N-glycans, such as HexNAc4Hex3DeoxyHex1 and HexNAc5Hex3DeoxyHex1, derived from both cell lines exhibited a significant increase in Plasmax. Among the O-glycans, significant differences were also observed. Both cell lines cultivated in EMEM had the lowest amounts of O-glycans expressed. The original work described the development of Plasmax, which improves colony formation, and resulted in transcriptomic and metabolomic alterations of cancer cell lines, while our results indicate that Plasmax can significantly impact protein glycosylation. This study also provides information to guide the selection of media for in vitro cancer cell glycomics studies.A complex physiological culture medium (Plasmax) was introduced recently, composed of nutrients and metabolites at concentrations normally found in human plasma to mimic the in vivo environment for cell line cultivation. As glycosylation has been proved to be involved in cancer development, it is necessary to investigate the glycan expression changes in media with different nutrients. In this study, a breast cancer cell line, MDA-MB-231BR, and a brain cancer cell line, CRL-1620, were cultivated in Plasmax and commercial media to reveal cell line glycosylation discrepancies prompted by nutritional environments. Glycomics analyses of cell lines were performed using LC-MS/MS. The expressions of multiple fucosylated  The nutrient compositions of culture media can profoundly affect the phenotypic behavior of cells, including their response to stress and stimuli, the epigenotype. However, most of the commercially available and widely used cell culture media may not be able to mimic the in vivo environment for cell lines. The compositions of the commercial media were not designed to reproduce a physiological cellular environment, such as human plasma, but rather to provide continuous cultivation of cells using a minimal amount of nutrients. A good example is Eagle’s Minimal Essential Medium (EMEM), a synthetic cell culture medium developed by Harry Eagle . In addiO-glycan containing a sialic acid α-2,6 linked to GalNAc α-O-Ser/Thr) antigen can inhibit cell adhesion and thus increase cell movement and spread 2+ = 915.4908, the EIC was extracted, with the full MS spectrum depicted by the inset. The composition was confirmed as HexNAc4Hex3DeoxyHex1, which is a potential fucose positional isomer. Then, the MS/MS spectrum in m/z = 468.3 ([M + H]+) represents the diagnostic fragment ion of core-fucosylation. It contains one fucose connected with one GlcNAc on the reducing end, which is the unique part of core-fucosylataed N-glycans. Therefore, this structure of HexNAc4Hex3DeoxyHex1 was confirmed as core-fucosylataed. Another example from the CRL cell line is shown in 4Hex5DeoxyHex1  was confirmed through full MS. The MS/MS spectra assigned the fucose to the core GlcNAc as the diagnostic ion of m/z = 468.3 was also observed for this structure. By applying the same strategy, more than 50 unique N-glycan structures were identified from each cell line cultivated in three media. Core-fucosylation was mostly found among the fucosylated N-glycans derived from the two types of cancer cell lines used for this study.Since the positional isomers are very common in N-glycans from the 231BR cell line and 51 N-glycans from the CRL cell line were identified  in the relative abundance when cultivated in Plasmax compared to DMEM and EMEM. On the other hand, 29 N-glycans, derived from the brain cancer cell line CRL, showed significantly different expressions between Plasmax and the two commercial media. Bar graphs  were upregulated in Plasmax. Interestingly, the expressions of the fucosylated structures with galactose at the end of the branches tended to be more active in DMEM  were also observed, with a higher abundance in Plasmax, including HexNAc4Hex5DeoxyHex1NeuAc1, HexNAc3Hex4DeoxyHex1NeuAc1, and HexNAc5Hex6DeoxyHex1NeuAc1. In addition, significant differences were also found among sialylated structures from the CRL cell line, such as HexNAc3Hex5NeuAc1, HexNAc5Hex5NeuAc1, and HexNAc5Hex6NeuAc2  and the pie charts in t-test was applied to assess the statistical significance between each two media . A relatively lower amount of sialylated glycans was observed in all three media, with 0.8% in DMEM, 1.5% in EMEM, and 1.0% in Plasmax. Sialo-fucosylated structures also had the highest abundance of 5.7% in EMEM, relative to 3.4% and 3.1% in DMEM and Plasmax, respectively.Based on the monosaccharide compositions, all identified wo media . The disN-glycans from the CRL cell line. High-mannose N-glycans showed the highest abundance in all three media. Both DMEM and EMEM have more than 50% of their N-glycans as high mannose, while the 48.3% in Plasmax is significantly lower than the 53.3% in EMEM (p = 3 × 10−4). The amounts of sialylated N-glycans were relatively higher compared to the 231BR cell line. A total of 3.9% and 4.1% sialylated structures in Plasmax and EMEM, respectively, is both significantly higher than the 2.0% in DMEM (p = 1 × 10−3). The highest fucosylation level was observed in DMEM at 38.0%, which is 4.3% higher than Plasmax (p = 0.04). In EMEM, however, the abundance of fucosylated N-glycans was 3.2% lower than that in Plasmax (p = 0.04). Sialo-fucosylated N-glycans showed the highest abundance in Plasmax of 6.5%, which is upregulated by 2.5% relative to DMEM (p = 9 × 10−4). Other structures accounted for 7.6% in Plasmax, which is significantly higher than the 6.2% in EMEM (p = 0.02) and 4.8% in DMEM (p = 1 × 10−3).O-glycans, the same data processing strategies were applied. First, we identified the O-glycan structures in two cancer cell lines cultivated by three media using the software MultiGlycan and manually checked the results using Xcalibur. A total of 27 and 31 O-glycans were identified and quantified from the 231BR and CRL cell lines, respectively . O-glycans in both cell lines. Each O-glycan is labeled by monosaccharide compositions, which were confirmed based on their m/z values. O-glycan structures, only 4 structures were detected in EMEM, including HexNAc2Hex7, HexNAc2NeuAc1, HexNAc5Hex6, and HexNAc2Hex3DeoxyHex1. In addition, the sialo-fucosylated structure HexNAc2DeoxyHex1NeuAc1 showed the highest relative abundance when cultured in Plasmax. However, this O-glycan was not detected in DMEM and EMEM, indicating that the nutritional environment of Plasmax might be more suitable for the expression of this glycan. Furthermore, HexNAc1Hex1NeuAc2, HexNAc2Hex6, and HexNAc3Hex1 were exclusively detected in DMEM. The abundances of fucosylated structures such as HexNAc3Hex2DeoxyHex1 and HexNAc5Hex6DeoxyHex2 were higher in Plasmax. The neutral structure HexNAc2Hex10 showed a higher abundance in DMEM compared to Plasmax.By comparing the relative abundance of each identified O-glycans of the CRL cell line. Similar to the results we found in the 231BR cell line, multiple O-glycans of CRL were absent in EMEM, suggesting that the nutritional environment of EMEM might not be very supportive of O-glycan expressions in cancer cell lines. Therefore, we focused on the expression differences between DMEM and Plasmax. Sialylated structures such as HexNAc6Hex5NeuAc1 and HexNAc1Hex1NeuAc1 exhibited significantly higher abundances in Plasmax (p < 0.005). However, four fucosylated structures, including HexNAc6Hex4DeoxyHex1, HexNAc6Hex3DeoxyHex1, HexNAc2Hex5DeoxyHex3, and HexNAc6Hex5DeoxyHex1, had lower relative abundances in Plasmax when compared to DMEM. In addition, another four sialo-fucosylated O-glycans, including HexNAc3Hex3DeoxyHex2NeuAc2, HexNAc3Hex3DeoxyHex1NeuAc1, HexNAc3Hex3DeoxyHex1NeuAc3, and HexNAc2Hex4DeoxyHex3NeuAc2, also had lower level of relative abundance in Plasmax than in DMEM. Other O-glycan structures, such as HexNAc2Hex6 HexNAc2Hex7 and HexNAc2Hex8, showed comparable abundances between Plasmax and DMEM. As reported by the previous study [N-glycans were detected after applying the same method to release O-glycan, thus we have a high confidence in identifying these structures as O-glycans. However, since the sample species were different in this study, we cannot completely exclude the possibility of N-glycans. This needs to be investigated in future studies. The significant up- and downregulation of O-glycans in the three media was also compared and is shown in heat maps in O-glycans and eight downregulated O-glycans in Plasmax. However, CRL cell line has 12 upregulated O-glycans and 9 downregulated O-glycans.us study , none ofO-glycans, the identified structures were grouped into four different types, including sialylated, fucosylated, sialo-fucosylated, and other structures. As shown in O-glycans from both cell lines cultivated in three media are presented by the pie charts, with p = 0.01). Fucosylated structures, with 22.4% in DMEM and 21.0% in Plasmax, were both higher than the 12.9% in EMEM. The highest sialylation level was observed in DMEM, which is 6.3% higher than Plasmax (p = 4 × 10−3). For other structures, no significant difference was observed between the media.To study the global effects of different media on the expressions of O-glycans are shown in p = 0.02 and p = 1 × 10−4, respectively). On the other hand, CRL cells cultured in Plasmax exhibited the highest expression level of sialylated O-glycans, which is 15.4% higher than DMEM (p = 0.01). In contrast, the fucosylation level in Plasmax was the lowest when compared to that in DMEM (p = 4 × 10−3) and EMEM (p = 5 × 10−5).For the CRL cell line, the different distributions of N- and O-glycans; therefore, the