To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission. To complete cell division, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by a machinery named “endosomal sorting complex required for transport” (ESCRT). The dissection of this machinery is important in basic biology and for investigating diseases in which cell division is altered. AKTIP, a factor discovered to be needed for chromosome integrity, shares similarities with a component of the ESCRT machinery named TSG101. Here we present evidence that AKTIP is part of the ESCRT machinery, as TSG101. More specifically, we show that AKTIP physically interacts with members of the ESCRT machinery and forms a characteristic circular structure at the center of the bridge linking the daughter cells. We also show that the reduction of AKTIP levels causes defects in the assembly of the ESCRT machinery and in cell division. In future work, it will be interesting to investigate the association of AKTIP with cancer, because in tumorigenesis cell division is altered and since an implication in cancer has been described for TSG101 and other ESCRT factors. To complete cytokinesis, cells need to cleave the intercellular bridge, a membrane structure enriched in microtubules linking the two daughter cells. This cleavage step, named abscission, is operated by the endosomal sorting complex required for transport (ESCRT) machinery ,2. BeyonDuring cytokinesis, the members of the ESCRT machinery are positioned at the midbody of the intercellular bridge. The ESCRT machinery comprises the ESCRT I, II and III modules. The ESCRT I module includes TSG101, VPS37, MVB12 and VPS28 . The ESCDrosophila which has no CEP55, ALIX and TSG101 recruitment to the midbody is promoted by the centralspindlin component Pavarotti  [The assembly of the ESCRT machinery at the midbody is initiated by CEP55, which recruits the ESCRT I component TSG101 and ALIX ,5,12,13.n MKLP1) .Super-resolution microscopy has permitted the interpretation at 100–200 nanometer scale of the organization of ESCRT and ESCRT associated factors supra-molecular complexes formed at the midbody –19. SuchDrosophila) is organized in foci enriched at the nuclear rim in interphase cells and that a reduction in AKTIP (or Peo) results in telomeric defects, aging and cancer aggressiveness [In our previous work, we have shown that a protein named AKTIP , α-tubulin and endogenous AKTIP and imaged by confocal microscopy. In interphase, AKTIP is detectable as discrete and abundant foci at the nuclear rim and within the nucleus. During the initial phases of mitosis, AKTIP moves along the microtubules and on the spindle midzone during anaphase. In late telophase, AKTIP accumulates on the intercellular bridge at the center of the midbody. At this stage, the AKTIP punctate signal is also visible at the reforming nuclear rim and in the nucleoplasm .To validate the specificity of the localization of AKTIP to the midbody, we compared the immunostaining obtained using the monoclonal anti-AKTIP antibody , which delivers ~120nm resolution . Cells wThese data taken together demonstrate a physical organization of AKTIP at the midbody similar to that of components of the ESCRT machinery.To investigate the biochemical association of AKTIP with the ESCRT machinery we carried out a yeast two hybrid screen on ESCRT I, II, III subunits and on the ESCRT associated factors. Yeast cells were co-transformed with a plasmid encoding AKTIP fused to the Gal4 DNA-binding domain, in combination with plasmids encoding ESCRT or ESCRT associated factors fused to the VP16 activation domain and the LacZ activity in co-transformants was measured. We observe that AKTIP significantly interacts with the ESCRT I subunit VPS28 .To validate the interaction between AKTIP and VPS28 in mammalian cells, VPS28 was cloned as a GST-fusion and AKTIP as a MYC- or HA-tagged fusion. 293T cells were co-transfected with control GST or GST-VPS28 and either MYC-AKTIP or HA-AKTIP. Pull down assays followed by Western blotting show that AKTIP co-precipitates with VPS28 Figs  and S2. We next analyzed in vivo the co-localization of AKTIP and VPS28. Spinning disk analyses and image reconstruction of HeLa cells stained with anti-AKTIP and anti-VPS28 antibodies show that the two signals are in proximity at the midbody . The sigSince TSG101 is known to bind VPS28 ,31–34, wTaken together, these data show that VPS28 can be in complex with both TSG101 and AKTIP. In vivo, AKTIP and TSG101 and AKTIP and VPS28 are concomitantly present in proximity at the center of the midbody.α positions equal to 1.9 Å (AKTIP and TSG101 sequences contain a ubiquitin E2 variant (UEV) domain, which in AKTIP is in its central region (aa 78–220) . To compto 1.9 Å . Betweento 1.9 Å .To define the AKTIP region implicated in the interaction with VPS28, we produced three GFP-tagged AKTIP truncations, including the N-terminus (aa 1–76), the C-terminus (aa 221–292) or the UEV (aa 78–220) portions of AKTIP. 293T cells were co-transfected with the different AKTIP constructs together with full length GST-VPS28. Pulldown experiments demonstrate that the central region of AKTIP interacts with VPS28 . To furtTo extend the characterization of AKTIP in relation with the ESCRT subunits, we tested AKTIP on the ESCRT I subunits VPS37A to D and on the ESCRT II subunits VPS25, VPS36 and VPS22. To this end GST- or GFP-tagged ESCRT subunits were tested on MYC-tagged AKTIP. Pull down assay shows no significant interaction between AKTIP and ESCRT I VPS37A to D or ESCRT II VPS25, VPS36 and VPS22 Figs  and S3B.Given the similarity between AKTIP and TSG101, we explored the possibility of an interaction between AKTIP and SEPT9. This idea comes from the fact that an interaction between SEPT9 and TSG101 was previously described and assigned to two N-terminal PTAP motifs in SEPT9 . We usedTogether these data show that AKTIP interacts with the ESCRT I module and shares with the ESCRT I subunit TSG101 the ability to bind the N-terminus of VPS28. However, at the condition tested and differently from TSG101, AKTIP binds VPS28 through its UEV domain and does not bind SEPT9 .The ESCRT I subunits require for the execution of abscission the ESCRT III module. To evaluate the relationship between AKTIP and the ESCRT III module, we carried out 3D-SIM co-analyzation studies of AKTIP and of the ESCRT III components CHMP4B, CHMP2A and IST1, for which the localization has been previously defined using super-resolution microscopy ,19,36.Co-staining of AKTIP and CHMP4B  or CHMP2We next analyzed the proximity of AKTIP to IST1. This analysis shows that in early forming midbodies neither AKTIP nor IST1 are yet organized in a supra-molecular structure. In the mid-stage, AKTIP and IST1 are detected as ring shaped structures. Two IST1 rings flank the central, single, larger, and thicker AKTIP ring at the midbody . In lateTriple staining of AKTIP, TSG101 and IST1 in HeLa cells shows that while AKTIP and TSG101 localize in close proximity, the IST1 rings flank the AKTIP-TSG101 signal . In agreThese results taken together show that the AKTIP circular supra-molecular structure is associated with the ESCRT I module that includes TSG101 and VPS28, and localizes in proximity, albeit not coincidently, with ESCRT III subunits IST1, CHMP2A and CHMP4B when these organize into double rings in the central area of the midbody.Since in mammalian cells at the earliest stages of abscission, CEP55 controls the recruitment of TSG101 and of the TSG101 associated factor ALIX ,5,12,13,To validate this last point, i.e. the independence of AKTIP from TSG101 and ALIX extrapolated from the data obtained in siCEP55 cells, we analyzed by immunofluorescence TSG101 in shAKTIP cells. This analysis shows that the level of TSG101 positive midbodies is not dependent on AKTIP . We nextWe next monitored by immunofluorescence the midbodies for the presence of ALIX in shAKTIP cells and reverse. We observe that AKTIP loss does not impact on ALIX recruitment to the midbody and viceversa .To complete the picture of the elements playing a role in the assembly of the early members of the ESCRT complex and given the biochemical interaction of AKTIP with VPS28, we checked whether VPS28 was affected in shAKTIP cells. Immunofluorescence analysis shows no significant differences of VPS28 in shAKTIP cells as compared to controls . The revAltogether, these results show that the recruitment of AKTIP to the midbody can happen independently of CEP55, TSG101, ALIX and VPS28.Drosophila the recruitment to the midbody of TSG101 and ALIX can be independent of CEP55, while it is promoted by the centralspindlin component Pav  [We next investigated whether AKTIP recruitment to the midbody was dependent on MKLP1. The idea for this came from data showing that in mammals) . We alsomammals) .To analyze the association of AKTIP and MKLP1, firstly we performed co-immunofluorescence and analyzed by confocal imaging the relative spatiotemporal distribution of the two factors. We observe that AKTIP and MKLP1 have similar distributions . To obtaAs a second step, to evaluate if the recruitment of AKTIP was MKLP1 dependent, we analyzed AKTIP localization at the midbody in siMKLP1 cells . The quaIn summary, these data provide evidence that MKLP1 not only localizes in close proximity to AKTIP but also, importantly, is needed for the recruitment of AKTIP to the midbody.The ESCRT I module contributes to the assembly of ESCRT III modules ,32. TherWe firstly analyzed the ESCRT III subunit CHMP4B in shAKTIP cells by immunofluorescence. The data show that the number of CHMP4B positive midbodies in shAKTIP cells are comparable to control cells, revealing that AKTIP depletion alone is not sufficient to affect CHMP4B recruitment to the midbody . Along wWe next asked whether AKTIP would influence CHMP4B by cooperating with TSG101. We produced siTSG101 and shAKTIP/siTSG101 cells by transient transfection of siTSG101 in control and shAKTIP cells, respectively. siTSG101 causes a reduction of TSG101 positive midbodies in both conditions . In lineWe next analyzed by immunofluorescence the presence of CHMP4B in either shAKTIP cells or in co-depleted shAKTIP, siALIX cells. We observe that the siALIX alone does not significantly affect CHMP4B recruitment, neither it does in combination with AKTIP reduction Figs .As a further step we looked at IST1 in shAKTIP, siTSG101 and shAKTIP/siTSG101 cells. We observe that both AKTIP and TSG101 are required for IST1 recruitment. Furthermore, we observe that the levels of the reduction of IST1 are comparable in shAKTIP, siTSG101 and shAKTIP/siTSG101 cells, suggesting that AKTIP and TSG101 act in a