|Year : 2018 | Volume
| Issue : 2 | Page : 328-334
The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines
Sibel Azizenur Ozturk1, Ebru Alp2, Atiye Seda Yar Saglam1, Ece Konac1, Emine Sevda Menevse1
1 Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Besevler, Ankara 06500, Turkey
2 Department of Medical Biology, Faculty of Medicine, Giresun University, Giresun 28200, Turkey
|Date of Web Publication||8-Mar-2018|
Dr. Sibel Azizenur Ozturk
Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Besevler, Ankara 06500
Source of Support: None, Conflict of Interest: None
Context: Thyroid cancers (TCs) are the most common endocrine malignancies. There were two problems with the current cancer chemotherapy: the ineffectiveness of treatment due to resistance to cancer cell, and the toxic effect on normal cells.
Aims: This study was aimed to determine the effects of thymoquinone (TQ) and genistein (Gen) phytotherapeutics on telomerase activity, angiogenesis, and apoptosis in follicular and anaplastic thyroid cancer cells (TCCs).
Materials and Methods: Cell viability, caspase-3 (CASP-3) activity, and messenger RNA (mRNA) expression levels of human telomerase reverse transcriptase (hTERT), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-kB), cyclin-dependent kinase inhibitor 1 (p21), and vascular endothelial growth factor-A (VEGF-A) genes were analyzed.
Results: It was found that TQ and Gen treatment on TCCs caused a statistically significant decrease of cell viability, and mRNA expression levels of hTERT, VEGF-A, and NF-kB genes, but a statistically significant increase of PTEN and p21 mRNA expression levels. In addition, TQ and Gen treatment also caused a statistically significant increase active CASP-3 protein level in TCCs. Moreover, our results demonstrated that, when compared with follicular TCCs, anaplastic TCCs were more sensitive to the treatment of TQ and Gen.
Conclusions: Based on these results, two agents can be good options as potential phytochemotherapeutics against TCCs.
Keywords: Anaplastic thyroid cancer, follicular thyroid cancer, genistein, thymoquinone, thyroid cancer
|How to cite this article:|
Ozturk SA, Alp E, Yar Saglam AS, Konac E, Menevse ES. The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines. J Can Res Ther 2018;14:328-34
|How to cite this URL:|
Ozturk SA, Alp E, Yar Saglam AS, Konac E, Menevse ES. The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines. J Can Res Ther [serial online] 2018 [cited 2019 Nov 12];14:328-34. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/328/202886
| > Introduction|| |
Thyroid cancers (TCs) are the most common endocrine malignancies, and there are three main types, namely, well-, poor- and undifferentiated TC. Follicular TC, which is a well-differentiated carcinoma, has a good prognosis because of the slow growth. On contrary with that, anaplastic TC, which is an undifferentiated carcinoma, has a relentless and aggressive clinic course with rapid progression, and its effective treatment has not been found yet., The fetal cell carcinogenesis hypothesis suggests that anaplastic TCs derived directly from the remnants of fetal thyroid cells rather than mature thyroid follicular cells. This hypothesis well-explains the clinical and biological features of thyroid carcinomas based on their gene expression profiles., The gene expression profiles of anaplastic TC cells (TCCs) demonstrated that these cancer cells express oncofetal fibronectin, a fetal protein, ALDH, CD133, CD44, CD24, and Oct4 transcription factor seen in the early thyroid development.,, According to these results, it is proposed that the cancer stem cell (CSC) feature of TCCs can support to clarify other limited understood questions in cancer biology, such as therapy-resistance, metastasis, and recurrence as well. An ideal treatment regime should kill thyroid CSCs selectively but also be nontoxic to normal cells.,
In recent years, phytochemical compounds have been investigated with increasing a great interest due to their minimal toxic effects on normal cells., Thymoquinone (TQ), the most abundant and active component of the extract of Nigella sativa, has been reported to have anti-inflammatory, antioxidant, anti-cancer, proapoptotic, anti-angiogenic, and anti-proliferative effects,, on various cancer cells, including osteosarcoma, myeloblastic leukemia, lymphoblastic leukemia,, ovarian, breast, lung, hepatocellular, laryngeal, colon, prostate carcinoma, and pancreatic carcinoma. However, TQ has not been studied in terms of its effects on TCs, and underlying molecular mechanisms of its anti-cancer properties have not been well-understood yet. Genistein (Gen), the most active and abundant flavonoids in soybeans, has been demonstrated to have multiple effects such as anti-proliferative, anti-angiogenic, antioxidant, proapoptotic, anti-cancer, anti-inflammatory, and anti-telomerase activity on different cancer cells, including breast, pancreatic, prostate and lung carcinoma, brain tumor, head-neck squamous cell cancers, and melanoma as well as thyroid carcinoma.
