIn the case of ambiguous signals originating from mixed virus populations, an additional quick screen was performed via Sanger sequencing of the ORF1/2 overlap (base pairs 4864 to 5298 in MNV-1 CW1), this to exclude the presence of potential recombinants or PCR chimeras from interfering with later calculations of WU20 to CW1 infectious virus ratios. 10 genomic copies over all measured time points, to be higher than those of CW1 in all but four of the resulting 24 viral progenies. Only short superinfection delays  with a WU20 to CW1 starting ratio of one to ten resulted in CW1 genomic copy numbers significantly higher than or equal to those of WU20 24 h post co- or superinfection at 3.23 (±0.96), 3.44 (±0.48), 3.74 (±0.09), and 3.93 (±0.06) log10, respectively  values of the qPCR reactions and normalised against GAPDH Ct values. This genomic quantification (5′ region 1 amplicon) revealed WU20 absolute genomic copy numbers, averaging 3.55 (±0.57) log10  24 h after either coinfection (t0) or superinfections with delays of half an hour (t0.5) to two hours (t2). From a superinfection delay of four hours (t4) onwards, WU20 mean genomic copy numbers ranging from 3.28 (±0.56) to 4.24 (±0.01) log10 (99.43 to 99.96% of the population) are juxtaposed against CW1 values of 1.43 (±0.81) to 0.07 (±0.32) log10.These absolute genomic copy numbers translate into relative ratios of genomic copies that reflect a disproportionate WU20 dominance within the majority of viral populations (10 (77.30%) WU20 to 2.49 (±0.54) log10 (22.69%) CW1 (t0) and 2.98 (±0.78) log10 (62.64%) WU20 to 2.89 (±0.59) log10 (37.36%) CW1 ratios (t0.5), are succeeded by a marked increase of the WU20 proportion, covering 4.27 (±0.11) (77.63%) (t1), 4.17 (±0.18) (89.26%) (t2), and 3.37 (±0.04) log10 (86.01%) (t4), and then reaching values of over 3.16 (±0.47) log10 (95%) from t8 onwards, while CW1 values are consistently at least one order of magnitude lower and never surpass 2.67 (±0.02) log10 from t4 onwards.At an equal WU20 to CW1 starting MOI of 1 (expected yield to 50% WU20 and 50% CW1 genome copies), 3.17 (±0.33) log10 to 3.23 (±0.96) log10 and 3.88 (±0.09) to 3.44 (±0.48) log10 WU20 to CW1 genome copies at t0 and t0.5, respectively (roughly 50–50 ratios), fulfilled expectations with 2.2 (±0.59) log10 WU20 to 3.74 (±0.09) log10 CW1 genome copies (6.16% WU20 to 93.84% CW1) at t1, after which WU20 genome copy numbers progressively increased to 3.76 (±0.01), 3.68 (±0.29), 4.16 (±0.3), 3.09 (±0.63), and 3.19 (±0.32) log10  at t2, t4, t8, t12, and t24, respectively. CW1 genome copy numbers correspondingly decreased. A WU20 to CW1 starting MOI ratio of 1 to 10 (expected to yield 10% WU20 and 90% CW1 genome copies) resulted in 3.66 (±0.24) logTo isolate and screen infectious progeny viruses present within the various viral populations 24 h after initial co-or superinfection, 36 viral plaques per condition were picked from a plaque assay, further propagated in RAW246.7 cells, and then analysed in parallel duplex qPCR runs to discriminate between MNV-1 CW1 and WU20 (as well as possible recombinant viruses) based on 5′ and 3′ SNPs. In three cases, additional ORF1/2 screening confirmed sequence kinship to either WU20 or CW1.Overall, the previously observed WU20 dominance, particularly following longer CW1 superinfection delays, is mirrored in the proportions of plaque picked infectious viruses . Thus, aAt an equal WU20 to CW1 starting ratio , initial 76% WU20 to 9% CW1 (plus 15% mixed) and 9% WU20 to 50% CW1 (plus 41% mixed) ratios at t0 and t0.5 are succeeded by a marked increase of the WU20 proportion. WU20 thus accounts for 67%, 71%, and 52% of infectious viral progenies at t1, t2, and t4, and consistently reaches values of over 94% from an eight-hour superinfection delay (t8) onwards. CW1 and mixed progeny proportions correspondingly decrease following the one-hour superinfection delay (t1). A WU20 to CW1 starting MOI ratio of 1 to 10 (expected to yield infectious progeny virus proportions of 10% WU20 to 90% CW1) resulted in WU20 proportions of 3% following coinfection (t0) and 6%, 0%, and 3% following early superinfection delays (t0.5 to t2). From t4 onwards, WU20 quantities are seen to progressively increase, accounting for 21% (t4), 46% (t8), and 100% (t12 and t24) of infectious virus progenies. Viral recombination has been identified as a key mechanism shaping the evolution and diversity of NoVs ,26,27. IHere, we determined the effect of a temporal separation of in vitro infections with the two homologous MuNoV strains MNV-1 WU20 and CW1 on the composition of nascent MuNoV populations. In utilising an in vitro system, we excluded both environmental and host influences and were thus able to examine only those effects mediated by the viruses themselves.Subsequent to initial WU20 and CW1 cell coinfections or superinfections with half-hour- to 24 h-delays and varying input MOIs , followed by a 24-h propagation step, individual viral progeny distributions were analysed via qPCR. This quantitative analysis revealed a disproportionate dominance of primary infecting WU20 genomic copies in the majority of resulting viral progenies. While the WU20 dominance appeared to be near-independent of the input MOI ratios of the two viruses (and indeed skewed expected genomic copy ratios throughout), it was markedly time-dependent; increasing CW1 superinfection delays from one to two hours onwards were associated directly with increasing WU20 genome copy fractions. While primary infecting WU20 is expected to have undergone a round of replication before addition of CW1 at superinfection delays of more than eight hours , input and expected ratios deviate significantly even at earlier time points where this effect cannot serve to explain the observed WU20 dominance.10 WU20 to CW1 genomic copy proportions. Vacillating levels of infectious virus and genomic copies have previously been associated with the presence of defective interfering (DI) RNAs or DI particles within NoV populations [Interestingly, the way in which higher-than-expected WU20 genomic copy numbers skewed expected genomic copy ratios even after coinfections or short superinfection delays may hint at the mechanism of the pronounced dominance following longer delays. Where input MOIs of 1:0.1, 1:1, and 1:10 were expected to yield WU20 to CW1 genomic copy ratios of 90% to 10%, 50% to 50%, and 10% to 90% following coinfection, these expectations were frustrated in the face of 3.40 (±0.43) to 1.79 (±0.75), 3.17 (±0.33) to 2.49 ±0.54), and 3.66 (±0.24) to 3.23 (±0.96) logulations . DI RNAsulations ,53. DI R, and 3.6Relative proportions of infectious viruses isolated from viral progenies following coinfection (t0) or short superinfection delays of up to two hours (t0.5 to t2), support a possible role of WU20 DI RNAs. Thus, e.g., coinfection with a one to ten WU20 to CW1 MOI ratio resulted in skewed genomic copy ratios of 45.57% WU20 to 54.43% CW1, but translated into infectious virus ratios of 3% WU20 and 91% CW1 (plus 6% mixed). Following asynchronous infection with longer delays (t4 to t24), a time-dependent WU20 dominance and corresponding CW1 decrease is evident within infectious virus progenies. Mixed populations registered subsequent to two-hour superinfection delays may indicate that that the barrier is established progressively and is, initially, not strong enough to completely repel superinfecting CW1, especially in the face of high input titres. Taken together, these results demonstrate that a time interval from one to two hours onwards between two consecutive in vitro MuNoV infections allows establishment of a barrier that progressively reduces or prevents superinfection. While viral interference, or superinfection exclusion, has hitherto not been described for NoVs, it is well documented for other positive sense, single-stranded RNA viruses, such as hepatitis C-, bovine viral diarrhoea-, and West Nile virus and may be established within 30 min to several hours of primary infection ,55,56,57In future investigations it will be interesting to leverage population-level deep sequencing to analyse how the viral interference effects pinpointed here may influence the generation of NoV RNA recombinants (and thus ultimately influence the chances of recombinant virus generation under the application of selective pressures). Further work should also focus on the mechanism of NoV interference  and will investigate whether the observed block can be overcome by superinfecting viruses.Understanding the influence that viral interference may have on NoV population dynamics has clear implications for NoV epidemiology and risk assessment. The phenomenon is thought to decrease the evolution of drug resistance and immune escape by limiting population variability and virus recombination . Identif