common path for the recruitment of IST1 to the midbody Figs .To further explore whether both AKTIP and TSG101 were needed for IST1 recruitment to the midbody, we tested the ability of AKTIP to rescue TSG101 deficiency. To this end we created a stable AKTIP-FLAG overexpressing cell line. We verified the presence of AKTIP-FLAG at midbody by immunofluorescence with an anti-FLAG antibody and monitored the expression level of AKTIP in AKTIP-FLAG cells . We nextTaken together, these results indicate that AKTIP and TSG101 cooperate in the recruitment of CHMP4B to the midbody, but act in two independent pathways. On the other hand, the absence of additivity and of rescue indicates, respectively, that AKTIP and TSG101 act in the same pathway on IST1 but are both needed for its recruitment to the midbody.Given that AKTIP loss causes a reduction of IST1 and that IST1 functions in cytokinesis , we askeFinally, since in our previous work we documented a role for AKTIP in telomere dysfunction , we nextAKTIP is a protein with similarities with the ESCRT I subunit TSG101 ,27. HereThe first experimental evidence linking AKTIP to the ESCRT complex is in the characteristics of its supra-molecular organization. We show that AKTIP forms a large multimeric circular structure around microtubules at the midbody where the ESCRT complex is recruited ,16. The A second set of experimental evidence links AKTIP to the ESCRT I module. AKTIP biochemically interacts with VPS28. VPS28 is an ESCRT I member required for the assembly of ESCRT complex ,32. VPS2Drosophila [Interestingly, the positioning of AKTIP at the midbody is not controlled by CEP55. Coherently with this experimental evidence, the sequence of AKTIP does not include the CEP55 binding residues 158–162 identified in TSG101 . AKTIP rosophila .The third piece of evidence functionally associating AKTIP with the ESCRT machinery is the impact of AKTIP on ESCRT III recruitment to the midbody. Notably, the combined depletion of AKTIP and TSG101 reduces the recruitment of the ESCRT III member CHMP4B. On the other hand, the single depletion is not sufficient to impair CHMP4B recruitment. These results show that AKTIP and TSG101 act in parallel arms on the recruitment of CHMP4B . PreviouA direct relationship between members of the ESCRT I module and IST1 has been previously described and indicated as a path modulating a subset of the ATPase VPS4 activity at the midbody . We repoGiven the function exerted by IST1 in the final stages of cell division , the AKTCollectively, the results presented demonstrate for the first time that AKTIP, a protein similar to the ESCRT I component TSG101, functionally associates with the ESCRT I module at the midbody and is required for the assembly of the ESCRT III module implicated in the final stages of cytokinesis. In future work, it will be interesting to explore if the AKTIP association with the ESCRT machinery has an impact on cancer, which has been indicated for TSG101  and more2 in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies) and 50U/ml penicillin and streptomycin (Life Technologies). For transient RNA interference, cells were cultured in 6-well plates and were transfected with 20μM siRNA oligonucleotides using Lipofectamine 2000 (Life Technologies) following manufacturer’s instructions. Cells were collected or fixed 48hrs post-transfection. To obtain cell lines stably interfered for AKTIP or TRF2 we used lentivirus (LV) mediated interference and the LV-shAKTIP (shAKTIP), LV-shTRF2 (shTRF2) and LV-scramble (shctr) vectors were described previously [HeLa cells (ATCC CCL-2) and 293T cells (ATCC CRL-11268) were grown at 37°C; 5% COeviously . The muleviously  at a moieviously . SeventyOne-week post-transduction, cells were lysed by addition of TRIzol reagent (Invitrogen) and RNA extracted according to the manufacturer’s instructions. After DNase treatment (Invitrogen), RNA was reverse transcribed into cDNA as already described . Q-PCR rAKTIP Forward 5’- TCCACGCTTGGTGTTCGAT-3’;AKTIP Reverse 5’-TCACCTGAGGTGGGATCAACT-3’;TRF2 Forward 5’- TCCTCACGATGGCCAAAAAG-3’;TRF2 Reverse 5’- GCTGTTTATCTTCCTTCCCTGTACT-3’;GAPDH Forward 5’-TGGGCTACACTGAGCACCAG-3’;GAPDH Reverse 5’-GGGTGTCGCTGTTGAAGTCA-3’–ΔΔCq method as previously described [and analyzed with the 2escribed . For Wesescribed  and quanHeLa cells were seeded onto glass coverslips in 6-well plates and fixed with 3.7% formaldehyde in PBS for 10min. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 5min, treated with PBS 1% BSA for 30min and then stained with primary antibodies in PBS 1% BSA for 1hr at RT. For AKTIP-TSG101 co-immunofluorescence, HeLa cells were seeded onto glass coverslips, fixed as previously described [For Structured illumination microscopy (3D-SIM) and Multispot-SIM (MSIM) imaging, HeLa cells were seeded onto glass coverslips  in 6-well plates and fixed with 3.7% formaldehyde in PBS for 10min at RT and then incubated in 50mM NH4Cl/PBS (15min). Cells were incubated with primary and secondary antibodies diluted in PBS-BSA 1% for 1hr at RT and washed in PBS. Acquisition of 3D-SIM images was performed using a DeltaVision OMX v4 Blaze microscope  equipped with the BGR-FR filter drawer for acquisition of 3D-SIM images, an Olympus Plan Apochromat 100×/1.4 PSF oil immersion objective lens and liquid-cooled Photometrics Evolve EM-CCD cameras for each channel. 15 images per section per channel were acquired with a z-spacing of 0.125μm ,48. Stru3D volume reconstructions and movies generation were done in Imaris . Image analysis and quantification was performed using Image J/FIJI , Excel (Yeast two hybrid assays were performed as previously described . BrieflyGST pull down were performed as previously described . Full leFor GFP-TRAP 293T cells were transfected with plasmids encoding GFP-tagged versions of ESCRT-subunits or SEPT9 isoforms and pCMV6-Entry-AKTIP-Myc-Flag (ORIGENE). pEGFPC2 plasmids encoding SEPT9 isoforms were kind gifts from Ulrike Eggert . Cells were then lysed in pull down lysis buffer , cleared by centrifugation and incubated magnetic agarose GFP-trap beads (Chromotek) for 10 minutes. Captured fraction separated by magnetisation and washed with pull down wash buffer . Eluted proteins were recovered with 2x LDS sample buffer and resolved by SDS- PAGE. Resolved proteins were transferred onto nitrocellulose by Western blotting and were probed with the indicated antibodies in 5% milk. HRP-conjugated secondary antibodies were incubated with ECL Prime enhanced chemiluminescent substrate  and visualized by exposure to autoradiography film. The following primary antibodies were used: anti-HA , anti-MYC , anti-GST , anti-GFP . The secondary antibodies used were goat anti-rabbit HRP-conjugated .AKTIP and TSG101 sequence alignment was performed with Jalview . The PDB AKTIP homology model  and the Statistical analyses were performed using Excel and Graphpad Prism software. Results are shown as mean ± SEM of at least three independent experiments. Data were analyzed using unpaired two-tailed Student’s t-test. p-values below 0.05 were considered significant and reported in figures as *p<0.05; **p<0.01; *** p<0.001. p values above 0.05 were considered not significant and were not reported in figures.S1 Fig(A) AKTIP localization at the midbody in HeLa cells using anti-AKTIP antibodies WH0064400M2 clone 2A11 and HPA041794. (B) Detection of exogenous AKTIP-FLAG in transfected cells using anti-FLAG antibody. (C) Immunofluorescence of HeLa cells transduced with a control lentivector containing a scramble interfering sequence (shctr) or with AKTIP interfering sequence (shAKTIP) stained for α-tubulin (green) and AKTIP (red). Scale bars, 5μm. (D) Western blotting of AKTIP expression in shAKTIP and shctr cells. Actin was used as loading control. (E) Representative Q-PCR of AKTIP mRNA expression in shAKTIP and shctr cells.(TIF)Click here for additional data file.S2 FigWestern blotting showing that AKTIP interacts with GST-VPS28 but not with GST alone. Cells were transfected with plasmids encoding the indicated fusion proteins. Purified VPS28-GST or GST alone were used to pull down interacting proteins; cell lysates and glutathione-bound fractions were then analyzed with HA antisera. GST-pull downs were repeated two times. In the HA-AKTIP blot we observe a major band at the correct molecular weight, in the pulldown fractions we observe also degradation products that retain the GST moiety.(TIF)Click here for additional data file.S3 Fig(A) Grey squares: identical residues in AKTIP and TSG101 sequences. Blue boxes: H1, H2, H3, and H4 TSG101 helices. Red boxes: H2, H3, H4, H5 and H6 helices predicted in AKTIP model. White arrows: β-strands. (B) Western blotting showing that AKTIP does not interact with VPS25, VPS22 and VPS36 and confirming its interaction with VPS28. Cells were transfected with the indicated fusion proteins. Purified VPS25-GFP, VPS22-GFP, VPS36-GFP, VPS28-GFP or GFP alone were used to trap interacting proteins; then cell lysates and GFP-trapped fractions were analyzed with MYC antisera. GFP-TRAP were repeated twice.(TIF)Click here for additional data file.S4 Fig(A-B) Representative images and quantification of sictr and siCEP55 transiently transfected HeLa cells stained for CEP55 (red) and α-tubulin (green). Scale bar, 2.5μm. For results in (B) at least 100 midbodies per condition were counted. (C-D) Representative images and quantification of multinucleated cells in sictr and siCEP55 HeLa cells. For results in (D) at least 200 nuclei per condition were counted. Scale bar, 5μm.(TIF)Click here for additional data file.S5 Fig(A-B) Representative images and quantification of sictr and siTSG101 transiently transfected HeLa cells stained for TSG101 (red) and α-tubulin (green). (C-D) Representative images and relative quantification of shctr and shAKTIP HeLa cells stained for ALIX (red) and α-tubulin (green) showing that ALIX is present at the midbody in shAKTIP cells. (E-F) Representative images and relative quantification of sictr and siALIX HeLa cells stained for AKTIP (red) and α-tubulin (green) showing that AKTIP is present at the midbody in siALIX cells. (G-H) Representative images and quantification of sictr and siALIX transiently transfected HeLa cells stained for ALIX (red) and α-tubulin (green). (I-J) Representative images and quantification of sictr and siVPS28 transiently transfected HeLa cells stained for VPS28 (red) and α-tubulin (green). Scale bars, 5μm. For results in  at least 80 midbodies per condition were counted.(TIF)Click here for additional data file.S6 Fig(A-B) Representative images and quantification of sictr and siMKLP1 transiently transfected HeLa cells stained for α-tubulin (green) and MKLP1 (red). For results in (B) at least 25 midbodies per condition were counted. Scale bar, 2.5μm.