transcriptomics analysis of glycosylation genes can provide deeper understandings of the alterations in Plasmax. In our study, we focused on the glycogenes that are associated with the glycan expression changes described above. All detected glycogenes are shown in N-acetylglucosaminyltransferase (MGAT) transcripts. Both MGAT1 and MGAT2 were more than 2-fold upregulated in Plasmax over EMEM, leading to activated GlcNAc addition and branching of N-glycans [N-glycans, respectively. The related transcripts of glycotransferases are labeled. Similar changes in MGATs were also observed in CRL cell lines as shown by N-glycans in Plasmax. In addition to that, a higher abundance of fucosyltransferases was also observed in Plasmax compared to EMEM  in different media, we observed obvious fold changes, which might contribute to the subsequent glycomics biosynthesis enzymes, thus regulating the glycan synthesis processes.Glycotransferase plays an important role during the synthesis processes of -glycans . Figure  to EMEM . SpecifiN- and O-glycomes profiling of cancer cell lines cultivated in Plasmax, and compared the results with the expression results in commercial media DMEM and EMEM. Furthermore, the transcriptomics of cells cultured in different media was also investigated, focusing on glycosylation gene expression. These glycosylation gene expressions were correlated with glycomic changes to acquire a deeper insight into glycosylation alterations in Plasmax.Cell line cultivation is a vital part of in vitro studies of disease marker candidate discovery, which is also one of the main strategies of current cancer research. As a major material for the experiment, the culturing media should be carefully taken into consideration to provide a suitable nutritional environment for cell lines, since it can determine the proliferation, transcriptional, and metabolic phenotypes of cells. The novel physiological medium, Plasmax, composed of nutrients and metabolites found in human plasma, has been reported to enhance colony formation and alter the metabolism of cells. Moreover, Plasmax can closely mimic the in vivo environment of tumors compared with the commercial medium DMEM-F12 , leadingN-glycan analysis, each run contained reduced and permethylated glycans after enzymatical release from 100 μg of proteins using PNGase F. On the other hand, O-glycans released from 300 μg of proteins were injected for each analysis. Unlike N-glycans, so far, there are fewer efficient and reliable enzymes for the cleavage of O-glycans from glycoproteins; therefore, chemical release methods such as β-elimination are frequently used for O-glycomics studies [β-elimination can suffer from significant sample loss during the cleaning step for the removal of extra salt. In our work, we followed an enzymatic/chemical approach, which minimizes the sample loss and improves the sensitivity of detection. Moreover, this method does not lead to detectable co-release of N-glycans according to the reported results [β-elimination process, thus releasing O-linked glycans. The following permethylation reaction converts the released O-glycans to their permethylated derivatives, which improves the structural stability and the ionization efficiency of glycans during the electrospray ionization (ESI) process in positive ion mode [After the cell harvest and protein extraction, the amount of proteins in each sample was normalized utilizing a BCA protein assay. For  studies ,51. Howe results . Startinion mode ,53. MoreN-glycan structures were identified and quantified in the 231BR and CRL cell lines, respectively. The relative abundances of the glycans were calculated and subjected to unsupervised PCA. The clusters of the triplicates cultivated in Plasmax were clearly separated from the commercial media in both cell lines, indicating the significantly different glycan expressions in this physiological media. In addition, the DMEM and EMEM clusters were also separate from each other, which could be caused by the higher concentrations of certain nutrients in DMEM, although the compositions of both commercial media are very similar. Moreover, the PCA plots from the two cancer cell lines exhibited different distributions of the three media in terms of the PC1 and PC2 scores as described above, suggesting that the influence of Plasmax on N-glycosylation is cell line specific. For O-glycans, overall, 27 and 31 unique structures were observed and studied in the 231BR and CRL cell lines, respectively. Regarding the O-glycan expressions in each media, the amounts of O-glycans identified in EMEM were relatively lower compared to Plasmax and DMEM. For both cell lines, around 10 structures identified in the latter two media were absent in EMEM, suggesting that EMEM might not be able to provide a suitable environment for the expression of O-glycomes. The limited O-glycan expression in EMEM is also the main reason for the different PC1 scores compared to Plasmax and DMEM shown in the PCA plots.A total of 52 and 51 unique p < 0.05) were investigated between Plasmax and the two commercial media. The differentially expressed 15 N-glycans of the 231BR cell line are all neutral structures without sialic acid, suggesting that Plasmax does not have a perceptible influence on the sialylation of N-glycans in this type of cancer cell line. According to the distribution of different types of N-glycans, no significant differences were observed regarding fucosylation. However, significantly higher abundances of fucosylated structures HexNAc4Hex3DeoxyHex1, HexNAc5Hex3DeoxyHex1, and HexNAc6Hex3DeoxyHex1 were observed in Plasmax than both DMEM and EMEM. In addition, the other fucosylated structures, such as HexNAc4Hex5DeoxyHex1, HexNAc4Hex4DeoxyHex1, HexNAc3Hex5DeoxyHex1, and HexNAc3Hex4DeoxyHex1, have lower relative abundances, which draws our attention to the galactose on the N-glycan branches. The overexpression of galactosylated N-glycans in commercial media, especially in DMEM, is associated with the upregulation of B4GALTs in the Golgi apparatus. For the fucosylated HexNAc4Hex3DeoxyHex1 and its non-fucosylated counterpart HexNAc4Hex3, both structures exhibited the lowest abundance in EMEM; similar results were also observed in another pair of N-glycans: HexNAc3Hex3DeoxyHex1 and HexNAc3Hex3. If we take the most abundant significant N-glycan HexNAc2Hex3DeoxyHex1 into consideration, we observe a complementary distribution of the relative abundance of this structure in the three media. Such results indicate a lower chance of adding N-acetylglucosamine (GlcNAc) onto the mannose of the N-glycan core structure when cultivating in EMEM, which also agrees with the pie charts, where the lowest abundance of other structures was identified in EMEM among the three media. The addition of GlcNAc in N-glycans is catalyzed by the enzyme N-acetylglucosaminyltransferase (MGAT) in the Golgi, and this process is predicted to be inhibited in the 231BR cell line cultivated by EMEM according to our transcriptomics results. Furthermore, the upregulation of tri-antennary HexNAc5Hex3DeoxyHex1 and tetra-antennary HexNAc6Hex3DeoxyHex1 in Plasmax suggest that GlcNAc addition and the branching processes of N-glycans are more active than both commercial media.The glycans with a significantly different relative abundance , was only detected in Plasmax. The fucosylated N-glycans in the CRL cell line showed a higher abundance in Plasmax than EMEM, which indicates increased stimulated fucosylation in the CRL cell line when cultivated in Plasmax. Combining the sialylation and fucosylation, several N-glycans with both sialic acid and fucose were found to be overexpressed in Plasmax, including HexNAc4Hex5DeoxyHex1NeuAc1, HexNAc3Hex4DeoxyHex1NeuAc1, and HexNAc5Hex6DeoxyHex1NeuAc1. These changes are driven by the combination of more activated sialyltransferases and fucosyltransferases in Plasmax compared with DMEM and EMEM.In contrast to the 231BR cell line, a total of 29 pparatus . This prO-glycans identified in both cell lines cultivated by EMEM, we do not recommend the use of EMEM as the culturing medium for O-glycomics studies of cell lines as it might limit the O-glycan expression or O-glycosylation. The comparisons between Plasmax and DMEM will mainly be discussed. For the 231BR cell line, a significant decrease in sialylated structures was observed in Plasmax. A good example is HexNAc1Hex1NeuAc2, which was only detected in DMEM. According to the transcriptomics results, several sialyltransferases were upregulated in DMEM, including both α2, 3- and α2, and 6-sialyltransferases (ST3GAL and ST6GAL), which stimulated the corresponding processes [O-glycan HexNAc2DeoxyHex1NeuAc1 was solely found in Plasmax. Since the fucosylation levels in Plasmax and DMEM are comparable, there is not enough evidence to correlate the expression change of HexNAc2DeoxyHex1NeuAc1 with fucosyltransferases that are involved in O-glycan expression. Moreover, because of the various core structures of O-glycans and the complex enzymatic activities that occur during the modification of sialo-fucosylated O-glycans, further study is required to build the connections between the expression changes of this type of O-glycan with enzymes in the 231BR cell line. On the other hand, the sialylation of O-glycans in the CRL cell line was increased in Plasmax (such as HexNAc1Hex1NeuAc1 and HexNAc6Hex5NeuAc1), while fucosylation was decreased compared to DMEM . Both changes are similar to those found in N-glycans of CRL. However, in contrast to N-glycans, the sialo-fucosylated