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. 2022 Feb 25;12(3):78. doi: 10.1007/s13205-022-03144-z

Chemical alternative for cell identification and cross-contamination detection

Dhouha Msalbi 1,✉, Jihene Elloumi-Mseddi 1, Bochra Hakim 1, Emna Sahli 1, Sami Aifa 1
PMCID: PMC8881561  PMID: 35251881

Abstract

Misidentification of human cell lines has previously led to confusing results during cell culture experiments. Although several enzymatic as well as molecular analysis approaches have been developed for cell-line authentication, these methods remain costly. In the present paper, we describe a simple chemical alternative based on known compound cell cytotoxicity. In addition to cisplatin, a pool of eight tamoxifen derivative compounds was used to compare the cytotoxic effects on three different breast cancer cell lines: MCF-7, T47D and MDA-MB-231. Our results show that four out of the eight cytotoxic-related compounds allowed to distinguish the different cell lines based on their IC50 (the half maximal inhibitory concentration) values which are cell type dependent. The remaining chemicals, particularly the most cytotoxic P15, showed close IC50 values for all the cell lines. Interestingly, flow cytometry experiments have identified notable differences among the three cell lines treated with P15. T47D and MDA-MB231 cells were blocked in SubG1 phase and S phase, respectively, while no significant change in cell cycle profile was noticed for MCF-7 cells. Differences were also noted at the level of caspase-3 activity and cell proliferation in P15-treated cells.

Keywords: Cytotoxic, Breast cancer, Authenticate cells, Tamoxifen derivatives, Cell cycle analysis

Introduction

Since 1948, the establishment of the first animal cell lines from mouse fibroblasts, called “L”, has been a revolution in biomedical research by Wilton R. Earle 1993 (Institute (U.S.) 1943). Three years later, the first human cell-line “HeLa” has been established by George Gey in 1951 (Scherer et al. 1953). This immortal human cell line was isolated from an aggressive adenocarcinoma of the cervix of an American cancer patient Henriettea Lacks. Few years later, many cell lines have been established, namely the CHO (Chinese hamster ovary) by Theodore T. Puck (Puck et al. 1958), a non-tumor cell line, derived from epithelial-like cells that opened the area of recombinant therapeutic mammalian proteins mainly monoclonal antibodies (mAb). Consequently, practical improvement of cell culture techniques, together with the development of specific labeling and a more sophisticated microscopy, have revolutionized cell biology studies and facilitated cell imaging. Moreover, specific cell lines have allowed several drugs testing as well as a detailed metabolic or signaling pathways investigation (Taylor 2014). Authentic therapeutic proteins are routinely produced by CHO or HEK293 cells (a human embryonic kidney cell line established in 1973).

Cell culture experiments are increasingly used in labs worldwide. However, the notable diversity of materials, the multidisciplinary of researchers and students are constantly increasing the rate of errors. These errors might occur in different levels, such as when labeling, storing and/or cultivating cells, which leads to the misidentification of cell lines. The misidentification of cell lines, with its multifactorial causes such as exchanging materials and neglecting details of security rules (Nims et al. 2010), represents a persistent problem in biomedical sciences, contributing to the growing concerns about false conclusions and irreproducible experiments (Horbach et Halffman 2017).

The problems with cell-line misidentification have been known for decades, starting with the first case caused by Hela cells that were generously distributed by George Gey in different world laboratories (Gartler 1967). Hela cells have been previously proved to contaminate 19 cell lines through detecting the same G6PD (glucose-6-phosphate dehydrogenase) and PGM (phosphoglucomutase) phenotypes (Gartler 1967). Therefore, one of the first initiative to remedy this problem was the chromosome banding that revealed the presence of Hela cells in at least 6 cross-contaminated cell lines (Nelson-Rees et al. 1974). Later, several different tests, using chromosome banding techniques, glucose-6-phosphate dehydrogenase (G6D), electrophoretic mobility and a specific prostatic acid phosphatase test, have shown that MA-160 cell line was originated from HeLa cell contaminant and not from prostatic epithelium (Webber et al. 1977). Since that, several new announcements of large-scale cell lines cross contamination with Hela cells, have been recently described on a human salivary gland cell line (Lin et al. 2018) and a 35 Chinese established cell lines (Huang et al. 2017). In addition to Hela cells described as “ghosts of Hela” (Horbach and Halffman 2017), numerous misidentified cell lines have been published and had continued to haunt biomedical research, of which the melanoma M14 and MDA-MB-451 breast cancer cell lines (Korch et al. 2018). Cross-contaminations have been also described in hematopoietic cell lines giving rise to misinterpretations and invalidation of research work (Drexler et al. 1999).

