Antiproliferative activity, cell-cycle arrest, apoptotic induction and LC-HRMS/MS analyses of extracts from two Linum species

Abstract Context Linum is the largest genus of the Linaceae family; the species of this genus are known to have anticancer activity. Objective In this study, ethyl acetate extracts of L. numidicum Murb. (EAELN) and L. trigynum L. (EAELT) were examined, for the first time, for their anticancer capacity. The secondary metabolites compositions were analysed by LC-HRMS/MS. Materials and methods The antiproliferative effect of EAELN and EAELT (0–10.000 μg/mL) against PC3 and MDA-MB-231 cell lines were  evaluated by the MTT assay after 72 h of treatment. Flow cytometer analysis of apoptosis (Annexin V-FITC/PI) and cell cycle (PI/RNase) was also performed after treatment with EAELN and EAELT at 250, 500, and 1000 μg/mL, for 24 h. Results EAELN had the highest antiproliferative activity against PC3 (IC50 133.2 ± 5.73 μg/mL) and MDA-MB-231 (IC50 156.9 ± 2.83 μg/mL) lines, EAELN had also shown better apoptotic activity with 19 ± 2.47% (250 μg/mL), 87.5 ± 0.21% (500 μg/mL), and 92 ± 0.07% (1000 μg/mL), respectively, causing cell cycle arrest of PC3 cells in G2/M phase, whereas arrest in G0/G1 and G2/M phases was observed after treatment with EAELT. LC-HRMS/MS profiling of the extracts revealed the presence of known compounds that might be responsible for the observed anticancer activity such as chicoric acid, vicenin-2, vitexin and podophyllotoxin-β-d-glucoside. Discussion and conclusions We have shown, for the first time, that EAELN and EAELT exert anticancer activity through cell cycle arrest and induction of apoptosis. EAELN can be considered as a source to treat cancer. Further studies will be required to evaluate the effect of the active compounds, once identified, on other cancer cell lines.


Introduction
Cancer is the second main cause of death in the world and remains one of the most difficult diseases to combat (Teles et al. 2018). Cancer is characterised by the uncontrolled proliferation of cells, loss of cell cycle control and insensitivity to apoptosis which often lead to the formation of malignant tumours, which can invade neighbouring parts of the organism (Seyfried and Shelton 2010).
The development of anticancer drugs and more effective treatment strategies to improve the quality of life of patients is of great importance in the field of oncology. Currently, cancer treatments, such as chemotherapy and radiotherapy have drawbacks, including strong systemic toxicities and local irritations (Shibata et al. 1990;Huncharek et al. 2001). In addition, resistance to anticancer drugs and adverse outcomes of radiotherapy (Jabir et al. 2018;Lee et al. 2018) emphasise the urgent need to discover new, less toxic agents with higher clinical efficacy (Lee et al. 2003;Newman and Cragg 2007).
Medicinal plants are known for constituting a rich source of clinically relevant anticancer compounds. In this context, the inhibition of cancer cell proliferation and induction of apoptosis by phytochemicals is considered a promising feature of chemotherapeutic drugs (Lowe and Lin 2000;Gurumurthy et al. 2001). Currently, many major anticancer drugs available are either natural products or their derivatives, such as taxol, vinblastine, vincristine, and camptothecin (Newman et al. 2003;Newman and Cragg 2007;Ojima 2008).
The Linum genus (Linaceae) includes more than 200 species distributed throughout the world (Rogers 1982). Several studies have reported that Linum species might inhibit the growth of various types of cancer cell lines through cell cycle arrest and induction of apoptosis (Amirghofran et al. 2006;Mohammed et al. 2010;Alejandre-Garc ıa et al. 2015;Akbari Asl et al. 2018). The investigation of the phytochemical composition of Linum species revealed the presence of lignan-type compounds (Vasilev et al. 2008). These types of compounds constitute an important group of natural products that exert different biological activities and can serve as lead compounds for the development of new therapeutic agents with antiangiogenic, antirheumatic, antipsoriasis, antiasthmatic, hypolipidemic, antifungal, and antiviral activity (Ayres and Loike 1990;Iwasaki et al. 1996). Moreover, cytotoxic and antitumor activities are of major interest for these types of lignans (Ayres and Loike 1990).
The present study determines the ability of the ethyl acetate extracts (EAELN and EAELT) of two Algerian Linum species, L. numidicum Murb. and L. trigynum L., respectively, to inhibit cancer cell proliferation, block the cell cycle and induce apoptosis. In addition, the secondary metabolites composition of the two extracts was analysed by LC-HRMS/MS to determine the relationship between their anticancer activity and their chemical composition.
According to the World Health Organisation (WHO) 2020, the highest incidences in the world are observed for breast and prostate cancers. We, therefore, decided, as proof of concept, to test the effect of our extracts on breast and prostate cancer cell lines. The aerial parts of L. numidicum and L. trigynum (900 g), previously dried and pulverised, were individually macerated at room temperature in a hydroalcoholic mixture (EtOH/H 2 O, 8:2, v/v) for a period of 72 h. The crude extracts obtained were taken up with distilled water (1000 mL). The latter underwent liquid/ liquid type extractions, using solvents of increasing polarity (petroleum ether, chloroform, ethyl acetate and n-butanol). The obtained organic phases were concentrated under reduced pressure to dryness.

