Thymoquinone and Costunolide Induce Extensive Apoptosis of Senescent Colon and Senescent Breast Cancer Cells

Several chemotherapeutic agents induce cancer cells’ senescence as monitored by enlargement of cell, cell cycle arrest, elevated activities of senescence-associated β-galactosidase (SA-β-gal), and upregulation of p53 and p21. Doxorubicin (Dox) is an anticancer chemotherapeutic drug, classi�ed as an anthracycline antibiotic, that induces senescence of numerous cancer cell types. The arrest of cancer cell growth is a bright face of senescence, while these senescent cancer cells relapse again if they are not eliminated. On this principle, we investigated the apoptotic effect of thymoquinone (TQ), the active ingredient of Nigella sativa seeds and costunolide (COS), the active ingredient of Costus speciosus, on senescent colon (Sen-HCT116) and senescent breast (Sen-MCF7) cancer cell lines in reference to their corresponding proliferative cells to rapidly eliminate the senescent cancer cells. The senescence markers of Sen-HCT116 and Sen-MCF7 were determined by signi�cant decrease in bromodeoxyuridine (BrdU) incorporation and signi�cant increases in SA-β-gal, p53, and p21 levels. After proliferative, Sen-HCT116, and Sen-MCF7 cells were subjected to either TQ (50 µM) or COS (30 µM), the Bcl2-associated X protein (Bax), B-cell lymphoma 2 (Bcl2), and their ratio were established. Results revealed that TQ signi�cantly increased the Bax/Bcl2 ratio in HCT116+Dox5+TQ, MCF7+TQ, and MCF7+Dox5+TQ compared with their corresponding controls. COS signi�cantly increased the Bax/Bcl2 ratio in HCT116+Dox5+TQ and MCF7+Dox5+TQ compared with their corresponding controls. The data revealed more sensitivity of senescent cells to TQ or COS than their corresponding proliferative cells. Thus, we may be able to use TQ or COS to rapidly


Introduction
The principal bioactive ingredient of Nigella sativa seeds is thymoquinone (TQ), which has a number of biological activities such as antioxidant, anticancer, and antitoxicant [1][2][3][4][5][6].Gali-Muhtasib et al [7] stated that TQ prompted apoptosis of a human colon cancer (HCT116) cell line via a p53 dependence.Also, Dastjerdi et al [8] revealed that TQ induced apoptosis in a human breast cancer cell line (MCF7) through upregulation of p53 in a time-dependent manner.Moreover, TQ inhibited B-cell lymphoma 2 (Bcl2), which is anti-apoptotic in both in vitro and in vivo models [9].Costunolide (COS) is a natural sesquiterpene lactone isolated from Costus speciosus roots that has anti-in ammatory, antioxidant, and anticancer effects [10].COS targets a large number of important macromolecules, such as mitogen-activated protein kinases, protein kinase B, nuclear factor-κ B, transcription signal transducer and activator and activator protein-1 [11].
Also, COS at doses of 40 and 80 µM induced apoptosis of human lung squamous carcinoma cell line (SK-MES-1) through increases of p53 and Bax expressions and decreases of Bcl2 expression [12].Similarly, Roy and Manikkam [13] observed signi cant upregulation of caspase 3 and caspase 9, which enhanced apoptosis of breast cancer cell lines (MCF7 and MDA-MB-

231).
Cellular senescence is an irreversible arrest of growth caused by various harmful stimuli [14].The senescent cells have a larger and attened cell form and elevation of senescence-associated β-galactosidase (SA-β-gal) activity [15,16].Also, in senescence, p53 protein phosphorylation with p21 upregulation was recognized as leading to arrest of cell cycle [17].Many chemotherapy medications at low doses change cancer cells' states and induce senescence features of treated cancer cells [18].Cisplatin and doxorubicin (Dox) are the rst chemotherapeutics reported to induce senescence in tumor cells [19,20].Dox is a frequently used chemotherapy in the treatment of numerous cancers and it induces cell growth arrest with senescence markers [21,22].The senescent cells lose proliferation, migration, and invasion, and secrete a set of senescence-associated secretory phenotype (SASP), which in uences normal cells and non-senescent cancer cells that induce tumor growth and recurring cancer cells [23].
Senescent cells have tumor-promoting effects on the surrounding microenvironment, as stated by Bavik et al [24] who found that coculture of senescent broblasts induced generation of preneoplasia of prostate epithelial cells.Also, Demaria et al [21] observed a relapse of tumor growth despite Dox-induced senescence and cell cycle arrest of tumors in a mice xenograft model.
For this reason, in the current study we determined the ability of TQ and COS to induce apoptosis of Sen-HCT116 and Sen-MCF7 cell lines compared with their proliferative cells.