The frequent occurrence of cell cross contamination has pushed many laboratories to develop more efficient methods to characterize cell lines and avoid misidentifications. In the early eighties, the allozyme genetic signature has been proposed as a definitive monitor of cell identity allowing the distinction of a determined cell line or a variety of biological tissue studies (O’Brien et al. 1980). Moreover, cytological characteristics such as gross chromosome morphology, C-banding, fluorescence, and the nucleolus organizer regions have been used for cell-line contamination identification (Pathak and Hsu 1985). Steube et al., has emphasized standardized isoelectric focusing (IEF) as an efficient and rapid method for routinely monitoring the cell lines authenticity (Steube et al. 1995).

Molecular methods based on DNA analysis have brought many solutions; meanwhile the costs could be sometimes considerable for a given laboratory. DNA fingerprinting has been reported to be highly reliable and robust enabling cell authentication independent from individual cell lines quantity (Drexler et al. 1999). Parodi et al. has previously adopted a PCR-based method (polymerase chain reaction) to identify or confirm the cell lines species origin belonging to the nine most common animal species in cell culture laboratories. They have used a panel of oligonucleotides that specifically anneal to DNA sequences of human, cat, dog, mouse, rat, horse, rabbit, African green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b (cyt b) gene of Chinese hamster (Parodi et al. 2002). Ramya et al. have also shown that the nested PCR technique for the Cyt b gene could potentially replace the isoenzyme analysis. Therefore, the PCR with its proved simplicity and sensitivity, has been considered as a reliable tool for cell-line authentication (Ramya et al. 2009).

Although short tandem repeat (STR) profiling has been used as a useful method to discriminate cell lines that have been misidentified by cross contamination (Yoshino et al. 2006), mismatch repair (MMR)-deficient cell lines could be misclassified. Therefore, a High-throughput signal nucleotide polymorphism (SNP)-based authentication tools applied on human cell lines has been developed (Castro et al. 2013) and SNP array profiling has been used to combat misidentification and mouse cell lines contamination (Didion et al. 2014).

The antibody variable-region sequencing was reported to be efficient in Hybridoma cell-line authentication (Koren et al. 2008). The proteomics was also used in cell identification as previously reported for fish cell lines (Wagg and Lee 2005). Recently, next generation sequencing data have been also involved and used in the identification of cancer cell lines through Uniquorn, a computational method based on variant profiles (Otto et al. 2017). In spite of all these initiatives and approaches to remedy the misidentification problem, misidentified cell lines are still being used under their false identities (Otto et al. 2017) causing doubts around the corresponding scientific conclusions (Masters et ASN-0002 2010).

In this work, based on our routine activity, we aimed to establish a chemical method for cell authentication. Therefore, we have conducted experiments by treating different cell lines with chemical compounds, notably the P15. Cell viability was evaluated by determining the IC50 values through an MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) simple test and, when necessary, we used cell cycle progression, migration, caspase activity and DNA synthesis.

Materials and methods

Chemicals and reagents

The present chemical compounds (Fig. 1) were a gift of Dr Siden Top (DREM-CNRS UMR 8232 UPMC, IPCM, Eq. ChemBio, France). A total of eight tamoxifen derivatives (P5, P15, P85, P41, P148, DP1, OH-Tam2 and OH-Tam3) were obtained. These tamoxifen derivatives have been previously described: P5 (Bruyère et al. 2014), DP1 (Plazuk et al. 2009) (Bruyère et al. 2014), P15 (Jaouen et al. 2000; Hillard et al 2010), P85 (Top et al. 2001), P41 (Hillard et al. 2007) and P148 (Top et al. 2003). Cisplatin and OH-TAM2 were purchased from Sigma Aldrich (Saint Luis, USA). OH-TAM3, an organic analog of OH-TAM2 including the same dimethylamino-terminated side, was prepared by Top et al. (Top et al. 2011). The compounds were solubilized in DMSO as stock solutions (100 mM) and serial dilutions were prepared with cell culture media just prior to use. Human breast cancer cells MCF-7 and T47 D are estrogen receptor positive (ER+) but MCF-7 contains more than 2.5 × of ER compared to T47D (Taylor et al. 1984) while MDA-MB231 is an ER− cell line. These cells were obtained from the Pasteur Institute of Tunis.

Fig. 1.