(TIF)Click here for additional data file.S7 Fig(A) Quantification of the percentage of midbodies with abnormally localized CHMP4B (black) or with two rings (red) and with cones or spirals (white) of CHMP4B referred to CHMP4B positive midbodies in shctr and shAKTIP HeLa cells stained for CHMP4B and α-tubulin ((B-C) Representative images and relative quantification of shctr, shAKTIP, siTSG101, and shAKTIP/siTSG101 HeLa cells, stained for α-tubulin (green) and TSG101 (red). (D-E) Representative images and relative quantification of shctr, shAKTIP, siALIX, and shAKTIP/ siALIX HeLa cells, stained for α-tubulin (green) and ALIX (red). For results in (C and E) at least 100 midbodies per condition were counted. Scale bars, 2.5μm.-tubulin . (B-C) R(TIF)Click here for additional data file.S8 Fig(A-B) Representative images and relative quantification of ctr, and AKTIP-FLAG HeLa cells, stained for α-tubulin (green) and FLAG (red). (C) Representative Q-PCR of AKTIP mRNA expression in ctr and AKTIP-FLAG HeLa cells. (D-E) Representative images and relative quantification of sictr, siTSG101, AKTIP-FLAG/siTSG101 HeLa cells, stained for α-tubulin (green) and TSG101 (red). (F) Representative Q-PCR of AKTIP mRNA expression in shctr, 3’UTR shAKTIP and AKTIP-FLAG/3’UTR shAKTIP HeLa cells. For results in (B and E) at least 80 midbodies per condition were counted Scale bars, 2.5μm.(TIF)Click here for additional data file.S9 Fig(A) Western blotting of AKTIP expression in siAKTIP and sictr cells. Actin was used as loading control. (B) Representative Q-PCR of TRF2 mRNA expression in shTRF2 and shctr HeLa cells. (C-D) Representative images and relative quantification of shctr, shAKTIP, and shTRF2 HeLa cells, stained for α-tubulin (green) and IST1 (red). For results in (D) at least 100 midbodies per condition were counted. Scale bar, 2.5μm.(TIF)Click here for additional data file.S1 Movie3D volume rendering of mid stage midbody imaged with 3D-SIM. HeLa cells were stained with antibodies against α-tubulin (green) and AKTIP (red). The volume rendering and the movie generation were performed with IMARIS software .(MP4)Click here for additional data file.S2 Movie3D volume rendering of late stage midbody imaged with 3D-SIM. HeLa cells were stained with antibodies against α-tubulin (green) and AKTIP (red). The volume rendering and the movie generation were performed with IMARIS software .(MP4)Click here for additional data file.S3 Movie3D volume rendering of midbody imaged with MSIM. HeLa cells were stained with antibodies against AKTIP (green), and TSG101 (red). The volume rendering and the movie generation were performed with IMARIS software .(MP4)Click here for additional data file.S4 Movie3D volume rendering of mid stage midbody imaged with 3D-SIM. HeLa cells were stained with antibodies against α-tubulin (green), IST1 (blue) and AKTIP (red). The volume rendering and the movie generation were performed with IMARIS software .(MP4)Click here for additional data file.S5 Movie3D volume rendering of late stage midbody imaged with 3D-SIM. HeLa cells were stained with antibodies against α-tubulin (green), IST1 (blue) and AKTIP (red). The volume rendering and the movie generation were performed with IMARIS software .(MP4)Click here for additional data file.S6 MovieTime-lapse microscopy of HeLa cells stably expressing mCherry-tubulin treated with siAKTIP. The movie generation was performed with Fiji software.(AVI)Click here for additional data file.S7 MovieTime-lapse microscopy of HeLa cells stably expressing mCherry-tubulin treated with siAKTIP. The movie generation was performed with Fiji software.(AVI)Click here for additional data file.S8 MovieTime-lapse microscopy of HeLa cells stably expressing mCherry-tubulin treated with ctr. The movie generation was performed with Fiji software.(AVI)Click here for additional data file.


Numerous natural products exhibit antiproliferative activity against cancer cells by modulating various biological pathways. In this study, we investigated the potential use of eight natural compounds  and two repurposed agents (fulvestrant and metformin) as chemotherapy enhancers and mesenchymal-to-epithelial (MET) inducers of cancer cells. Screening of these compounds in various colon, breast, and pancreatic cancer cell lines revealed anti-cancer activity for all compounds, with curcumin being the most effective among these in all cell lines. Although some of the natural products were able to induce MET in some cancer cell lines, the MET induction was not related to increased synergy with either 5-FU, irinotecan, gemcitabine, or gefitinib. When synergy was observed, for example with curcumin and irinotecan, this was unrelated to MET induction, as assessed by changes in E-cadherin and vimentin expression. Our results show that MET induction is compound and cell line specific, and that MET is not necessarily related to enhanced chemosensitivity. Cancer is the second leading cause of death worldwide and about one in six deaths are due to cancer . ChemothNutrition and diet are effective in reducing cancer risk. Numerous dietary natural products have shown potential in prevention and/or treatment of cancers ,8,9. AccCleus forskohlii, was shown to induce tumor-initiating cells to undergo mesenchymal-to-epithelial transition, and to cause them to lose their tumor-initiating ability [In line with these observations, modulators of EMT, combined with chemotherapeutics and/or targeted drugs, may possibly improve clinical outcomes in cancer. Most studies aiming to address this issue, however, were conducted either in single cell lines, or only in one cancer type . Hence,  ability . Procyan ability . Additio ability ,32. It w ability . Fulvest ability ,35,36,37 ability ,51,52,53We first sought to characterize the inhibitory effect of natural products and repurposed agents on various breast, colon, and pancreatic cancer cell lines. We selected eight naturally occurring molecules, an estrogen antagonist, and an anti-diabetic drug that were previously shown to inhibit and/or reverse EMT. The cells were designated resistant if an IC50 value was not observed within the concentration ranges tested. For the breast cancer cell lines, curcumin exhibited a remarkable inhibition of cell viability, with IC50 values ranging from 12 to 40 μM among the cell lines tested . AdditioSince diet is an important factor in colon cancer risk, studying the effects of natural products on colon cancer may provide new insights into cancer prevention and treatment . In a siPancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancer types, and a number of naturally occurring molecules in preclinical studies exhibit promising pharmacological features ,57. We eIn total, 12, 13, and 15 cancer cell lines (out of 16) were resistant (IC50 could not be determined within the concentration ranges tested) to forskolin, procyanidin B2, and fulvestrant, respectively, suggesting that these compounds were not effective inhibitors of cancer cell viability. Apigenin, curcumin, and resveratrol were the only three compounds for which IC50 values could be generated for all of the cell line types tested. Among these compounds, the lowest mean IC50 was obtained for curcumin for all three cancer types, with values of 23.8 μM, 10 μM, and 15 μM for breast, colon, and pancreatic cancer cells, respectively.We treated six cell lines from three cancer types with natural compounds and evaluated changes in the epithelial/mesenchymal phenotypes . We usedOverall, we noted transcriptional changes suggesting a shift towards a more epithelial phenotype in four out of the six cell lines tested upon treatment with at least one compound. The MET induction effects summarized in We next asked whether the treatment with natural compounds would result in chemosensitization. For this purpose, the KM12 and WiDr colon cancer cell lines were treated with various doses of natural products, alone or in combination with two doses (1 μM and 10 μM) of 5-FU, a commonly used chemotherapeutic in colon cancer, after twenty-four hours of cell seeding. Cell viability assays were then performed 72 h after the treatments. In KM12, we observed MET induction with 20 uM of fisetin and two doses (10 μM and 30 μM) of forskolin. However, only the 10 uM 5-FU and forskolin combination treatments resulted in synergy, with the combination index (CI) score ranging from 0.91 to 0.37 . AdditioWe next investigated the combinatory effects of irinotecan, used in pancreatic cancer, and some widely studied natural compounds, curcumin and resveratrol, in pancreatic cancer cell lines, AsPC-1, BxPC-3, MIA PaCa-2, and SU8686 . Most ofGemcitabine is a standard agent for the treatment of pancreatic cancer, either alone or in combination ,63. We tInnate or acquired resistance of cancer cells to chemotherapeutics is responsible for tumor growth and metastasis ,66. SeveA wide range of natural products have been reported as being cytotoxic for cancer cells and therefore useful in cancer therapy. Natural products can act as therapeutic, preventive, or chemosensitizer agents. Enhancement of chemotherapy sensitivity in tumor cells using naturally occurring biomolecules is a new and promising strategy aiming to promote the efficacy of conventional chemotherapeutics and targeted drugs . SeveralIn the present study, we used eight natural compounds and two repurposed agents, that were previously shown to modulate EMT in different types of cancer cell lines, to test their capability to sensitize cancer cells to chemotherapy in a comprehensive manner. Although there are studies supporting the modulatory effects of all these compounds on EMT, only a few of them were able to induce MET in specific cancer cell lines in our experiments, suggesting a highly selective cell-line specific action. Interestingly, none of the compounds were able to induce MET in the Caco-2 cell line, which can easily be induced to undergo EMT and MET under various other conditions . In factDifferent transcriptomic signatures have been evaluated for determining the EMT status of cancer cells ,72,73. EAmong the compounds we examined in this study, almost none were found to both trigger MET and induce chemosensitization. In KM12, although fisetin and forskolin induced MET, the fisetin and 5-FU combination did not show a synergistic effect. On the other hand, urolithin A synergized with 5-FU in KM12 in the absence of MET induction. Furthermore, slight MET induction in MIA PaCa-2 with natural compounds did not lead to improved