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.


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 (


Malignant pleural effusion (MPE) is an independent determinant of poor prognostic factor of non-small cell lung cancer (NSCLC). The course of anchorage independent growth within the pleural cavity likely reforms the innate molecular characteristics of malignant cells, which largely accounts for resistance to chemotherapy and poor prognosis after the surgical resection. Nevertheless, the genetic and transcriptomic features with respect to various drug responses of MPE-complicated NSCLC remain poorly understood. To obtain a clearer overview of the MPE-complicated NSCLC, we established 28 MPE-derived lung cancer cell lines which were subjected to genomic, transcriptomic and pharmacological analysis. Our results demonstrated MPE-derived NSCLC cell lines recapitulated representative driver mutations generally found in the primary NSCLC. It also exhibited the presence of distinct translational subtypes in accordance with the mutational profiles. The drug responses of several targeted chemotherapies accords with both genomic and transcriptomic characteristics of MPE-derived NSCLC cell lines. Our data also suggest that the impending drawback of mutation-based clinical diagnosis in evaluating MPE-complicated NSCLS patient responses. As a potential solution, our work showed the importance of comprehending transcriptomic characteristics in order to defy potential drug resistance caused by MPE. It is also the most prevailing cause of malignant pleural effusion (MPE), accounting for approximately 30% of such effusions3. Although recent advances in prevention and early detection have contributed to the general increase in the survival rate, the occurrence of MPE in the context of lung cancer designates terminal stage and an independent prognostic parameter of the poorer survival rate4. Lung cancer patients with MPE mostly exhibit insensitivities to certain chemotherapies mainly due to high tumor burden6. Besides, the course of acquiring anoikis resistance alters multiple tumorigenic pathways, which eventually brings about insensitivity to certain chemotherapies7. Nevertheless, the genetic and transcriptomic features with respect to various drug responses of MPE-complicated NSCLC have not been thoroughly studied yet, which makes the implication of drug insensitivities of MPE-derived tumor cell still hypothetical.Lung cancer is one of the most prevalent cause of cancer-related death worldwide, with an estimated one million deaths annually. Within Korean population, it is the most lethal disease and the third common types of cancer8. Nevertheless, the widely accepted oncogenic association of secondary or tertiary alternations by the second line chemotherapy has been focused on the primary NSCLC, and oncogenic impact of chemotherapy-derived molecular changes in MPE remains unclear.Moreover, several studies have indicated that acquired resistance to second line chemotherapy such as crizotinib is associated with aggressive disease progressionsEGFR) and anaplastic lymphoma kinase (ALK) fusion with other domains, but also demonstrated the presence of distinct translational subtypes in accordance with the mutational profiles, which was consistently reflected to the drug responses of certain targeted chemotherapies. Our data also suggest that the imminent downside of mutation-based clinical diagnosis in assessing MPE-complicated NSCLS patient responses. As a potential solution, our data showed the importance of comprehending transcriptomic characteristics in order to defy potential drug resistance caused by MPE.To obtain a clearer overview of the MPE-complicated NSCLC in accordance with heterogeneous drug responses, we established 28 lung cancer cell lines derived directly from the MPE, and two crizotinib-resistant sublines which were then subjected to genomic, transcriptomic and pharmacological analysis. Our results not only showed that MPE-derived NSCLC cell lines recapitulated representative genetic aberrations of the primary NSCLC including epidermal growth factor receptor  and targeted sequencing (CCP) on the 28 MPE-derived cell lines. The overall mutational spectrum of the MPE-derived NSCLC cell lines such as mutant-allele tumor heterogeneity (MATH) analysis and relative contribution of mutational signatures was analyzed using WES data. The targeted sequencing as well as direct Sanger sequencing re-confirmed the presence of key driver mutations