Sample preparation
Dried acetate extracts from both plants (EAELN and EAELT) were dissolved in dimethylsulphoxide (DMSO, Sigma Aldrich) followed by an RPMI medium. The DMSO content in the solution did not exceed 0.1%. The mixture was then centrifuged to remove insoluble ingredients and the supernatant was passed through sterilisation filters. The solution was diluted with an RPMI medium and prepared at different concentrations.

MTT test
The antiproliferative activity of EAELN and EAELT against the two selected cell lines was evaluated by the MTT assay (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich), according to Mossman (1983). PC3 and MDA-MB-231 lines (5000 cells/well) were seeded in 96-well plates and incubated with different concentrations of EAELN and EAELT (2.44, 9.77, 39.06, 156.25, 625, 2500, and 10.000 lg/mL). Cells without treatment were used as controls. After 72 h of exposure to the extracts, the wells were emptied, washed with PBS and then 100 mL of MTT (5 mg/mL) was added to each well. The plates were then incubated for an additional 2 h at 37 C and 5% CO 2 . After that, the medium was carefully aspirated and the formed formazan crystals were dissolved in DMSO. The absorbance in each well was measured at 570 nm using a microplate reader (Varioskan LUX, Thermo Scientific).

Apoptosis assay
Cell apoptosis induced by EAELN and EAELT was detected by Annexin V-FITC/PI staining according to the manufacturer's instructions (BD Pharmingen TM FITC Annexin V Apoptosis Detection kit I). PC3 cells (10 5 cells/well) were treated with EAELN and EAELT after seeding in 96-well plates with different concentrations (250, 500, and 1000 mg/mL) for 24 h. Cells were then washed with PBS buffer, centrifuged, and stained with 5 lL of Annexin V and 5 lL of propidium iodide (PI). After 15 min of incubation at room temperature, 400 mL of binding buffer was added. Stained cells were determined using Accuri TM-C6 flow cytometry (BD-Biosciences).

Cell cycle analysis
The cell cycle was determined using a cell cycle detection kit (BD Pharmingen TM PI/RNase Staining Buffer). PC3 cells (10 6 cells/well) were treated with EAELN and EAELT after seeding in 6-well plates with different concentrations (250, 500, and 1000 mg/mL) for 24 h. At the end of the treatment, cells were collected and washed with cold PBS and fixed in cold 70% ethanol (À20 C) for 2 h. The cells were then centrifuged and the cell pellet was resuspended with 0.5 mL of a mixture containing propidium iodide and RNase. After 15 min of incubation at room temperature, the DNA content of the cells was quantified using an Accuri TM-C6 flow cytometer (BD-Biosciences).

LC-HRMS/MS analyses
LC À ESI À DDA À HRMS/MS spectra were obtained using a Dionex Ultimate 3000 liquid chromatography (HPLC) system equipped with an Ultimate 3000 RS pump hyphenated to a Thermo Instruments MS system (LTQ Orbitrap XL). The HPLC analysis was performed on a Water BEH C18 column (1.7 lm, 2.1 mm Â 150 mm) and using the following solvents: Solvent A 0.1% formic acid H 2 O, Solvent B 0.1% formic acid acetonitrile. The gradient used was 5% B for 5 min, 5-100% for 20 min, and 100% for 7 min. The flow rate used was 0.150 mL/min and the column temperature was at 45 C. MS/MS spectra were realised using MZmine 2.53 with a mass detection noise level set at 2E4 and 2E, respectively in both positive and negative ion modes. The compound identification was aided by using the Global Natural Product Social Molecular Networking platform (GNPS).