sensitivity to irinotecan, gemcitabine, and gefitinib in almost all combinations.These findings show that synergy between natural compounds and chemotherapeutics/targeted agents can be observed only for some cell lines and that the underlying mechanism is likely not to be the simple induction of MET, but possibly a combination of several other mechanisms. The fact that synergistic effects were also quite variable across cancer types as well as in cell lines within a given cancer type suggests that the mechanisms underlying these observations are highly cell line specific.2.Breast cancer cell lines, CAL51, MDA-MB-157, MDA-MB-231, MDA-MB-436, MDA-MB-453, and MDA-MB-468 were cultured in DMEM medium . Colon cancer cell lines, Caco-2, HCT-8, KM-12, LS513, LoVo, SW837, and WiDr were cultured in RPMI-1640 medium . Pancreatic cancer cell lines, AsPC-1, BxPC-3, and SU8686 were cultured in RPMI-1640 medium, while MIA PaCa-2 cells were cultured in DMEM medium. The respective media were supplemented with 10% fetal bovine serum (FBS) , 1% penicillin-streptomycin , and 1% 200 mM L-Glutamine  at 37 °C with 5% COChemotherapeutics, 5-FU (S1224), gemcitabine (S1149) and irinotecan (S2217), were purchased from Selleckchem . Targeted cancer therapy drugs, gefitinib (S1025) and selumetinib (S1008) were purchased from Selleckchem, while lapatinib (11493) was purchased from Cayman Chemical . Fulvestrant  and metformin  were used as repurposed agents. Natural products, apigenin (S2262), curcumin (S1848), fisetin (S2298), and forskolin (S2449) were purchased from Selleckchem, while (-)- epigallocatechin gallate (70935), procyanidin B2 (19865), resveratrol (70675) and urolithin A (22607) were purchased from Cayman Chemical. All chemicals had more than 98% purity. Stock solutions were prepared according to the manufacturers’ instructions.Cancer cell lines were seeded at 2000 cells per well in 96-well plates . After incubation for 24 h, cells were treated with chemotherapeutics, targeted drugs, and natural compounds using 6 different concentrations: apigenin, curcumin, EGCG, fisetin, forskolin, procyanidin B2, resveratrol, urolithin A, fulvestrant: 0.1–100 μM; metformin: 0.4–60 mM (in colon and pancreatic cancers); metformin: 0.1–100 μM (in breast cancer). Cell viability was analyzed in quadruplicates using the CellTiter-Glo Luminescent Cell Viability Assay , according to the manufacturer’s protocol, after 72 h of treatment. IC50 values were obtained by a method previously described . DiffereTo quantitate synergy combination treatments of natural compounds, chemotherapeutics and targeted drugs were analyzed with the Chou-Talalay methods using CompuSyn Software  which generates a combination index (CI) which was calculated with the cell viability values from combination treatments. CI < 1 indicates synergy, CI = 1 indicates additive effect, while CI > 1 indicates antagonism.Total RNA was isolated using Purezol  according to the manufacturer’s instructions. Two micrograms of total RNA were reverse-transcribed into cDNA using the Revert-aid first strand cDNA synthesis kit  according to the supplier’s protocol using a random hexamer. Quantitative profiling of EMT marker genes CDH1 and VIM by qRT-PCR was performed in triplicates using a LightCycler 480 II real-time PCR system  and iTaq Universal SYBR Green Supermix . Real-time PCR reactions were run under cycling conditions according to the manufacturer’s instructions. GAPDH was used as an endogenous control in qPCR reactions in triplicates and gene expression signals were normalized to GAPDH. Relative expression values of CDH1 and VIM were calculated by the ddCt method according to samples with least expression as previously described . qPCR prp-values less than 0.05 were considered as statistically significant.Statistical analysis was performed using R Statistical Software  and different treatment groups were compared using the Mann–Whitney U test.


Testicular cancer (TC) is the most frequent type of cancer among young men aged between 15 and 34 years. TC is treated using cisplatin, but 3%–5% of TC patients fail to respond to cisplatin, with a very bad to fatal prognosis. Accordingly, it is most important to quickly and readily identify those TC patients who are resistant to cisplatin treatment.This study seeks to investigate changes in the glycosylation associated with cisplatin resistance to TC cell lines.A specific glycoprofiling of human chorionic gonadotropin (hCG) was analysed in three TC cell lines and one cell line of female origin. A typical calibration curve for hCG glycoprofiling showed a dynamic range up to 50 ng/ml, with a limit of detection of 0.3 ng/ml and assay reproducibility represented by relative standard deviation of 3.0%. Changes in the glycan signatures on hCG were analysed in cisplatin‐sensitive cell lines and in their cisplatin‐resistant sub‐lines using an enzyme‐linked lectin assay (ELLA) protocol. An immobilised antibody was applied to a selective capture of hCG from a cytoplasmic fraction of cell lysates with final incubation using a lectin from a panel of 17 lectins.Dolichos biflorus agglutinin (DBA) can selectively discriminate sensitive TC cell lines from resistant TC cell lines. Moreover, there are additional lectins which can provide useful information about the strength of cisplatin resistance.The results suggest that one particular lectin  50 values for the sensitive cell lines and for the resistant cell lines.This is the first study focused on the use of a specific glycoprofiling of hCG using a panel of lectins for the investigation of glycosylation changes associated with TC or associated with cisplatin resistance. We identified DBA lectin as the most perspective one to evaluate resistivity of TC lines. Moreover, we found some lectins, which can be applied for positive correlation between lectin binding to hCG and cisplatin IC This drug is also effective in curing TC patients having metastases with cure rates of up to 90% and with a general survival rate of up to 95%.Cisplatin  which cannot be repaired by the DNA repair mechanisms, resulting in a disruption of synthesis of DNA, mRNA and proteins, promoting the accumulation of reactive oxygen species; activating signalling pathways and finally resulting in cell death.N‐acetylglucosaminyltransferase V (GnT‐V), which synthesises β6GlcNAc branched N‐glycans,N‐glycans, were determined in a radioresistant nasopharyngeal carcinoma cell line.N‐glycans synthesised by upregulated FUT4 fucosyltransferase are also behind multidrug resistance of breast cancer cells.Glycomics is an emerging scientific discipline studying the involvement of glycans (complex carbohydrates) in the physiological and pathological processes including cancer development and progression.O‐glycan on Ser132 of the β‐subunit of hCG), which is known as the hyperglycosylated form of hCG (hCG‐H), in relation to various physiological and pathological conditions.We have not yet identified any study focusing on an investigation of glycan changes associated with cisplatin resistance of TC cells lines. Hence, the main aim of this study was to investigate changes in the glycan expression of four different cell lines together with their cisplatin‐resistant sub‐lines. We wanted to investigate glycosylation changes in hCG associated with cisplatin resistance and to identify lectins able to provide information about the presence of cisplatin resistance. The main reason for choosing hCG for glycoprofiling is the fact that the level of hCG is routinely used as a TC biomarker to monitor therapy efficiency.22.12.1.1The following antibodies were purchased from Abcam and were used in the study: anti‐hCG beta core fragment antibody [INN‐hCG‐106] (ab11382); anti‐hCG beta 1 epitope antibody [INN‐hCG‐2] (ab11388) and anti‐hCG beta 2 epitope antibody [INN‐hCG‐106] (ab11389).2.1.2Three different hCG proteins purchased from Abcam were used in the study to identify proper hCG standards to be recognised by the antibody, while being glycosylated: native human hCG beta protein (ab126653), native human hCG protein (ab77874) and recombinant human hCG beta 7 protein (ab164958).2.1.3Aleuria aurantia lectin , Concanavalin A , Dolichos biflorus agglutinin , Narcissus pseudonarcissus (Daffodil) lectin , Datura stramonium lectin , Galanthus nivalis lectin , Hippeastrum hybrid (Amaryllis) lectin , Lens culinaris agglutinin , Maackia amurensis agglutinin II , Phaseolus vulgaris erythroagglutinin , Phaseolus vulgaris leucoagglutinin , Peanut agglutinin , Pisum sativum agglutinin , Ricinus communis agglutinin I , Sambucus nigra agglutinin , Wisteria floribunda agglutinin  and Wheat germ agglutinin .The following biotinylated forms of lectins for the enzyme‐linked lectin assays (ELLA) were provided from Vector laboratories: 2.1.4All the following chemicals were obtained from Sigma: bovine serum albumin , albumin from human serum , hydrogen peroxide solution , phosphate‐buffered saline , phosphate‐buffered saline with Tween  and o‐phenylenediamine .Streptavidin‐conjugated HRP (ready‐to‐use) was obtained from Abcam (ab64269).2.22.2.1Aleuria aurantia lectin ; Maackia amurensis agglutinin II ; Phaseolus vulgaris erythroagglutinin ; Phaseolus vulgaris leucoagglutinin  and Sambucus nigra agglutinin .The following lectins were used to determine whether hCG standard is glycosylated and can be used in a standardisation process within an enzyme‐linked lectin assay (ELLA) format. All the following non‐conjugated lectins were obtained from Vector Laboratories: 2.2.2All the reagents used in SPR experiments were purchased from GE Healthcare, including HBS‐P+ , amine coupling kit (BR100050), EDC (0.4 M), NHS (0.1 M) and ethanolamine hydrochloride . For regeneration, the following regeneration buffers were tested : NaOH  and glycine/HCl ; acetate 5.0 (BR‐1003‐51) was used for coupling. In order to regenerate the SPR chip following binding with lectins, the following elution solutions were used (Vector Laboratories): that is, solutions for eluting mannose/glucose‐binding lectins (ES‐1100‐100); galactose/GalNAc‐binding lectins (ES‐2100‐100); fucose/arabinose‐binding lectins (ES‐3100‐100); GlcNAc/chitin‐binding lectins (ES‐5100‐100) and sialic acid‐binding lectins (ES‐7100‐100).SPR assays were run on Biacore X100  using a sensor chip CM5 (29‐1496‐04) under a constant flow rate of 30 μl/min at 25°C. Original SW Biacore X100 Control Software was used to operate the instrument.2.2.3In this examination, hCG was used as a ligand to be immobilised on the SPR chip. The SPR chip was activated using EDC/NHS (ratio 1+1) amine coupling with the chip activated for 420 s. Then, hCG  was diluted