minimizing the possible false-positive sequencing errors  in order to avoid misestimating the mutation frequency due to inflated germinant/benign mutations. The pathogenicity of the representative driver mutations was manually inspected, and all marked mutations for calculating the frequency of mutated tumor genes in Fig. Several studies have shown that MPE-derived tumor cell lines maintain the mutational profiles of the lung adenocarcinoma10 which was composed of a total of 63 lung cancer cell lines  compared to CCLE cohort (14%). It has been reported that the EGFR mutation frequency in lung adenocarcinoma is approximately 10% as much larger cohort (TCGA)11 indicated  compared to CCLE (35%) and TCGA (30%) cohorts, which was unexpected since mutant KRAS is reported to promote malignant pleural effusion formation5. Previous study indicated that EGFR is more frequently aberrated than KRAS in Korean NSCLC population13, which suggest that the geographic factor partially shaped the mutational landscape of our cohort. Nine patients harbored driver fusion genes including EML4-ALK, KIF5B-RET and CD74-ROS1 in our cohort nes Fig. A. As a f cohort 1%. It has16. This analysis allowed to estimate the dispersion of mutant allele frequencies (MAFs) within a cell population, which reflected the intra-tumor genetic heterogeneity. The cell lines with mutant EGFR had higher MATH score than two other groups, suggesting that higher level of genetic heterogeneity is causative factor for EGFR mutation ion Fig. B. Then, ion Fig. . Except ion Fig. D.EGFR aberration, fusion genes, and others. We have applied these groups for further analysis of transcriptome and drug screening.Overall, comprehensive mutational analysis indicated that there is a distinct separation of genomic drivers in our cohort: EGFR mutation were specifically clustered with less degree of dispersion on the PCA plot compared to the fusion group, which suggests that EGFR-mutated NSCLCs in MPE display characteristic transcription patterns. Hierarchical clustering analysis also displayed that cell lines with the fusion genes were clustered distinctly from the cell lines with EGFR mutation  to identify pathways that are specifically enriched in each group. Only spermatogenesis pathway was significantly enriched in EGFR group compared to other two groups (p < 0.05). Multiple tumor driver genes that are associated with spermatogenesis pathway such as mTOR, EZH2, NF2, DCC and MLF1 had high enrichment score in the EGFR group , Hypoxia (p < 0.01), TNFα signaling (p < 0.05), Angiogenesis (p < 0.05). Genes with high enrichment scores for each signaling pathway are summarized in Supplementary Table We then verified that the pattern of mRNA expression reflects the distinctive genomic aberrations. The principle component analysis (PCA) of the mRNA expression indicated that MPE-derived cell lines were likely grouped in accordance with their genetic factors Fig. A. Cell lion Fig. B. The otoup Fig. C. We furoup Fig. D: InflamALK mutations or alternative pathway changes cause a fraction of the tumors to be resistant to ALK inhibitors18. We further validated that the acquired resistance to crizotinib, the first generation of the ALK inhibitor, alters the transcriptional patterns of MPE-derived NSCLC cell lines harboring EML4-ALK fusion gene. We established two crizotinib-resistant sublines (SNU-2550CR and SNU-2563CR) by long-term exposure to increasing concentrations of crizotinib, and performed RNA-seq. Both sublines exhibited distinct insensitivities to crizotinib, and a list of differentially expressed genes and their log fold changes together with signaling pathways topology were identified in order to detect the pathways most relevant to the crizotinib resistance. SPIA two-way analysis of RNA-seq revealed multiple signaling aberrations including p53 signaling and focal adhesion  fusion gene. However, secondary We assembled an 18-compound library for drug screening. In total, 30 cell lines including two crizotinib resistant sublines from 28 patients were successfully screened in experimental triplicate, generating > 500 measurements of cell line-drug interactions.EGFR mutations such as SNU-2556, SNU-2643, SNU-2708.1, SNU-2741, SNU2867, SNU-2887, SNU-2964 and SNU-3023 as well as EGFR inhibitors including gefitinib, erlotinib and dacomitinib were tightly clustered together. Moreover, MPE-derived NSCLC cell lines with EML4-ALK fusion and its targeted drugs including crizotinib, alectinib, and ceritinib were