O-glycans showed a lower abundance in Plasmax, such as HexNAc3Hex3DeoxyHex2NeuAc2, HexNAc3Hex3DeoxyHex1NeuAc1, and HexNAc3Hex3DeoxyHex1NeuAc3. One potential reason is that the expression of this type of O-glycans is mainly driven by the level of fucosyltransferases [N-glycans [O-glycans. In this study, focusing on the expression changes of glycans, we cannot draw a certain conclusion regarding whether these two enzymes are the main reasons for the abundance changes in sialyl-fucosylated glycans.Due to the fewer rocesses . On the sferases , which i-glycans . It has N- and O-glycosylation changes in cancer cell lines cultivated in a newly designed physiological medium Plasmax in relation to the two commercially available media DMEM and EMEM. For the breast cancer cell line MDA-MB-231BR, GlcNAc addition and the branching of N-glycans were more activated in Plasmax. However, the galactosylated N-glycans showed a higher relative abundance in the commercial media. The O-glycans of the 231BR cell line exhibited lower sialylation but higher sialo-fucosylation levels in Plasmax, indicating that Plasmax has different impacts on the enzymatic behaviors involved in O-glycosylation compared to N-glycans. On the other hand, the activation of the GlcNAc addition and branching of N-glycans was also observed in the brain cancer cell line CRL-1620, suggesting that these changes were more likely general effects on cancer cells cultivated in Plasmax. In addition, the sialylation levels and fucosylation levels of CRL N-glycans in Plasmax were significantly different than DMEM and EMEM, which resulted in higher expressions of sialo-fucosylated N-glycans in Plasmax than both commercial media. Compared to DMEM, both sialylated O-glycans and fucosylated O-glycans from the CRL cell line showed the same regulation trends as N-glycans. The glycan expressions changes were also correlated with the transcriptomics results, indicating that the unique environment of Plasmax can induce alterations in glycotransferases. For EMEM specifically, the lower number of unique O-glycan structures detected in this medium renders EMEM inadequate for cell line O-glycomic analysis. The glycomics analyses in this study provided complementary information to the original work that focused on colony formation, transcriptomics, and metabolomics. Since aberrant glycosylation has been related to a variety of diseases such as cancer, and significant glycan expression alterations were observed in this study, it is necessary to consider the influence of nutrient compositions of the culturing media and the selection of a suitable media for in vitro cancer cell glycomics.In this study, we investigated


To increase the intracellular concentration of DCA, thereby potentiating its effect, DCA-loaded poly(lactic-co-glycolic acid) nanoparticles were produced. At higher DCA concentrations, encapsulation was found to increase its toxicity. These results may help find a new treatment strategy through combined therapy, which could open doors to new treatment approaches.Antiglycolytic agents inhibit cell metabolism and modify the tumor’s microenvironment, affecting chemotherapy resistance mechanisms. In this work, we studied the effect of the glycolytic inhibitors 3-bromopyruvate (3BP), dichloroacetate (DCA) and 2-deoxyglucose (2DG) on cancer cell properties and on the multidrug resistance phenotype, using lung cancer cells as a model. All compounds led to the loss of cell viability, with different effects on the cell metabolism, migration and proliferation, depending on the drug and cell line assayed. DCA was the most promising compound, presenting the highest inhibitory effect on cell metabolism and proliferation. DCA treatment led to decreased glucose consumption and ATP and lactate production in both A549 and NCI-H460 cell lines. Furthermore, the DCA pretreatment sensitized the cancer cells to Paclitaxel (PTX), a conventional chemotherapeutic drug, with a 2.7-fold and a 10-fold decrease in PTX IC Non-small cell lung cancer is one of the most common malignant tumors in men and women and a leading cause of mortality worldwide ,2. The h6H12O5, dichloroacetate (DCA)—C2HCl2O2—and 3-bromopyruvate (3BP)—C3H3BrO3—have been assayed as putative antitumor agents that target glycolysis, hampering this metabolic pathway or, in the case of DCA, redirecting pyruvate to acetyl-CoA synthesis  × 100. The DL (drug loading) was calculated taking into account the total dry weight of PLGA NPs using the following equation: DL (%) = [/Total dry weight of NPs] × 100.® 100 RP-18 (5 mm) (250 mm × 4.6 mm) (Merck). Chromatographic analysis was performed in an isocratic mode where the mobile phase consisted of 5% acetonitrile and 95% of 2% phosphoric acid in ultrapure water. The eluent flow rate was 1.0 mL/min. The column was maintained at room temperature, and the injection volume was 20 μL. Detection was performed by UV at 214 nm. All samples were run in triplicate, and the total area of the peak was used to quantify DCA.Free DCA in the supernatant was quantified by high-performance liquid chromatography (HPLC) in a Shimadzu UFLC Prominence System equipped with two LC-20AD pumps, a SIL-20AC autosampler, a CTO-20AC oven, a DGU-20A degasser, a CBM-20A system controller and a LC solution version 1.25 SP2. The UV detector was a Shimadzu SPD-20A, and the column used was a LiCrospherThe cells were seeded in 96-well plates as described and, after adhesion, incubated for 24 h with medium containing DCA-loaded PLGA NPs or free DCA at different concentrations . The influence of empty PLGA NPs on cell viability was also tested and adjusted according to the concentrations of DCA-loaded PLGA NPs. At least three independent assays were performed in triplicate, and cell viability was determined by the SRB assay, assuming 100% viability for untreated cells in each case.p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.The results presented correspond to the average of triplicates of at least three independent experiments. Results were expressed as means ± SD. For the statistical analysis, GraphPad Prism 8.3.1 software was used. All the assays were analyzed using one-way ANOVA, considering significant values to be * MDR is one of the major causes of treatment failure in cancer. This phenotype can be associated with several causes, including the energetic metabolism of cancer cells, which mainly relies on glycolysis, either in aerobic or anaerobic conditions. In this study, we aimed to exploit the effect of GIs on cancer cell properties, namely by investigating how they can overcome such a phenomenon of resistance to conventional drugs, opening doors for new therapeutic strategies. Thus, our overall objectives were: (i) to analyze the effect of the GIs 3BP, DCA and 2DG on lung tumor cell line properties; (ii) to verify their ability to reverse the MDR phenotype, when used in combination with PTX, a conventional drug used in lung cancer therapy; and (iii) to increase the efficiency of DCA delivery to tumor cells after its encapsulation into polymeric NPs, which may overcome limitations regarding the maximum dose of GIs that can be used.50, using the SRB assay , and determined the respective ICRB assay .50 values for these GIs but a higher IC50 value for DCA , HKII . Further50 values of each GI. Flow cytometry analysis showed that the mechanism of cell death depended on the cell line and on the compound used.To assess the cell death mechanism induced by the GIs 3BP, DCA and 2DG, Annexin V/PI and caspase-3 assays were performed. Concerning the A549 cell line, untreated cells (control) showed a basal level of around 0.5% and 10% of apoptotic and necrotic cell death, respectively. Both DCA and 2DG, but not 3BP, induced an increase in apoptotic levels, whereas necrosis was stimulated by DCA and 3BP but not by 2DG. DCA induced the greatest effect, resulting in 40% of cell death, mainly by necrosis (approximately 36 ± 4.31% of necrosis and 3% of apoptosis). The treatment with 2DG induced an increase in apoptotic cells only (around 3%) and 3BP in necrotic cells only (around 20%).An increase in caspase-3 activity was not observed in A549 cells treated with 3BP, DCA or 2DG , which mConcerning the other cell line, NCI-H460, untreated cells (control) showed a basal level of around 10% and 4% of apoptotic and necrotic cell death, respectively. In this case, and differently from A549 cells, DCA induced apoptosis only, whereas 3BP induced both mechanisms of cell death. No effect, both in apoptosis and necrosis, was observed in 2DG-treated cells. In this cell line, it was 3BP that produced the greatest effect, resulting in 70% of total cell death (45% of apoptosis and 25% of necrosis), whereas DCA significantly induced an average of 38% of total cell death (about 35% of apoptosis and 3% of necrosis). In agreement with these results, treatment with 3BP revealed an increase in caspase-3 activity. However, the increase in apoptotic cells, determined by the annexin assay for DCA-treated cells, was not confirmed, suggesting that the annexin assay is more sensitive than the caspase-3 assay. For 2DG, no increase in apoptotic rate was observed, neither through the annexin assay nor through the caspase-3 activity assay. As such, these results may suggest that 2DG induces cell death by another mechanism—likely autophagy. In fact, some authors have reported that, in vitro, 2DG induces autophagy in different tumor cell types ,56.In summary, for both cell lines, an increase was observed for both apoptosis and necrosis, depending on the cell