Topoisomerases, targets of inhibitors used in chemotherapy, induce DNA breaks accumulation leading to cancer cell death. A newly synthesized copper(II) indenoisoquinoline complex WN197 exhibits a cytotoxic effect below 0.5 µM, on MDA-MB-231, HeLa, and HT-29 cells. At low doses, WN197 inhibits topoisomerase I. At higher doses, it inhibits topoisomerase IIα and IIβ, and displays DNA intercalation properties. DNA damage is detected by the presence of γH2AX. The activation of the DNA Damage Response (DDR) occurs through the phosphorylation of ATM/ATR, Chk1/2 kinases, and the increase of p21, a p53 target. WN197 induces a G2 phase arrest characterized by the unphosphorylated form of histone H3, the accumulation of phosphorylated Cdk1, and an association of Cdc25C with 14.3.3. Cancer cells die by autophagy with Beclin-1 accumulation, LC3-II formation, p62 degradation, and RAPTOR phosphorylation in the mTOR complex. Finally, WN197 by inhibiting topoisomerase I at low concentration with high efficiency is a promising agent for the development of future DNA damaging chemotherapies. Adenocarcinomas are the most diagnosed cancers. Among them, breast and cervix, respectively the first and fourth most represented cancers in women, and colorectal cancers the second and third most represented cancers respectively in women and men . CurrentTopoisomerases (Top) regulate DNA topology during replication, transcription, and chromosomal segregation –6. To reTop inhibitors display different action mechanisms. Poisons target the DNA/topoisomerase cleavage complex, form a ternary complex  inhibiting DNA religation, and result in persistent DNA breaks . CatalytSince the discovery of platinum anticancer properties and the use of cisplatin, a platinum-based alkylating agent, and its derivatives in chemotherapy –39, othe50 below 0.5 µM) on the three cell lines and significantly below the value of human non-tumorigenic epithelial cell line MCF-10A (IC50 1.08 µM). WN197 acts as a Top1 poison and displays DNA intercalation properties. The action mechanism of WN197 is further deciphered to bring insights into its efficiency. DNA damage is detected by the presence of a rapid increase in nuclear phosphorylated H2AX (after 30 min of treatment with 0.5 µM) and the main DDR kinases are activated by phosphorylations. Cell cycle arrest in the G2 phase is confirmed by the inhibitory phosphorylation of Cdk1 on tyrosine 15, an accumulation of cyclin B, and the unphosphorylated form of histone H3. Furthermore, the cell cycle is halted in G2 by inhibitory phosphorylation of Cdc25C on serine 216 associated with a binding to the 14.3.3 chaperon. Cancer cells halt in G2, die by autophagy detected through an increase in Beclin-1, and a decrease in the LC3-I/LC3-II ratio and the p62 marker. Moreover, the RAPTOR component in the mTORC1 complex is phosphorylated on serine 792, a feature of autophagic-induced cell death.As a part of an ongoing effort to develop new efficient anticancer organometallic drugs and to palliate limitations in drug resistances and/or side effects, the synthesis of a novel copper(II) complex of indenoisoquinoline ligand, named WN197, is established based on previous studies , 55. Thi® PLUS . The molar conductance measurement was carried out using a CDRV 62 Tacussel electronic bridge, employing a calibrated 10-2 M KCl solution and 10-3 M solutions of compounds in DMSO. Purities of all tested compounds were ≥95%, as estimated by HPLC analysis. High Resolution Mass Spectrum (HR-MS) was measured at REALCAT (Université de Lille) on a Synapt G2Si (Waters) equipped with an ion mobility cell.All commercial reagents and solvents were used without further purification. Cisplatin is purchased from Alfa Aesar ; rapamycin from Abcam ; doxorubicin, nocodazole and DMSO from Sigma-Aldrich . Stock solutions were prepared in DMSO. Melting points were determined with a Barnstead Electrothermal (BI 9300) capillary melting point apparatus and are uncorrected. Elemental analyses were performed with a varioMICRO analyser. Thin layer chromatography (TLC) was carried out on aluminium-baked  silica gel 60. Column chromatography was performed on silica gel (230-400 mesh). The electronic absorption spectra were acquired on a UV-Vis double beam spectrophotometer SPECORD2  in MeOH (7 mL). After stirring at room temperature for 10 h, the reaction mixture was filtered off to yield an orange precipitate which was washed with MeOH and dried under vacuum (8 h at 100°C). Yield: 132 mg (70%). Decomposition at 194°C. Anal. Calcd. for C44H54Cl2CuN6O8 (%): C, 56.86; H, 5.86; N, 9.04: Found C, 56.76; H, 5.89; N, 9.22. FT-IR (neat) : 1650 (C=O), 1549 (C=C), 490 (Cu-N). UV-vis in DMSO-H2O (19/01), λ/nm (ϵ/M−1cm−1): 625 (156), 463 (4500) (9800), 353 (17620), 350 (18100), 328 (16440). ΛM  (S cm2 mol-1): 24. HRMS (ESI) m/z: calcd for [M]+ C44H46ClCuN6O4 820.2565; Found 820.2332.The equations should be inserted in editable format from the equation editor.WN170 was synthesized according to the literature procedure . To a so2 in DMEM medium  supplemented with 10% fetal bovine serum , 1% Zell Shield  and 1% non-essentials amino-acids . MCF-10A were maintained in MEBM medium  supplemented with MEGM . All cell lines culture media were added with 1% Zell Shield .HeLa, MDA-MB-231, HT-29 and MCF-10A cell lines originate from ATCC , and were maintained at 37°C in a humidified atmosphere with 5% CO® AQueous One Solution Cell Proliferation Assay . 2.103 cells well were seeded in 96-well plate for 24 h before treatment with 0 to 100 µM of WN197, WN170 or cisplatin for 72 h. After a 2 h incubation with 20 µL of CellTiter solution at 37°C in 5% CO2, the production of reduced MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) in formazan was measured at 490 nm . IC50 were calculated using GraphPad Prism V6.0 software. Statistical differences between WN197 and WN170 were ascertained by a Student t-test (**p<0.01 and ****p<0.0001).Cell viability was determined using CellTiter 965 cells seeded on glass coverslips were treated with 0.5 µM of WN197 or WN170, 5 µM of doxorubicin, 20 µM of cisplatin as positive controls, or 0.1% DMSO as a solvent control for 30 min or 24 h. Fixation was performed with 4% paraformaldehyde  for 5 min and followed by permeabilization with 0.1% Triton in PBS  for 10 min and saturation of unspecific sites with 1% BSA in PBS (Sigma) for 1 h at room temperature. Anti-γH2AX mouse antibody  was incubated overnight at 4°C, washed 3 times with 1% BSA/PBS. Cells were incubated with secondary anti-mouse IgG  for 1 h at room temperature in the dark, washed 3 times before nuclei were stained with DAPI . Images were captured under a Leica fluorescent microscope, and γH2AX foci were counted with ImageJ  on 30 cells from 3 independent experiments and quantified with GraphPad Prism V6.0 software. Statistical significances (mean ± SD) were performed by a two-way ANOVA followed by Dunnett’s multiple comparison test .2.105 cells were seeded for 24 h and treated with 0.5 µM of WN197 or WN170, 20 µM of cisplatin, 5 µM of doxorubicin, or 0.1% DMSO (solvent control). After 24 h, they were lysed in RIPA buffer  supplemented with 1% of protease inhibitor cocktail  and phosphatase inhibitors .7.5.105 cells were seeded for 24 h and treated for 3 h, 16 h, 24 h or 48 h with 0.5 µM of WN197, and for 24 h or 48 h with 5 µM of doxorubicin as positive control. Cells were lysed in a glass grinder at 4°C in homogenization buffer .For cytochrome C analysis, 7.5.10Samples were centrifuged for 10 min at 12,000 G and protein concentration of supernatants were determined using the Bradford assay  at 595 nm . Proteins were denatured in 2X Laemmli buffer  at 75°C for 10 min. 15 µg of proteins were separated on 4-20% SDS PAGE gels , for 1 h at 200 V in denaturing buffer . Proteins were transferred onto nitrocellulose membrane  by wet transfer , for 1 h at 100 V. Membranes were saturated with 5% low fat dry milk in TBS added with 0.05% Tween , and incubated overnight at 4°C with specific primary antibodies: rabbit polyclonal antibodies were against ATM , 1/1000), ATR , phosphorylated ATR , Beclin-1 , Cdc25C , phosphorylated Cdc25C , phosphorylated Cdk1 , phosphorylated Chk1 , phosphorylated Chk2 , cleaved caspase 3 , phosphorylated H2AX , histone H3 , phosphorylated H3 , phosphorylated p53 , p53 , p21 , LC3 , mTOR , RAPTOR , phosphorylated RAPTOR ; mouse monoclonal antibodies against phosphorylated ATM , Santa Cruz, CA, USA, 1/200), Chk1 , Chk2 , Cdk1 , 14-3-3 , cyclin B2 , p62 ; goat polyclonal antibodies against β-actin ; and cocktail antibodies against cleaved PARP . After three washes of 10 min in TBS-Tween, nitrocellulose membranes were incubated 1 h with the appropriate horseradish peroxidase-labeled secondary antibodies: anti-rabbit or anti-mouse antibodies  or anti-goat antibodies . Secondary antibodies were washed in TBS-Tween three times for 10 min and the signals were revealed with a chemiluminescent assay  on hyperfilms . β-actin or histone H3 were used as loading controls. Signals were quantified with Image J , and normalized to respective loading control. The means of 3 independent experiments were calculated.Topoisomerase activities were examined in assays based on the relaxation of a supercoiled DNA into its relaxed form. Topoisomerase I (Top1) activity was performed using the drug screening kits protocol . The reaction mixture was composed of supercoiled pHOT1 DNA (250 ng), 10X TGS buffer , 5 units of Top1, a variable amount of compound to be tested, and a final volume adjusted to 20 µL with H2O. WN197 was tested at concentrations ranging from 0.2 to 2 µM. Camptothecin (10 µM) was used as a positive control (poison inhibitor of Top1 activity), etoposide (100 µM) as negative control (inhibitor of Top2 activity), and 1% DMSO alone as vehicle control. Relaxed pHOT1 DNA (100 ng) was used as migration control. The addition of proteinase K (50 µg/mL) for 15 min at 37°C allowed Top1 degradation to visualize the cleavage products (nicked DNA). Reaction products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 µg/mL) for 1 h at 100 V in TAE  buffer.Topoisomerase II Relaxation Assay Kit  was used to measure topoisomerase II (Top2) activity. The reaction mixture was composed of supercoiled pBR322 DNA (1 µg), 10X assay buffer (50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 100 µg/mL albumin), 30 mM ATP, 5 units of Top2α or Top2β, variable amount of compound to be tested, and a final volume adjusted with H2O to 30 µL. Etoposide  was used as positive control, and camptothecin (10 µM) as negative control. The mixtures were incubated at 37 °C for 30 min and the reactions stopped by the addition of 5 µL 10% SDS. Reaction products were separated by electrophoresis in a 1% agarose gel for 1 h at 100 V in TAE buffer, and stained with ethidium bromide (0.5 µg/mL) for 15 min. After destaining in water, the DNA migratory profiles were visualized under UV light .2PO4, 6 mM Na2PO4, 1 mM EDTA, pH 7.1) in the presence or not of 20 µM DNA from calf thymus . Absorbances were measured at 260 nm (Uvikon 943 coupled to Neslab RTE111) every minute over the range of 20 to 100°C with an increment of 1°C per minute. All spectra were recorded from 230 to 500 nm. Tested compound results are referenced against the same DNA concentration in the same buffer. The Tm values were obtained from the first derived plots.Melting temperatures were obtained as described , 55. 