Thymoquinone Inhibits Proliferation and Induces Apoptosis in Immortalized Keratinocytes via Upregulation of p53 Expression

Background:Nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science. Earlier studies have been reported that thymoquinone (TQ), an active compound of its seed, contains anticancer properties. Previous studies have shown that TQ induces apoptosis in breast cancer cells but it is unclear the role of P53 in the apoptotic pathway. Hereby, this study reports the potency of TQ on expression of tumor suppressor gene P53 and apoptosis induction in breast cancer cell line Michigan Cancer Foundation-7 (MCF-7).Methods:MCF-7 cell line was cultured and treated with TQ, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out for evaluating the half-maximal inhibitory concentration (IC50) values after 24 h of treatment. The percentage of apoptotic cells was measured by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate the messenger RNA expression of P53 in MCF-7 cell line at different times.Results:The IC50 value for the TQ in MCF-7 cells was 25 μM that determined using MTT assay. The flow cytometry and real-time PCR results showed that TQ could induce apoptosis in MCF-7 cells, and the P53 gene expression was dramatically up-regulated by ascending time, respectively. Hence, there was significant difference in 48 and 72 h.Conclusions:Our results demonstrated that TQ could induce apoptosis in MCF-7 cells through up-regulation of P53 expression in breast cancer cell line (MCF-7) by time-dependent manner.

Lung cancer has been associated with the highest cancer-associated mortality rate in the world. Chemotherapeutic management of cancer necessitates introducing new promising agents. Plants represent a rich source of new antineoplastic and chemotherapeutic agents. Thymoquinone (TQ), the main constituent of Nigella sativa (black seed or black cumin), has shown potent antioxidant and anti-inflammatory activities so far. The purpose of the current study was to evaluate the antineoplastic potential of TQ and their underlying mechanisms in A549 cells (human lung cancer cell line). The A549 cells were treated with the different concentrations of TQ for three following days. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Necrosis and apoptosis were assessed by fluorescence-activated cell sorter analysis through propidium iodide and annexin V staining and also by assessing caspase-3 and -9 activation. DNA fragmentation was monitored by gel electrophoresis. TQ decreased the viability and increased apoptotic cell death in A549 human lung tumor cells. TQ treatment significantly elevated the Bax/ Bcl-2 ratio in the lung cancer cells. TQ also upregulated p53 expression, another apoptotic modulator in A549 cancer cells. TQ also activated caspase-dependent apoptosis by the activation of caspases-3 and -9. Our results proposed that TQ may be a potential new therapeutic agent for the management of lung cancer. TQ promoted apoptosis in A546 lung cancer cells by the activation of p53 and caspase cascade dependent pathways.

Colorectal cancer is one of the worldwide common malignancies with a high mortality level. The major constituent of natural oil of black seed "Nigella sativa" is Thymoquinone (TQ), which has a magical anti-proliferative activity against colorectal cancer cells. This study evaluates the potential cytotoxic effect of TQ on human colorectal cancer cells (HCT-116). Results showed that TQ cytotoxicity was significantly increased (p< 0.001) in the cell culture containing TQ (HCT-116/TQ+) as compared to controls without TQ (HCT-116/TQ-), where maximum effect reached at 60 μM of TQ. Additionally, expression of three important tumor associated gene studied in parallel (p53, caspase 3 and Bcl-xl genes). Both p53 and caspase 3 genes show significant increase in cell cultures containing TQ (HCT-116/TQ+) than controls without TQ (HCT-116/TQ-). Conversely, Bcl-xL gene show significant decrease. All these observations concluded that thymoquinone (TQ) was cytotoxic towards HCT-116 cells in a dose-dependent manner and induced apoptosis via caspase 3 and p53-dependent pathway.