We aimed to investigate the effects of TQ and Gen treatment on telomerase activity, apoptosis, angiogenesis and survival in CAL-62, human anaplastic TCC line and CGTH-W1, and human follicular TCC line for determination of differences between treatment responses of anaplastic and follicular cancer cells.
| > Materials and Methods|| |
Human thyroid cell lines derived from anaplastic carcinoma (CAL-62, ACC 448), and follicular carcinoma (CGTH-W1, ACC 360) were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultured in appropriate media as indicated by the supplier: CAL-62 in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (FBS) and CGTH-W1 in Roswell Park Memorial Institute (RPMI)-1640 medium plus 10% FBS. All the media were supplemented with 2 mM glutamine and 100 U/mL penicillin-100 mg/mL streptomycin (HyClone, Thermo, USA) in a humidified incubator at 37°C in atmosphere of 5% CO2.
3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay
Cell growth and viability were assessed by 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) assay. The cells were seeded in a 96-well cell culture plates at a density of 1 × 104 cells/mL and treated with TQ (Sigma-Aldrich) at various concentrations on CAL-62 cells (0.5–20 μM) and CGTH-W1 cells (10–150 μM), respectively. Similarly, cell lines were treated with Gen (Sigma-Aldrich, St. Louis, MO, USA) at various concentrations on CAL-62 cells (10–200 μM) and on CGTH-W1 cells (10–300 μM), respectively. In addition, a group of cells in a fixed concentration of Gen (25 μM) were treated with varying concentrations of TQ. Then, 10 μl MTT (5 mg/mL) was added into each well for another 4 h incubation, after cells were incubated further at 37°C for 48 and 72 h, individually. Finally, MTT formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO) (Amresco, USA), and the absorbance was measured using Spectramax M3 Microplate Reader (Molecular Devices, USA) at 570 nm. All of the experiments were performed in triplicate for each concentration.
RNA isolation, complementary DNA synthesis, and quantitative real-time polymerase chain reaction analysis
Total RNA was isolated from each 6-well plate using the High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions, combined with DNase I treatment (Roche Diagnostics, Mannheim, Germany) to eliminate potential genomic DNA contamination. The total RNA concentration and purity (only samples with an A260/A280 ratio from 1.8-2) of each sample were measured the absorbance at 260/280 nm using the Nanodrop spectrophotometer (NanoDrop ND-1000, Montchanin, DE, USA). Total RNA (1 μg) from each sample was reverse-transcribed in 20 μL reaction mixture using random hexamers and Transcriptor High Fidelity complementary DNA (cDNA) Synthesis Kit (Roche Diagnostics GmbH, Germany) according to manufacturer's protocol. Human telomerase reverse transcriptase (hTERT), vascular endothelial growth factor-A (VEGF-A), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-κβ), and cyclin-dependent kinase inhibitor 1 (p21) messenger RNA (mRNA) expression levels were measured using a quantitative real-time polymerase chain reaction (PCR) method. Gene-specific intron spanning primers and probes were designed with the Universal Probe Library (UPL) Assay Design Center (https://www.universalprobelibrary.com). The gene-specific primer sequences and probe numbers for each gene are shown in [Table 1]. The 20 μL reaction mixture contained 1X Light Cycler 480 Probes Master mixture (Roche Diagnostics, Germany), 2.5 pmol of each primer, 1 pmol UPL probe (Roche, Germany), 4 mM MgCl2, and 1 μL cDNA, prepared in 96-well plates. All PCR reactions were performed in the LightCycler® 480 Instrument (Roche Diagnostics, Germany). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene to normalize expression levels. Each sample was tested in triplicate.
Measurement of active caspase-3 level
Caspase-3 (CASP-3) activity was measured using PathScan® Cleaved CASP-3 (Asp175) Sandwich Elisa Kit (Cell Signaling Technology Inc., Beverly, MA, USA) according to the manufacturer's instructions. The purpose was to detect proapoptotic effects of different concentrations of TQ and Gen alone and/or together at specified incubation periods, given that this family of protease plays key effector roles in apoptosis in mammalian cells. Briefly, adding the reagent to the wells resulted in cell lysis, followed by CASP cleavage of the substrate, and generation of a luminescent signal produced by luciferase, which is proportional to the amount of analyzed by reading the absorbance at 450 nm with Spectramax M3 Microplate Reader (Molecular Devices, USA). Each experiment was performed in quadruplicate.