20 50 values for ethidium bromide (EB) displacement were calculated using a fitting function incorporated into GraphPad Prism 6.0 software. The apparent binding constants were calculated using the equation Kapp = (1.26 (Kapp(EB)/IC50) with Kapp(EB) =107 M−1 and IC50 in μM.Fluorescence titrations were determined as described , 55. Eth5 cells plated for 24 h were treated with 0.5 µM WN197 or WN170, 20 µM of cisplatin (S phase arrest control), 83 nM of nocodazole (M phase arrest control), or 0.1% DMSO (solvent control). For the dose titration experiments, cells were treated for 24 h with increasing concentrations of WN197. For kinetic experiments, cells were treated with 0.5 µM of WN197 or WN170 from 4 to 48 h. Cells were detached using trypsin , centrifuged at 1,000 G for 10 min, resuspended in PBS, and fixed with 70% ethanol at -20°C for 24 h, before they were centrifugated , resuspended in PBS, and treated for 15 min at room temperature with RNase . Finally, incubation with propidium iodide  at 4°C for 30 min was performed before flow cytometry  analysis. For each sample, 10,000 events (without cell doublets and cellular debris) were considered. The cell cycle repartition was analyzed with Graphpad Prism V6.0 software. Statistical significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett’s multiple comparison test .7.5.10Cell lysates were obtained as described in the Western blot section. Samples were pre-cleared with protein A sepharose  for 1 h at 4°C under gentle rocking. After brief centrifugation, supernatants were incubated with antibodies against 14.3.3 , Cdc25C  or mTOR  at 4°C for 1 h under rotation and followed by incubation with protein A sepharose  for 1 h at 4°C under rotation. Samples were rinsed 3 times with RIPA buffer. Pellets were collected by brief centrifugation, resuspended in 2X Laemmli buffer, and heated at 100°C for 10 min before SDS-PAGE and Western blots were performed.2. After purification, WN197 was obtained in 70% yield.The synthesis of WN197 is described in 50 obtained are respectively 0.144 µM, 0.22 µM, and 0.358 µM for WN197 below the cisplatin IC50 values ranging from 10 to 40 µM. The copper-free indenoisoquinoline ligand, WN170, affected cell viability at higher doses , showing that the presence of the copper metal significantly enhances the anticancer effect of the indenoisoquinoline core for MDA-MB-231 and HeLa cell lines. A significantly higher IC50 (1.080 µM) is obtained on MCF-10A compared to the adenocarcinoma cell lines (Cells viability was assayed on the triple-negative breast cancer cells (MDA-MB-231), the cervix cancer cells (HeLa), and the colorectal cancer cells (HT-29) Table 1.foci were visualized in the nucleus at 0.5 µM of WN197, a concentration close to the IC50 determined previously, in MDA-MB-231, HeLa, and HT-29. After 24 h of treatment, the average number of γH2AX foci per cell were respectively 99, 98, and 70 for MDA-MB-231, HeLa, and HT-29 cells , and higher than the number of γH2AX foci triggered by an alkylating agent, cisplatin (average of 55 foci per cell). WN197 induced more DNA damage than the indenoisoquinoline WN170 (average of 23 foci per cell). Controls with DMSO solvent showed a low number of foci (average of 9 foci per cell for the 3 adenocarcinomas) compared to treated conditions  test relies on the ability of Top1 to relax supercoiled DNA, and the absence of relaxed DNA implies inhibition of Top1 activity. In the presence of Top1, supercoiled DNA showed a relaxed profile (To determine whether the Cu(II)-complex WN197 is a topoisomerase inhibitor, Top1 inhibitors can act either as catalytic inhibitors by DNA intercalation at the Top1 fixation site or as poisons, forming a ternary complex (DNA + Top1 + compound) , 25, preThe effect of WN197 on Top2α and Top2β activities were also assayed. The same principle based on the inhibition of topoisomerase-induced DNA relaxation was used  against thermal denaturation was used as an indicator of the capacity of indenoisoquinoline derivatives to bind and stabilize the DNA double helix. The Cu(II) indenoisoquinoline complex WN197 displayed a slightly higher ΔTm value compared to the metal-free indenoisoquinoline WN170 , showing a better binding affinity with DNA Table 3.7 M−1) is higher compared to the original ligand value (2.436 ± 0.883 107 M−1). These results are in agreement with the ΔTm values showing that the complexation of indenoisoquinoline ligand by copper allows a stronger interaction with DNA  complex , while p21 was highly increased in HT-29 cells (by a factor 8.3) compared to MDA-MB-231 and HeLa (respectively 1.3 and 2.2). The WN170 values are slightly identical except for p53 and p21 in MDA-MB-231 (respectively factors 0.4 and 1.0). In doxorubicin and cisplatin treated cell lines, p53 and p21 were not increased except for p53 in MDA-MB-231 and p21 in HT-29 cells.p53 facilitates cell cycle arrest by targeting p21The cell cycle repartition following the DDR pathway activation was monitored by flow cytometry in cells exposed for 24 h to different treatments , 58, indTo determine the lower dose necessary to induce a G2/M phase accumulation, flow cytometry experiments were performed with increasing concentrations of WN197 and results are shown in A kinetic of treatment with WN197 (0.5 µM) was realized on the three adenocarcinoma cell lines by flow cytometry to determine the earliest-induced G2/M accumulation  as indenoisoquinoline derivatives  complex with DNA can be attributed to the π-cation interaction between the base pairs and the atom of Cu(II) coordinated with ligands, but also to the capability to increase the π-π interaction between the base pairs of DNA and a second ligand molecule  indenoisoquinoline complex WN197 displays an anti-cancerous activity at low doses inhibiting Top1. MDA-MB-231 (triple negative breast cancer cells), HeLa (cervix cancer cells), and HT-29 (colon cancer cells), cancer cells accumulate DNA breaks and arrest in the G2 phase of the cell cycle. This arrest is characterized by the inactivation of the Cdc25C phosphatase through phosphorylation on serine 216 and binding to 14.3.3 that consequently leaves in its inactive form the MPF (a phosphorylated form of Cdk1 associated to accumulated cyclin B). Autophagy is further processed by the RAPTOR effector phosphorylation in the mTOR complex, and associated to p21 overexpression. WN197 appears as a new efficient drug to counteract cancer cells when used at low doses. The action mechanism of the copper complex is summarized in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceptualization: CM, LP, KC, and AM. Performing experiments: CM, NW, TB, LP, KC, and AM. Manuscript reviewing: A-SV-E. Writing and editing: CM, LP, KC, and AM. All authors contributed to the article and approved the submitted version.CM is a recipient of a doctoral fellowship from the French ministry. This work was supported by the CNRS, the University of Lille, and by grants from the “Ligue Contre le Cancer, Comités Nord et Aisne” (AM).Author NW was employed by AGAT Laboratories, Intertek.