Statistical analyses
Data analyses were performed using GraphPad PRISM software (version 8.0.2). All experiments were performed in triplicate. One-way or 2-way ANOVA with Bonferroni's post hoc comparisons was used as indicated in the legends. Data are presented as mean ± SD. Significant differences were set at a p-value < 0.05.

Results
Antiproliferative effect of EAELN and EAELT on the PC3 and MDA-MB-231 cell lines To study the effect of EAELN and EAELT on the growth of human prostate and breast cancer cells, PC3 and MDA-MB231 cells were treated with different concentrations of extracts for 72 h, and cell viability was determined using the MTT assay.

Induction of apoptosis by EAELN and EAELT in PC3 cells
To explore the mechanism by which EAELN and EAELT inhibit cell proliferation, a flow cytometer apoptosis analysis, using Annexin V-FITC/PI double staining, was performed after a 24 h exposure period to different extract concentrations (250, 500, and 1000 lg/mL).
EAELN and EAELT induced apoptosis of PC3 cells, which was evidenced by the accumulation of early and late apoptotic cells, and by the decrease of viable cells, with a higher effect at a dose of 500 and 1000 lg/mL ( ÃÃÃÃ p < 0.0001) compared with the negative control ( Figures 3A,B and 4A,B).

Induction of cell cycle arrest in PC3 cells by EAELN and EAELT
To better understand the mechanism by which EAELN and EAELT inhibit cell proliferation, flow cytometry analysis using PI staining was performed to assess cell cycle distribution in the PC3 cell line after 24 h of exposure to 250, 500, and 1000 lg/mL of extract. After treatment with EAELN, the percentage of cells in the G0/G1 and S phase was reduced, while that in the G2/M phase was significantly increased ( ÃÃÃÃ p < 0.0001).
The percentage of cells in the G0/G1 phase in the three groups of samples treated with EAELN was 75.60 ± 0.47%, 73.75 ± 0.22%, and 71.79 ± 0.01 ( ÃÃÃ p < 0.001) for 250, 500, and 1000 lg/mL, respectively, while that of the control was 74.46 ± 0.49%. The percentage of cells in S phase increased from 19.29 ± 1.39% (control) to 5.66 ± 0.53% (250 lg/mL), 7.96 ± 0.20% (500 lg/mL), and 10.55 ± 0.55 (1000 lg/mL), all ÃÃÃÃ p < 0.0001 compared with control, while that in G2/M phase went from Figure 1. Dose-dependent effect of EAELN and EAELT on the viability of PC3 cancer cells. Cell viability was determined by an MTT assay and was expressed as a percentage. Cells were treated with tow extracts at different concentrations for 72 h. Data are expressed as mean ± SD (n ¼ 3), ÃÃÃ p < 0.001, ÃÃÃÃ p < 0.0001, EAELN versus EAELT. Two-way ANOVA followed by Bonferroni's correction. Figure 2. Dose-dependent effects of EAELN and EAELT on the viability of MDA-MB-231 cancer cells. Cell viability was determined by an MTT assay and was expressed as a percentage. Cells were treated with tow extracts at different concentrations for 72 h. Data are expressed as mean ± SD (n ¼ 3), ÃÃ p < 0.01, ÃÃÃÃ p < 0.0001, EAELN versus EAELT, Two-way ANOVA followed by Bonferroni's correction. 7.50 ± 1.36% (control) to 18.73 ± 0.67% (250 lg/mL), 18.29 ± 0.12% (500 lg/mL), and 17.72 ± 0.64% (1000 lg/mL), all p < 0.0001 compared with control ( Figure 5A, B). Treatment with EAELT showed different effects in different phases of the cell cycle, with the percentage of cells in the S phase being reduced, while that of G0/G1 and G2/M phase increased significantly ( Figure 6A, B).

LC-HRMS/MS analyses of EAELN and EAELT
With the aim to analyse the phytochemical composition of EAELN and EAELT we conducted UHPLC-DAD-ESI/HRMS-MS. The latter revealed the presence of 75 compounds (Table 2 and Figure 7A, B) in each extract. The metabolites were identified using the GNPS platform. EAELN and EAELT showed the presence of polyphenols, terpenoids, alkaloids, polyketides, fatty acids, and carbohydrates. The most important classes were flavones (23 compounds) and hydroxycinnamic acids (17 compounds).