grouped as well , which recapitulated the role of the increased nuclear localization of β-catenin caused by the elevated DKK1 on the crizotinib resistance.Among the MPE-derived NSCLC cell lines harboring fusion genes, two cell lines with the EGFR-mutated cell lines, but also revealed that cell lines harboring the EML4-ALK fusion displayed significant insensitivities to EGFR targeted therapy. As indicated earlier, cell lines with CD74-ROS fusion were specifically sensitive to MK-5108 as well as cyclopamine.We further integrated the genetic factors of the MPE-derived NSCLC cell lines with 18 drugs using Wilcox ranked sum test Fig. B. Gene-dEGFR mutations, yet still positioned adjacent to cell lines with EGFR mutations on the transcriptomic analysis. This mRNA expression pattern is reflected by the notably good responses of these cell lines to EGFR TKIs, which suggests that the shape of transcriptomic landscape can be a determinant factor for EGFR-targeting therapy  and AUCs of each drug. The AUC value of cyclopamine was in direct proportion to the DEPTH score  to find which drugs accorded with the three different transcriptional subtypes. MANOVA indicated that EGFR TKIs were significantly associated with the mRNA expression patterns  compared to those without effusions (about 12–18 months)23. In the tumor-node-metastasis (TNM) staging classification of lung cancer, patients with MPE are categorized as category M1a (stage IV) as it is a sign of metastatic dissemination24. Although few studies have focused on malignant tumor cells from pleural fluids, only genomic analysis including mutational aspects and copy number variations was conducted27. Giuseppe Roscilli et al. integrated the mutational characteristics of human lung adenocarcinoma cells derived from MPEs to the patients chemosensitivity6, yet the number of included drugs and patients were not enough to draw solid conclusion.Nearly 20% of lung cancer patients develop malignant pleural effusions (MPEs)EGFR mutations and fusion events compared to the lung adenocarcinoma-originated cell lines. Supporting the notion of distinguished EGFR and fusion aberrations, our transcriptomic analysis demonstrated that MPE-derived cell lines were subtyped in parallel with the genetic aberrations. The transcriptomic subtyping of EGFR-mutant NSCLC has been performed in other studies as well29. Our study has demonstrated that MPE-derived NSCLC cell lines also retain the transcriptional characteristics of EGFR-mutant NSCLC, which were distinctive to NSCLC cell lines with other driver mutations such as KRAS or fusion genes.In this study, we present 28 MPE-derived lung cancer cell lines and two crizotinib resistant sublines that were comprehensively characterized in accordance with multiple drug responses. These in vitro models retained the representative lung cancer mutations in parallel with mutational profiling of other external datasets consisting of lung cancer cell lines (CCLE) as well as tumor samples (TCGA). Our data revealed that MPE-derived lung cancer cell lines involves higher rates of 30. We further inspected if MET aberrations occurred in our two crizotinib sublines as well. We confirmed that MET was clearly amplified in SNU-2550CR cell line compared to its parental SNU-2550 cell line regardless of crizotinib treatment, yet no MET amplification was found in SNU-2563 and SNU-2563CR cell lines. This suggested that MET amplification indeed accounted for acquired crizotinib resistance in SNU-2550 set, and the SNU-2563CR cell lines had different resistance mechanism such as nuclear localization of β-catenin. No MET mutation was found in both resistant sublines.We suggested that the nuclear localization of β-catenin in MPE-derived NSCLC cell lines through increased DKK1 expression potentially caused crizotinib resistance. The acquire crizotinib resistance has been also associated with both MET amplification and exon 14 skipping mutation in lung cancerOur work also enabled the integration of highly prevalent genetic and transcriptional characteristics of MPE-derived lung cancer cell lines to various drug responses. We re-confirmed that targeted therapies, especially EGFR TKIs showed high sensitivities in accordance with the presence of key mutations using Wilcox ranked sum test Fig. B and MANIn conclusion, our work emphasized the significance of integrating the mutational status with transcriptomic subtypes using statistical models such as Pearson correlation coefficient and MANOVA in order for the accurate