line, mainly upon 3BP and DCA treatment. Our results are in agreement with other reports that also showed that 3BP induces apoptosis and necrosis and that DCA induces mainly apoptosis ,57. In fIn order to understand if the effect of GIs on cell viability was due to metabolic disturbance in cancer cells, glucose consumption and lactate production, as well as ATP levels, were assessed in A549 and NCI-H460 cell lines .50 values of 3BP, DCA and 2DG. After treatment, extracellular glucose and lactate and intracellular ATP were quantified and normalized against total biomass or protein, respectively  and, in the most sensitive cell line, NCI-H460, this reduction was even more evident (from around 55 mg/dL to 10 mg/dL). DCA treatment also led to the depletion of cellular ATP in both lung cancer cell lines, with a decrease to less than half in A549 and a decrease of approximately 40% in NCI-H460. Accordingly, DCA treatment lowered lactate levels in both cell lines: in A549 cells, lactate produced was reduced from 30 mg/dL (control) to approximately 13 mg/dL (treated cells) and, in NCI-H460 cells, from 35 mg/dL (control) to 10 mg/dL (treated cells). These results indicate that glucose oxidation switched from fermentative glycolysis toward oxidative mitochondrial metabolism. Since DCA can reverse the Warburg effect through PDH activation, DCA-induced stimulation of oxidative metabolism interrupts the metabolic advantage of tumor cells. Due to the frequent occurrence of mutations in their mitochondrial DNA, tumor cells often present dysfunction of the respiratory chain. As a result, they become unable to sustain energy demand . FurtherDifferent results were obtained when the cell lines were treated with the GI 3BP. As previously noted, the A549 cell line was less sensitive to this compound, with a non-significant reduction of the glucose consumed and ATP cell content. However, a significant decrease in lactate production was observed, similarly to the DCA treatment. Consistently with the results that indicate that the A549 cell line has higher rates of oxidative metabolism, the 3BP treatment did not affect the energetic yield of this cell line. In fact, there can be cases where cancer cells also rely on oxidative metabolism. Moreno-Sanchez described the contribution of OXPHOS in a model of lung cancer, where the majority of ATP was produced during OXPHOS ,62. ThisIn NCI-H460, the glucose consumed  was decreased by half after treatment with 3BP, while ATP production was significantly reduced to less than half. However, this alteration was not accompanied by a decrease in lactate production. Due to the metabolic plasticity exhibited by tumor cells, it is not unexpected that these cells could develop resistance to inhibition of a specific pathway through the upregulation of alternative pathways . It is kWhen 2DG was used, the ATP content was not reduced in both cell lines. However, this GI was shown to be able to decrease lactate production, both in A549 and NCI-H460 cells, which is in agreement with its inhibitory effect on the glycolytic pathway.These results show that lung cancer cell lines treated with GIs, namely with DCA, suffer a disruption in their metabolism, with a significant decrease of energy, particularly the NCI-H460 cell line, which is also the most sensitive to the drugs. Furthermore, DCA was the only GI capable of disturbing ATP production in the most resistant cancer cell line, A549.50 value of 3BP, DCA (24 h) or 2DG (48 h)  . The treAs shown in In both cell lines, the highest effect was observed for DCA, where 50% of cell proliferation was inhibited. In effect, the use of glucose supplies cells with intermediates used in other pathways, like lipid, nucleotide and amino acid biosynthesis ,67. As sMigration is a process that offers valid targets for intervention in important physiological and pathological phenomena, such as wound healing and cancer metastases . To stud50 of 3BP. Therefore, the results suggest that 3BP seems to influence the migratory capacity of cells, and such ability may contribute to its anticancer effect. In the A549 cell lines, again, an increase in cell migration was unexpectedly observed with 2DG treatment, consistently with its effect on cell proliferation.The NCI-H460 cell line exhibited a slightly higher migratory capacity . HoweverPTX is one of the most commonly used anticancer drugs in therapies against solid tumors, although the disease relapses frequently due to the development of resistance to the drug . Such reCells expressing MDR proteins, such as Pgp, are known to require ATP as the energy source to pump out drug substrates . Thus, i50 value decreased when the cells were pre-incubated with DCA, showing that this GI can sensitize cells to PTX. However, this effect is less evident in the nontumor cell line HPAEpic. In fact, although this cell line is more sensitive than the A549 cell line to two of the GIs studied, the effect of potentiation appears to be more specific in tumor cell lines. In contrast, for both cancer cell lines, such effect was very evident, even with a lower concentration of DCA (½ IC50), but much more pronounced in the NCI-H460 cell line. In A549, the most resistant cell line to PTX and to GIs, the IC50 value decreased 2.7-fold, whereas, in the NCI-H460 cell line, the IC50 value decreased 10.1-fold. ATP depletion and exported lactate should affect proteins putatively involved in chemoresistance, which can be present in cancer cells. In fact, the cancer cell line treatment with DCA had almost the same effect on metabolism in both cell lines (as assessed through cellular ATP levels and lactate production), for which a similar effect could be expected for the decrease in PTX IC50 in both cell lines after DCA pretreatment. However, the decrease in PTX IC50 did not parallel the effects on metabolic parameters, being more pronounced in NCI-H460 cells. These were shown to be intrinsically more sensitive to GIs, which suggests that other metabolic parameters and/or membrane transporters and proteins involved in drug resistance may contribute to cell line sensitivity to PTX.The three untreated cell lines presented similar sensitivity to PTX and, in all of them, the ICOur results demonstrate that the biological activity of DCA is mainly due to its ability to decrease tumor cell metabolism. However, there are disadvantages to a metabolism-based approach in cancer therapy, since the metabolic pathways required for cell survival are also present in normal cells. Thus, metabolism-based treatment can face a major hurdle of non-specific toxicity . TherefoThe average size is within the 125–130 nm range. The encapsulation of DCA into PLGA NPs did not change the particle size. However, there was an increase in the polydispersity index  and a decrease in the zeta potential (from −4.31 to −8.99). The increase in PdI and the decrease in the zeta potential may be indicative of some aggregation and fusion of nanoparticles . HoweverThe formulations, represented as DCA and DCA-loaded PLGA NPs, showed a concentration-dependent effect on the viability of lung cells. After 24 h, no significant differences were found in all samples at concentrations between 10 and 100 mg/mL. Viability results showed that PLGA NPs did not lead to significant cytotoxicity in lung cells, which is in agreement with the literature . Howeverp < 0.01 for A549 and NCI-H460 cell lines and p < 0.001 for the HPAEpic cell line), which might be interpreted as a result of increased intracellular deposition of the drug. The decrease in viability might be associated with the fact that DCA has been internalized by lung cancer cells, binding to the intracellular components, namely PDK. By blocking PDK, DCA shifts pyruvate metabolism from glycolysis to OXPHOS, allowing a decrease in cell viability. Indeed, our study demonstrated that encapsulation was successfully achieved, enabling an observable biological effect on lung cancer cells. Although not explored here, it may be expectable that the surface modification of NPs with an active targeting fraction would increase target specificity [Even so, this concentration is much lower than those used in the previous assays. The results showed that DCA-loaded NPs allowed a decrease in cell survival , treateux pumps . This, tOur results with DCA were quite promising, given that the decrease in cell viability upon DCA pretreatment was higher for the tumor cell lines than for the normal cell line. However, the fact that the effect on cell viability was not absent for HPAEpic underlines the need to enhance drug targeting to tumor cells. Since the inhibition of the metabolism of healthy cells and the significant drug accumulation outside the tumor cells could lead to serious adverse effects , we aimeTumor cell biology is extremely complex, and an array of factors can be involved in the MDR phenotype, thus compromising chemotherapy outcomes. Many other components , complementary to those assayed in this work, putatively represent valuable targets of anticancer therapies and are being explored as part of new therapeutic approaches. In this work, the effect of a standard drug already in use (PTX) has been intensified by exploring the reprogrammed metabolism as the ‘Achilles heel’ of cancer cells through the use of a GI (DCA). The effect was further potentiated by NPs encapsulating the DCA. Thus, the results herein presented demonstrate the potential of “all in one” therapeutic approaches, combining multiple strategies  as the key to efficiently and selectively targeting tumor cells.


Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia including head and neck squamous cell carcinoma (HNSCC). In HNSCC, 5-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic and prognostic biomarkers and as therapeutic targets. Nevertheless, HNSCC-specific glycome is to date largely unknown. Herein, we tested six established HNSCC cell lines to capture the qualitative and semi-quantitative N-glycome using porous graphitized carbon liquid chromatography coupled to electrospray ionisation tandem mass spectrometry. Oligomannose-type N-glycans were the predominant features in all HNSCC cell lines analysed (57.5–70%). The levels of sialylated N-glycans showed considerable cell line-dependent differences ranging from 24 to 35%. Importantly, α2-6 linked sialylated N-glycans were dominant across most HNSCC cell lines except in SCC-9 cells where similar levels of α2-6 and α2-3 sialylated N-glycans were observed. Furthermore, we found that HPV-positive cell lines contained higher levels of phosphorylated oligomannose N-glycans, which hint towards an upregulation of lysosomal pathways. Almost all fucose-type N-glycans carried core-fucose residues with just minor levels (< 4%) of Lewis-type fucosylation identified. We also observed paucimannose-type N-glycans (2–5.5%), though in low levels. Finally, we identified oligomannose N-glycans carrying core-fucose residues and confirmed their structure by tandem mass spectrometry. This first systematic mapping of the N-glycome revealed diverse and specific glycosylation features in HNSCC, paving the way for further studies aimed at assessing their possible diagnostic relevance.The online version contains supplementary material available at 10.1007/s00216-022-04376-x. Head and neck cancer (HNC) refers to a heterogeneous group of malignant neoplasms, with over half a million new cases diagnosed annually across the globe , 2. MajoHuman papillomaviruses  are small, double-stranded, circular DNA viruses that are responsible for a global epidemic of a subset of HNSCC, mainly originating in the lymphoid mucosa of the oropharynx  . Currenty) and sialyl Lewis X (SLex) epitopes, changes which are known to promote EGFR phosphorylation in OSCC cell lines  to establish the first N-glycan map of the most widely used HNSCC cell lines , of which 2A3 and VU-147 T considered as HPV ( +) cells. In addition, we have also investigated whether HPV infection affect the relative distribution of N-glycans. While the HNSCC cells analysed exhibited general similarities, distinct cell line-specific N-glycosylation traits were identified that make each cell line an individual research resource. Distinct differences were identified in sialic acid linkage distribution. Various levels of phosphorylated oligomannose, oligomannose and sialylated N-glycans were determined. We also confirmed that oligomannose N-glycans can act as a substrate for FUT8, as demonstrated by the presence of several core-fucosylated oligomannose-type N-glycan structures. This systematic N-glycome map of the most widely used HNSCC cell lines provides the first HNSCC-glycome reference dataset that builds the foundation for future glyco-biomarker research in HNSCC.3 in H2O, sodium borohydride (NaBH4), potassium hydroxide (KOH), trifluoroacetic acid (TFA), LC–MS grade, water, fetuin from foetal calf serum, RPMI medium and foetal bovine serum (FBS) were obtained from Sigma-Aldrich. AG50W-X8 cation-exchange resin was purchased from BioRad. ZipTip with C18 resin and Immobilon-PSQ (0.45 µm pore size) PVDF membranes were obtained from Millipore. RIPA buffer and Pierce™ BCA protein assay kit were purchased from Thermo Fisher Scientific. PNGase F was from New England Biolabs. Porous graphitized carbon columns were obtained from New Objectives . The SpeedVac concentrator was from Thermo Fisher Scientific. Acetonitrile LC–MS grade was purchased from Merck.Ethanol, methanol, glacial acetic acid and direct blue 71, polyvinylpyrrolidone (PVP40), ammonium hydroxide solution (≥ 25% (vol/vol) NH2, 5% CO2. Briefly, 0.6–0.7 million cells were seeded into T75 mL flasks and incubated with medium consisting of RPMI-1640, 10% foetal bovine serum and penicillin/streptomycin, of which 2A3 cells were incubated with media consisting of hGlucose DMEM-10%FBS + 2 nM glutamine + G418 200 ug/mL. Once cells density reached to 85–90% confluence, we washed the cells twice with ice cold PBS. Cells were lysed and harvested (by scraping) in cold RIPA buffer with freshly added protease inhibitor cocktails. The next step was to vigorously vortex the cells thrice for 30 s followed by ultrasonication (an ultrasonic bath for 10 min). Cell lysates were centrifuged at 14,000 × g for 15 min at 4 °C. Supernatant was collected, and protein concentrations were measured using the BCA protein assay kit as per the manufacturer’s instruction. After protein quantitation, (glyco)proteins were precipitated using ice cold (− 20 °C) acetone. The resultant protein pellet was allowed to air dry at room temperature. The sample was resuspended using 8 M urea by intensive vortexing.All the HNSCC cell lines were STR profiled and authenticated. This gave us the confidence that our cell lines are correctly identified, and not cross-contaminated with other cells. In addition, we have also performed mycoplasma testing using Lonza’s MycoAlert® Mycoplasma Detection Assays and were confirmed to be of mycoplasma free. Six HNSCC cell lines, including two HPV positive, were purchased from ATCC (American Type Culture Collection) or a generous gift (Supplementary Table 50 µg of (glyco)proteins were immobilised onto a 0.45 µm pore size PVDF, washed and stained with direct blue 71. N-glycans were released from glycoproteins using PNGase F as described previously , 17, 18.https://glyconnect.expasy.org/compozitor/) was used to visualise the glycan network [N-glycan structures were determined as previously described , 18, 19. network . The relhttps://glycopost.glycosmos.org/preview/7702599506310db315e4be PIN CODE: 4365 [The data is made available via GlycoPOST, accession number GPST000279, URL, glyConnect compozitor network analysis to understand the biosynthetic connections between the identified N-glycans and to uncover any potential gaps in the acquired dataset  lacto series glycosphingolipid biosynthesis, led to the unexpected depletion of α2-6 sialylated N-glycans due to the lack of ST6GAL1 expression in the ∆B3GNT5 cells [While the overall level of complex-type N-glycans differed significantly between cell lines Table , almost T5 cells . It is cWith exception of SCC-25, where slightly higher levels of neutral complex-type N-glycans were identified (3.7%), the levels of neutral, complex-type N-glycans were around 1% or lower in all other cell lines Fig. . Thus, sFucose plays a major role as a component of cancer-associated glyco-epitopes such as (but not limited to) Lewis X or Lewis Y , 33. CorAlmost all paucimannose-type N-glycans were core-fucosylated Fig. . In Cal-Mannose 6-phosphate (Man6P) is a common modification important to guide glycoproteins towards the lysosome via the Man-6-P receptor (M6PR) . Thus, tMGAT1-deficient (Lec1) Chinese hamster ovary (CHO) [MGAT1-independent core fucosylation on oligomannose-type N-glycans can occur in otherwise unmodified human cells in low levels   and HEK2ry (CHO) , as wellry (CHO)  and on hry (CHO) . Yang etry (CHO) . In the els Fig. .Fig. 6CoDespite the increasing prevalence of HNSCC and its known association with HPV infection, the N-glycome of the cell lines most widely used in HNSCC research has not been studied. For the first time, we demonstrated that while these cell lines exhibit similar general profile, significant quantitative differences in their N-glycosylation features exist that make each of them unique research resources Fig. . As repoSialylation of tumour tissues has been correlated with cancer progression, metastatic spreading and poor prognosis across many different cancer types , 45, 46.The interplay between the different sialyltransferases known to add NeuAc residues onto N-glycans, such as ST6GAL1 or the ST3GAL4/5/6, clearly plays a major role in this context of the tumour microenvironment. The inhibition of α2-3 NeuAc expression has been demonstrated to suppress the migration and metastasis in melanoma cells . ST6GAL1This study provides for the first time a comprehensive mapping of the N-glycome from in six widely used HNSCC cell lines. Our data constitute the basis for further studies aiming at better understanding how changes in the HNSCC glycome may contribute to the pathogenesis of these highly heterogenous cancers. Moreover, identification of HNSCC-specific glycome modifications may be exploited to improve the predictive and prognostic definition of these patients and provide novel targets for improved treatments.Supplementary file1 (DOCX 1.39 MB)Supplementary file2 (XLSX 1527 KB)Below is the link to the electronic supplementary material.


Cell lines have been considered excellent research models in many areas of biomedicine and, specifically, in the study of carcinogenesis. However, they cease to be effective models if their behavior changes. Although studies on the cross-contamination of cell lines originating from different tissues have been performed, little is known about cell lines derived from cervical neoplasia. We know that high-risk HPV (HR-HPV) is associated with the development of this type of cancer. This link between HPV infection and cancer was first established over 35 years ago when HPV16 DNA was found to be present in a large proportion of cervical cancer biopsies. The present review paper aims to report the status of the establishment, authenticity, and characterization of cervical cancer (CC) cell lines. This is a systematic review of articles on the establishment, authenticity, and characterization of CC cell lines, published from 1960 to date in the databases and in cell repository databases. 