to 16.3 μg/ml (428 nM) in an acetate immobilisation buffer of pH 5.0 and was immobilised on the CM5 sensor chip for 1000 s. Finally, the SPR chip was blocked with ethanolamine (contact time of 420 s). A typical level of bound hCG was 1735 RU. The chip preparation was completed by washing the cell with a running buffer (HBS‐P+) for 10 min with application of five pulses of 20 mM NaOH for 30 s; finally, the chip was re‐equilibrated with a HBS‐P+ running buffer.A recombinant hCG  or a native hCG  was immobilised on the sensor chip CM5 for 600 s into cell 1 and cell 2, respectively, using a protein concentration of 16.3 μg/ml (428 nM).Interactions between the ligand immobilised on the chip (hCG) and lectins were measured by a multi‐cycle kinetics mechanism (MCK). Association and dissociation phases were set to 120 and 500 s, respectively. Lectins were diluted in a HBS‐P+ buffer to concentrations of 300, 150, 75, 37.5, 18.8 and 0 nM (lectins at a concentration of 18.8 nM were measured twice). Chip regeneration after each cycle was performed using 60 s pulse with the suitable glycoprotein eluting solution (Vector Laboratories) described above followed by 30 s pulse with 20 mM NaOH; finally, the chip was re‐equilibrated with an HBS‐P+ running buffer.Interactions between the ligand immobilised on the chip (hCG) and antibodies were also measured by MCK. Sample contact time and dissociation time were set to 120 and 750 s, respectively, for interaction with antibodies diluted in a HBS‐P+ buffer to concentrations of 73.3, 36.7, 18.33, 9.16, 4.58 and 0 nM (antibodies at a concentration of 4.58 nM were measured twice). Chip regeneration after each cycle was performed by surface washing using a 30 s pulse with a glycine/HCl regeneration buffer pH 2.5 and using 30 s pulse with 20 mM NaOH; finally, the chip was re‐equilibrated with an HBS‐P+ running buffer.via EDC/NHS chemistry and then blocked with ethanolamine . Chip preparation was completed by washing its surface for 10 min with a running buffer (HBS‐P+) and by five pulses of 20 mM NaOH for 30 s each. Interactions between antibodies immobilised on the chip and hCG were measured by MCK. Sample contact time and dissociation time were set to 120 s and 750 s, respectively. An analyte was diluted in an HBS‐P+ buffer to concentrations of 239, 119.5, 59.8, 29.9, 14.9 and 0 nM (hCG at a concentration of 14.9 nM was measured twice). Chip regeneration after each cycle was performed by washing the surface with a glycine/HCl regeneration buffer pH 2.5 for 30 s and for 30 s with 20 mM NaOH solution; finally, the chip was re‐equilibrated with an HBS‐P+ running buffer.In order to double check the bioaffinity interaction between hCG and an antibody, an anti‐hCG antibody was also immobilised onto the SPR chip. An anti‐hCG beta 2 epitope antibody , diluted to 2.86 μg/ml (19.0 nM) in an acetate immobilisation buffer (pH 5.0) was immobilised to a target level of 400 RU onto the sensor chip CM5 Interaction in a sandwich configuration was studied in two different forms by an MCK mechanism. In the first, we used a feature of the Biacore X100 Control Software to start each cycle with a capture. hCG (ab77874) diluted in a running buffer to 81.6 nM was repeatedly captured for 120 s over the immobilised antibody on the CM5 chip. In the next step, the lectin binding was investigated using a common MCK mechanism. In this case, sample (lectin) contact time and dissociation time were set to 120 and 500 s, respectively, and chip regeneration was performed with glycine pH 2.5 for 30 s and 20 mM NaOH for 30 s (i.e. both lectins and hCG were released from the surface). In the second approach, the hCG was injected over the antibody‐immobilised surface just once. Then the lectin binding to hCG captured over the anti‐hCG layer was monitored using MCK. Chip regeneration was performed using a suitable glycoprotein‐eluting solution for 30 s (i.e. only the lectin was released from the surface). In both versions, the lectin concentrations investigated were 300, 150, 75, 37.5, 18.8 and 0 nM.The data thus obtained were evaluated using the original SW Biacore X100 Evaluation Software with pre‐set preferences.2.3o‐phenylenedimamine) as a substrate. OPD and hydrogen peroxide were dissolved in a citrate‐phosphate buffer (pH = 4.6) to concentrations of 7.4 mM and 0.52 M, respectively. To each well, 100 μl of this OPD‐peroxide solution was applied. The absorbance at 450 nm was measured after 15 min incubation in dark and the reaction was stopped with 3.6 M H2SO4.The ELLA assay was based on a common protocol of a conventional sandwich ELISA. Briefly, 100 μl of an anti‐hCG antibody diluted to 1 μg/ml with PBS was added into the wells of the plate . After overnight incubation in a refrigerator, the plate was washed three times with 215 μl of PBST buffer. Unoccupied places in wells were blocked with 200 μl of HSA (1 mg/ml) for 45 min using a plate shaker . The ELISA plate was then incubated with 100 μl of sample and, after washing, 100 μl of lectins diluted by 1 mg/ml of HSA solution to concentration of 1.5 μg/ml were added and incubated for 1 h under shaking. After washing, 100 μl of 75× diluted streptavidin HRP in HSA (1 mg/ml) was added to the wells and incubated for 60 min. Then the plate was rinsed 3× with 215 μl of PBST buffer and once again with PBS. The detection was accomplished using OPD .2.4Since lectins are able to bind to the glycans present in the Fc fragment of the antibody , it is advisable to de‐activate such a glycan recognition site by glycan oxidation.2O2 solution, the signal was read at 490 nm after a blank subtraction.Glycans of anti‐hCG antibodies were modified to prevent binding of lectins, which would interfere with the glycoprofiling of hCG. Prior to oxidation, the following two reaction solutions were prepared: solution A (150 mM sodium acetate of pH 5.5) and solution B (25 mM sodium metaperiodate in solution A). An antibody ab11389 was diluted to 0.1 mg/ml with solution B and incubated at 4°C in dark for 30 min. The solution was then desalted using previously equilibrated columns (Zeba Spin 7000 MWCO). Freshly prepared 2 mM propionic acid hydrazide in solution A was added to an antibody solution at a 1:1 (v/v) ratio. The reaction mixture was incubated at ambient temperature in dark for 2 h. The final desalting was performed using a new desalting column and the oxidised and blocked antibody was stored at −80°C in aliquots. ELLA was used to control efficiency of oxidation of anti‐hCG antibody glycans. The wells of the ELISA plate were incubated with intact and chemically oxidised antibodies. The surface was subsequently blocked and left to interact with a panel of 17 biotinylated lectins. After 60 min incubation with streptavidin peroxidase (HRP) conjugate and OPD/H2.5Four cisplatin‐sensitive TC cell lines  and their cisplatin‐resistant variants (JEG‐3 CisR (D) VOL II, NCCIT CisR (D) VOL II, NTERA‐2 CisR and TCam‐2 CisR) together with a control non‐cancerous testis cell line (Hs 1.Tes) were used in the study.® CRL‐2073™) were maintained in RPMI 1640 medium (GIBCO® Invitrogen) containing 10% FBS, 10.000 IU/ml penicillin, 5 μg/ml streptomycin, 2.5 μg/ml amphotericin and 2 mM glutamine. The human embryonal carcinoma cell line NTERA‐2 (ATCC® CRL‐1973™) and the JEG‐3 choriocarcinoma cell line (ATCC® HTB‐36™) were cultivated in high‐glucose (4.5 g/L) DMEM (PAA Laboratories GmbH) supplemented with 10% FBS (GIBCO® Invitrogen), 10.000 IU/ml penicillin (Biotica), 5 μg/ml streptomycin, 2.5 μg/ml amphotericin and 2 mM glutamine (PAA Laboratories GmbH). Finally, the human normal testis cell line Hs 1.Tes (ATCC® CRL‐7002™) was grown in high‐glucose (4.5 g/L) DMEM (PAA Laboratories GmbH) with the addition of 10% FBS (GIBCO® Invitrogen).The TCam‐2 human seminoma cell line  as well as the human embryonal carcinoma cell line NCCIT (ATCCCisplatin‐resistant sub‐lines (JEG‐3 CisR (D) VOL II, NCCIT CisR (D) VOL II, NTERA‐2 CisR and TCam‐2 CisR) were generated in our laboratory over a period of 6 months. Cisplatin‐resistant sub‐lines used in this study were derived by a long‐term propagation of matched parental cells in sub‐lethal concentrations of cisplatin  in the culture medium described previously.2. The characteristics of the cell lines investigated are summarised in Table All the used parental as well as cisplatin‐resistant cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.63 cells/100 µl  and of 4 × 103 cells/100 µl for NCCIT cell line, respectively, per well, were placed in 96‐well white‐walled plates (Corning Costar Life Sciences) and treated with cisplatin (0.01–5 µg/ml) overnight. The relative viability of the cells was measured using the CellTiter‐Glo™ Luminescent Cell Viability Assay (Promega Corporation) according to the manufacturer's protocol and evaluated by the LUMIstar GALAXY reader (BMG Lab Technologies) after 6–7 days  VOL II cells) or after 3 days of treatment (NCCIT and NCCIT CisR (D) VOL II cells). Each concentration was measured in quadruplicates. IC50 values were calculated by CalcuSyn 1.1 software (Biosoft).For the determination of chemosensitivity, cisplatin‐sensitive and cisplatin‐resistant TC cells of 2 × 102.76 cells  or 8 × 106 cells  were centrifuged at 700 × g for 5 min. Pellets of cells were washed with 5–10 ml of ice‐cold PBS and centrifuged again at 700 x g for 5 min. Supernatants were removed and the pellets thus obtained were re‐suspended in 400 µl of Cytosol Extraction Buffer Mix (CEB Mix containing DTT and Protease Inhibitor Cocktail). After 20 min incubation of the samples on ice, the analysed samples were centrifuged again at 700 × g for 5 min. The isolated supernatants, representing the cytosolic protein fraction, were stored at −80°C.A cytosolic protein fraction from TC cell lines was prepared using Fraction PREP TM Cell Fractionation kit (Bio Vision) according to the manufacturer's protocol. Briefly, 4 x 1033.1Investigation of the lectin binding to hCG was performed using surface plasmon resonance (SPR) involving a panel of five lectins, three hCG standards and using injections of lectins at seven different concentration levels . In total, at least 107 SPR sensorgrams were recorded and evaluated. Recombinant hCG (ab164958) was not glycosylated since SPR did not show any binding to a panel of five lectins (data not shown) and this form of hCG was excluded from further assays. Hence, the glycosylation of only two hCG forms was investigated.D and RUmax) regarding the strength and intensity of the lectin binding to the hCGs immobilised on the SPR chip, are summarised in Table The results indicate that hCG (ab77874) generally shows a higher level of binding