Changes of hTERT, VEGF-A, PTEN, NF-κβ, and p21 mRNA expression levels were compared through Relative Expression Software Tool (REST) 2009 version 2.0.13 (Qiagen, Hilden, Germany). Other parameters were compared using one-way analysis of variance with Tukey's post hoc tests using SigmaStat version 3.5 software (Systat Software, Inc, Point Richmond, CA, USA). Data were expressed as the mean ± standard deviation from a representative experiment, and P values lower than 0.05 were considered as statistically significant.
| > Results|| |
Effects on the cell viability of genistein and thymoquinone treatment on CAL-62 and CGTH-W1 thyroid cancer cells
CAL-62 and CGTH-W1 cells were treated with various concentrations of Gen and TQ to determine the concentration-dependent response of the cells. As shown in [Figure 1]a, TQ caused a concentration-dependent decrease in cell viability as measured by MTT assay. The viability of CAL-62 cells was found to be 19 and 22%, whereas the viability of CGTH-W1 cells was found to be 25 and 13% after treatment with the highest concentration of TQ (20 μm and 150 μm) for 48 and 72 h, respectively [Figure 1]a. According to our results, half maximal inhibitory concentration (IC50) values of TQ were found to be approximately 10 μM for 48 and 72 h in CAL-62 cells and 100 μM for 48 h, 80 μM for 72 h in CGTH-W1 cells. Based on these results, viability of CAL-62 cells, when compared to CGTH-W1 cells with TQ, was found to affect at lower concentrations.
|Figure 1: Cell viability rates after (a) TQ, (b) Gen, and (c) TQ plus Gen incubation of the CAL-62 and CGTH-W1 cells. These cells were treated with various concentrations of TQ and Gen, and cell viability was determined using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. *P < 0.05, versus control cells for 48 h. #P < 0.05, versus control cells for 72 h. Gen=Genistein, TQ=Thymoquinone, DMSO (vehicle)=Dimethyl sulfoxide|
Click here to view
Similarly, Gen alone treatment decreased the viability of CAL-62 and CGTH-W1 cells for 48 and 72 h [Figure 1]b. At the highest concentration of Gen (200 μM), cell viability of CAL-62 cells was found to be 35% and 17% for 48 and 72 h, respectively [Figure 1]b. On the other hand, at the highest concentration of Gen (300 μM), the cell viability of CGTH-W1 cells was found to be 39%, and 36% for 48, and 72 h, respectively [Figure 1]b. The IC50 values of Gen were found to be approximately 150 μM at the end of 72 h in CAL-62 cells and 250 μm for 48 and 72 h in CGTH-W1 cells. Accordingly, cell viability rate of the CAL-62 and CGTH-W1 was decreased depending on the concentration of Gen. CAL-62 cell viability compared to CGTH-W1 cells were observed more affected by increasing concentrations of Gen.
As shown in [Figure 1]c, when TQ combined with Gen treatment diminished cell viability of CAL-62 and CGTH-W1 cells for 48 and 72 h. TQ and Gen treatment reduced the growth of CAL-62 and CGTH-W1 cells concentration-dependently. According to our results, IC50 value of CAL-62 cells was determined to be approximately 10 μM TQ + 25 μM Gen after incubation for 48 and 72 h. Moreover, IC50 value of CGTH-W1 cells was found to be 80 μM TQ + 25 μM Gen after incubation for 48 and 72 h.
Effect of genistein and thymoquinone treatment on human telomerase reverse transcriptase, vascular endothelial growth factor-A, PTEN, nuclear factor-kappa B, and p21 messenger RNA levels
Alterations of gene expression in CAL-62 and CGTH-W1 cells treated with TQ and Gen alone and TQ plus Gen at the mRNA level were verified for hTERT, VEGF-A, PTEN, NF-κβ and p21 genes. We determined statistically significant decrease for hTERT mRNA levels after 10 μM TQ treatment in CAL-62 cells and 80 μM TQ treatment in CGTH-W1 cells for 72 h (P< 0.05). In addition, hTERT mRNA expressions significantly decreased at 25 or 50 μM Gen in CAL-62 cells and 50 μM Gen after 72 h Gen treatment in CGTH-W1 cells (P< 0.05). mRNA levels of hTERT gene were significantly decreased approximately 6.5-fold for 10 μM TQ + 25 μM Gen treatment in CAL-62 cells and approximately 3-fold for 80 μM TQ + 25 μM Gen treatment in CGTH-W1 cells at 72 h (P< 0.05). Low concentrations of Gen plus TQ also caused a decrease in hTERT mRNA levels in CGTH-W1 cells, but this reduction was not statistically significant [Figure 2].