assessment of drug responses in lung cancer patients with MPE.The research protocol was reviewed and approved by the institutional review board of the Seoul National University Hospital (IRB No. 1102-098-357). The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients enrolled in this study.http://cellbank.snu.ac.kr) at the initial passage in order to be distributed to researchers worldwide.Cell lines were established from malignant pleural effusion (MPE) derived from pathologically proven non-small cell lung cancer (NSCLC). A total of 28 MPE specimens of human NSCLC from 28 different patients who underwent a pleural intervention for the palliation of dyspnea were obtained from Seoul National University Hospital . MPE samples were directly transferred from the operating room to the laboratory for cell culture. Tumor cells were spun down by centrifugation the MPE sample at 300 rpm for 5 min, and re-suspended with Opti-MEM I  supplemented with 5% fetal bovine serum  and 1% Penicillin/Streptomycin. Gathered cells were then seeded into T-25cm2 flasks . Confined-area trypsinization or scraping method was used to attain a pure tumor cell population when stromal cells like mesothelial cells or fibroblasts grew in the initial culture. Established cell lines were sustained in RPMI 1640 medium with 10% fetal bovine serum and 1% (v/v) penicillin and streptomycin . Cultures were maintained in humidified incubators at 37 °C in an atmosphere of 5% CO2 and 95% air. The initial passage was assigned when substantial tumor cell growth was detected, and successive passages were given at sub-confluence after trypsinization. When one culture population contains both floating and adherent cells, floating cells were gathered by centrifuging the medium and dispersed by pipetting. All cell lines introduced in this study will be deposited in the Korean Cell Line Bank  was added, the plate was incubated at 37 °C for 2 h, and the optical density of each well was calculated by Multiskan™ GO Microplate Spectrophotometer  at 450 nm wavelength. Acquired growth rate values were calibrated with GraphPad Prism 5 . The morphology of each cell lines was obtained using phase-contrast microscopy. Mycoplasma contamination was checked by the 16S-rRNA-gene-based polymerase chain reaction (PCR) amplification method using e-Myco Mycoplasma PCR Detection Kit .Genomic DNA extraction was performed using QIAamp DNA Mini kit (Qiagen). Genomic DNA extracted from each lung cancer cell line was amplified using an AmpFlSTR identifiler Polymerase Chain Reaction (PCR) Amplification Kit . A single cycle of PCR amplified 15 short tandem repeat markers  and an Amelogenin gender-determining marker containing highly polymorphic microsatellite markers. Amplified PCR products were analyzed by an ABI 3500XL Genetic analyzer (Applied Biosystems).To obtain RNA, TRIzol  was added to cell pellet acquired from cultured lung cancer cell line. After cell lysis was happened, chloroform was added. Then, the sample was vortexed and 4 °C, 12,000 g centrifugation. Each tube containing the sample was divided into aqua phase, interphase, and organic phase. In those aqua phase was transferred into new tube. Isopropanol alcohol was mixed with an equal part of the aqua phase. Mixed sample was centrifuged at 4 °C, 12,000 g after incubated at − 20 °C. RNA was acquired from pellet. The pellet was precipitated by ethanol. RNA was melted in 4th Distilled water containing RNase inhibitor (DEPC). Complementary DNA (cDNA) was synthesized from extracted RNA by reverse transcription kit . Generally measured concentration of RNA was adjusted to 1ug diluted with gDNA wipeout buffer. Extracted RNA concentration was measured nano-spectrometer . Each 1ug RNA was mixed with RT buffer, RT primer mix, and RTase. Those compounds were heated 42 °C for 30–45 min purposed on annealing, extension. Heated compound was additionally heated 95 °C for 3 min to denature synthetic cDNA. The list of primers we have used are as follows.For cDNA synthesis, QuantiTect Reverse Transcription Kit (Qiagen) was used. One microgram of total RNA, 2 μL of gDNA Wipeout Buffer, and RNase free water up to 14 μL were mixed together and incubated at 42 °C for 2 min. The mixture was mixed with Quantiscript RT Buffer, RT Primer Mix, and Quantiscript Reverse Transcriptase and incubated at 42 °C for 45 min. Then, the mixture was incubated at 95 °C for 2 min and cooled down to room temperature.PCR product was precipitated by 5% sodium acetate buffer (Sigma-Aldrich) and 95% ethanol mixed solution. Then washed product was set on ice for 10 min and centrifuged at 4 °C, 14,000 rpm. Supernatant was discarded and the product was rinsed this time by 70% ethanol and centrifuged 14,000 rpm. Supernatant was discarded then the products were dried using vacuum concentrator (Eppendorf). 