to a panel of lectins Figure  top leftIn the next experiment, the binding of antibodies to the two glycosylated forms of hCG was investigated. In this particular case, we investigated the binding of three different antibodies at seven different concentration levels  to the two different hCGs immobilised on the SPR chip, that is, investigating at least 42 SPR sensorgrams. Some experiments were performed in a configuration with an antibody immobilised on the SPR chip with hCG present in a running buffer to cross‐check antibody–hCG bioaffinity interactions (i.e. 63 SPR sensorgrams). A summary from these investigations is provided in Table The results suggest that the best bioaffinity interaction was achieved between the native human hCG protein (ab77874) captured over the immobilised anti‐hCG beta 2 epitope antibody (ab11389) Table .In the final SPR experiment, we investigated the possibility of binding lectins to hCG (ab77874) bioaffinity captured on the SPR chip modified by the immobilised anti‐hCG beta 2 epitope antibody (ab11389). In other words, it was important to determine whether a biorecognition between hCG and an antibody occurs at the epitope, which is distant from the glycan‐containing epitope on hCG making it possible to form an Ab/hCG/lectin sandwich configuration, which is required for a specific glycoprofiling of hCG.The results showed that MAA, SNA and PHAE lectins  and an oxidised anti‐hCG was performed in an ELISA‐like analysis format. The experiment indicates four lectins, in particular, significantly binding to the non‐oxidised anti‐hCG antibody. Of those four lectins, two bind to fucose (AAL and LCA) and the other two lectins bind to mannose glycan units (ConA) or terminal 2,6‐SA (SNA) on the non‐oxidised antibody. Oxidation of the glycan on the anti‐hCG resulted in a significant decrease in binding of all the lectins investigated Figure . On the In the next sequence, we used the oxidised form of the antibody taking into account that the binding of ConA and AAL to the antibody was not fully suppressed. So the results obtained by the glycoprofiling of hCG using ConA and AAL should be treated with caution.3.350 values for sensitive and resistant variants of the cell lines is provided in Table In this study, we used four different cell lines  with cisplatin‐sensitive and cisplatin‐resistant variants and one control cell line (CTRL i.e. Hs 1.Tes cell line) Table . We inveThe binding of selected lectins to hCG immunocaptured by the anti‐hCG antibody in the ELLA experiment is shown in Figure 3.3.1The NTERA‐2 cell line is a primary male testicular cell line, representing a malignant pluripotent embryonal carcinoma isolated from lung metastases with non‐seminoma‐related cells.In general, it may be stated that no significant changes in hCG's glycans are observed between the sensitive NTERA‐2 and the CTRL cell lines, when considering mannose‐recognising lectins such as ConA, HHL, DFL, GNL and PSA. Insignificant glycan changes when compared with the CTRL cell line were also observed for other lectins such as DBA, RCA, PNA, WGA, DSL, PHAL and PHAE Figure .A significant difference between the sensitive NTERA‐2 and the CTRL cell lines was observed using the LCA lectin  and the AAL lectin (recognising core and antennary fucose (aFuc)) with such glycans being present in lower amounts in the sensitive NTERA‐2 cell line compared to the CTRL one. Significantly lower binding to hCG from the sensitive NTERA‐2 cell line than for the CTRL was also observed for the WFL, SNA and MAA lectins Figure .Accordingly, we may conclude that the sensitive NTERA‐2 cell line does not synthesise a higher level of glycans than the CTRL cell line, which accords with the published data.3.3.2TCam‐2 cell line represents a testicular seminoma‐like cell line of male origin.While there is no difference in ConA binding, a significant increase in HHL and DFL binding to hCG for the cancerous‐sensitive cell line over the CTRL cell line was observed Figure . This inA significant increase in the binding of lectins to hCG from the cancerous‐sensitive cell line over the CTRL cell line was further observed for WFL, AAL, RCA, DSL, WGA, PHAL and SNA.N‐glycans (PHAL) or 2,6‐SA (SNA)‐containing glycans in the sensitive TCam‐2 cell line than in the CTRL cell line. An increased binding of SNA to hCG from the cancerous cell line TCam‐2 over the control cell line is in agreement with findings in the literature, where increased levels of sTn and sT antigens were shown in the tissues of the patient with seminoma type of TC.Hence, we may conclude that there is an increased expression of glycans containing pMan (HHL and DFL), aFuc (AAL), GalNAc (WFL), Gal/GalNAc (RCA), Gal‐GlcNAc (DSL), GlcNAc (WGA), branched N‐glycans in TC serum samples performed by another group of researchers revealed that, in particular, levels of tri‐/tetra‐antennary glycans (PHAL) with terminal sialylation (SNA and/or MAA), galactosylation (RCA and/or DSL) or terminal GlcNAc (WGA), were increased in GCT patients.Analysis of 3.3.3JEG‐3 cell line represents choriocarcinoma of placenta cell line of female origin.The JEG‐3 cell line exhibits lower ConA (Man), DFL (pMan), GNL (Man), LCA (cFuc), AAL (aFuc), WFL  and SNA 2,6‐SA) binding than in the CTRL cell line  of male origin.Hence, a lower expression of various types of glycans on hCG from the NCCIT cell line than for the CTRL cell line is similar to the JEG‐3 and NTERA‐2 cell lines.3.4The binding of selected lectins to hCG immunocaptured by the anti‐hCG antibody in the ELLA experiment is shown in Figure 3.4.1a antigen, GalNAc‐Gal) and SNA  binding to hCG expressed by the resistant cell line over the sensitive one  binding to hCG from the resistant NTERA‐2 cell line when compared with the sensitive NTERA‐2 cell line with a significant increase in DBA  and the resistant  cell lines versus cisplatin IC3.5.150 values is shown in Figure 50 values for the following lectins: AAL, DFL, DSL, HHL, PHAE, PNA, SNA and WFL with a quasi‐exponential and strong pattern of correlation for HHL .If we hypothesise that the changed glycosylation on hCG results from an exposure of cells to cisplatin, then any type of correlation between lectin binding and cisplatin IC50 value  activity (formation of Sda antigen) in colon, gastric cancer and embryonal carcinoma.a antigen to the enzymatic action of fucosyltransferases expressing sLea or sLex antigens on glycans, which are very important glycan determinants of induction of a metastatic process.a level is directly proportional to a decrease in the level of sLea or sLex antigens (aFuc) and vice versa.a antigen) to hCG was observed  to hCG of the sensitive cell line was observed , HHL , DFL , AAL (TCam‐2 and NCCIT), WFL (TCam‐2), WGA (TCam‐2), SNA (TCam‐2 and NCCIT) and MAA (NCCIT). This is in agreement with the study showing an increased binding of DBA lectin to hepatocellular carcinoma cells treated with the multikinase inhibitor sorafenib, while the binding of lectins such as MAA, PHAL, RCA and SNA was significantly reduced for the cells treated with sorafenib on a lectin microarray using a cytosolic cell fraction.w = 90–150 kDa for a resistant sub‐line than for a sensitive one.On the other hand, the binding of several lectins to HCG's glycans was lower for the cisplatin‐resistant cell lines than for the sensitive cell lines including SNA lectin. This is in agreement with the study, where it was observed that a resistant serous ovarian cancer cell line exhibited a lower binding of SNA, WFL and seven other lectins.N‐glycans.The level of fucosylated glycans was lower in the resistant ovarian cancer cell line than in the sensitive one, applying mass spectrometry to a cytosolic fraction of cells with released N‐ or O‐glycome using a lectin microarray or a MS‐based approach. Accordingly, it is important to note that in these studies the authors did not identify proteins with particularly changed glycans. Only one study indicates that a particular glycan signature can be associated with one protein or subset of proteins and the other particular glycan change can be associated with another protein or another subset of proteins.Lycopersicon esculentum lectin, increase in binding) and SNA (decrease), when using a lectin microarray. A lectin dot blot revealed that LCA bound to glycoproteins with Mw of 70–180 kDa; ConA bound glycoproteins with Mw of 50–180 kDa; LEL bound to glycoproteins with Mw of 50–110 kDa, Mw of 40–50 kDa, and with Mw above 180 kDa; while SNA bound to glycoproteins with Mw of 50–180 kDa.It should be observed that all the published studies discussed so far analysed the whole N‐glycans or O‐glycans significantly affects the binding of lectins recognising the terminal parts of the glycans   or CAD antigenThe results thus obtained lead us to hypothesise that the addition of GalNAc to ) Figure . Based o) Figure , cells a) Figure  or TC ce) Figure  with an Furthermore, lectins such as AAL, DFL, DSL, HHL, PHAE, PNA, WFL and PSA could potentially be used for the identification of a pre‐target resistant mechanism, while lectin SNA could be used for the identification of a post‐target resistant mechanism (i.e. to identify TC patients resistant to cisplatin treatment after a first round of chemotherapy). This hypothesis, however, needs to be verified using serum/plasma samples of TC patients.550 values for the sensitive cell lines and for the resistant cell lines. The results observed in the current study require validation using serum/plasma samples of TC patients in order to corroborate the clinical potential of specific glycoprofiling of hCG to identify cisplatin‐resistant TC patients, who have a poor prognosis.This is the first study focused on use of a specific glycoprofiling of hCG using a panel of lectins for investigation of glycosylation changes associated with TC or associated with cisplatin resistance. We identified the DBA lectin as the best prospect for evaluating the resistance of TC lines. Moreover, we identified some lectins to be used for positive correlation between lectin binding to hCG and cisplatin ICThe authors declare no conflict of interest.MH & EJ: investigation, data curation and writing ; KK: investigation, resources and writing original draft; MC & MM: conceptualisation, resources and writing ‐ review and editing; PK: funding acquisition, project administration and writing ‐ review and editing; TB: formal analysis, visualisation and writing  and JT: conceptualisation, formal analysis, funding acquisition, methodology, supervision and writing .The work with TC cell lines did not require ethics approval.Supplementary MaterialClick here for additional data file.