Fig. 1

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Chemical structure of the tamoxifen derivatives used in this study

Cell culture

The human breast cancer cell line MCF7, MDA-MB-231 was grown in DMEM while T47D cells were maintained in RPMI. Cell culture media were supplemented with 10% fetal bovine serum (FBS) and 200 U/ml of penicillin–streptomycin. Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere.

MTT assay

Cell viability was tested by MTT assay, cells were seeded in 96-well plates at 8000 cells/well (in 100 µl medium) and incubated overnight in full media (DMEM, RPMI containing 10% FBS). Cells were then treated with various concentrations of tamoxifen derivatives. After 48 h the media was removed and MTT solution (5 mg/ml) was added to each well containing 100 µl fresh media. After 4 h the media was removed and 100 µl of SDS 10% was added to dissolve the formazan precipitate. The absorbance of solubilized formazan was read at 570 nm using ELISA reader (Varioskan Thermo Fisher). The IC50 values were than calculated using the following formula: Cell viability ratio (%) = (OD treated/OD control) × 100%

Caspase 3 assay

The downstream executioner caspase-3 activities were evaluated using EnzChek Caspase-3 Assay kit#1(Invitrogen, catalog number: E13183) allowing the detection of apoptosis by testing the increase of caspase-3 activity. Cells were exposed to IC50 concentration of P15 for 48 h. Control cells were incubated with absence of P15. According the manufacturer instructions, cells were lysed and Z-DEVD-AMC substrate was added to the lysates. After a 30 min incubation, the released caspase-3 cleaved Z-DEVD-AMC and generated a bright fluorescent. Fluorescence was measured in a microplate reader (Varioskan, ThermoFisher) using excitation and emission filters (342/441 nm).

Cellular proliferation assay

The 5-Bromo-2’-deoxy-uridine (BrdU) Labeling and Detection Kit III (Roche, catalog number: 11 444 611 001) was used for the determination of cellular proliferation. BrdU is usually incorporated into freshly synthesized DNA instead of thymidine. Cancer cell lines were cultured in a 96 microtiter well plate with a final volume of 100 μl culture medium and treated with P15. After incubation, the BrdU labeling and detection was conducted according to the manufacturer instructions. BrdU labeling solution was added to the culture medium, then cells were fixed and DNA was partially digested by nucleases. The anti-BrdU-POD was used to detect BrdU incorporation through the colored reaction product in the presence of the peroxidase substrate ABTS. Samples were measured in microplate reader at 405 nm with a reference wavelength at 490 nm.

Cell cycle

After treatment with P15 compound, cells were trypsinized and rinsed with PBS than fixed with 70% cold alcohol overnight. After fixation, cells were washed twice with cold PBS and incubated with 500 µl of PBS, 12µlof propidium iodide (PI) 20 mg/ml, 2.5 µl of RNaseA (20 mg/ml) for 60 min at 37 °C in obscurity. Samples were than incubated at 4 °C until Flow Cytometry analysis. Cells were analyzed with Attune Nxt flow acoustic focusing cytometer (Thermo Fisher) equipped with a 488-argon laser. For each sample data were collected for 10.000 events, using linear amplification, at a flow rate of 100 μl/ min.

The red fluorescence corresponding to PI fluorescence signal was collected at wavelength at 695 ± 40 nm (channel BL3). Unstained and untreated cells were used as negative and positive controls, respectively. Cell cycle distribution was determined using BL3-A peak versus counts and each assay was performed in Triplicate.

Cell migration assay

To evaluate whether P15 inhibited cell migration, a classic migration assay was performed with breast cancer cell lines. A straight scratch was made, cells were incubated with IC50 concentration of P15 then cell ability to wound healing was detected by CytoSMART live cell imaging System.

Statistical data analysis

All experiments were performed in triplicate. Data were exposed as mean ± SD. Statistical analyses were performed by Student’s test. The normality and Levene tests for homogeneity of variances were applied prior to one way analysis of variance ANOVA and multiple mean comparisons were performed with Duncun test at p values < 0.05 to investigate the significance difference of factors at a confidence level of 95% between groups.