10 μL of distilled water was added to dilute precipitated sample. When the product is all diluted in distilled water, cyclic PCR was carried out. Two separate mixtures for forward and reverse sequences were made where they each include 5X sequencing buffer (Applied Biosystems), Big Dye (Applied Biosystems), forward or reverse primer, distilled water, and product from the previous step. Cyclic PCR was carried out with denaturation step at 96 °C, annealing temperature at 55 °C, and elongation at 60 °C for 25 cycles. The cyclic PCR product was then precipitated with 5% sodium acetate buffer and 95% ethanol mixed solution and set on ice for 10 min then it was centrifuged at 4 °C and supernatants were carefully discarded and the final product was dried using the vacuum concentrator. 10 μL Hi-Di formamide (Applied Biosystems) was added to dilute the dried product. This final product was transferred to 96 well PCR plate and denatured at 95 °C for 2 min before taken to 3500xL Genetic Analyzer (Applied Biosystems) for sequencing.CCP (Comprehensive Cancer Panel) is technology using Ion Ampliseq (Thermofisher Scientific). CCP panel could detect 409 genes mutations on exomes. There are various mutation types about driver-genes. According to the manufacturer’s instructions as described, gDNA was extracted from cell pellet specimen using QIAamp DNA Mini kit. Quantity and quality of obtained DNA samples was evaluated using the Qubit fluorometer and the Qubit dsDNA HS assay kit . AmpliSeq library was assembled with NEBNext® Ultra DNA Library Prep Kit for Illumina (NEB #E7370S/L) and the Ion AmpliSeqTM Library Kit 2.0 . And then, the amplicons were purified with AMPure XP reagent . The products were processed in end repair by NEBNext® End Prep kit (NEB #E7442). NEBNext® Multiplex Oligos for illumina (NEB #E7335 or NEB #E7500) were ligated with DNA ligase. Adapter-ligated products were then purified by AMPure XP reagent , and PCR-amplified for a total of 7 cycles. The complete library was purified by AMPure XP reagent (Beckman Coulter) and GeneRead size selection kit(Qiagen). Size and concentration of the library were analyzed by Agilent 2100 BioAnalyzer and Agilent BioAnalyzer DNA High-Sensitivity LabChip (Agilent Technologies). The amplified libraries’s quality was proved by capillary electrophoresis . Qualified libraries were mixed in index tagged in equimolar amounts of the pool, after QPCR that was taken by using SYBR Green PCR Master Mix (Applied Biosystems). Sequencing was completed using an Illumina NextSeq 500 system following referred protocols for 2 × 150 bp sequencing.2 × 10^5 to 4 × 10^5viable cells from each cell line were seeded into well of 96 well plate in triplicate to measure drug sensitivity of 18 compounds. After 72 h-incubation at 37 °C, 10ul EZ-Cytox solution was added to well of each seeded lung cancer cells. After 2 h-incubation at 37 °C, optical density of EZ-Cytox-treated cells was calculated by Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific). These steps were repeated in triplicate. The half maximal effective concentration (EC50) were measured by GraphPad Prism 5 (GraphPad Software). The maximum concentration and solvent are as follows.Cultivated cells that had full confluency were harvested with cell scrapper. Cell pellet was treated by EzRIPA buffer  after washed by cool PBS. Whole protein was extracted by this step. Protein concentration of each cell line was determined by SMART micro BCA protein assay kit (Intron biotechnology). Proteins that fixed into equal concentration were loaded on a 4–12% Bis–Tris gel (Invitrogen) at 70 V for 3 h and then proteins on loaded gel were transferred to a PVDF membrane (Invitrogen) by electro-blotting in condition under a constant current of 80 mA at 4 °C overnight. Proteins of transferred membrane was blocked by incubating in 1.5% to 2.0% skim milk and 0.05% Tween 20-TBS buffer including 1 mM MgCl2 for an hour at room temperature. Primary antibodies were used against DKK1, β-catenin, E-cadherin, and Vimentin and β-actin. Mouse or rabbit IgG 2nd antibody  (1:5000) conjugated with peroxidase that matched with used 1st antibody was added to membrane. After chemiluminescent working solution, WESTZOLTM (Intron biotechnology) was treated to the membrane, the membrane was exposed to Fuji RX film  for 1–5 min. The raw and unprocessed blotting images are provided with supplementary data file. The blotting image for DKK1 was closely cropped near the membrane edges, and we provide replicates performed as well. The MET protein level was confirmed with ChemiDoc Touch Imaging System  using consecutive exposing mode with 1-min interval for 5 min.Cells were seeded on chambered coverglass  with a desirable cell confluency. The chambered coverglass was designed to be hydrophilic and no ECM component was treated before seeding. 