52 cell lines were identified in the literature. Only 25 cell lines were derived from cervical neoplasia, of which only 45.8% have a reported identity test (genomic fingerprint). Despite the increase in the establishment of cell lines of cervical neoplasia and the standards for the regulation of these study models, the criteria for their characterization continue to be diverse. Strategies for prevention, timely diagnosis, and therapeutic interventions have been developed thanks to cancer research based on models using cell lines. The HPV life cycle is intrinsically linked to the programmed epithelial differentiation of the cervix. The majority of the focus has been on high-risk HPVs (HR-HPVs), which have been linked to cancer development.3 This link between HPV infection and cancer was first established over 35 years ago when HPV16 DNA was found to be present in a large proportion of cervical cancer biopsies.5 Cell lines are used in multiple areas of biomedicine, specifically in the study of carcinogenesis. A cell line is “an in vitro culture of cells that achieve indefinite survival and can be frozen and retrieved a theoretically infinite number of times.”6 When the right conditions are met, these cells will keep dividing and keep many of the characteristics of the cell type or tissue from which they came.7 The European Collection of Animal Cell Cultures, 2012,8 defines two types of cell lines according to the capacity of the cells to continue dividing; finite or senescent, which die after a fixed number of duplications, and continuous, which can be propagated indefinitely. Every cell line comes from a specific tissue or by cloning an already established line; each tissue has specific characteristics . When a cell line is established, many of these characteristics are lost, such as cell-cell (or cell-tissue) junction, cell communication, and regulation.9 The characteristics of a cell line vary from one person to another. However, they all share certain features.Even though cervical cancer (CC) may be preventable, it continues to occupy fourth place in the incidence of neoplasms worldwide, with 604,127 cases per year. In Mexico, it is the second most common cancer in the female population, with 9,439 cases per year.10 Multiple studies have demonstrated these issues, and it is estimated that between 18 and 36% of cell lines are contaminated or misidentified,11 with only 43% of cell lines considered “well identified.12 The cost of cell line contamination is high. For example, in the United States in 2013, investment in breast cancer research amounted to 370,000 million dollars, and it is estimated that at least 100 million dollars were lost in studies using cell lines that did not come from this tissue.13 In this case, not only are investment costs at stake, but also the credibility of the researchers and the inability to reproduce and transfer the results they come up with.However, working with these models has a limited efficiency due to their behavior, i.e., this can change for two main reasons: contamination with other lines and misidentification.14 demonstrated by electrophoretic analysis of the enzymes Glucose 6 Phosphate Dehydrogenase (G6PD) and Phosphoglucomutase (PGM). He evaluated 30 different cell lines, finding that 18 of them, despite coming from the Caucasian race, presented the same polymorphism (A) as the HeLa cells, which come from the African-American race, concluding that it was contamination of these lines and introducing the concept of biochemical polymorphisms. Since then, multiple studies15–20corroborating his results have been conducted. Recently, the International Cell Line Authentication Committee (ICLAC) has reported 486 lines as misidentified, which has harmed at least 32,000 published articles.21Dr. Gartler published the first report of contamination in cell lines in 1968,22 Other researchers corroborate this fact since there is an error when describing cell lines according to the tissue of origin.23–25 For example, the ECV304 line is said to come from normal endothelial cells when in fact it comes from bladder cancer. The KB line is said to come from laryngeal carcinoma when in fact it is a clone of modified HeLa cells that have been grown in a lab. It’s also possible to mix up culture samples, cross-contaminate cell lines with each other, and get biological contamination from microorganisms like mycoplasma and fungi.26Capes-Davis et al. (2012) point out that a cell line is misidentified when its DNA profile is no longer consistent with that of the donor from which it was first obtained.27 indicates that there are three fundamental characteristics to ensure the quality of work with cell lines: 1) purity, that is, that they are free of microorganisms; 2) identity, which refers to the cells’ being who they say they are; and 3) stability, indicating that the genotype and phenotype must remain unchanged during growth and in vitro passages. Other criteria highlighted by cell culture researchers are the verification of viability, karyotyping, confirmation of the species of origin, specific cell identification (genomic fingerprinting), cell markers, genetic expressions, pluripotency (in the case of stem cell tissues), as well as quality controls in culture.27 Cell morphology (phenotypic changes) and ploidy (genotypic changes) are also things to keep an eye on in cell lines to make sure they have the right biological properties.28Stacey (2007)30The criteria for authentication and characterization of a line are very diverse among researchers. Therefore, international organizations such as the International Committee for Cell Line Authentication, the European Society for Animal Cell Technology, the World Biological Standards Institute, or the UK Cancer Research Coordinating Committee, among others, issue guidelines on the use, maintenance, development, and deposition of cell lines. Among the norms described by these organizations, the ethical aspects stand out, such as the need for the approval of the study by a research ethics committee, obtaining the informed consent of the donor, the transfer of rights to the biological material, etc. The data obtained on the tissue and the donor, the methods suggested to verify the authenticity of the cell line by DNA analysis, the criteria for its deposit in repositories, and even data on the establishment and characterization of the line should be included in the publications.31 Genomic fingerprinting exploits the variability found in non-coding regions of the human genome, which is organized into repeated sequences (variable numbers of VNTR tandem repeats) of two types: minisatellites (10 to 100 bp) and microsatellites (2 to 5 bp), also called short tandem repeats (STRs). The analysis of these regions is necessary because the probability of two unrelated individuals having the same combination at a specific locus is less than 1%9 In addition, advances in technology have favored the evaluation of cell lines. From morphological observation of cells in culture by transmission or scanning electron microscopy, to the implementation of genomic analysis methods and molecular cytogenesis, researchers now have more accurate methods to assess the authenticity of cell lines and their growth characteristics in culture.Also, the techniques used in the characterization and authentication of cell lines are varied. For example, isoenzyme analysis determines the origin of the species of a given line and allows the detection of cross-contamination through the electrophoretic mobility of different isoenzymes. Cytogenetic analysis allows chromosomal markers to be identified.32–36 and the development of these biological models has increased significantly in recent decades. However, despite this, there is a lack of information on appropriately identified cervical neoplasia-derived cell lines. So, this review will show how cervical cancer (CC) cell lines are made, how they are authentic, and how they are characterized.In summary, studies on cross-contamination of cell lines originating from diverse tissues have been performed,To show how cervical cancer (CC) cell lines have been set up, authenticated, and characterized over time.37 The concepts and strategies for the systematic search to locate information from Pozos and Garrocho (2012) were also taken up.38The present study summarizes the current knowledge concerning the establishment and characterization of CC cell lines established between 1960 and 2020. It is a systematic review based on the phases of the Preferred Reporting Items for Systematic Reviews and Partial Analyses (PRISMA).A search for publications concerning the establishment and characterization of CC cell lines from 1960 to 2020 was performed in the databases PubMed, Web of Science, Natura, SpringerLink, EBSCO, ScienceDirect, Ovid, Redalyc, PLOS, BMC, SciELO, PMC, Google, Google Scholar, BIG  and Academic Source. These cell repositories, including the American Type Culture Collection (ATCC), European Collection of Authenticated Cell Cultures (ECACC), Accegen, and Cellosaurus, were also looked at.All titles that presented at least one of the following keywords: establishment, characterization, authenticity of cervical cancer cell lines were reviewed. Initially, papers that did not respond to any of the keywords, articles written in languages other than Spanish, English, or French, and titles without an abstract or text were eliminated. Next, abstracts of articles on the establishment and/or characterization of CC cell lines were obtained, excluding those in which the lines had originated from animal models, from other already established lines , or those derived from Low-Grade Intraepithelial Lesions (LGEI).Subsequently, references to CC lines were obtained and analyzed for their integration or exclusion in the study. The HeLa cell line was first ruled out because it had been reported as a contaminant of other cell lines and because no specific papers on how it was made or how it was characterized were found. However, many papers on some of its characteristics were found.The documents were then obtained in PDF format, and the data was organized under two headings: 1) the establishment of CC cell lines and 2) the characterization of the lines. An EXCEL database was constructed considering the following: year of publication, authors, journal, names of the established cell lines, general data , characterization methods , and ethical aspects.To verify that the lines were derived from cervical neoplasia, they were examined for: a) karyotype, b) HPV genotyping , and c) tissue of origin. A second document review of each line was performed to identify missing data regarding these three conditions. The data on the methods used to verify the authenticity and characterization of each line were subsequently organized into tables . FinallyA total of 378 abstracts were reviewed from which 140 scientific articles were obtained. In these papers, the establishment of 52 CC cell lines from 1960 to 2020 was reported.In terms of origin, we found that the countries reporting the highest percentage (53.7%) of established lines are in Asia, followed by North America (24.1%) and Latin America (11.1%), while European countries report the lowest percentage (9.3%) of established lines. Only one of the lines (DoTc2) did not report this data. According to Regarding the type of neoplasm, 76% of the lines are described as epithelial carcinomas and only 12% as adenocarcinomas , 8% of the authors do not specify whether it is squamous or glandular (CA and SKG-III), and only one line (4%) is said to come from a lymphoepithelial type cancer (HUUCLE) determined by histology. Concerning the degree of progression of the neoplasm, we found great variability among the authors since the majority (80%) only refer to the stage according to the FIGO classification and only 4% include the grade, while 16% do not refer to the progression of cancer. The lines classified as stage I occupied 28% of the total, those included in stages II and III were 20%, and only 16% came from metastatic cervical neoplasms (stage IV).Because of the association of cervical cancer with high-risk HPVs, viral genotyping has become an important element in cell line specificity. Of particular interest for study in carcinogenesis are those that have been reported: a) positive