Results

Compounds and cell viability

Tamoxifen and tamoxifen derivatives have been widely described as potent cytotoxic chemicals against tumor cell lines particularly the breast cancer cells. A range of 8 cytotoxic tamoxifen derivative compounds (OHTAM2, OHTAM3, P15, P5, P85, P41, and P148) were kindly obtained from the laboratory of Dr. Gérard Jaouen (France). In this study, we evaluated the effect of these compounds on three breast cancer cell lines (MCF-7, T47D and MDA-MB-231). Cells were exposed to a wide range of concentrations (from 0.3 to 100 µM) for 48 h. Cell viability was evaluated by MTT assay as detailed in “Materials and methods” section. The obtained results showed that the cells viability was reduced in a dose and compound-dependent manner (Fig. 2) with specific IC50 values (Table 1). Results on MCF-7 cells showed that all the tamoxifen derivative compounds, except the P41, were more toxic than cisplatin, particularly P148 (IC50 = 4.9) and P15 (IC50 = 5.28). In addition, the P15 was the most toxic compound on both T47D and MDA-MB-231 cells (IC50 = 5.59 and 7.05, respectively). Therefore, P15 demonstrated the highest cytotoxic effect on all three cancer cell lines with a comparative IC50 values.

Fig. 2.

Fig. 2

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The effect of tamoxifen derivatives on breast cancer cell lines; A T47D, B MDA-MB-231, C MCF-7 was evaluated using MTT assay. Cells were treated with different compound concentrations ranging from 0.3 µM to 100 µM. Data were presented as mean of three independent experiments ± standard deviation (SD), all values with subscripts (a–g) are significantly different at p ≤ 0.05, ANOVA; post hoc test Duncan

Table 1.

IC50 values (µM/ml) of breast cancer cell lines: MCF-7, T47D and MDA-MB-231 treated with different concentrations of tamoxifen derivatives for 48 h

Cell lines Compounds
OH-TAM2 OH-TAM3 P85 P41 P148 P15 DP1 P5 Cisplatin
MCF-7 33.09 ± 1.54c 21.9 ± 0.79e 13.46 ± 0.49f  > 100 4.9 ± 0.19 g 5.28 ± 0.2 g 58.1 ± 2.41b 29.98 ± 1.03d 83.35 ± 3.99a
T47D 30.37 ± 1.4d  > 100  > 100 49.77 ± 2.2b 76.8 ± 3.07a 5.59 ± 0.23e 51.76 ± 2.1b 29.35 ± 1.2d 40.80 ± 0.97c
MDA-MB-231 40.74 ± 1.92b 15.04 ± 0.62e 27.24 ± 1.21d  > 100  > 100 7.05 ± 0.29f 67.22 ± 3.01a 32.99 ± 1.49c 38.60 ± 1.72b
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IC50 values are means of three independent experiments (n = 3, mean ± SD) all values with subscripts (a–g) are significantly different at p ≤ 0.05, ANOVA; post hoc test Duncan. If we don’t reach the 50% of dead treated cells, we considered the compound not interesting in tumor cytotoxicity (IC50 >100 µM)

Cell cycle progression

The cellular DNA content was analyzed using PI to examine the effect of P15 on cell cycle regulation. The cell cycle was evaluated within treated breast cancer cell lines with IC50 concentration for 24 h and 48 h (Table 2) (Fig. 3). The evaluation of cell subpopulation showed a different cell cycle distribution. We revealed a statistically significant number of T47D cells in sub G1 after 48 h of exposure, while MCF-7 cells showed a slight increase in S phase compared to control cells and a decrease in G0/G1 phase after 48 h. The profile of MDA- MB-231 cells revealed a significant increase in the percentage of cells in S phase (Fig. 4).

Table 2.

Effect of P15 on cell cycle distribution T47D, MCF-7 and MDA-MB-231 cells were treated with IC50 concentration of P15 for 24 and 48 h

T47D MCF7 MDA MB-23
Sub G1 G0/G1 S G2/M Sub G1 G0/G1 S G2/M Sub G1 G0/G1 S G2/M
Control 0b 36.53 ± 0.1d 34.21 ± 1.1c 12.5 ± 0.3b 0b 72.351.1a 15.53 ± 0.3e 12.04 ± 0.36b 0b 67.68 ± 0.39a 14.86 ± 0.15c 17.33 ± 0.17a
P15 24 h 0b 28.64 ± 0.6e 60.28 ± 0.48a 11.08 ± 0.04b 0b 70.40 ± 0.66b 19.8 ± 0.47d 9.53 ± 0.44b 0b 71.93 ± 1.15a 15.74 ± 0.36e 11.79 ± 0.49b
P15 48 h 54.86 ± 0.6a 23.27 ± 0.17e 15.81 ± 0.5e 5.96 ± 0.35c 0b 53.15 ± 1.2c 30.87 ± 0.65c 15.67 ± 0.47a 0b 36.56 ± 0.45d 47.16 ± 0.58b 15.63 ± 0.78d
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DNA content was detected by flow cytometry using PI staining. Experiments are performed in triplicate; the values are expressed as mean ± SD, all values with subscripts (a–e) are significantly different at p ≤ 0.05, ANOVA; post hoc test Duncan

Fig. 3.