Discussion
Medicinal plants are important sources for the development of effective anticancer agents. Currently, many drugs available on pharmacy shelves are either natural products or their derivatives (Mukherjee et al. 2001;Khan 2014;Ijaz et al. 2018).
Species of Linum genus are known to induce anticancer activity (Hartwell 1982). The present study examined, for the first time, the ability of EAELN and EAELT to inhibit cancer cell proliferation, block the cell cycle and induce apoptosis which is known to be the most promising routes to treat cancer (Hanahan and Weinberg 2000;Ghobrial et al. 2005). In addition, the secondary metabolites of EAELN and EAELT were analysed by LC-HRMS/MS to determine the relationship between their anticancer activity and their chemical composition.
The results of our study showed, for the first time, that EAELN and EAELT inhibited the proliferation of PC3 and MDA-MB-231 cells significantly in a concentration-dependent manner (Figures 1 and 2). This could be explained by the presence of polyphenols such as flavonoids, phenolic acids, and lignans in both extracts. Polyphenols could exert anticancer effects through different mechanisms, including modification of cell signalling, inhibition of cell proliferation, induction of cell cycle arrest, and apoptosis (Spatafora and Tringali 2012;Abbas et al. 2017). Therefore, the current study suggests that polyphenols, detected in the extracts, may exhibit anticancer activity by inducing apoptosis and blocking the cell cycle at different stages. EAELN had the highest antiproliferative activity against PC3 (IC 50 133.2 ± 5.73 lg/mL) and MDA-MB-231 lines (IC 50 156.9 ± 2.83 lg/mL) which could be due to the difference in chemical contents and their concentration in the two extracts.
Apoptosis plays an essential role in cellular homeostasis; nevertheless, abnormal apoptosis is a pathological process (Xiang et al. 2016). Tumour formation is the result of the loss of balance between cell proliferation and apoptosis. Induction of apoptosis is known to be a promising strategy to treat cancer (Schulze-Bergkamen and Krammer 2004). Previous research has shown The percentage of apoptotic cells was calculated. Each bar represents the mean ± SD (n ¼ 3), Ã p < 0.05, ÃÃÃÃ p < 0.0001, compared with control. One-way ANOVA followed by Bonferroni's correction.
that the antiproliferative effect of naturally occurring products is associated with the induction of apoptosis in cancer cells (Chidambara Murthy et al. 2011;Park et al. 2011;Zhong et al. 2011). To determine whether the antiproliferative effect of EAELN and EAELT was due to apoptosis, treated PC3 cells were stained with Annexin V and PI and analysed by flow cytometry. Analysis of PC-3 cells treated with EAELN and EAELT suggested apoptotic activity, evidenced by the accumulation of early and late apoptotic cells.
The present study showed that EAELN and EAELT induce a significant antiproliferative effect associated with apoptosis. The apoptotic effect of EAELN on the PC3 line was higher than that of EAELT ( Figures 3A, B and 4A, B). The effect induced by EAELN can be attributed to its major bioactive compounds and other compounds present in the extract by a direct and also synergistic effect. These data strongly support previous research on Linum species as new sources for candidate anticancer drugs The cell cycle plays a primary role in controlling cancer cell proliferation and cell cycle dysregulation is a fundamental feature of cancers (Vermeulen et al. 2003;Otto and Sicinski 2017). Natural products that can disrupt the cell cycle are the most commonly used anticancer drugs (Paier et al. 2018).
Therefore, the ability of EAELN and EAELT to block the cell cycle of PC3 cells was examined. Our results showed that EAELN induced a significant cell cycle arrest in the G2/M phase ( Figure 4A, B), while an arrest in the G0/G1 and G2/M phases was observed in PC3 cells after exposure to various concentrations of EAELT, for 24 h ( Figure 5A, B). The two extracts exhibited a different cell cycle arrest pattern.
The different observed patterns of cell cycle arrest as a pharmacological endpoint indicate the involvement of several mechanisms of action, suggesting the involvement of compounds in each extract in mediating this activity. Our results support the idea that the extracts inhibit cancer cell proliferation by inhibiting cell cycle progression, raising the possibility that the extract may be a potential therapeutic agent.
The anticancer effect of EAELN was higher than that of EAELT, this could be explained by its richness in chicoric acid (8.19%), vicenin-2 isomer 1 (8.08%), vitexin (7.53%), and podophyllotoxin-b-D-glucoside (4.41%) compared to EAELT, which presented lower levels of these compounds known for their anticancer activities. Indeed, it has been reported that chicoric acid has a strong growth inhibitory effect against HCT-116 colon cancer cells and effectively induces apoptosis, characterised by DNA fragmentation, caspase-9 activation, PARP cleavage, and b-catenin downregulation (Tsai et al. 2012). Its anticancer activity on the MCF-7 cell line has also been noted (Huntimer et al. 2006). Vicenin-2 has also been reported to have numerous pharmacological properties, including antioxidant, anti-inflammatory and anticancer effects (Ku and Bae 2016;Yang et al. 2018). Studies by Nagaprashantha et al. (2011) have also shown that vicenin-2 induces antiangiogenic, pro-apoptotic and antiproliferative inhibition of prostate cancer cells. Vicenin-2 also inhibits HT-29 colon cancer cell proliferation by inhibiting the WNT/b-catenin signalling pathway, inducing apoptosis, and leading to the arrest of HT-29 cells in the G2/M phase. Furthermore, treatment with vicenin-2 increases the expression of apoptosis-associated proteins Bax, cytochrome c, and caspase-3, and decreases that of Bcl-2 (Yang et al. 2018). Vitexin is a naturally occurring flavonoid compound that exhibits antioxidant , anticancer and neuroprotective properties (Zhu et al. 2016). Recently, vitexin has attracted the attention of many researchers for its potential antitumor properties. This compound has shown an antitumor effect against various cancers, including breast, prostate, and ovarian cancers, by inhibiting proliferation and promoting apoptosis of cancer cells (Zhou et al. 2009;He et al. 2016;Ganesan and Xu 2017). Zhou et al. (2009) reported that vitexin inhibited the proliferation of prostate, breast and ovary cancer cells and induced apoptosis by activating caspases and decreasing the Bcl-2/Bax ratio. Vitexin has been shown to induce G2/M phase cell cycle arrest and apoptosis by affecting the Akt/ mTOR signalling pathway in human glioblastoma cells and nonsmall cell lung carcinoma Liu et al. 2019). Moreover, podophyllotoxin glycosides and their derivatives are considered to lead compounds for anticancer drug development. The podophyllotoxin-b-D-glucoside compound exhibits more potent cytotoxic activity than the control drug (etoposide) in various cancer cell lines (PC-3, HeLa, HCT-116, HEK-293, and MCF-7) (Zilla et al. 2014). It has also been noted for its cytotoxic activity in other human cancer cell lines, HL-60, SMMC-7721, A-549, and SW480 (Zi et al. 2019). Podophyllotoxin and its derivatives exhibit anticancer activity, mainly due to its ability to    inhibit tubulin polymerisation into microtubules (Kamal et al. 2014). It is obvious that the observed anticancer activity of the extract could be due to one or a mixture of the compounds highlighted above. Moreover, in silico studies regarding the interaction of the identified compounds with potential biological targets involved in cell proliferation could provide valuable information.