Osteosarcoma (OS) is known as the malignant tumors in the bone. Cyanidin 3-OGlucoside (C3G) has a potential to induce the apoptotic cell death in different cancer cells; however, the mechanisms of action for C3G have not been clarified yet. In this study, the apoptotic effects of C3G on three different osteosarcoma cell lines including Saso-2, MG-63, and G-292 (clone A141B1) were investigated. The 24-hr IC50 of C3G for Saso-2, G-292, and MG-63 cells was evaluated by the MTT assay. Apoptosis induction in these cell lines after treatment with the C3G was approved by the Annexin V/PI flow cytometry. Changes at the mRNA expression level of PPARγ, P21, Bax, and Bcl-xl genes were investigated by real-time Polymerase Chain Reaction (PCR) technique, and P21 expression was further confirmed by the western blotting. The MTT assay results demonstrated that the 24-hr IC50 of C3G was equal to 110μg/ml for Saso-2 and G-292 cells while it was about 140μg/ml for the MG-63 cells. The results of real-time PCR clearly showed that treatment of the cells with 24hrs IC50 of C3G caused the upregulation of PPARγ, P21, and Bax genes. Moreover, western blot analysis confirmed that P21 protein overexpressed endogenously after treatment of the cells with the C3G, and it was more upregulated in the MG-63 cells compared to the other cell lines. According to the findings of the study, the C3G is a novel anti-osteosarcoma agent with the ability to induce the apoptosis in different osteosarcoma cells through upregulation of the PPARγ and P21 genes.

Cellular senescence is a process of physiological growth arrest that can be induced by intrinsic or extrinsic stress signals. Some cancer therapies are associated with senescence of cancer cells with a typical cell cycle arrest. Doxorubicin (Dox) induces senescence by a p53-dependent pathway and telomere dysfunction of numerous cancers. However, cellular senescence induces suppression in proliferation activity, and these cells will remain metabolically active and play an important role in tumor relapse and development of drug resistance. In the current study, we investigated the apoptotic effect of curcumin (Cur), caffeine (Caff), and thymoquinone (TQ) on senescent colon cancer HCT116 and breast cancer MCF7 cell lines treated with Dox. Results showed typical senescence markers including decreased bromodeoxyuridine incorporation, increased accumulation of senescence-associated β-galactosidase (SA-β-gal), cell cycle arrest, and upregulation of p53, P-p53, and p21 proteins. Annexin-V analysis by flow cytometry revealed 2- to 6-fold increases in annexin-V–positive cells in Dox-treated MCF7 and HCT116 cells by Cur (15 µM), Caff (10 mM), and TQ (50 µM; P < .001). In comparison between proliferative and senescent of either HCT116 or MCF7 cells, Caff at 15 mM and TQ at 25 µM induced significant increases in apoptosis of Dox-treated cells compared with proliferative cells (P < .001). Data revealed that Cur, Caff, and TQ potentially induced apoptosis of both proliferative and senescent HCT116 and MCF7 cells. In vivo and clinical trials are of great importance to validate this result.

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.

Thymoquinone (TQ) is the active ingredient extracted from the essential oil of Nigella sativa. A number of studies implicated TQ as an antitumor agent. In this study, cytotoxic effects of the oil of N. sativa and TQ were evaluated on human cervical cancer cell line, HeLa cells. IC50 value was ~0.125 μl/ml for N. sativa oil preparations and 12.5 μM for TQ. TQ strongly inhibited wound healing at all concentrations ranging from 12.5 to 100 μM in a scratch wound healing assay. Additionally, induction of apoptosis by TQ was assessed by Giemsa staining and TQ was found to induce apoptosis in cancer cells especially at concentrations of 50 and 100 μM. TQ-mediated transcriptional regulation of 84 genes involved in apoptosis was studied using a PCR array. At low dose (12.5 μM), TQ was found to induce expression of four pro-apoptotic genes: BIK (~22.7-fold), FASL (~2.9-fold), BCL2L10 (~2.1-fold), and CASP1 (~2-fold). TQ was also found to reduce the expression of an anti-apoptotic gene implicated in NF-kappa-B signaling and cancer: RELA (~8-fold). At high dose (100 μM), TQ mediated the expression of 21 genes implicated directly in apoptosis (6 genes), TNF signaling (10 genes), and NF-kappa-B signaling (3 genes) such as BIK, BID, TNFRSF10A, TNFRSF10B, TNF, TRAF3, RELA, and RELB. In conclusion, this study implicates the role of TQ in the inhibition of cancer cell proliferation and migration. At the same time, our results strongly suggest that TQ intervenes with TNF and NF-kappa-B signaling during TQ-mediated induction of apoptosis in cancer cells.