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hence, this study aims to investigate the effects of a diarylpentanoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13) on cytotoxicity, anti-proliferative, apoptosis-inducing, anti-migration properties, and the underlying molecular mechanisms on treated androgen-independent prostate cancer cells, DU 145 and PC-3. A cell viability assay has shown greater cytotoxicity effects of MS13-treated DU 145 cells (EC50 7.57 ± 0.2 µM) and PC-3 cells (EC50 7.80 ± 0.7 µM) compared to curcumin . In addition, MS13 exhibited significant anti-proliferative activity against AIPC cells compared to curcumin in a dose- and time-dependent manner. Morphological observation, increased caspase-3 activity, and reduced Bcl-2 protein levels in these cells indicated that MS13 induces apoptosis in a time- and dose-dependent. Moreover, MS13 effectively inhibited the migration of DU 145 and PC-3 cells. Our results suggest that cell cycle-apoptosis and PI3K pathways were the topmost significant pathways impacted by MS13 activity. Our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration as well as inducing apoptosis in AIPC cells.Diarylpentanoids exhibit a high degree of anti-cancer activity and stability  Prostate cancer (PCa) is the most common malignancy and the second-leading cause of cancer-related death among men worldwide . AndrogeCurcuma longa that demonstrated a variety of pharmacological activities, including anti-tumour, anti-inflammatory and chemopreventive properties (in vitro and in vivo (Curcumin (diferuloylmethane)  is a natoperties . Curcumi in vivo . Cell-ba in vivo . Curcumi in vivo . Also, c in vivo . Despite in vivo , its app in vivo . These l in vivo .in vitro over its parent compound and other diarylheptanoids in a wide range of cancerous cells (in vitro and in vivo models (Diarylpentanoids (DAPs) have gained interest to researchers due to the high degree of anti-cancer activity and stability us cells . These aus cells  and imprus cells . DAPs hao models . Studieso models . Severalo models , WZ35 -1,4-pentadien-3-one), a DAP, , cervicaCurcumin analogue, MS13 -1,4-pentadien-3-one), with 326 molecular weight, is a chemically purified diarylpentanoid synthesized by coupling the appropriate aromatic aldehyde with acetone under base catalysed aldol condensation, using a 1:2 ratio of ketone to aldehyde. The compound was determined based on the analysis of spectroscopic data and the comparison of the data with the related compounds. Curcumin was purchased from Sigma Aldrich . The stock solution of both compounds was prepared by dissolving in dimethyl sulfoxide  to a final concentration of 50 mM.® HTB-81™ and PC-3, ATCC® CRL-1435™) cells and human epithelial hepatocytes cell line  cells were purchased from American Type Culture Collection . DU 145 and WRL 68 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) meanwhile, PC-3 cells were cultured in Ham’s F-12 medium (F-12K) . These cells were incubated at 37°C in a humidified atmosphere of 5% CO2. All the cell lines were maintained in the respective media supplemented with supplemented with 10% (v/v) fetal bovine serum (FBS)  and 1% penicillin (100 U/mL)/streptomycin (100 μg/ml) .The androgen-independent human prostate cancer cell lines  and diluted in the culture media to working concentration 200 μM. Curcumin was used as a positive control was also prepared in a similar manner. A two-fold serial dilution was performed for each sample in triplicates down the columns of the plate, yielding a final volume and concentration of 100 μL in 0.2% DMSO in each well. Control cells were treated with media containing 0.2% of DMSO. The cell viability and anti-proliferative assay were determined by using 3--2,5-diphenyltetrazolium bromide (MTT) assay  in triplicates at a density of 1 × 10T) assay . Followi50) value was generated using GraphPad Prism version 7.0 . EC50 value represents the concentration at which the tested compound caused 50% growth inhibitory effect averaged from triplicates absorbance values. Selectivity index (SX) is the degree of selectivity of the compound tested against the cancerous cell, in which values larger than “100” indicates the compound is selective towards cancerous cells and conders minimal toxicity toward non-cancerous cells  and incubated overnight until they reached 80–90% confluency. Cells were exposed to two different concentrations of MS13 for 24, 48 and 72 h, and each experiment included a set of control cells. A fresh clean pellet of the harvested cells was re-suspended in 150 ml ice-cold 1 × PBS. A mixture of the fluorescent dye staining solution (1:1), comprising 50 μg/ml Acridine Orange and 50 μg/ml Propidium iodide were prepared. A total of 10 µL of AO and PI were added to the pellet and incubated at room temperature in the dark for 5 min. The freshly stained cell suspension was dropped onto a glass slide and observed under a UV-fluorescence microscope (Olympus BX41) attached with Leica LAS X software. Fluorescent detection was observed under the fluorescence microscope using a dual filter set for FITC (green) and rhodamine (red). The different morphological criteria were used to clarify healthy live cells, early or late apoptotic and necrotic cells. The viable cells show an intact green nucleus with round intact structure, an early apoptosis display a dense bright green fluorescent cells exhibits distinct morphological changes like membrane blebbing, shrinkage of the cells and chromatin condensation. On the other hand, late apoptotic cells and necrotic cells will stain with both AO and PI. Hence, late apoptosis exhibits a yellowish orange to bright red cells with bright yellow beads nucleus whilst necrosis appeared as uniformly stained red with intact nuclei  and propidium iodide (PI) double staining. This method was used to differentiate between viable, non-viable cells and cells that undergo early or late apoptosis and necrosis. Cells were seeded in T25 cmt nuclei . The per2 flasks (Nunc) and then treated with MS13 at different concentration (EC50 and 2x EC50) at various time point 24, 48 and 72 h. Each experiment included a set of control cells (media with DMSO). Harvested cells were resuspended in chilled Cell Lysis Buffer, and the concentration of the protein lysate was determined by using Pierce BCA Protein Assay Kit  based on manufacturer’s protocols. The 2x Reaction Buffer (containing 10 mM DTT) was added to each sample for each assay. The reaction was started by the addition of 5 μL of the 4 mM DEVD-pNA substrate incubated at 37°C for 2 h. The absorbance was measured at 400 nm using a microplate spectrophotometer . The data was presented in the fold-change of absorbance from treated cells against absorbance from control based on the equation below:Caspase-3 activity assay was performed using the Caspase-3 Colorimetric Assay Kit , following the manufacturer’s instruction. This assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labelled substrate DEVD-pNA, the pNA light emission can be quantified using a spectrophotometer at 400 or 450 nm. Firstly, cells were seeded in T75 cm2 flasks (Nunc) and treated with different concentration of MS13 (EC50 and 2x EC50) at 24, 48 and 72 h. Each experiment included a set of control cells (media with DMSO). The concentration of protein lysate was also determined by using Pierce BCA Protein Assay Kit . The Bcl-2 concentrations of the samples were obtained by comparing the absorbance obtained against the standards at 450 nm by using a microplate spectrophotometer . Data was presented in the fold-change of absorbance from treated cells against absorbance from untreated cells (control) based on the equation below:Bcl-2 cellular protein concentration was quantified using the Human Bcl-2 Platinum ELISA Kit  based on the manufacturer’s instruction. The kit is able to detect the human Bcl-2 by solid-phase sandwich ELISA which is designed to measure the amount of target bound between a matched antibody pair. The cells were seeded in T75 cm50 and 2× EC50), incubated at 37°C, and photographed under a microscope for 0, 6, 12, 24, and 30 h. The control well was added with media and DMSO. The anti-migration ability of MS13 was determined by using in vitro scratch wound assay and analysed by using the ImageJ software based on the method described by Cells were seeded in triplicates in 24 well plates  overnight at 37°C in a humidified incubator to a nearly confluent cell monolayer. The monolayer cells then carefully scratched using a yellow pipette tip to draw a liner “wound” of each well, and the debris was removed. The cells were treated with MS13 .All samples were measured in triplicates from three independent experiments. Comparison between sets of data was performed by using one way and two-way analysis of variance (ANOVA) followed by Dunnett’s multiple group comparison test. Results presented as mean ± standard deviation (SD). Statistically significant differences between groups were accepted at 7 cells in T75 cm2 flasks (Nunc) and incubated overnight to allow cells attachment at 37 °C in a humidified incubator with 5% CO2. The cells were incubated with 16 μM of MS13 for 24 h duration based on the EC50 values. Meanwhile, the untreated cells received only media with only DMSO. After 24 h treatment, the treated cells were harvested by pre-warmed accutase® Cell Detachment Solution , washed with 2 ml of 1× PBS (Corning®) and centrifuged twice to obtain a fresh clean pellet. Then, the fresh pellet was re-suspended in 1,000 ml of 1x PBS (Corning®), kept on ice and further to RNA extraction. The experiment was performed in three biological replicates.In order to examine the gene expression profiling affected by MS13 on the prostate cancer cell lines, both DU 145 and PC-3 cells were seeded at the density of 1 × 10® analysis. A nCounter PanCancer Pathway Panel  was used. Triplicate wells were assayed for each experiment, and three independent experiments were performed.Total RNA was extracted from MS13-treated DU 145 and PC-3 cells and untreated controls using the RNeasy Mini Kit  as per the manufacturer’s instructions. The QIAshredder spin columns  was used for homogenization. RNA concentration and purity were determined by using a NanoPhotometer . A ratio of absorbance at 260/280 nm ∼2.0 was accepted as pure and was used for Nanostring® nCounter Technology , using the nCounter PanCancer Pathway Panel , which consists of a panel 770 genes associated with 13 pathways of cancer. Results were