72 h after cell seeding, cells were washed with cold DPBS for 15 min three times. Then, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ . After cells were washed with washing solution (BD science), DPBS containing 2% FBS  was applied for an hour for blocking. After cells were washed with cold DPBS β-catenin antibody  diluted in 0.05% of PBS.T was applied for a 1.5 h in room temperature. Thereafter, cells were washed with 0.05% of PBS.T, and Alexa 488 and Alexa 568 secondary antibodies  diluted in 0.05% of PBS.T were applied for an hour in room temperature. 1 × DAPI and Rhodamine-conjugated Phalloidin  were diluted in distilled water and applied for 30 min in room temperature. The cells were washed with DPBS three times, and pictured under confocal microscope. LSM800 Confocal Laser Scanning Microscope and ZEN software  was used to examine cells. Diverse magnifications were used for different growth patterns and sizes of cells. The intensity of each channel was fixed for the comparison of target protein expression between samples. Digital resolution, scan speed and the number of pictures averaged were set to 1024 × 1024, 40 s per one channel, and 8 pictures respectively. The pictures were focused on the very bottom of the fixed cells for investigating protruding region of cell colonies and the location of β-catenin.Total RNA was isolated from cell lysate using Trizol (Qiagen) and Qiagen RNeasy Kit (Qiagen). Sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Kit. Fifty-one million reads were obtained from the cell lysates. Following base-calling and alignment with the Tuxedo Suite, rejected reads were analyzed using FusionMap, ChimeraScan, and Defuse with default parameters for RNA and alignment to GRCh37.72. The output was filtered to include in-frame fusions, with at least one rescued read and two unique seed reads, and exclude known, recurrent artifacts.SureSelect sequencing libraries were prepared according to manufacturer’s instructions  using the Bravo automated liquid handler. Three micrograms of genomic DNA were fragmented to a median size of 150 bp using the Covaris-S2 instrument . The adapter ligated DNA was amplified by PCR, and the PCR product quality was assessed by capillary electrophoresis . The hybridization buffer and DNA blocker mix were incubated for 5 min at 95 °C and then for10 minutes at 65 °C in a thermal cycler. The hybridization mixture was added to the bead suspension and incubated for 30 min at RT while mixing. The beads were washed, and DNA was eluted with 50 ml SureSelect elution buffer (Agilent). The flow cell loaded on HISEQ 2500 sequencing system (Illumina).p < 0.05 was considered statistically significant. For hierarchical cluster analysis on a set of dissimilarities, each object was assigned to its own cluster, which an algorithm proceeds through iteratively. Two of the most similar clusters are joined at each stage until there is a single cluster. Distances between clusters are recomputed at each stage by the Lance–Williams dissimilarity update formula according to the particular clustering method being used. Clustering methods include: Ward's minimum variance method, complete linkage method, k-means method, and single linkage method.Statistical analysis was performed using R program version 3.3.1  with various packages including maftools, PerformanceAnalytics, survminer, survival, iplot, gplot, and lattice. Fisher’s exact test was used to analyze GO analysis of various genes. A multivariate analysis of variance (MANOVA) model was applied to the drug response data matrix with various factors such as the mutational status and the three different transcriptional subtypes. Approximate F value, p-value and Pillai’s trace score were obtained for each of the factors/drug pairs. A value of Supplementary Information 1.Supplementary Table S3.Supplementary Table S5.