Low Concentrations of Curcumin Induce Growth Arrest and Apoptosis in Skin Keratinocytes Only in Combination with UVA or Visible Light

Healthy mitochondria and mitochondrial quality control are essential for vital cell activities. Cell health is fundamentally maintained by the coordinated regulation of processes such as mitochondrial fusion, fission, and mitophagy. Their disruption plays a role in cancer pathogenesis, as well as in many diseases. This study investigated the effects of thymoquinone (TQ), a bioactive compound from Nigella sativa, on mitochondrial dynamics and quality control in Hepatocellular Carcinoma Cells (HepG2) and Human Dermal Fibroblasts (HDF). Results from molecular techniques such as the MTT assay, colony formation assay, wound healing assay, DAPI staining, and JC-1 staining, as well as Real-Time Polymerase Chain Reaction (RT-PCR) and Western blot analysis, were evaluated. TQ treatment caused dose-dependent decreases in cell viability and migration in both cell types, according to the MTT and wound healing assay results. While nuclear morphology assessments with DAPI staining served as a parameter for apoptotic changes, JC-1 analysis revealed a significant loss of mitochondrial membrane potential (ΔΨm) in HepG2 cells, while a relatively milder decrease was observed in HDF cells. At the molecular level, TQ exposure increased Cytochrome c (Cyt c) and Transcription Factor EB (TFEB) levels in both cell lines, but Dynamin-Related Protein 1 (DRP1) upregulation was more pronounced in HDF cells. Specifically, Western blot results showed an increase in PTEN-Induced Kinase 1 (PINK1) protein in HepG2 cells, but not in HDF cells. These findings suggest that TQ can trigger the mitochondrial stress response in HepG2 cells through DRP1-dependent fission, TFEB-associated lysosomal activation, and PINK1-associated mitophagy signaling. The stronger suppression of ΔΨm and PINK1 induction in HepG2 suggests an increased likelihood of activation of the intrinsic apoptotic pathway, while the partial preservation of mitochondrial integrity in HDF cells suggests a mild adaptation to stress. Further studies on mitophagy flux, Cyt c intracellular distribution, and TFEB nuclear translocation will be needed to define the mechanisms underlying these cell-type-specific responses.

Platycodin D-induced apoptosis through nuclear factor-κB activation in immortalized keratinocytes

Objective To investigate the role of activation of Akt/mTOR pathway in denfense against UVB-induced apoptosis in cultured human skin keratinocyte cell line HaCaT. Methods HaCaT cells were irradiated with UVB at different doses for various durations. Western blotting was performed to detect dynamic changes of Akt/mTOR pathway-related signaling molecule, such as phosphorylated-epidermal growth factor receptor (EGFR), -Akt, -4EBP1, etc; apoptosis was estimated by staining with DNA dye Hoechst 33342. To evaluate the role of signaling molecules in defense against UVB-induced apoptosis, HaCaT cells were pretreated before irradiation with EGFR inhibitor (PD153035), PI3K inhibitor (LY294002), mTOR inhibitor (rapamycin) followed by the detection of expressions of signaling molecule and apoptosis. Results UVB could activate Akt/mTOR pathway in a dose- (5 ～ 30 mJ/cm2) and time- (5 ～ 30 min) dependent manner. PD153035,LY 294002 and rapamycin could inhibit UVB-induced activation of the Akt/mTOR pathway. The apoptosis rate in HaCaT cells was upregulated by pretreatment with rapamycin and LY294002. Conclusion The activation of Akt/mTOR signaling pathway could inhibit the UVB-induced apoptosis in cultured HaCaT cells. Key words: Ultraviolet rays; Akt/mTOR; Signal transduction; Apoptosis