Conclusions
Our results demonstrated for the first time that ethyl acetate extracts of two Linum species, L. numidicum (EAELN) and L. trigynum (EAELT), inhibited the proliferation of PC3 and MDA-MB-231 cells in a dose-dependent manner. EAELN had the highest antiproliferative activity against both lines tested. EAELN and EAELT induced apoptosis of PC-3 cells; the apoptotic effect of EAELN was higher than that of EAELT extract. EAELN induced a significant cell cycle arrest in the G2/M phase, while an arrest in the G0/G1 and G2/M phases was observed after treatment with EAELT. In this study, we showed, for the first time, that EAELN and EAELT exert anticancer activity by inducing apoptosis and blocking the cell cycle. This could be due to the presence of phenolic compounds such as flavonoids, lignans and phenolic acids which were detected by LC-HRMS/MS. The anticancer effect of EAELN was higher than that of EAELT. The effect induced by EAELN could be attributed to its major bioactive compounds such as chicoric acid, vicenin-2, vitexin, and podophyllotoxin-b-D-glucoside and other compounds present in the extract by a direct and/or synergistic effect. EAELN can be consered as a source of phytochemicals to treat cancer. Owing that most of the detected compounds can be obtained from commercial sources, it is intended to test those of them that are known for their anticancer activity.