Background: It is estimated that one in five people worldwide faces a diagnosis of a malignant neoplasm during their lifetime. Carvacrol and its isomer, thymol, are natural compounds that act against several diseases, including cancer. Thus, this systematic review aimed to examine and synthesize the knowledge on the antitumor effects of carvacrol and thymol.Methods: A systematic literature search was carried out in the PubMed, Web of Science, Scopus and Lilacs databases in April 2020 (updated in March 2021) based on the PRISMA 2020 guidelines. The following combination of health descriptors, MeSH terms and their synonyms were used: carvacrol, thymol, antitumor, antineoplastic, anticancer, cytotoxicity, apoptosis, cell proliferation, in vitro and in vivo. To assess the risk of bias in in vivo studies, the SYRCLE Risk of Bias tool was used, and for in vitro studies, a modified version was used.Results: A total of 1,170 records were identified, with 77 meeting the established criteria. The studies were published between 2003 and 2021, with 69 being in vitro and 10 in vivo. Forty-three used carvacrol, 19 thymol, and 15 studies tested both monoterpenes. It was attested that carvacrol and thymol induced apoptosis, cytotoxicity, cell cycle arrest, antimetastatic activity, and also displayed different antiproliferative effects and inhibition of signaling pathways (MAPKs and PI3K/AKT/mTOR).Conclusions: Carvacrol and thymol exhibited antitumor and antiproliferative activity through several signaling pathways. In vitro, carvacrol appears to be more potent than thymol. However, further in vivo studies with robust methodology are required to define a standard and safe dose, determine their toxic or side effects, and clarify its exact mechanisms of action.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=176736.This systematic review was registered in the PROSPERO database (CRD42020176736) and the protocol is available at  It is estimated that one in five people worldwide faces the diagnosis of some malignant neoplasm during their lifetime, and the number of people with cancer is forecast to double by the year 2040 . In factTaxus brevifolia Nutt. species . There are many medicinal plants whose pharmacological properties have already been described and scientifically proven . However species . More re species .Origanum vulgare L.), thyme (Thymus vulgaris L.) and “alecrim-da-chapada” (Lippia gracilis)  and its thymol isomer (2-isopropyl-5-methylphenol), classified as natural multi-target compounds, deserve attention. Both are monoterpenoid phenols, the main components present in essential oils obtained from several plant species of the Lamiaceae and Verbenaceae families, such as oregano (racilis) , which hracilis) , includiracilis) . In addiracilis)  and antiracilis)  activitiracilis) . Hence, in vitro) or in animal models of cancer? The review followed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (The purpose of this systematic review was to answer the following question: Do carvacrol and thymol exhibit an anti-tumor effect on cancer cells ((PRISMA) .in vitro and in vivo.A PICOS strategy  was used based on: P: Animals with cancer or tumor cells; I: Treatment with carvacrol or thymol; C: No treatment, healthy cells or placebo (vehicle); O: Cytotoxic and antitumor effects, induction of apoptosis and inhibition of proliferation; S: Pre-clinical studies in vitro, in vivo, carvacrol or 5-isopropyl-2-methylphenol and thymol or 5-methyl-2-propan-2-ylphenol  using a combination of health descriptors, MeSH terms and their synonyms, such as antitumor, antineoplastic, anticancer, cytotoxicity, apoptosis, cell proliferation, in vitro studies of cancer cell lines, in vivo study of animals with cancer; cytotoxic effect, antitumor effect, inhibition of proliferation and apoptosis and experimental studies (in vitro and in vivo). The exclusion criteria were: experiments with derivatives of the carvacrol or thymol, association of the two compounds with other substances or in mixtures composing essential oils and extracts, animals with other diseases in addition to cancer, review articles, meta-analyses, abstracts, conference articles, editorials/letters and case reports. A manual search of the reference lists of all selected studies was also conducted, in order to identify additional primary studies for inclusion.Two independent reviewers (L.A.S. and L.T.S.P.) analyzed the research results and selected potentially relevant studies after reading their title and abstract, using the systematic review application, Rayyan . We usedWe extracted the following data from the included articles: author, year, country, data about the monoterpene , concentration and/or dose, type of animal or cell line, results , proposed mechanisms involved in the antitumor effect and conclusion. The authors of the included studies were contacted when necessary (whenever any data or article was not available).in vivo experimental studies  (ICnit (μM)  and wereκ = 0.813) among the reviewers, after selecting the titles and abstracts.The initial search resulted in 1,170 records, of which 594, 169, 194, and 213 were found in PubMed, Web of Science, Scopus and Lilacs, respectively. Of these, 388 were excluded due to duplication. After screening the title and abstract, 706 reports were excluded, and 1 report was sought for retrieval, as it met the criteria after reading the full text, resulting in 77 studies. Of these, three were excluded after reading the full text (two for presenting the same results and one that tested the anti-cancer effect on bacteria) and four for not having access to the full text, resulting in 70 articles. In addition, 9 studies were identified after a manual search of the references and two studies obtained from other sources, but only seven studies were added , finally resulting in 77 included studies . There wn = 14), China (n = 14), Turkey (n = 13), the Republic of Korea (n = 5), Slovakia (n = 5), Iran (n = 5), Morocco (n = 2), Brazil (n = 2), Greece (n = 2), Iraq (n = 2), The United States of America (n = 2), Canada (n = 2), Egypt (n = 2), Croatia (n = 1), Lithuania (n = 1), Italy (n = 1), Spain (n = 1), The United Kingdom (n = 1), Peru (n = 1), Belgium (n = 1). Asia (51.9%) was the continent with the largest number of publications on the subject, followed by Europe (33.7%), the Americas (9.3%) and Africa (5.1%), with a greater trend of publications on this subject in the last three years (in vitro experiments and 10 (12.9%) were in vivo studies. Likewise, 43, 19, and 15 publications tested only carvacrol, thymol and both compounds, respectively. They were mostly obtained commercially, provided by Sigma Aldrich (n = 52), Fluka (n = 5), Aldrich Chemical (n = 2), Western Chemical (n = 1), Agolin SA (n = 1), Alfa Aesar (n = 1). In only five studies were the compounds isolated from essential oils. Remarkably, ten studies did not report the source of the tested compounds. A detailed description of the included studies is shown in in vitro and in vivo studies.The selected studies were carried out in different countries: India  was able to inhibit the viability and proliferation of lung cancer cells (A549 cell line), in addition to inducing early apoptotic characteristics  and reduIn relation to hepatocarcinomas (HepG2 cell line), carvacrol exhibited anticancer effects, provoking cell death and antiproliferative effects in a concentration-dependent manner . The inhColorectal cancer (Caco-2 cell line) also exhibited reduced cell viability and a significant increase of early apoptotic cells after carvacrol incubation (115 μM) . There wIn respect of breast cancer, treatment with carvacrol decreases MDA-MB231  and MCF-It was also observed that the administration of carvacrol provoked cytotoxic and apoptotic effects on HeLa and SiHa cervical cell lines . In factCarvacrol also induced cytotoxicity and apoptosis (via caspase-3 and reactive oxygen species—ROS) of human oral squamous cell carcinoma (OC2 cell line) in a concentration-dependent manner . In tongIn a prostate cancer cell line (DU 145), carvacrol showed a significant reduction of cell viability and proliferation in a concentration and time dependent manner, marked by a cell cycle arrest, resulting in the accumulation of cells in the G0/G1 phase, and apoptosis, related to the increased activity of caspase-3, production of ROS and loss of mitochondrial membrane potential . PC-3 ceRegarding human choriocarcinoma (JAR and JEG3 cell lines), carvacrol was able to inhibit proliferation and induce cell cycle arrest. The results showed that carvacrol reduced cell proliferation and provoked apoptosis mediated by mitochondrial membrane potential depolarization, increased mitochondrial calcium and activation of Bax and Cytochrome C expression. In addition, treatment with this monoterpene promoted an accumulation of cells in the sub-G1 phase, indicating that changes in intracellular calcium and ROS generation are related to the antiproliferative effects observed. Additionally, there was a marked phosphorylation of ERK1/2 and also inhibition of the PI3K/AKT signaling pathway, indicating that carvacrol regulates signaling pathways by inhibiting MAPK and PI3K .In murine B16-F10 and A375 melanoma cell lines, carvacrol reduced cell viability and induced cytotoxicity . The ant50 of 421 and 229.68 μM in 48 and 72 h, respectively (50 = 60.01 μg/mL) concentrations and antitumor effects (apoptosis and inhibition of cell proliferation) at higher concentrations (>IC50 = 60.01 μg/mL)  . ElshafiThymol also showed concentration-dependent cytotoxic effects and reduced the proliferation of Caco-2 cells . In contBreast cancer cells (MDA-MB 231 cell line) also exhibited a reduction in cell viability after thymol treatment . The inhThe administration of thymol to bladder cancer  provoked inhibition of cell proliferation and decreased the cell viability in a concentration and time dependent manner, along with marked cell cycle arrest in the G2/M phase and induction of apoptosis through the intrinsic pathway, together with the activation of caspase-3 and -9, JNK and p38, release of cytochrome C, negative regulation of Bcl-2 