|Figure 2: Effects of indicated concentrations of TQ, Gen, and TQ plus Gen treatment on qRT-PCR mRNA levels of human telomerase reverse transcriptase gene in CAL-62 and CGTH-W1 cells. Expression levels of the target genes were normalized to the mRNA expression level of GAPDH. *P < 0.05 compared with control. mRNA=Messenger RNA, qRT-PCR=Quantitative real-time polymerase chain reaction, Gen=Genistein, TQ=Thymoquinone|
Click here to view
VEGF-A mRNA levels also significantly decreased after treatment with TQ and Gen alone for 72 h in both thyroid cells (P< 0.05). The mRNA levels of VEGF-A gene significantly decreased after exposure to 2.5 or 10 μM TQ alone and 25 or 50 μM Gen alone in CAL-62 cells. In 10 μM TQ + 25 μM Gen concentration, which is the highest concentration used, VEGF-A mRNA level was seen to decrease about 18-fold for 72 h in CAL-62 cells (P< 0.05). Furthermore, the mRNA levels of VEGF-A gene also significantly decreased after exposure to 80 μM TQ alone and 25 or 50 μM Gen alone in CGTH-W1 cells. We showed that statistically significant decrease (about 10-fold) for VEGF-A mRNA levels was found after 80 μM TQ + 25 μM Gen treatment for 72 h in CGTH-W1 cells (P< 0.05) [Figure 3].
|Figure 3: Effects of indicated concentrations of TQ, Gen, and TQ plus Gen treatment on qRT-PCR mRNA levels of vascular endothelial growth factor-A gene in and CAL-62 CGTH-W1 cells. Expression levels of the target genes were normalized to the mRNA expression level of glyceraldehyde 3-phosphate dehydrogenase. *P < 0.05 compared with control. mRNA=Messenger RNA, qRT-PCR=Quantitative real-time polymerase chain reaction, Gen=Genistein, TQ=Thymoquinone|
Click here to view
In CAL-62 and CGTH-W1 cells, TQ and Gen alone treatments significantly increased the expression levels of both p21 and PTEN genes when compared with the control cells (P< 0.05). We showed statistically significant increase for p21 and PTEN mRNA levels after combination treatment for 72 h in CAL-62 and CGTH-W1 cells (P< 0.05) [Figure 4]. 10 μM TQ + 25 μM Gen treatment caused 3 folds increase in p21 mRNA levels and 4.5 folds increase in PTEN mRNA levels in CAL-62 cells when compared to control cells for 72 h. Similarly, when using highest concentration combination treatment, p21 mRNA levels were observed to increase approximately 2.5 folds, and PTEN mRNA levels were increased about 3 folds in CGTH-W1 cells (P< 0.05) [Figure 4].
|Figure 4: Effects of indicated concentrations of TQ, Gen, and TQ plus Gen treatment on qRT-PCR mRNA levels of PTEN, nuclear factor-kappa B and p21 genes in CAL-62, and CGTH-W1 cells. Expression levels of the target genes were normalized to the mRNA expression level of GAPDH. *P < 0.05 compared with control. mRNA=Messenger RNA, qRT-PCR=Quantitative real-time polymerase chain reaction, Gen=Genistein, TQ=Thymoquinone|
Click here to view
In CAL-62, mRNA expression levels of NF-κβ gene were also significantly decreased 10 μM TQ and 50 μM Gen at 72 h (P< 0.05). Meanwhile, 2.5 μM TQ + 25 μM Gen treatment also caused significant decrease in NF-κβ mRNA levels in CAL-62 cell lines for 72 h (P< 0.05) [Figure 4]. After treatment with 10 μM TQ + 25 μM Gen concentration, which is the highest concentration, NF-κβ mRNA levels were observed to decrease approximately 4.5 folds in CAL-62 cells. Moreover, NF-κβ mRNA levels were also decreased after treatment with TQ and Gen alone for 72 h in CGTH-W1 cells when compared to control cells, but no statistically significant changes were observed (except 50 μM Gen) (P > 0.05) [Figure 4]. We showed that statistically significant decrease for NF-κβ mRNA levels occurred after 40 μM TQ + 25 μM Gen treatment for 72 h in CGTH-W1 cells. After treatment with the 80 μM TQ + 25 μM Gen concentration which is the highest concentration, NF-κβ mRNA levels were observed to decrease approximately 3.5 folds in CGTH-W1 cells (P< 0.05) [Figure 4].