analysed using the Nanostring nSolver 4.0 and Advanced Analysis Module, plugin (Ver.2) with R statistical software and normalization of housekeeping genes were obtained from the geNorm algorithm. Differential expression of the treated cells was compared with untreated cells (control), with the fold change and p values calculated using nSolver default settings. Genes were considered as significant differentially expressed genes (DEGs) based on the criteria of fold-change (FC) ≥ 2, p. adjusted ≤0.05, and a false discovery rate (FDR) ≤ 0.05.Gene expression profiling was performed by Nanostringin vitro dose-dependent cytotoxicity and selectivity index effects, DU 145, PC-3 and WRL 68  cell lines were incubated with different concentration of MS13. Overall, MS13 induced cytotoxic effects against DU 145 and PC-3 cells in a dose-dependent manner. MS13-treated DU 145 cells has shown a significant reduction of cell viability beginning from 3.1 μM onwards to about 97%. The cell viability was reduced to 78% at 6.3 μM, followed by less than 10% from 12.5 to 100 μM. Meanwhile, MS13-treated PC-3 cells has shown a significant reduction of cell viability by approximately 88% at 12.5 μM and continue to decrease below than 4% from 25 to 100 μM. Besides, curcumin showed a significant cell inhibitory effect at 50 μM with 25% cell viability in DU 145. However, in PC-3 treated cells, 57% cell viability was noted at 25 μM, while 10% was observed at 50 μM  while curcumin displayed a higher value of EC50 . Furthermore, MS13 also exhibited lower EC50 value for WRL 68 cells (9.31 ± 0.2 μM) compared to curcumin (26.45 ± 3.3 μM). (MS13 has exhibited lower EC3.3 μM).  this indSelective index values of MS13 and curcumin were also evaluated where the value greater than 100 suggests that the compounds are less toxic to normal cells but greater toxicity towards PCa cells. MS13 showed higher SX values in both PCa cells  compared to curcumin . In summThe evaluation of the anti-proliferative activity has shown that MS13 inhibited the proliferation of DU 145 and PC-3 cells in a dose- and time-dependent manner. The percentages of cell viability of the cells were decreased as the concentration of MS13 increased and prolonged exposure time. MS13-treated DU 145 cells have displayed significant inhibition of cell proliferation at 12.5 μM onwards for 24, 48 and 72 h. At 24 h, the cell viability was significantly reduced by approximately 29% and about 66 and 91% at 48 and 72 h, respectively. A significant gradual reduction of cell proliferation was observed when treated with 25–100 μM by 47–98% for all time points . LikewisThe apoptotic changes in terms of colour and morphology in MS13-induced DU 145 and PC-3 cells were analysed at 24, 48 and 72 h by using acridine orange (AO) and propidium iodide (PI) double staining technique. In MS13-treated DU 145, under untreated condition, viable cells are characterized by intact green-coloured cells exhibited a spherical structure, indicating no signs of apoptosis. After 24 h of incubation with 7.57 µM of MS13, a dense bright green fluorescent cells exhibited distinct morphological changes like membrane blebbing, shrinkage of the cells and chromatin condensation were increased, signifying a shift from viable to early apoptotic cells. As the concentration of MS13 increased to 15.14 μM, a higher proportion of a yellowish orange to bright red cells were observed, indicating the majority of the cells underwent late apoptosis. Meanwhile, after 48 h of incubation with 7.57 μM, a heterogeneous population of early and late apoptotic cells was noted, exhibited by a higher intensity of a dense bright green and a yellowish orange to bright red fluorescence cells. This indicated an increasing number of apoptotic cells with an increasing length of incubation. An increased number of cells turned from a dense yellowish orange to bright red cells was observed as the concentration of MS13 increased. Upon 72 h of incubation, as the mixture of cells emits dense yellowish orange to bright red cells and distinct apoptotic bodies increased, a proportion of reddish cells that defined necrotic cells were also increased. A higher proportion of uniform red or orange-stained cells were observed as the MS13 dose was increased .A similar pattern of cell proportion was observed in MS13-treated PC-3 cells . At the The percentage of viable, apoptotic and necrotic cells were quantitatively determined. An early and late apoptosis groups were combined to indicate an apoptotic activity for this analysis. In MS13-treated DU 145 cells, the percentage of viable cells was significantly decreased at 24 h in a dose-dependent manner when treated with 7.57 μM (39%) and 15.14 μM (36%) compared to the control (66%). After 48 h incubation, there was a greater reduction in the cell viability to 29 and 17% for 7.57 and 15.14 μM, respectively. The percentage of viable cells was further reduced significantly to 14% for both doses after 72 h. Untreated cells exhibited a high percentage of viable cells, with 57 and 45% after 48 and 72 h. The percentage of cells that underwent apoptosis at 24 h was relatively low at all treatment doses; 7.57 μM (58%) and 15.14 μM (59%). However, the percentage of apoptotic cells increased after 48 h with 68 and 72% when treated with 7.57 and 15.14 μM, respectively. The untreated cells that underwent apoptosis were relatively low for 24 h (32%) and 48 h (41%), including after 72 h (50%). The percentage of the necrotic cells was relatively low at 24 h, approximately 4% for all doses and increased from 3 to 11% at 48 h. Upon 72 h, the percentage of apoptotic cells showed 66 and 63% for both doses at 72 h. Although apoptotic was relatively high at 72 h but, necrotic cells were noted to increase at this time up to approximately 20–30% for 7.57 and 15.14 μM, respectively .The same dose-dependent pattern was shown in MS13-treated PC-3 cells. A significantly higher apoptotic activity was shown at 48 h when treated with 7.80 μM (61%) and 15.60 μM (80%) with relatively low necrotic cells, 8 and 9%, respectively. At 72 h, the percentage of apoptotic cells increased when treated with 7.8 μM (67%), with a slight increment following treatment with 15.60 μM (70%). The necrotic cells were highest at 72 h compared to 24 and 48 h with 20% for 7.8 μM and 24% for 15.60 μM. Besides, the viable cells were decreased as the doses increased compared to the control . In summIn MS13-treated DU 145 cells, the results showed a significant increase of caspase-3 activity at 48 h when treated with 7.57 μM. However, caspase-3 activity was slightly decreased as the dose increased to 15.14 μM. A similar trend was observed in MS13-treated PC-3 cells incubated with 7.80 and 15.60 μM at 48 h. There were no significant differences in caspase-3 activity at 72 h for both doses compared to untreated cells in both cells. In PC-3 cells, treatment with 7.80 μM showed no significant increase of caspase-3 activity at 24 and 72 h, but an increase was observed with 15.6 μM treatment for 24 h compared to untreated cells. The results indicated that the MS13 induces the highest caspase-3 activity at 48 h in both DU 145 and PC-3 cells .On the other hand, data analysis showed a significant and progressive decrease in Bcl-2 concentration from 24 to 72 h when treated with 7.57 and 7.80 μM of MS13 in DU 145 and PC-3 cells, respectively. However, increased doses of MS13 to 15.14 μM for DU 145 and 15.60 μM for PC-3 with a similar incubation period, a further reduction of Bcl-2 concentration was noted . Hence, A wound healing assay was performed to assess the effect of MS13 on cell migration. The results have shown that the control filled the entire wound gap after 24 h for DU 145 and PC-3 cells . Quantit50) were used for both DU 145 and PC-3 cells, incubated for 24 h. Genes with a filter of two-fold-cut-off, p. adjusted ≤0.05, and FDR ≤0.05 were defined as significant differentially expressed genes (DEGs). Overall, the data revealed that MS13 affected mutually regulated DEGs which associated with 12 canonical cancer pathways, including MAPK, JAK-STAT, PI3K, RAS, cell cycle-apoptosis, Wnt, DNA damage repair, driver gene, transcriptional misregulation, chromatin modification, notch and TGF-ß pathways.Anti-cancer properties of MS13 on DU 145 and PC-3 was further investigated using nCounter PanCancer Pathway Panel  gene expression analysis. Based on the apoptosis induction studies, the doses of MS13 at 16 µM (2x ECHowever, our findings indicated that the cell cycle-apoptosis and PI3K pathways were the most significantly dysregulated following MS13 treatment. A total of 18 mutually DEGs was associated with cell cycle-apoptosis pathway, of which 14 were downregulated, and 4 were upregulated . Besides50 values of MS13 for PC-3 and DU 145 cells were 7.80 ± 0.7 μM and 7.57 ± 0.2 μM, respectively, much lower than curcumin. Overall, these results are in agreement with previously published data (50 value compared to curcumin in multiple cancer cell lines, including colorectal (Based on the results, MS13 has shown to decrease the percentage of viable cells in both DU 145 and PC-3 in a dose-dependent manner. The EChed data . Besideshed data . An earlhed data . Moreovehed data . Additiohed data . Also, ohed data . Previoulorectal , nasophalorectal , breast lorectal , gastriclorectal  and panclorectal  cancer cApoptosis is closely related to the survival of cancers and has become an important factor in the