for viral load and b) infected with more than one HPV type most of the lines (56%) are described as HPV16, followed by those characterized as HPV18  and HPV negative . Only one line was reported as HPV59 (HHUS) and one as HPV56 (HUUCLEC). In the line named CUMC-6, both type 16 and type 18 viral genomes were identified.All lines are tumorigenic, so the karyotypes showed aberrations in different chromosomes and were referred to as aneuploid (60%), triploid (12%), diploid or hyperdiploid (8%), respectively. A few have been described with other chromosomal alterations, such as SiHa (hypertriploid), CUMC-3 (hypotetraploid), and, most notably, CX, which was reported as euploid (46 chromosomes), despite structural alterations in the XX chromosome.Although many of the data reported in the studies were in general, some of the most constant ones were the time in which the cell population in culture doubles its population, as well as the confirmation of the tissue of origin. However, a considerable percentage (24%) of the total lines report 5 or fewer characteristics (some only two-TC140-); the majority (68%) include 6 to 7 criteria in their characterization (including authenticity and free of microorganisms), while only 8% include 8 to 9 .Among the lines with accelerated growth (24 h) were XH1, CX, and CA, followed by those with intermediate growth (24–48 h), which corresponded to 48%, while 24% of the lines presented slow growth times (48–76 h). Two lines (8%) - CaLo, INBL-were reported with the slowest cell division times (more than 76 h) and two others  did not report the data. The number of times cells were subcultured varied from line to line, from 34 passages (SiSo) to 300 (CUMC-6); only a few lines  do not have the report.One of the criteria rarely found in the publications was the inhibition of cell growth by contact. Only in 11 lines (44%) was this feature described; in seven of them their cells do not stop dividing upon contact with others, as happens in neoplastic tissues, while in four lines  contact inhibition was documented. Regarding the density at which the cells grow, in 36% (9 cell lines), this characteristic has been reported using qualitative  and quantitative (various) scales as shown in the table.On the other hand, the characterization of tumorigenic capacity was established in 21 lines (84%); the privileged model for this was murine (18 lines), followed by 3D cultures (4 lines), while in three cell lines  no publications were found; only one line (HUUCLEC) reported the absence of carcinogenesis in a murine model after inoculation. Regarding the establishment of line specificity and identity, methods based on DNA analysis  were recurrent (52%) among the investigators (13 lines), whether used alone or in combination with RNA analysis (transcriptome) or protein expression (proteome). However, in only 44% of the lines, no other genetic tests were done other than karyotyping.Likewise, the isoenzymatic analysis of biochemical polymorphisms seems to be a common criterion for the lines established before the 1990s, since in 11 lines (44%), established in that period, this data was documented, while for the rest it was not; the most studied enzymatic polymorphisms were in G6PD, PGM1 and 3, AK-1 and ACP1. The only line that didn’t report on this was C33. All the other lines reported on either a) specific epithelial tissue antigens , specific neoplastic tissue antigens (carcinoembryonic antigen), histochemical or morphological studies, or a combination of these.On the contrary, and even though the problem of cross-contamination between cell lines, as well as contamination by biological organisms, has been exposed since 1968, in nine lines (36%) no evidence was found for the absence of intra-or interspecies contamination, in 12 lines (48%) only contamination with mycoplasma or with another microorganism (Epstein-Barr virus) was evaluated, in three (12%) the absence of contamination with HeLa cells was reported, and only in one line (SW756) contamination with mycoplasma and HeLa cells were evaluated together.Finally, in 76% of the lines identified, no ethical aspects were mentioned although the inclusion of these aspects has become an indispensable requirement in the publication of studies on the establishment of cell lines, in the procurement and use of tissues, as well as in the manipulation of genetic material. Likewise, ethical criteria are particularly important concerning the procurement of biological tissues, the transfer of rights, and the approval of the study by an ethics committee.6178.8% of cervical cancer cell lines reported between 1960 and 2020 emerged from developed countries, such as Canada, the USA, Japan, the United Kingdom, and South Korea. A likely consequence of its high investment in research and development (R & D), however, is that the USA is the nation that invests the most in this concept , followed by the European Union (19%) and Japan (10%). The remaining lines (21.2%) were developed in countries considered in transition , where R & D investment is not so strong.63However, only 48% of the lines that were reported came from cervical tissue, so they can be used as biological models of how malignant cells behave in real life. Culturist researchers say that it is important to know where the cell line came from so that future research can show how the system model works.Different cervical cancer cell lines are important because they allow us to know how the cells will react to a stimulus or treatment when they are grown in xenograft cultures in vivo. This is because we need to know how the cells will react to a stimulus or treatment when they are grown in xenograft cultures.64 According to international organizations, only one cell line had its origin confirmed by the donor.65 At least 12 cell lines, established after the 1990s, lack an ethical review, i.e., obtaining biological material and consent or rights from the donor. In this case, if an ethics committee had given its approval to the methods for getting the tissue, that would be something that is not talked about.In another respect, 9 of 25 cervical cancer cell lines were established between 1960 and 1990, so the obtaining of the cells was understood as an extension of the diagnostic process in which the patient did not need to be consulted.66The American National Standards Institute (ANSI) and the American Type Culture Collection (ATCC) and other international agencies  promote the use of genomic analysis techniques  for identity verification of human cell lines (ASN-0002) that are unique and unrepeatable. STR analysis is the most common method used by the authors. It is a standard method that is fast, cheap, and has been tested by cell banks. The data is also consistent.6768Other techniques that corroborate the uniqueness of cell lines are HPV integration sites, microsatellite stability, HLA typing, polymorphisms, as well as mutations and gene expression. In our study, few researchers reported these latest data. One disadvantage of this is that some cell lines tend to undergo genetic changes with continuous passages in culture. In malignant cells, there may be loss of heterozygosity and increased instability of microsatellites.Also, persistent high-risk papillomavirus infection, i.e., HVP16, has been significantly associated with neoplastic progression. However, the determinants of viral persistence and clearance are not yet well understood. Many studies have been done to compare the responses of different types of CC cell lines to the administration of antineoplastic substances, but the most common models have been genotyped with HPV16 or 18. This means that little is known about how neoplastic cells react to another or no viral type.69–73 Multiple infections appear to be more common in women under the age of 30, whereas viral type 16 excels in single and multiple infections associated with high-and low-risk HPV. A single viral genotype has been detected in a single sample.66 Some studies have shown that having a lot of HPV can make it more likely that you’ll get high-grade lesions and get cancer, but they aren’t sure yet.In recent studies conducted in Europe, North America, and Latin America, in more than 1000 women with positive samples for HPV, they show the presence of multiple infections at high and low risk in more than 30% of the positive cases studied.In our review, a single cell line reports the viral presence of both HPV16 and 18 in a woman over 30 years of Asian origin. The development of models with these characteristics could help us learn more about how many infections affect cervical carcinogenesis, which would help us figure out who is at risk in groups.75 their data are not conclusive if these lines were found or not contaminated; the International Committee for Authentication of Cell Lines,65 reports five lines of CC that have been found as contaminants of other-HeLa, ME180, SKGII, C33A, and TCO2–. The above data shows that it’s important to report how to keep cultures healthy and cell lines true.At least five lines of cervical cancer do not report the data and, although few studies on cell line contamination include these five lines of cervical cancer,32Knowing the morphological characteristics of cells in culture allows identifying changes in response to different environmental conditions  that require constant monitoring to prevent the invalidation of research work.1 phase of the cell cycle and deteriorate very little. Tumor cells result in morphological deterioration but continue to proliferate beyond confluence. Freshner32 suggests expressing cell density in cell/cm2 to avoid ambiguities in interpretations such as high, medium, and low of the correct seeding density and subculture interval is done by performing a growth curve to establish the deviations from this pattern and prevent cell deterioration.Normal cells usually stop dividing at a high cell density. They block in the G76 Viability and karyotype have also been used to check the genetic stability of cell cultures and to see if cells have changed.77Likewise, chromosome content in cancer lines is aneuploid  and heteroploid (variability in chromosome number in the same cell population) as a result of alteration of tumor suppressor proteins, i.e., p53 and Rb, leading to genetic instability throughout the subcultures.Phenotypic changes can occur during different passages of the cultures or by intra-and interspecies contamination, so it is important to report the number of passages that have had the established line, as well as the purity tests used in the laboratory. Karyotype is the most commonly used method to monitor the genetic instability of culture and determine the malignancy of the cell line, yet almost half of the identified lines do not report it.Ultimately, the results of the study indicate a significant increase in the establishment of cervical cancer cell lines in recent decades. However, despite the regulations of international agencies, authentication and characterization criteria remain very diverse among researchers and some dates, i.e., ethical review, are even unaffordable for the majority.