Cell Lines
Human colon HCT116 and breast MCF7 cancer cell lines were purchased from ATCC (Manassas, VA, USA).Each cell line was grown in a recommended medium, McCoy's 5A medium for HCT116 and low glucose Dulbecco's Modi ed Eagle Medium for MCF7, supplemented with 10% fetal bovine serum and penicillin/streptomycin solution.

Bromodeoxyuridine Incorporation
HCT116 or MCF7 cells were cultured in 12-well plates (3 × 10 4 per well) and incubated for 24 h at 37°C in a 5% CO 2 incubator.
After 24 h, cells were treated with bromodeoxyuridine (BrdU, Sigma-Aldrich, MO, USA).The extent of BrdU incorporation in cells was examined using a uorescence microscope (Nikon, Melville, NY, U.S.A) after mounting with DAPI [2].BrdU positive cells were counted with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Senescence-Associated β-Galactosidase Assay
The amounts of SA-β-gal in Sen-HCT116 and Sen-MCF7 cells for 5 days were determined according to the method of Dimri et al [25].Cells were examined using the uorescence microscope after mounting with DAPI [2].SA-β-gal positive cells were counted with ImageJ software.The IC 50 of TQ and COS were determined by seeding of HCT116 or MCF7 cells in 12-well plates (3 × 10 4 per well) and incubated for 24 h at 37°C in a 5% CO 2 incubator.At day 5 of 100 nM Dox treatment, cells were treated with TQ (0, 5, 10, 25, 50, 60, 75, or 100 µM dissolved in DMSO) or COS (0, 10, 25, 50, 75, 100, or 200 µM dissolved in DMSO), incubated for 24 h, and then treated with MTT reagent (1.25 mg/ml) and incubated for 2 h.The resulting formazan crystals were dissolved in 1 ml DMSO and the optical density was determined using a microplate reader at 570 nm [2].

Flow Cytometric Assessment
HCT116 or MCF7 cell lines were cultured in T25 asks (30 × 10 4 cells per ask) for 24 h and then treated with 10 ml of TQ (50 µM) or COS (30 µM) in each ask for proliferative cells.For Sen-cells, the same counts of cells were cultured and treated with 100 nM of Dox (10 ml) for 5 days after which 10 ml of TQ (50 µM) or COS (30 µM) were added.Treated cells with their corresponding control asks were trypsinized, washed with PBS and suspended in 70% ethanol until ow cytometry assessment of senescence (p53 and p21), apoptotic (Bax), and anti-apoptotic (Bcl2) protein markers using a BD Accuri C6 Plus ow cytometer (Becton Dickinson, San Jose, CA, USA).Cells were treated with FITC-conjugated and mouse-raised anti-p53 (Cat # 554298), anti-p21 (Cat # 612236), and anti-Bcl2 (Cat # 340650) produced by Becton Dickinson and anti-Bax (Cat # sc-7480) produced by Santa Cruz Biotechnology (Dallas, TX, USA) .Data were analyzed using Accuri C6 software.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
HCT116 or MCF7 cell lines either proliferative or senescent were cultured in T25 asks and treated the same way as in the ow cytometry assessment.Total RNA in pelleted cells was extracted using a Total RNA Extraction Kit (iNtRON Biotechnology, Inc., Gyeonggi-do, South Korea).cDNA reverse transcription of the extracted 1 μg of RNA was performed with a TIANScript RT Kit (Tiangen, Shanghai, China).RT-PCR was performed with iCycler-iQ Optical System Software version 3.0 A (Bio-Rad, CA, USA).The following primers were used for the quantitative RT-PCR analysis: for caspase 3 (forward): CATGGAAGCGAATCAATGGACT and reverse: CTGTACCAGACCGAGATGTCA, and the housekeeping GAPDH forward primer sequence is GAAGGTGAAGGTCGGAGTCA and the reverse sequence is TTGAGGTCAATGAAGGGGTC.
PCR ampli cation was done with RealMOD™ Green FAST qPCR Master Mix (S) (Tiangen) using the following conditions: 1 cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, 1 cycle at 95°C for 5 s.The relative mRNA expression levels of caspase 3 gene were calculated using the comparative (Ct) method [26].