Fig. 3

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Representative profiles of cell cycle distribution phases in breast cancer cell lines A T47D, B MCF-7and C MDA-MB-231. Cells were exposed to IC50 concentration of P15 for 24 and 48 h then DNA content was measured by flow cytometry (x axis: DNA content; y axis: cell number). Experiments were performed in triplicate

Fig. 4.

Fig. 4

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Changes of cell cycle phases distribution in A T47D, B MDA-MB-231 and C MCF-7cells after exposure to IC50 concentration of P15 for 24 and 48 h. Data were shown as mean ± SD of three independent experiments compared to control (*p < 0.05; **p < 0.01; ***p < 0.001)

Inhibitory effect of P15 on breast cancer cell lines migration and invasion

To assess whether P15 promotes cell migration and invasion a wound-healing scratch assay was performed. Cells were incubated with IC50 concentration of P15 (MCF-7:5.28 µM, T47D:5.59 µM, MDA MB-231:7.05) for 72 h. Scratch closer was compared in treated and untreated cells. Images were captured at 0, 24, 48 and 72 h. After 72 h the P15 completely inhibited cell proliferation for T47D and MCF-7 cells while for the MDA-MB-231 cells motility was reduced without total inhibition of the proliferation (Fig. 5).

Fig. 5.

Fig. 5

Fig. 5

Fig. 5

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Scratch wound-healing assay was performed in breast cancer cell lines; A T47D, B MDA-MB-231, C MCF-7. Scratch closer for traded cells with IC50 concentration of P15 was compared to untreated ones. Images were taken at 0, 24, 48 and 72 h

Caspase-3 activity

Caspase-3 is a very important executioner enzyme known to be implicated in the later degradation step of apoptosis. To understand the effect of P15 on cancer cells signaling, the activity of caspase 3 was measured at different time points. Results showed an increase in the level of released caspase-3 in all the cell lines with less extent in MCF-7 cells meanwhile the maximum of activity was reached in T47D cells (Fig. 6A) after 2 h (Fig. 6).

Fig. 6.

Fig. 6

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Detection of caspase 3 activity in AT47D, B MDA-MB-231 and C MCF-7 cells using EnzChek® Caspase-3 Assay Kit. Cells were treated with IC50 concentration of P15. Fluorescence was measured in a fluorescence microplate reader using excitation at 342 nm and emission detection at 441 nm. Values represent the means ± SD (n = 3), (*p < 0.05; **p < 0.01; ***p < 0.001)

DNA synthesis

To understand the mechanism of P15 inhibition on cells, the BromodeoxyUridine (BrdU) was added as a marker of DNA synthesis. All the cells showed a decrease in BrdU incorporation proportional to the increase of P15 concentration. This decrease is more pronounced in MCF-7 followed by T47D cells (Fig. 7).

Fig. 7.

Fig. 7

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The effect of P15 on breast cancer cell proliferation was evaluated using BrdU incorporation assay. AT47D, B MDA-MB-231 and C MCF-7 cells were exposed to different concentration of P15 for 48 h. cell proliferation was measured, results were presented as mean ± SD from three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001)

Discussion

Cell culture is a worldwide practice in biological laboratories that is still hindered by cross contamination of cell lines. Our laboratory is interested in screening tumor specific cytotoxic compounds and uses multiple cancer cell lines. We have noticed that several tested cytotoxic chemicals presented reproducible IC50 values cell type specific, hence the idea of using these recorded values for the cell lines identification. The 8 Hydroxytamoxifen derivatives, previously shown to be highly potent anticancer molecules, have been chosen for this study. Hydroxytamoxifen is an active metabolite of tamoxifen showing a greater affinity to the estrogen receptor (30–100 times) than tamoxifen. It exhibits a greater antiestrogenic effect (Ahmad et al. 2010).