Cancer stem cells are contributors of chemotherapeutic resistance. We have previously shown that resveratrol (RSV) potentiates grape seed extract (GSE) induced apoptosis and suppression of proliferation in human colon cancer cell lines, but not in normal colonocytes, via p53 dependent mitochondrial apoptotic pathway. However, no information is available on whether grape compounds elevate apoptosis in the cancer stem cells. We hypothesized that RSV‐GSE combination will elevate apoptosis in human colon cancer stem cells (CD34+, CD44+, CD133+, ALDH1b1+), even the presence of IGF‐1 (a mitogen growth factor elevated during obesity), via elevation of p53 dependent pathway. RSV‐GSE combination elevated (p&lt;0.05) apoptosis (Caspase 3/7 Glo and TUNEL assay) in the colon cancer stem cell line similar to that of 5‐fluorouracil (5‐FU), a standard colon cancer chemotherapeutic drug. P53 inhibition by pithifrin‐α (150 μM), a transcriptional inhibitor, and p53 shRNA (2 × 105 lentiviral particles) suppressed (p&lt;0.05) RSV‐GSE and 5‐fluorouracil induced apoptosis. RSV‐GSE elevated (p&lt;0.05) apoptosis, even in the presence of IGF‐1, indicating its potential efficacy in obese condition. This study demonstrates RSV‐GSE induced apoptosis in colon cancer stem cells via p53 dependent pathway. Results suggest that consumption of RSV‐GSE could contribute towards colon cancer prevention.

Osteosarcoma (OS) is a primary bone sarcoma with a high recurrence rate and poorer prognosis. The application of natural agents in combinational therapies can increase the efficacy of treatment and decrease the side effects. Herein, we aimed to evaluate the effects of Thymoquinone (TQ) combined with Cisplatin on apoptosis and its underlying mechanisms in the Saos-2 cells. The effects of TQ and Cisplatin on Saos-2 cell viability were measured using an MTT assay. Western blotting was applied for the measurement of γH2AX protein expression. The expression levels of 8-Hydroxy-2'-deoxyguanosine (8-oxo-dG) were evaluated by enzyme-linked immunosorbent assay (ELISA). DCFH-DA fluorescence dye was used to detect reactive oxygen species (ROS) formation. For evaluation of apoptosis, flow cytometry was employed. TQ dramatically promotes the cytotoxic effects of Cisplatin. TQ considerably enhanced the expression levels of 8-oxo-dG and γ-H2AX in Saos-2 cells. After TQ treatment, ROS levels were increased; furthermore, TQ treatment resulted in the potentiation of Cisplatin-induced apoptosis in Saos-2 cells compared to either TQ or Cisplatin treated cells. In general, TQ plus Cisplatin resulted in potentiated cellular cytotoxicity by increasing ROS level and inducing oxidative DNA damage, leading to the potent induction of apoptosis in tumor cells.

Effects of a novel carbocyclic analog of pyrrolo[2,3-d]pyrimidine nucleoside on pleiotropic induction of cell death in prostate cancer cells with different androgen responsiveness.

Thymoquinone (TQ) is likely responsible for the chemotherapeutic effects of N. sativa extract; however, the cellular mechanisms remain ill-defined. TQ-induced cytotoxicity was investigated using canine osteosarcoma (COS31), its cisplatin-resistant variant (COS31/rCDDP), human breast adenocarcinoma (MCF7), human ovarian adenocarcinoma (BG-1) and Madin-Darby canine (MDCK) cell lines. TQ-induced cytotoxicity was determined using a proliferation assay (MTT assay) and apoptosis assays. Effects of TQ on the cell cycle were determined using flow cytometry. COS31/rCDDP resistant cells were the most sensitive cell line to TQ and MDCK cells were the least sensitive. TQ (25 micro M) induced apoptosis of COS31 cells 6 h after treatment and decreased the number of COS31 cells in S-phase and increased cells in G1-phase, indicating cell cycle arrest at G1. These results suggest that TQ kills cancer cells by a process that involves apoptosis and cell cycle arrest. Non-cancerous cells are relatively resistant to TQ.