development of anti-cancer therapeutic agents . MS13 haCaspases play an important in the execution of apoptosis and lead to the characteristic of morphology changes of the cells undergoing apoptosis, including cell shrinkage, chromatin condensation, and DNA fragmentation . Among tCell migration is a key feature of metastatic progression for cancers . Herein,For gene expression analysis, our results demonstrated that several DEGs are mutually regulated despite the different biological properties of DU 145 and PC-3 cells. PC-3 cells were derived from bone metastasis with high metastatic potential, harbouring wild-type p53, while DU 145 cells were derived from brain metastasis with moderately metastatic harbouring mutant p53. Treatment on DU 145 and PC-3 cells demonstrated that several DEGs were associated with cell cycle-apoptosis and PI3K pathways, as the most significantly impacted pathways indicating the anti-cancer activity of MS13. Despite numerous pathways that may potentially contribute to the progression of PCa, cell cycle-apoptosis and PI3K pathways were the most frequently altered in primary and metastatic prostate cancer .s (CCNA2, CCNB1, CDC25B, CDC25C, and CDC7 were downregulated in MS13-treated DU 145 and PC-3 cells. CCNA2 regulates the G1/S and the G2/M checkpoints . In PCa,s . In PCa,s . This suSkp2 ubiquitin ligase complex, is frequently overexpressed in many human cancers (In addition, SKP2, AKT1, PKMYT1, TTK and PRKDC were downregulated following MS13 treatment in DU 145 and PC-3 cells. SKP2, a critical component of the SCF cancers . SKP2 ov cancers . The dow cancers . This su cancers . Curcumi cancers . This ma cancers . PKMYT1  cancers . In agre cancers . This su cancers . Thus, i cancers . The PRK cancers . Thus, t2+-mediated signals and may has a pro-tumorigenic role in PCa (Besides, MS13 treatment in DU 145 and PC-3 cells has shown the downregulation of PPP3CB, TFDP1, SMAD3, and ENDOG, which the anti-cancer activity have not been reported in PCa but noted in other cancers. PPP3CB, which plays an essential role in the transduction of intracellular Cae in PCa . PPP3CB e in PCa . This sue in PCa . In agree in PCa . Thus, te in PCa . Inhibite in PCa . This sue in PCa . Inhibite in PCa . Moreovee in PCa . Thus, tOn the other hand, multiple genes, including CDKN1A, PIK3R3, GADD45B and GADD45A, were upregulated when exposed to MS13 treatment. CDKN1A acts as a potent cyclin-dependent kinase inhibitor, whereby its inhibition in PCa leads to cell cycle progression inhibition at G1 and induced G2 arrest . CurcumiMS13 treatment on DU 145 and PC-3 cells also led to the downregulation of several mutually regulated DEGs associated with PI3K pathway, including IRS1, ITGA3, ITGA6, and LAMA3. IRS1 is the principal effector of the insulin and insulin-like growth factor signalling pathways, by which its expression often linked to tumorigenesis and radioresistance . Upregulin vivo tumour growth, angiogenesis, and metastasis of lung, brain, and colon tumours (In contrast to the downregulated genes, several DEGs associated with PI3K pathway, including HSP90B1, PGF, and PPP2CB, were upregulated following MS13 treatment. HSP90B1 is a molecular chaperone protein in which its downregulation is associated with prostate carcinogenesis and metastasis . Therefo tumours . Thus, t tumours . Hence, The diarylpentanoid MS13 has demonstrated greater cytotoxicity, growth inhibitory and anti-migratory effects on androgen-independent prostate cancer cells than the parent compound, curcumin. In addition, MS13 mediates apoptosis confirmed by morphological observation, increased caspase-3 activity, and reduced Bcl-2 levels. The gene expression study revealed that mutually regulated DEGs were associated with cell cycle-apoptosis and PI3K pathways, as the most significant pathways targeted by MS13. Thus, our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration and inducing apoptosis in AIPC cells.


Cannabis sativa L. has been traditionally used for centuries as an analgesic and anti-inflammatory remedy also in different gastrointestinal disorders. This study aims to investigate the effects of different C. sativa isolated compounds in an in vitro model of intestinal epithelium. The ability of treatments to modulate markers of intestinal dysfunctions was tested on Caco-2 intestinal cell monolayers. Our results, obtained by evaluation of ROS production, TEER and paracellular permeability measurements and tight junctions evaluation show Cannabidiol as the most promising compound against intestinal inflammatory condition. Cannabidiol is able to inhibit ROS production and restore epithelial permeability during inflammatory and oxidative stress conditions, suggesting its possible application as adjuvant in IBD management.The relevance and incidence of intestinal bowel diseases (IBD) have been increasing over the last 50 years and the current therapies are characterized by severe side effects, making essential the development of new strategies that combine efficacy and safety in the management of human IBD. Herbal products are highly considered in research aimed at discovering new approaches for IBD therapy and, among others,  The gastrointestinal epithelium forms the body’s larger interface between the external environment providing a functional epithelial barrier that regulates the bi-directional flow of water, ions and macromolecules between the lumen and the host. The barrier selectively allows absorption of water and nutrients, while limiting the permeation of toxins and antigens . A breacThe selective barrier is provided by precise physical approximation between enterocytes, which is reinforced by tight junctions (TJs), the main responsible for paracellular permeability regulating apical cell-cell adhesion. Moreover, the intestinal barrier is furnished with immune and non-immune molecules, including mucosal immunoglobulins that contributed to the maintenance of gut homeostasis .The prevalence of IBD is around one in 1,000 people in Europe, with increasing incidence in westernized and industrialized countries . The twoThe current therapeutic choices for the management of IBD are focused on the symptomatology to induce and maintain the suppression of aberrant immune response. The pharmacological treatment includes corticosteroids, immunosuppressant agents and biological therapies with anti-tumor necrosis factor-alpha (TNFα) antibody as the mainstream treatment for down-regulating immune responses and inflammatory cascades . NeverthIt is therefore of particular interest to identify and develop new therapies that combine efficacy and safety for the management of these inflammatory chronic pathologies.Cannabis sativa L. has been used for many centuries to treat a variety of gastrointestinal conditions such as inflammation, infections, pain, disorders of motility and vomiting , which activates CB1 receptors in brain , are free from this kind of central effects, having low affinity for both CB1 and CB2 receptors  extracts were provided by a local producer. The scCO2 extract was obtained from the aerial parts of Cannabis sativa L. cultivated in the North-Eastern region of Italy (Veneto region), using scCO2 at 280 bar and 42°C. The decarboxylated extract was obtained by heating the scCO2 extract at 150°C for 5 h and controlling the modification of the extract composition by TLC and HPLC.CBD, THC, cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) analytical standards were purchased from Sigma Aldrich. Stock standard solutions were prepared in methanol at concentrations of 1 mg/mL and stored in the dark at −20°C; the working standard solutions of CBD and THC were diluted in methanol with a concentration of 100, 50, 10 and 1 μg/mL to prepare the calibration curves.The total extract was analyzed by HPLC-DAD (high-pressure liquid chromatography coupled with diode array detector) to quantify the content in active compounds. The sample was prepared dissolving 40 mg of the extract in 25 mL of ethanol with an ultrasonic treatment for 20 min. After centrifugation  the supernatant was transferred in 1.5 mL vials for the analysis. The main cannabinoids were identified using reference literature , and ideFor the HPLC analysis, an Agilent 1260 binary pump equipped with a 1260 auto-sampler, column oven and DAD 1260 series detector was used. Separation was achieved using an Agilent Eclipse XDB C-18  column as stationary phase. The binary gradient of elution using aqueous formic acid 0.1% (A) and acetonitrile (B) was as follows: from 65 to 100% of B in 30 min, then to 65% of B in 1 min and isocratic up to 36 min. The flow rate was set at 1 mL/min and injection volume was 10 μl.For preparative HPLC, the sample was prepared dissolving the extract in ethanol with a final concentration of 10 mg/mL, using an ultrasonic bath for 20 min.The preparative HPLC system consisted of a Varian 920 HPLC with quaternary pump equipped with UV-Vis detector. The chromatographic separation was performed on an Agilent Zorbax SB C-18 column . The mobile phase was delivered at a flow rate of 5 mL/min. The chromatographic run was performed with a binary, linear A/B gradient . The program was as follows: 0 min, 65% B; 30 min, 100% B; 34 min, 100% B; 35 min, 65% B, and isocratic up to 38 min. The injection volume was 30 μl. Each peak was collected, and the obtained fractions were evaporated to dryness under vacuum on a rotary evaporator at 60°C. The dry residues were re-dissolved in deuterated chloroform for characterization by NMR analysis.1HNMR, COSY, TOCSY, HSQC, and HMBC spectra were recorded on a Bruker Avance III spectrometer (400 MHz), using standard pulse sequences. NMR analyses were performed on the whole scCO2 extract and on all the purified fractions. 