In developed countries, breast cancer as a multifactorial disease is a major health concern, and its incidence is constantly rising in low and middle-income countries. Numerous studies have demonstrated that phytochemicals such as carotenoids inhibit breast cancer growth and induce apoptosis. We recently enhanced the solubility of capsanthin in water by encapsulating it in diosgenin polyethylene glycol succinate, a novel non-ionic surfactant. Thus, this study aims to evaluate the cytotoxicity of water-soluble capsanthin-loaded micelles in MDA-MB-231 cells Capsicum annuum). Capsanthin-loaded diosgenin polyethylene glycol succinate 1000 (cap-DPGS-1000) micelles were prepared from capsanthin extract (cap) and diosgenin polyethylene glycol succinate 1000 (DPGS-1000) using the solid dispersion method. The capsanthin extract and cap-DPGS-1000 micelles were characterized by UV-visible spectroscopy, high-performance liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size distribution, polydispersity, and scanning electron microscopy (SEM). The effects of capsanthin extract and cap-DPGS-1000 micelles on a human triple-negative breast cancer cell line (MDA-MB-231) were tested to check the cell viability, proliferation and cytotoxicity of the micelles.In the current study, capsanthin, a hydrophobic carotenoid, is extracted from sweet red pepper  μg/mL, which is much lower than of capsanthin extract ((81.1±1.5) μg/mL). Capsanthin extract and capsanthin-loaded micelles are promising drug candidates to induce apoptosis and increase reactive oxygen species (ROS) in cancer cells.The solubility of encapsulated cap-DPGS-1000 micelles in water is greatly enhanced and leads to an increased scope for localized drug delivery, a better delivery option for treating residual cancerous tumours. The encapsulated capsanthin showed a sustained release in simulated intestinal fluid (pH=6.8). Our research proposes a sustained drug delivery system that ensures effective and controlled release to the affected site. The characterization data revealed no change in the structure and functional groups in the encapsulated capsanthin. The ICCapsicum annuum) showed remarkable cytotoxic effect on the triple-negative MDA-MB-231 cell line.The result shows the cytotoxic effect of capsanthin and capsanthin-loaded micelles on MDA-MB-231 cell line for the first time. Capsanthin from sweet red pepper ( Cancer, abnormal cell growth, can affect any organ or tissue of the body, and it is a multi-stage process that causes biological damage as a result of biochemical and molecular changes at the cellular level. The mortality rates depend on the prevention, early detection and effective treatment (Capsicum species) is unique for its diversified carotenoid profile and composition  and capsanthin-loaded micelles (hydrophilic) on cell apoptosis of human triple-negative breast cancer cell line (MDA-MB-231).Diosgenin polyethylene glycol succinate 1000 (DPGS-1000) was provided by Phytosol India Pvt Ltd., Bangalore, India. MDA-MB-231, the triple-negative breast cancer cells were purchased from the NCCS . The capsanthin reference standard was procured from Sigma-Aldrich, Merck . Dulbecco's modified Eagle’s medium, acridine orange, ethidium bromide, propidium iodide, Hoechst 33342, rhodamine 123, dichlorodihydrofluorescein diacetate, MTT -2,5-diphenyltetrazolium bromide), foetal bovine serum (FBS) and phosphate-buffered saline (PBS) were all procured from Merck Lifesciences, Bangalore, India.Capsicum annuum fruits were extracted with n-hexane at a temperature ranging from 40 to 60 °C (n-hexane layer was filtered through a 0.5-µm filter cloth and concentrated under a vacuum (0.05 mPa).The dried The obtained oleoresin was further enriched by supercritical fluid extraction (40–60 °C and 25–50 mPa). The enriched extract was saponified using 50% alcoholic potassium hydroxide at 80–85 °C for 2 h. The alcohol was removed under vacuum, and the product was washed with hot water until neutral pH. Ethyl acetate was used to extract the saponified capsanthin, and water-soluble impurities were removed by washing with water. Anhydrous sodium sulfate was used to dry the ethyl acetate and capsanthin extract was concentrated under a vacuum .The samples were dispersed in deionized and distilled water followed by sonication for 5 min at room temperature. A scanning electron micrograph (SEM) of the aqueous dispersion was recorded on a scanning electron microscope  by spreading the micelle dispersion over a carbon tape and drying it under a nitrogen stream.ṽ=400–4000 cm−1.Attenuated total reflectance Fourier-transform infrared (FTIR) spectra of capsanthin extract and cap-DPGS-1000 were analyzed to confirm any change in the structure and functional groups after capsanthin loading using an FTIR spectrophotometer . The wavenumber range was set at θ/°.The crystallographic nature of capsanthin extract and cap-DPGS-1000 was evaluated by X-ray diffraction (XRD). Using a multipurpose diffractometer , the test items were scanned at a voltage of 40 kW and 30 mA. The scanning rate was 3 °C per min, and the angle was maintained at 2The cumulative drug release of cap-DPGS-1000 was studied on a calibrated dissolution apparatus  with paddle stirring speed 100 rpm, and the bath temperature was set at (37±0.5) °C with a dissolution volume 900 mL. A drug release experiment was conducted using 500 mg cap-DPGs-1000 in each bowl. The drug was dissolved for the first 2 h in simulated gastric fluid (buffer at pH=1.2) followed by 6 h in simulated intestinal fluid (buffer at pH=6.8). The buffer at pH=1.2 was prepared by mixing 250 mL of 0.2 M potassium chloride solution with 425 mL of 0.2 M HCl and diluted to 1000 mL with water.The phosphate buffer at pH=6.8 was prepared by mixing 200 mL of 0.2 M potassium phosphate monobasic with 89.6 mL of 0.2 M sodium hydroxide, diluted to 1000 mL with water. At predetermined intervals, 5 mL of dissolution medium was removed for quantification and replaced with the same volumes of similar medium (φ(FBS)=10%) and 1% (m/V) antibiotics (penicillin/streptomycin) at 37 °C in a humidified atmosphere with 5% CO2 and 95% oxygen  supplemented with foetal bovine serum (4 cells per well in 96-well plates and incubated overnight. The next day the cells were treated with different concentrations of γ(capsanthin extract)=0.5, 1, 2, 4, 6, 8, 10, 25, 50, 75 and 100 μg/mL and γ(cap-DPGS-1000)=0.5, 1, 2, 4, 6 and 8 μg/mL for 24 h. After completing the incubation time, 20 μL of freshly prepared MTT (5 mg/mL in PBS) were added to each well, followed by incubation in a CO2 incubator (Thermo Fisher Scientific) for 3 h. The medium was removed thoroughly, and 200 μL of dimethyl sulphoxide were added to each well to dissolve the formazan crystals. Finally, the absorbance of the 96-well plates was read at 590 nm using a microplate reader .MTT -2,5-diphenyltetrazolium bromide) assay was performed to assess the cytotoxicity of capsanthin extract and cap-DPGS-1000 against human triple-negative cell breast cancer cells. MDA-MB-231 cells were seeded at a density of 10The relative cell viability (in %) after treatment with capsanthin extract and cap-DPGS-1000 was normalized with control. The cell viability was calculated using the following equation:5 cells per well) were grown in 6-well plates and incubated with half maximal inhibitory concentration (IC50) of capsanthin extract and cap-DPGS-1000 for 24 h.The morphology and membrane permeability of MDA-MB-231 cells treated with capsanthin extract and cap-DPGS-1000 were analyzed using the acridine orange/ethidium bromide (AO-EB) dual staining method  for 5 min. The cells were visualized under a fluorescence microscope  at 20× magnifications.5 cells per well) were cultured and treated with IC50 value of capsanthin extract and cap-DPGS-1000 for 24 h. The media were removed, and sufficient Hoechst 33342 stain (10 mg/mL) and PI (10 g/mL) were added separately to cover the cells. After incubation for 10–12 min in the dark, the media were removed, washed with PBS twice, and visualized under a fluorescence microscope  at 20× magnification.The cell-permeant nuclear stain Hoechst 33342  were washed with PBS, stained with Rho 123 for 1 h in the dark at 37 °C, and visualized under a fluorescence microscope  at 20× magnification.Rhodamine 123 (Rho 123) staining was used to evaluate mitochondrial membrane potential. The fluorescent Rho 123 dye binds to metabolically active mitochondria. MDA-MB-231 cells were grown and treated with IC5 cells per well were seeded into 6-well plates (grown overnight) and incubated with IC50 value of capsanthin extract and cap-DPGS-1000 for 24 h. The medium was removed, washed with PBS and stained with 100 μL DCF-DA (50 μM) for 25–20 min in the dark at room temperature. Fluorescence was detected using a fluorescence microscope  at 20× magnifications.The intracellular reactive oxygen species  accumulation within MDA-MB-231 cells was analyzed by 2′,7′dichlorodihydrofluorescein diacetate (DCF-DA) staining. For this, 10t-test using GraphPad Prism v. 7.04 software , and a broad -OH stretching band at 3436 cm-1  nm, almost 80 times lower than of the capsanthin extract (d=(31993±5) nm). Particle size and their distributions in a sample can be defined by polydispersity index (PDI).Particle size is an important factor for insoluble drugs  and 1.0 (multiple particle size). Usually, the acceptable value for a micelle is 0.2 and below. From the PDI value for cap-DPGS-1000 (0.3±1.1), we can conclude that the particle size is almost uniform, but for the free capsanthin, the PDI is 0.6±0.9, which is a characteristic of multiple particle size groups , MDA-MB-453 (triple-negative), and MDA-MB-231 (triple-negative) cell lines. Furthermore, lutein significantly reduced colony numbers in MCF-7 and MDA-MB-468 cells  is crucial for normal cell development and turnover, but the apoptosis pathway is deregulated in cancer. Triple-negative breast cancer is defined by the lack of estrogen and progesterone receptor expression and HER2  overexpression/amplification, and represents 10-15% of all breast cancer patients (50 at 24 h for capsanthin and capsanthin-loaded micelles were (81.1±1.5) and (3.1±1.1) μg/mL, respectively . The ICectively . Our stuectively , in capsectively  and cap-ectively . The celThe occurrence of apoptosis was further confirmed by the DNA-binding dye Hoechst 33342, which detects chromatin condensation, a hallmark of apoptotic cell death  generation is usually associated with cell apoptosis  comparedCapsicum annuum) fruit and developed capsanthin-loaded diosgenin polyethylene glycol succinate 1000 (cap-DPGS-1000) micelles successfully the first time, and the micelles exhibited suitable physicochemical properties. A solid dispersion technique was employed to prepare the cap-DPGS-1000 micelles as a delivery system for the hydrophobic capsanthin. The FTIR and other characterization data confirmed no changes in the capsanthin structure and functional groups in the capsanthin-loaded micelles. The capsanthin-loaded micelles were nanosized with low polydispersity, indicating the size uniformity. In vitro release behaviour of capsanthin was controlled for 240 min. Finally, capsanthin extract and capsanthin-loaded micelles were evaluated for their cytotoxic activity on triple-negative breast cancer cells MDA-MB-231. The results of the study show that both capsanthin extract and cap-DPGS-1000 had cytotoxic effects on MDA-MB-231 breast cancer cells. The IC50 value of the cap-DPGS-1000 micelles against MDA-MB-231 breast cancer cells was 3.1 μg/mL, which is much lower than of capsanthin extract (IC50=81.1 μg/mL). Capsanthin extract and capsanthin-loaded micelles are promising drug candidates to induce apoptosis and increase reactive oxygen species in cancer cells.In this study, we have extracted capsanthin from sweet red pepper (