Statistical Analysis
Statistical analyses were done with GraphPad Prism 5 (San Diego, CA, USA) using One-way ANOVA (Tukey's Multiple Comparison Test).The IC 50 values were determined with nonlinear regression using log (inhibitor) vs. response-variable slope equation.Data are presented as a mean ± SD.A value of P < 0.05 was considered statistically signi cant.All data show the mean results from at least three independent experiments.
The percentages of SA-β-gal positive cells indicates the extent of accumulation SA-β-gal in senescent cells that leads to enlargement of the senescent cells, and the percentages of BrdU positive cells at Dox 5 of the senescent cells is low due to the low cell division extent.
In the same manner, 30 µM of COS signi cantly increased (P 0.01) the relative expression fold change levels of caspase 3 mRNA in Sen-HCT116 in comparison with control (untreated proliferative HCT116), Dox5, and HCT116+TQ (Figure 4C).

Bax and Bcl2 Proteins
Bax protein levels of HCT116 either proliferative or senescent are presented in Figure 5A, 5B.Bax levels were signi cantly decreased (P 0.05) in Dox5, while signi cantly increased (P 0.01) in HCT116+Dox5+TQ compared with control.Also, its expressions were signi cantly increased in HCT116+Dox5+TQ compared with Dox5 and HCT116+TQ.
When the data of each cell line was calculated to their corresponding control of either proliferative or senescent cells (Table 1), TQ (50 µM) signi cantly increased (P < 0.001) Bax and signi cantly decreased (P < 0.001) Bcl2 in Sen-HCT116 and consequently increased Bax/Bcl2 ratio in Sen-HCT116 (1.72).TQ by the same concentration signi cantly increased (P < 0.001) Bax and signi cantly decreased (P < 0.001) Bcl2 in MCF7+TQ compared with control, while in Sen-MCF7, TQ signi cantly decreases (P < 0.001) Bcl2 compared with Dox5.Therefore, the Bax/Bcl2 ratio in proliferative and Sen-MCF7 were increased 1.69 and 1.33, respectively, compared with their corresponding control.From these results we can conclude that TQ (50 µM) induced apoptosis in both proliferative and Sen-MCF7 especially in proliferative MCF7 that exhibited greater Bax/Bcl2 ratio than Sen-MCF7.
Bax is a pro-apoptotic protein that is responsible for causing cell death and Bcl2 is an anti-apoptotic protein that regulates the mitochondrial membrane function and protects cells from apoptosis [56].Both Bax and Bcl2 are regulated by the tumorsuppressor protein p53 [57].Therefore, an increased Bax/Bcl2 ratio is a factor in predisposing apoptosis [58,59].In the current study, Bax levels were signi cantly increased in HCT116 + Dox5 + TQ compared with their control senescent (Dox5), and Bcl2 levels were signi cantly decreased.These data indicate the sensitivity of Sen-HCT116 to TQ-induced apoptosis compared with proliferative HCT116.Also, MCF7 + Dox5 + TQ exhibited signi cant decreases compared with Dox5 and MCF7 + TQ that favored apoptosis of both Sen-HCT116 and Sen-MCF7.Sensitivity of Sen-HCT116 and Sen-MCF7 to TQ compared with their corresponding proliferative cells has been stated in our previous study [2].Also, COS induced increases in Bax/Bcl2 ratio in HCT116 + Dox5 + TQ and MCF7 + Dox5 + TQ compared with their corresponding Dox5 cells.These results indicate the sensitivity of both Sen-HCT116 and Sen-MCF7 to COS compared with their corresponding proliferative cells.

Conclusion
Dox is a commonly used chemotherapeutic agent for cancer treatment and to induce cellular senescence of both HCT116 and MCF7 cell lines.To prevent the relapse of these senescent cells to proliferative cells, we intervened with TQ and COS treatments to rapidly eliminate the senescent cells in comparison with their proliferative cell type.Both TQ and COS induced more apoptosis of Sen-HCT116 and Sen-MCF7 in relation to their proliferative cells by different extents and cell speci city.Based on the current data, we conclude that TQ and COS induced senolysis of Sen-HCT116, and TQ induced apoptosis of both proliferative and Sen-MCF7.Conversely, COS induced slight apoptosis of Sen-MCF7 more than proliferative.We suggest that future in vivo and clinical trials include either TQ or COS in the Dox treatment schedule of cancer.

Declarations
Con of interest: The authors declare no con ict of interest.