family proteins and production of ROS. In addition, a considerable decrease in the expression of cyclin A, B1 and CDK2, as well as an increase in the expression of p21 were observed after treatment with thymol, suggesting that its antitumor effect occurs by inhibiting the PI3K/Akt signaling pathway, via MAPKs, and generation of ROS . In humaHuman glioblastoma cells (DBTRG-05MG) showed reduced viability in a concentration-dependent manner when treated with carvacrol (200–600 μM), induced apoptosis and necrosis by ROS production and caspase-3 activity . In a raRegarding thymol, treatment at concentrations of 100 and 200 µM induced a significant reduction in cell viability and inhibited the migration of glioma cells (C6 cell line) through phosphorylation of PKCα and ERK1/2, that resulted in decreased expression of MMP-9 and MMP-2 . In addiTreatment with carvacrol in leiomyosarcoma cells exhibited antiproliferative effects in a concentration dependent manner and also inhibition of cell growth . In addiIn relation to thymol, there were concentration-dependent cytotoxic effects and interruption of the cell cycle progression in the G0/G1 phase in P-815 cells . In humaCarvacrol showed cytotoxic effects against human myeloid leukemia cells (K-562 cell line)  and agai2O2, and Bax protein levels; and activation of caspase-8, -9, -3 and PARP  that at a concentration of 300 µM of carvacrol the KG1 cell lines were more sensitive compared to the HL60 cell line, and at 400 µM the K-562 cell line showed resistance after 48 h of treatment. Regarding thymol (50 µM), the KG1 cell line was also more sensitive when compared to the other two and at the 100 µM dose, thymol was able to induce complete cell death in the KG1 and HL60 cell lines (It was observed by Bouhtit ll lines .When using mouse myoblast cells (CO25 cell line) transformed with human N-RAS oncogene, 50 (μM) values determined 24 h after the incubation of the studied cells with carvacrol or thymol. It was possible to verify that, in general, carvacrol  is more potent than thymol , with difference between means of 103.8 (±106.9). The lowest IC50 values for carvacrol were against prostate carcinoma , vascular endothelial growth factor (VEGF) and decreased expression of the gamma glutamyl transferase (GGT) gene  gene .Carvacrol supplementation significantly improved the weight gain and growth rate of animals with colon cancer induced by 1,2-dimethylhydrazine (DMH), exhibiting a lower incidence of tumors and pre-neoplastic lesions, along with a reduction in oxidative stress damage , suggesting that carvacrol presents chemopreventive effects .in vivo models to investigate the effect of thymol on cancer progression. For the colorectal cancer model, HCT116 xenograft was injected (i.p.) into BALB/c mice, which after 7–10 days (when the tumors grew to approximately 100 mm3) were treated with thymol (75 or 150 mg/kg every other day). A significant reduction in cancer growth, a greater number of necrotic lesions and a lower level of Ki-67 expression were observed, which reflects cell proliferation. As for the lung metastasis model, HCT116 cells were injected into the tail vein of each mouse and then received treatment with thymol (75 or 150 mg/kg every other day). After 6 weeks, they found that the average number of tumor nodules on the lung surface of the two treatment groups was significantly lower, revealing an anti-metastatic effect, probably due to the inhibition of the Wnt/β-catenin signaling pathway  of the studies recorded reported randomness when taking photos of selected areas regarding apoptotic activity assays. However, 97% of the studies applied the same conditions  for in vitro assays, ensuring a low risk of bias  record random allocation of the animals in the study  were classified as uncertain due to the lack of information in the articles. In contrast, 30%  reported that carvacrol decreased CDK4 protein expression in human tongue squamous cell carcinoma (Tca-8113 cells) (n = 10) of in vitro studies also represents a promising target of cancer treatment, since natural compounds can act as modulators, interrupting the cell cycle and, therefore, killing cancer cells (n = 38) of the studies reported induction of apoptosis after treatment with carvacrol or thymol in their in vitro studies and only 40% (n = 4) in in vivo studies.Neoplasia can be described as a disease of unchecked cell division, and its progression is related to abnormal activity of cell cycle regulators. The cell cycle consists of four discrete phases in which the cell increases in size and cellular content is duplicated (gap 1 or G1 phase), DNA is replicated , it prepares to divide , and then divides, creating two identical daughter cells . As a cell moves through each phase, stimulated by growth and transcription factors, it passes through several checkpoints, which ensure that mitosis occurs only when the cellular genome has been precisely replicated, avoiding mutations and generation of transformed cells . The cel3 cells)  and in b3 cells)  and only3 cells) . CDK inh3 cells) , such as3 cells)  and brea3 cells) . In addier cells . The uncer cells , that iser cells   and can interrupt DNA replication and cell division through cell cycle arrest (G1/S checkpoint), in order to restore genetic integrity, thereby preventing genetically transformed cell proliferation . When rein vitro studies (n = 17) and 10% of in vivo studies showed an increase in Bax levels after treatment with carvacrol or thymol. Additionally, 26% (n = 18) of in vitro studies reported negative regulation of the anti-apoptotic protein Bcl-2 (n = 23) of in vitro studies reported an increase of caspase activity, more specifically caspase-3  of the studies that tested carvacrol or thymol showed that they were able to increase in vitro JNK phosphorylation in colon cancer (n = 3) of the studies in vitro induced phosphorylation of p38 in hepatocarcinoma cells  of in vitro studies using thymol in human promyelocytic leukemia cell lines (n = 5) of in vitro studies that used carvacrol in colon cancer  (in vivo study (10%) reported a decrease in the levels of MMP-2 and MMP-9 in liver cancer after treatment with carvacrol (n = 6) were in vitro studies. It is important to note that carvacrol prevented metastasis in vivo, demonstrated by tumors less likely to spread through intrahepatic veins  . On the ression) , colorecression) , ovarianression) , and wasression) . In addiression)  serving ression) . The incression) . The decression) . Togetheression) . High MMression)  and brearession) , oral (Lression) , bladderression) , skin (Fression) , larynx ression)  cancer. arvacrol , the majic veins  and previc veins , and thy pathway . HoweverIn vitro, carvacrol and thymol increased the generation of reactive oxygen species in 24.63% (n = 17) of the studies, a fact that is also observed in chemotherapeutics such as doxorubicin , these are a group of molecules that contain reduced forms of oxygen with short life and that are more energetically reactive than molecular oxygen . The genorubicin , cisplatorubicin  and bleoorubicin , which an cancer  and hepan cancer  increasen cancer . Similarn cancer . Howevern cancer . In thisn cancer . FurtherPost-treatment with carvacrol also promoted an increase in the expression of the DAPK1 gene and a decrease in the enzyme PPP2R2A . DAPK1 iin vitro studies, we found that carvacrol proves to be more potent than thymol, and appears to have a greater cytotoxic effect for some cell lines, such as carcinomas (prostate and stomach)  . Thymol in vivo studies, on the other hand, the effect of carvacrol has been predominantly studied against hepatocarcinoma, with few studies for breast and colon cancer. In a way, it can be seen that the studies in this area do not seem to evolve toward a specific target, with low complementarity of the screenings carried out in vitro for the studies developed with experimental animals. This is a great barrier advances in this area of knowledge and helps to explain the discrepancy in the number of studies published in vitro (n = 69) and in vivo (n = 10) over the last two decades, as well as the absence of clinical trials. Thus, it is necessary to consider the studies already carried out for these compounds before conducting new primary studies on this topic, in order to evolve the research stages to the next level of drug development: in vivo studies and clinical trials.In 50 of 138.1 ± 8.7 μM of carvacrol   (Seven studies used a positive control to compare the antitumor effect of carvacrol  and/or t/100 µL) , 5-fluor/100 µL) , doxorub/100 µL)  (1.0 μg//100 µL)  and (2 μ/100 µL) , camptot/100 µL)  and in v2 mg/kg) , were thin vivo studies, particularly in respect of thymol, are needed. Further studies are required to help unravel and define the mechanisms of action of the two compounds against cancer cells, as well as studies that further explore their chemopreventive and anti-metastatic effects. It is of note that several studies suggested that signaling pathways (PI3K/AKT/mTOR and MAPKs) may be the main mechanism of action of these monoterpenoids, but more advanced studies are needed to elucidate this issue. There is also still a wide variation in the doses used, requiring the establishment of a consensus in respect of defining potentially effective and safe doses. In addition, further studies are also needed to clarify the effects of these two compounds on normal/healthy cells in vitro, and in appropriate in vivo models.In summary, we observed that more in vitro studies that carvacrol seems more potent than thymol, and was shown to have a greater cytotoxic effect for some cell lines. Moreover, further animal studies should be encouraged, as they have as yet made limited progress, and most of the current results are based on in vitro studies.The knowledge obtained through the reviewed studies provides strong evidence of the antitumor and antiproliferative activity promoted by carvacrol and thymol. However, there are still gaps regarding the standard or ideal dose, the exact mechanism of action and safety of the two compounds, revealing challenges for future studies. In addition, it was found through