Despite their documented antitumor effects, thienopyrazole-based compounds remain an underexplored class of molecules. In this study, a screening of 2000 novel molecules revealed a unique thienopyrazole derivative, Tpz-1, that elicited potent and selective programmed cell death in human blood, breast, colon, and cervical cancer cell lines when compared to non-cancerous human fibroblast (Hs27) cells. Furthermore, in HL-60 leukemia cells, Tpz-1 interfered with components of signaling pathways and the cytoskeleton that are important to cell shape, internal organization, growth, and division. These findings encourage the continued investigation and development of Tpz-1 and other thienopyrazole derivatives to target and treat cancers.In recent years, the thienopyrazole moiety has emerged as a pharmacologically active scaffold with antitumoral and kinase inhibitory activity. In this study, high-throughput screening of 2000 small molecules obtained from the ChemBridge DIVERset library revealed a unique thienopyrazole derivative (Tpz-1) with potent and selective cytotoxic effects on cancer cells. Compound Tpz-1 consistently induced cell death at low micromolar concentrations (0.19 μM to 2.99 μM) against a panel of 17 human cancer cell lines after 24 h, 48 h, or 72 h of exposure. Furthermore, an in vitro investigation of Tpz-1’s mechanism of action revealed that Tpz-1 interfered with cell cycle progression, reduced phosphorylation of p38, CREB, Akt, and STAT3 kinases, induced hyperphosphorylation of Fgr, Hck, and ERK 1/2 kinases, and disrupted microtubules and mitotic spindle formation. These findings support the continued exploration of Tpz-1 and other thienopyrazole-based compounds as potential small-molecule anticancer agents. Small-molecule agents have become a mainstay of targeted cancer treatment in the past 20 years. Unlike macromolecule drugs, small molecules can penetrate cells and bind a wide array of intra- or extracellular targets; additionally, they can be selective or broad-spectrum agents ,2,3.Pyrazoles and thiophenes are well-studied pharmacophores known for their diverse and remarkable biological activity, particularly in cancer, and are common scaffolds in small-molecule drug design ,5. PyrazThienopyrazoles are a bicyclic combination of the pyrazole and thiophene moieties and have been endorsed as antiproliferative, antiviral, antimicrobial, and anti-inflammatory agents ,11,12. TThienopyrazoles are less-explored than the other isomers, despite growing evidence detailing their significant phosphodiesterase 7A (PDE7A) ,14, puriFormally named N’-(2-methoxybenzylidene)-3-methyl-1-phenyl-1H-thienopyrazole-5-carbohydrazide, we found that this compound was also identified as cytotoxic and a mitotic inhibitor in two previous studies under the aliases 5248881  and MTPCLeukemias are a leading cancer type in children and adults ,22. In aIn this study, the cytotoxicity of Tpz-1 was evaluated against a panel of sixteen cancer cell lines and one non-tumorigenic human cell line. All cell lines were obtained from the American Type Culture Collection .2 humidified 37 °C environment.Ten of the cell lines were of blood cancer origin: CCRF-CEM , HL-60 , Ramos , Jurkat , MOLT-3 , NALM6 , MM.1S , MM.1R , U266 , RPMI 8226 ; and were cultured in RPMI-1640 medium complete with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. An exception was made for HL-60, which required supplementation with an additional 10% FBS. In addition, two colorectal adenocarcinoma cell lines, COLO 205 (ATCC CCL-222) and HT-29 (ATCC HTB-38), were identically maintained in RPMI-1640 complete medium. Two triple-negative breast adenocarcinoma cell lines, MDA-MB-231 (ATCC CRM-HTB-26) and MDA-MB-468 (ATCC HTB-132), and estrogen-receptor-positive (ER+) cell line MCF7 (ATCC HTB-22) were cultured in DMEM medium complete with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin; cervical adenocarcinoma cell line HeLa (ATCC CRM-CCL-2) and non-tumorigenic foreskin fibroblast Hs27 (ATCC CRL-1634) cells were also maintained in this medium. Lastly, dopaminergic neuroblastoma cell line SH-SY5Y (ATCC CRL-2266) was cultured in DMEM/F12 medium with 10% FBS and antibiotics added as described above. All cells were cultivated in a 5% COBefore use in experiments, the viability of each cell culture was quantified by flow cytometry following propidium iodide (PI) staining or visualized by trypan blue exclusion . Only ce2O2) was alternatively diluted in 1× phosphate-buffered saline (PBS).All compounds described herein were commercially purchased. Experimental compounds from the ChemBridge DIVERset library were obtained pre-diluted to 10 mM in dimethyl sulfoxide (DMSO). This ChemBridge stock of N’-(2-methoxybenzylidene)-3-methyl-1-phenyl-1H-thienopyrazole-5-carbohydrazide , also known as Tpz-1, was used for the experiments detailed below. Lyophilized reference compounds Paclitaxel and Cytochalasin-D were similarly prepared in DMSO; and hydrogen peroxide  is a live cell imaging assay that uses two nuclear fluorescent dyes to distinguish between live and dead populations based on cell permeability . To achiv/v DMSO (vehicle control), 1 mM H2O2 (positive control), or left untreated (UNT) for 24–72 h. Four independent measurements were used for the samples, except for the vehicle control in which eight DMSO-treated independent measurements were used to ensure the average total cell count was representative for each experiment series. One to two hours before the end of treatment, a 1 μg/mL final concentration of Hoechst 33342 and PI was quickly added to each well and incubated until the remaining time elapsed. Four contiguous images, taken in 2 × 2 montages, were captured via GE InCell Analyzer 2000 with a 10x objective from the two fluorescent channels corresponding to Hoechst and PI emission. After analysis, the captured high-content images were segmented using the GE InCell Analyzer Workstation 3.2 software to obtain live and dead cell counts and percentages within each well. Due to technical problems, for the MCF7 cell line, the BD Pathway 855 bioimaging system was used to capture a similar 2 × 2 montages per well, and images were segmented with its associated AttoVision v1.6.2 software . To account for the lower total cell count in Tpz-1 or H2O2-treated (Treatment) samples caused by cell destruction (disappearance from count) over the course of incubation, cell death percentages were normalized to the vehicle control (DMSO) average total cell count by the following equation:For DNS analysis, cells were first seeded in 96-well plates at a density of 10,000 cells/well in 100 μL of complete medium and incubated overnight to facilitate cell attachment and acclimation. The next day, cells were exposed to Tpz-1 concentrations ranging from 0.01 to 10 μM, 1% 50 values.Normalized percentages of dead cells per treatment replicate were subsequently averaged and used to estimate CC50) for each cell line indicates the average concentration (μM ± S.D.) needed to kill 50% of the cell population at a given time point. From the percentage of cell death for each technical replicate at a concentration above and below 50% cell death, CC50 for each cell line was calculated via linear interpolation and averaged to obtain the reported values pyrazole derivative with potent and specific anticancer activity in vitro. Despite compelling evidence of the diverse biological activity of thienopyrazoles, these findings have brought to light a scarcity of publications focused on this unique scaffold. While the contribution of the thienopyrazole moiety to Tpz-1’s biological activity remains unknown, our data parallels previously studied thienopyrazoles. Research to further elucidate Tpz-1’s mechanism of action is ongoing. Until then, we hope that this work inspires our colleagues to consider thienopyrazole-based compounds in small-molecule anticancer drug formulations.


Academic procrastination is a specific sub-type of procrastination, assessing the tendency to delay academic tasks in connection with the preparation of courses or exams.To determine the impact of academic procrastination on studies and academic performances and identify associated factors.We conducted a cross-sectional descriptive study in students from three different universities: a medical school, a law school and an engineering school. Socio-demographic, clinical and academic data were collected. Procrastination was assessed using the Academic Procrastination Scale. We further administered the Short Version of the impulsive behaviour scale, the Satisfaction with Life Scale, the Perfectionism scale, and the one item Self-esteem Scale.Our sample consisted of 1019 students. The mean age was 22 ± 2.25 years, and 62% were females. About one third of study participants used tobacco or alcohol, and 10% used drugs (cannabis or others). We found a significant positive correlation between procrastination and academic failure (r=0.22 p= 0.00) and a negative correlation with academic success (r= -0.27 p=0.00). Multivariate regression analysis showed the following risk factors for academic procrastination: alcohol consumption , study field , and impulsivity .Academic procrastination has a negative impact on academic achievement and performance. This impact appears to differ depending on the field of study. It also seems closely related to impulsiveness and alcohol use. Our findings might contribute to find new ways of helping students to improve academic performance.No significant relationships.