Active caspase-3 levels
After incubation with indicated concentrations of TQ and Gen on CAL-62 and CGHT-W1 cells for 72 h, the active CASP-3 levels were determined using ELISA method. Cleaved CASP-3 protein levels elevated after TQ and Gen alone treatment in a concentration-dependent manner in both TCCs. Statistically, significant increase was obtained at 10 μM TQ and 50 μM Gen concentrations in CAL-62 cells and at 80 μM TQ and 50 μM Gen concentrations in CGTH-W1 cells for 72 h (P< 0.05). CASP-3 activity significantly increased at combination treatment with TQ and Gen in both cells at 72 h (P< 0.05). The amounts of active CASP-3 were observed to increase approximately 2.8 fold and 2.7 fold for 10 μM TQ + 25 μM Gen and 80 μM TQ + 25 μM Gen combinations, which are the highest concentration used, in the CAL-62 and CGTH-W1 cells, respectively (P< 0.05) [Figure 5].
|Figure 5: Effects of TQ, Gen, and TQ plus Gen treatment on active caspase-3 protein levels in CAL-62 and CGTH-W1 cells determined by ELISA assay. *P < 0.05 compared with control. ELISA=Enzyme-linked immunosorbent assay, Gen=Genistein, TQ=Thymoquinone|
Click here to view
| > Discussion|| |
In recent years, there is an increased interest to natural products due to potential chemotherapeutic effects on human tumors and lower cytotoxic effects on healthy cells. Therefore, we tested the effects of telomerase activity, apoptosis, angiogenesis, and survival of TQ and Gen treatment on TCCs. In our study, we used anaplastic and follicular TCCs. Thus, these two phytotherapeutic agents were comparatively investigated on two human TCC lines. In our study, it was observed that TQ and Gen decreased the viability of CAL-62 and CGTH-W1 cells in dose-dependent manner. Our results demonstrated that anaplastic TCCs were more sensitive to treatments of TQ and Gen than follicular TCCs.
In literature, it has been reported that TQ has telomerase inhibitory effect on glioblastoma cells, and Gen has suppressing effect on the gene expression levels of hTERT on prostate cancer cells and brain tumor cells. In addition, it has been reported that Gen allows reorganization of the hTERT promoter through methylation on breast cancer cells. In our study, we observed that TQ, Gen, and TQ + Gen combinations ensured a quite significant reduction in hTERT mRNA expression levels on CAL-62 cells at dose-dependent manner for 72 h, besides the reduction at high concentrations on CGHT-W1 cells. Maximum reduction of hTERT expression was seen at 10 μM TQ + 25 μM Gen combination on CAL-62 cells. These results indicated that TQ and Gen are significantly more effective in anaplastic CAL-62 cells.
In various studies, it has been reported that these agents have anti-angiogenic effect, because of that TQ, and Gen, inhibited VEGF levels in many cancer cells. In our study, we observed that VEGF mRNA levels significantly reduced at dose-dependent manner in the result of TQ and Gen treatments on both cell lines. Our results were also consistent with these reports. We also observed that individual and combination treatments of TQ and Gen were significantly much more effective (in especially the 10 μM TQ + 25 μM Gen concentrations with approximately 18-fold reduction) to the VEGF mRNA levels in CAL-62 cells. It was determined that combined concentration was the most effective in CGTH-W1 cells (80 μM TQ + 25 μM Gen combination with the approximately 10-fold reduction).
In some studies, it has been shown that PTEN expression levels were increased by Gen and TQ. In one study, it has been demonstrated that silenced PTEN resulted in suppression of TQ-induced apoptosis on breast carcinoma cells. In our study, we found that PTEN mRNA levels significantly increased in both TCCs at high concentrations of TQ. In particular, we showed the significant increase in PTEN mRNA levels after combined treatments of TQ and Gen on TCCs.