2D spectra were processed with Topspin 4.0.6 (Bruker) using zero filling to 1024 in F1 dimension, squared sine-bell apodization in both dimensions, prior to Fourier transformations.l-glutamine and 1% penicillin/streptomycin. Cells were maintained at 37°C under a humidified atmosphere of 5% CO2 in air. Experimental inflammatory condition in Caco-2 cell monolayers was induced by exposure for different times according to the assays, to 10 ng/mL recombinant human IFN-γ (Sigma-Aldrich) for 3 h and then 10 ng/mL TNF-α (Sigma-Aldrich) or to 500 μM H2O2, as previously described  supplemented with 10% FBS, 1% escribed . A 24 h 3) were seeded in 96-multiwells culture plates and treated with CBD, CBDA, THC and THCA (0.01–10 μg/mL) for 24-48-72 h. Cells were then washed, and fresh medium was added. MTT (5 mg/mL) was added to each well and incubated for 4 h at 37°C. Cells were lyzed with acidic isopropanol and the formazan absorbance was measured at 570 nm, using a Multilabel Plate Reader VICTOR™ X3 (Perkin Elmer).Cell viability was determined by the 3--2,5-diphenyltetrazolium bromide  assay, as previously described . Briefly2-DCF-DA, Sigma-Aldrich), as previously described  upon cleavage of acetate groups by intracellular esterase and oxidation. Briefly, Caco-2 cells (5 × 103) were seeded into 96-well plates and allowed to adhere overnight. 24 h treatment with CBD, CBDA, THC and THCA (0.01–10 μg/mL) was applied, followed by addition of 50 μM H2-DCF-DA, incubation for 30 min at 37°C and wash with phosphate-buffered saline (PBS). DCF fluorescence intensity was measured at excitation 485 nm—emission 535 nm, using a Multilabel Plate Reader VICTOR™X3  in absence or presence of 500 μM H2O2. Fold increase in ROS production was calculated using the equation: F is the fluorescence reading.ROS were quantified using 2′,7′-dichlorofluorescein-diacetate (Hescribed . The H2−4) were seeded on Transwell™ polyester membrane cell culture inserts  in 24-well plates and incubated with DMEM at 37°C in a humidified atmosphere and 5% CO2. Culture media was replaced every two days until confluent monolayer was obtained. The integrity of the cell monolayers was monitored by measuring the transepithelial electric resistance (TEER) from day 14th to day 21st after seeding. A 24 h pre-treatment was done adding CBD or THC (0.01–0.1 ug/mL) in the apical chamber. The TEER assay was performed in Hanks’ Balanced Salt solution  with 10 mM Hepes and 10 mM d-glucose (pH = 7.4), after an equilibration period at RT (® ERS meter (Millipore Corporation) connected to a pair of chopstick electrodes were inserted in the donor and receiver chambers and the 24 h-time courses of TEER variation was recorded (1-3-6-21-24 h). TEER was expressed as percentage of resistance, normalized to initial value  at excitation 480 nm—emission 530 nm.5 cells were seeded on glass coverslips precoated with gelatin  in 24-well plates, allowed to attach and reach the confluence for 5 days. Cells were then pretreated for 24 h with CBD and THC (0.1–1 μg/mL) following by addition of inflammatory stimulus (IFNγ 10 ng/mL for 3 h and TNFα 10 ng/mL for 21 h). At the end of treatment, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained for 1 h at 37°C with mouse monoclonal anti-occludin antibody (Invitrogen Life Technologies), rabbit anti-ZO1 antibody (Invitrogen Life Technologies). After PBS wash, cells were incubated with secondary antibodies/fluorescein isothiocyanate  for 1 h at 37°C. After washing, cells were incubated for 20 min with Hoechst  at RT. Th coverslips were then mounted on glass slides by using Mowiol 40–88 (Sigma-Aldrich) and images were acquired through confocal microscope LSM 800, magnification ×60, software ZN 2.1 blue Edition .15 × 10t-test was used to compare ROS values, TEER values and paracellular permeability; p values < 0.05 were considered statistically significant.The statistical analysis was performed using GraphPad Prism version 3 for Windows . Results are presented as mean ± SEM. Standard ANOVA procedures were performed for all the cell viability; the unpaired Student’s 2 extract were quantified by HPLC-DAD. A representative chromatogram is reported in 2 extract are reported in Cannabinoids in scCO2 extract using a semi-preparative HPLC method. The chromatographic gradient was maintained the same utilized for the characterization of cannabinoids in the scCO2 extract, to reproduce the elution of the peaks associated to each cannabinoid. The four isolated compounds, namely CBD, CBDA, THC and THCA, were characterized by 1HNMR, comparing the spectra with previously published literature  with those at δ 146.8 (C-4′), δ 112.0 (C-5′) and δ 103.5 (C-3′). Other significant correlations could be observed between the singlet at δ 6.26 (H-5′) and the signals at δ 114.5 (C-1′) and δ 175.0 (COOH). Diagnostic HMBC correlations of the core cyclohexane are those between the signal at δ 1.80 (CH3 in position 7) and those at δ 123.7 (C-2), δ 30.8 (C-4) and δ 139.5 (C-3). The signal at δ 5.56 assignable to the H-2 proton correlates with those at δ 31.7 and δ 37.4, ascribable to C-5 and C-1 carbons, respectively. Finally, other correlations are observed between the sp2 CH2 of the cyclohexane ring  and C-5, C-6 and C-10 carbons .The first compound, CNP-1, was assigned to CBDA. Characteristic signals in the NMR spectrum , and five CH, among which two sp2. Diagnostic HMBC correlations could be observed between the signal at δ 1.81 (attributable to the CH3 in position 7) and those at δ 139.4, δ 123.8, and δ 29.3, assignable respectively to the carbons C-5, C-8 and C-4. Overall, these data support the assignment of CBD for CNP-2 fraction , and the signals at δ 1.27 and δ 1.32 attributable to the intra-chain CH2 moieties. HSQC-DEPT analysis allowed to assign the signal at δ 0.88 to the proton H-5″ bonded to the carbon at δ 15.07, confirming its assignation. On the same way, H signals at δ 1.10 and δ 1.69 were associated to H-9 and H-3 bonded to carbons at δ 18.94 and δ 77.6, respectively. HMBC allowed to correlate the signal at δ 0.88 with the signals at δ 21.8 and δ 30.92 corresponding to C-4″ and C-3″, respectively, and the one at δ 1.27 with the carbon signal at δ 29.50 (C-2″), confirming the presence of the aliphatic chain. Diagnostic HMBC correlations for methyl groups were also observed: H-9 (δ 1.10) is correlated to C-8 (δ 29.08), C-6 (δ 45.7), and to a signal at δ 77.6 attributable to C-7. The proton in 8 (δ 1.42) correlates with C-6 (δ 45.7) and C-7 (δ 77.6), while the protons of the CH3 in position 7 (δ 1.69) correlate with C-4 (δ 31.02), C-2 (δ 123.6) and C-3 (δ 133.9).Finally, the spectrum of compound CNP-4 showed the characteristic signals of THC at δ 6.31, δ 6.28 and δ 6.15 integrating one proton each, and three singlets at δ 1.69, δ 1.42 and δ 1.10 integrating for three protons . The comThe NMR spectra and HPLC run also confirmed the purity of isolated fractions.As reported in Cannabis extracts on ROS production is depicted in Consistent increase of reactive oxygen and nitrogen species (ROS-NOS) production and increased epithelial permeability are among the factors implicated in IBD pathogenesis . The eff2O2) which induce a significant increase in ROS level . Only the treatments which resulted in a reduction of ROS production in both basal and oxidative condition  were applied.TEER and paracellular permeability were evaluated in Caco-2 monolayer in basal condition and after exposure to oxidative or inflammatory stimulus . Images in aloe vera, Boswellia serrata, tormentil extracts, mastic gum, etc. and some have been proven promising in IBD treatment  has been used as in vitro intestinal epithelial model and the effect of several cannabinoid extracts were tested in inflammatory-mimicking conditions. Beside the others, cannabinol resulted to be the most effective compound isolated from C. sativa at our purpose.The ability to modulate intestinal permeability during inflammation may be an important aspect to consider for therapeutic options to restore a leaky paracellular barrier. Thus, given the traditional benefic effect of cannabinoids in inflammatory intestinal conditions and the observational positive data, in this study we investigated the pharmacological activity of different cannabinoids isolated from Cannabis extract and its main constituents to modulate ROS levels of cells monolayer. CBD reduces oxidative stress both in basal and in oxidative stress conditions being able to counteract the overproduction of ROS species that are detrimental at epithelial level, inducing intestinal injury. Being the alteration of epithelial permeability a well-known factor involved in IBD development and maintenance, we tested the effect of Cannabis compounds on TEER and paracellular permeability of the intestinal monolayer. Interestingly, CBD has been shown to prevent tight junctions alterations in inflammatory conditions, allowing a better maintenance of intestinal epithelial barrier. TEER decrease and paracellular permeability increase, which are hampered by inflammatory stimulus, are prevented by cannabidiol treatment suggesting its protective role in gut homeostasis.Abnormally high levels of ROS are produced in IBD and could be a major contributing factor to tissue injury . The admOur study is located in the panorama of research aimed to elucidate the effectiveness of new herbal medicine strategies for IBD. Data acquired in this work underline the role of CBD as a potential modulator of markers of gut inflammation such as ROS production, alterations in the paracellular permeability and transepithelial resistance.in vitro and in acute and chronic animal models of inflammation (Several studies demonstrate that the non-psychotropic phytocannabinoid CBD may represent the most promising candidate for clinical utilization due to its lack of psychoactive actions . Data coammation  further