In this current study, we have used two hydroxytamoxifen compounds: OHATAM2 and OHATAM3. The main difference between both compounds is the presence of an additional –CH2 group in OHTAM3 (Fig. 1). Our results showed that, at 100 µM, the OHTAM3 caused more pronounced and specific cytotoxicity particularly against MDA-MB-231 cells compared to the T47D cells where no toxic effect have been detected (Table 1). For the OHTAM2, this hydroxytamoxifen compound exerted a cytotoxic effect against all the cell lines with close IC50 values, except for MDA-MB-231 cells, where a slight resistance has been noticed. The differences between OH-TAM3 and OH-TAM2 have been also observed on cytosolic and mitochondrial thioredoxin reductases activities in Jurkat cells (Schneider et al. 1977). In addition, OH-TAM 2 induced a slight increase of tamoxifen-like activity, while no effect has been observed with OH-TAM3 (Scalcon et al. 2017).

The ferrocenyl derivatives of tamoxifen have been developed to overcome the resistance problems of hormonal cancer to tamoxifen and OH-TAMs. Indeed, they are known by their cytotoxic effects on both ER+ and ER− breast cancer cells (Jaouen et al. 2015). The 6 ferrocifen compounds, in the present investigation, showed heterogeneous cytotoxic profiles; P85 seems to be more cytotoxic on MCF-7 cells without a significant effect on T47D cells; P41 is the less toxic compound without a significant toxicity on MVF-7 and MDA-MB-231 cells which confirms the previously described results by Hillard et al. (Elizabeth Anne Hillard et al. 2007). P148 displayed the lowest IC50 value on MCF-7 cells without a significant toxicity on MDA-MB-231 cells, however, a more pronounced cytotoxicity against all cell lines in presence of P15 compound have been noticed. Additionally, we showed that cytotoxicity decreased when treating cells with DP1 particularly against MDA-MB-231 cells. These results were in discordance with earlier findings describing a very low IC50 for MDA-MB-231 cells (Plazuk et al. 2009) which might be explained by the different incubation periods (5 days versus 48 h in our experiments). Moreover, P5 preserves almost the same effect as OH-TAM2, but slightly more toxic on MDA-MB-231cells which in concordance with the results reported by Plazuk et al. (Plazuk et al. 2009). These three compounds seem to be unable to distinguish between the three cell lines when just comparing the generated IC50 values. Based on our results, P148 represents a potent anticancer compound against MCF-7 cells.

Both P85 and OH-TAM3, did not affect the T47D cells growth, thus they can be used for a possible identification of this cell line. However, at 1 µM, these compounds inhibited the growth of MCF-7 less than P15 as previously reported (Top et al. 2001). Our findings show that P15 seem to be unable to distinguish between the present cell lines, however it represents the most cytotoxic compound for all tested breast cancer cell lines and thus merits deeper investigations as a potential anticancer drug. Meanwhile, following other experimental analysis, we have identified different behavior of the three cell lines after P15 treatment; flow cytometry analyses showed that T47D cells were accumulated in SubG1 phase, while MCF-7 cells displayed slit change in S phase after 24 h which has significantly increased after 48 h. Interestingly, MDA-MB-231 cells showed an increase in the percentage of S phase. Furthermore, all three cell lines were similarly inhibited by P15 in cell migration experiments, and the caspase-3 activity released after the treatment was significantly increased. Moreover, the incorporation of BrdU experiment showed less activity in MDA-MB-231 cells. Our results are consistent with the findings of Vessière et al. (Hillard et al. 2010). P15 (Fc-OH-TAM) allows cell cycle progression in the presence of ERα, with a subsequent blockage inG0/G1, while blocking cells on S phase in the absence of ERα.

In conclusion, since all these compounds showed reproducible cytotoxic effects with a constant IC50 values, they will enable an easy identification of each cell type when applied on misidentified cell stock of the studied breast cancer cell lines. In addition, even the compounds that seem to be equally cytotoxic on all the cell types, they can reveal differences in their effects with supplemental experiments such as flow cytometry and caspase-3 activity. Our findings clearly shows that drugs could be used in the authentication of cell lines through simple cytotoxicity tests or more advanced analysis, when necessary, to identify a specific cell line. The present chemical alternative method of cell identification could be more adequate at least for laboratories studying cell toxicity without extra investigations in enzymatic or molecular protocols.

Our results would also be used in the classification of the recently established cell lines belonging to the same cancer type. The newly established cell lines that record different IC50 values of the adopted cytotoxic compounds will be retained for further evaluation of their sensitivity against known cancer drugs and screening for novel anticancer chemicals. Moreover, the classification of tumor primary cells according to their degree of cytotoxicity could help in predicting the therapeutic tools.

Acknowledgements

The present work was supported by the Tunisian Ministry of High Education and Scientific Research.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

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