Breast cancer has been known as cancer with high mortality rates. It has been studied that MEX3A (Mex-3 RNA Binding Family Member A) is involved in carcinogenesis by accelerating cancer proliferation and migration. Therefore, this research aimed to study how MEX3A regulates the biological behaviors of breast cancer. Firstly, we used GEPIA and KM-plotter databases to evaluate MEX3A expression in human breast cancer tissue compared to adjacent normal tissue. Immunohistochemistry was employed to assess MEX3A protein expression in clinical specimens. MEX3A mRNA expression level was assessed through quantitative real-time PCR (RT-qPCR). Western blotting was used to detect protein expression. Moreover, Cell Count Kit-8 (CCK-8) assay, wound healing assay and transwell invasion assay were used to determine the proliferation, migration and invasion of breast cancer cells, respectively. Our study found that MEX3A expression level was much higher in human breast cancer tissues as compared to adjacent normal tissues. Similarly, breast cancer cell lines showed higher expression of MEX3A as compared to the normal breast cells. This higher expression of MEX3A was linked with the poor survival of breast cancer. Moreover, we found that overexpression of MEX3A stimulated proliferation and migration in the breast cancer cells. However, inhibition of MEX3A significantly reduced the proliferation and migration of breast cancer cells. In addition, we determined that MEX3A could activate RhoA/ROCK1/LIMK1 signaling in the breast cancer cells. Overall, our study concluded that MEX3A promotes its migration and proliferation in breast cancer cells via modulating RhoA/ROCK1/LIMK1 signaling pathway. Breast in vitro . MEX3A cin vitro . In addiin vitro .However, MEX3A dependent regulation of breast cancer has not been fully elucidated. Therefore, we designed this study to evaluate the expression of MEX3A in human breast cancer tissues and cell lines. Moreover, we also aimed to determine the functions of MEX3A in breast cancer cells. Finally, we assessed the MEX3A dependent regulation of RhoA/ROCK1/LIMK1 signaling pathway involved in the progression of breast cancer.2.2.1.All participants signed an informed consent form before their operation started. This study was approved by the ethics committee of our hospital, and carried out under the terms of the Helsinki Declaration. Forty-three breast cancer cases from June 2017 to March 2019 in our hospital were selected. We collected samples from mastectomy specimens from women undergoing surgery for breast cancer, and adjacent cancerous tissue specimens were obtained at least 5 cm away from the tumor. All breast cancer patients did not receive any treatment before surgery. All clinical specimens were kept at −196°C in liquid nitrogen until they were used for RNA extraction and other experiments .2.2.oC with 5% CO2 and 95% humidity [Four breast cancer cell lines  and human breast epithelial cell line (MCF-10A) were bought from BeNa Culture Collection Biological Technology Co. Ltd. . Cells were cultured in Dulbecco’s modified Eagle’s medium  added with 10% fetal bovine serum  and 1% penicillin-streptomycin . Cells were incubated at 37 humidity .2.3.Small interfering RNA (siRNA) of MEX3A (si-MEX3A) and the negative control siRNA (si-NC), pcDNAMEX3A (MEX3A) and the negative control pcDNA (NC) were synthesized by Genepharma (China). The transfection was facilitated with Lipofectamine® 3000 reagent following the manufacturer’s instructions  .2.4.2O2 at 25°C for 10 min. Following that, slides were soaked in distilled water for 10 min, blocked with 10% BSA at 25°C for 30 min, incubated with the primary antibody MEX3A  overnight. Afterward, tissue sections were rinsed 2 times for 5 min with PBS. Following that, these sections were incubated at for 60 min at 25°C. These tissue sections were added with diaminobenzidine (DAB) and then stained with hematoxylin for 30 s. Next, ethanol was used to dehydrate tissue sections and slides were mounted. Finally, the images of these tissue sections were taken. IHC results were independently scored by two senior pathologists in our hospital [Tissue sections (4 μm) were heated at 60°C for 30 min and next subjected to 3% Hhospital .2.5.Protein samples were collected from cell lysates. Protein was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and then were transferred to polyvinylidene fluoride (PVDF) membranes . At room temperature, these membranes were blocked in PBS containing 5% nonfat milk for 1 h, and then incubated with primary antibodies overnight at 4°C. The following were the primary antibodies in this research: anti-MEX3A , anti-RhoA , anti-ROCK1 , anti-LIMK1 , anti-β-actin , anti-RhoA (phospho S188) , anti-phospho-ROCK1 (Tyr913)  and anti-phospho-LIMK1 (Thr508) . After being washed with TBST, these membranes were incubated with secondary antibody at 25°C for 1 h. Protein blots were visualized using enhanced chemiluminescence  [2.6.The total RNA was extracted from the cells using TRIzol reagent . Then, the extracted RNA was reversely transcribed into cDNA using the ReverTra Ace qPCR RT Kit . RT-qPCR was carried out via SYBR Premix Ex Taq II . Three independent experiments were carried out to analyze each sample. β-actin was employed as an endogenous control. The specific primer sequences are mentioned in 2.7.3 cells/well) were placed onto 96-well plates. Then, CCK-8 solution (10 µL) was added into each well. Next, the plates were incubated for 2 h at 37°C. The absorbance value was measured at 450 nm with a microplate reader  [CCK-8 assay  was employed to measure cell proliferation. In short, cells  .2.8.4 cells/well) with 80% confluency were plated onto a six-well plate. We next used a sterile plastic micropipette tip to scrape the cells to make an artificial wound. Pictures were taken before the scratch and at 24 h after the scratch was created. Nikon NIS-Element ImagePro Plus 6 was used to measure wound closure [In short, cells (5 × 10 closure .2.9.5 cells/mL. We took 200 μL of cell suspension and inoculated cells in Transwell chambers (8 μm pore size), and 600 μL of the culture medium was placed at the lower chamber. After these chambers were cultured for 36 h at 37°C, the culture medium at the upper chamber was discarded. Next, a cotton swab was employed to carefully wipe cells in the upper layer of the chamber that did not pass through the filter membrane. After the upper chamber was washed with PBS, these membranes were fixed with 4% paraformaldehyde solution for 30 min. Following that, 0.1% crystal violet solution was utilized to stain these membranes for 30 min. Under microscope, cells passing through the membrane were counted in 5 fields, and average value was taken to reflect the migratory property of each group [Cell suspension was prepared with the culture medium, and the cell density was at 2 × 10ch group .2.10.SPSS 22.0 software  was utilized for us to analyze our data, and our data were expressed as mean ± standard deviation (SD). Paired t test was used for comparison between the two groups, and one-way analysis of variance was used for multiple groups. P value < 0.05 was considered statistically significant .3.3.1.http://gepia.cancer-pku.cn), MEX3A expression in breast cancer tissues was found to be much higher, in contrast to the control group . Furtheol group . Similarol group ). Moreovol group . These r3.2.http://kmplot.com/analysis/) was utilized to explore the correlation between MEX3A and the prognosis of breast cancer. The overall survival of breast cancer patients with high MEX3A expression was significantly shorter than that of patients with low MEX3A expression  [C. elegans. Four MEX3 proteins are mainly concentrated in cellular cytoplasm and can pass through the CRM1 channel between cytoplasm and nucleus. MEX3A gene is one of the four human homologous MEX3 genes, and it is involved in the regulation of mRNA [MEX3 is a KH-domain protein, which was first discovered in elegans) . Human M of mRNA ,10. Stud of mRNA ,17. MEX3 of mRNA . RNA-bin of mRNA . In gast of mRNA . In thisRhoA is an important member of the Rho family of small G proteins which are expressed in cellular cytoplasm and membrane, and it can regulate the cytoskeleton structure of cells . Rho-rel5.This study demonstrated that MEX3A expression is increased in breast cancer. This upregulation of MEX3A accelerates the proliferation and migration of breast cancer cells. Moreover, inhibition of MEX3A decreased cell proliferation and migration in breast cancer cell lines. Furthermore, MEX3A can regulate RhoA/ROCK1/LIMK signaling pathway in breast cancer cells. Overall, our research indicates that MEX3A and RhoA/ROCK1/LIMK signaling may be new targets for breast cancer treatment.