In various studies, it has been reported that Gen and TQ caused cell cycle arrest through p21 in cancer cells.,,, In our study, it was found that p21 mRNA levels caused a significant increase of high concentrations of TQ and Gen at dose-dependent manner in both cell lines. Moreover, the most effective results on TCCs was observed in combined concentrations of two agents, and the effects of drug groups gave similar results.
In some studies, it has been reached to the opinion that Gen is a powerful inhibitor of the NF-κβ signaling pathway, important for cell viability, and cancer development., It has been demonstrated that TQ inhibited NF-κβ effects., In our study, we observed that combined concentrations of TQ and Gen were more effective and reduced significantly NF-κβ mRNA levels at dose-dependent manner in both TCC lines.
In different in vivo and in vitro studies, it has been demonstrated that treatments of TQ,,,, and Gen, caused to cell death by CASP-3 activation as a result of induction of apoptosis. In our study, as compatible with the literature, we determined that the levels of activated CASP-3 increased significantly after treatments of combined doses and individual high doses of TQ and Gen for 72 h on both cell lines and that combined concentrations of these agents have highest effects. Moreover, TQ and Gen treatments caused CASP-3 activation at almost equal proportions in anaplastic and follicular TCCs. These results have indicated that anaplastic thyroid cells did not develop any resistance to TQ and Gen in terms of apoptosis.
| > Conclusion|| |
Consistent with the previous studies, our results showed that TCCs are very sensitive to apoptosis induced by TQ and Gen. In addition, our study indicated the significant reduction in hTERT mRNA expression levels after the treatment of combined doses of TQ and Gen on TCCs. Meanwhile, we observed that anaplastic TCCs were hypersensitive to TQ, Gen, and combined concentrations. Besides, hypersensitivity of anaplastic cells against the TQ and Gen was found also for VEGF mRNA levels. It is necessary to understand the complexities of anaplastic cells for development of more targeted and personalized treatment approaches in TCCs. Our preliminary results suggest that combined treatments of TQ and Gen are more effective than alone treatments of their in terms of progression to apoptosis of both TCCs. Therefore, these two natural plant compounds might be the potential candidates as chemotherapeutic agents for the treatment of these two TCCs. However, further studies using both in vitro and in vivo models are needed to confirm these findings. Meanwhile, differences between the two cells should be investigated (especially whether they are CSC features or not). In addition, it is required to be clarified with further researches of regulation mechanisms about hTERT and VEGF expression of TQ and Gen on TCCs.
Financial support and sponsorship
This study was supported by the Gazi University Scientific Research Projects Coordination Unit (Project No: 01/2010-68).
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Kondo T, Ezzat S, Asa SL. Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nature 2006;6:292-306.
Takano T. Fetal cell carcinogenesis of the thyroid: Theory and practice. Semin Cancer Biol 2007;17:233-40.
Takano T. Fetal cell carcinogenesis of the thyroid: A modified theory based on recent evidence. Endocr J 2014;61:311-20.
Klonisch T, Hoang-Vu C, Hombach-Klonisch S. Thyroid stem cells and cancer. Thyroid 2009;19:1303-15.
Nagayama Y, Shimamura M, Mitsutake N. Cancer stem cells in the thyroid. Front Endocrinol (Lausanne) 2016;7:20.
Gali-Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: A promising anti-cancer drug from natural sources. Int J Biochem Cell Biol 2006;38:1249-53.
Gurung RL, Lim SN, Khaw AK, Soon JF, Shenoy K, Mohamed Ali S, et al.
Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS One 2010;5:e12124.
Sethi G, Ahn KS, Aggarwal BB. Targeting nuclear factor-kappa B activation pathway by thymoquinone: Role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol Cancer Res 2008;6:1059-70.
Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, et al.
Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Ther 2008;7:1789-96.
Alhosin M, Abusnina A, Achour M, Sharif T, Muller C, Peluso J, et al.
Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1. Biochem Pharmacol 2010;79:1251-60.
Hussani IM, Amos S, Sirupson K, Redpath CI, Lyas C, Dipierro C. Nigella sativa
and thymoquinone induce caspase-9/3 activation and glioblastoma cell death. Neuro Oncol 2011;13:107-20.
Arafa el-SA, Zhu Q, Shah ZI, Wani G, Barakat BM, Racoma I, et al.
Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Mutat Res 2011;706:28-35.
Woo CC, Kumar AP, Sethi G, Tan KH. Thymoquinone: Potential cure for inflammatory disorders and cancer. Biochem Pharmacol 2012;83:443-51.
Hassan SA, Ahmed WA, Galeb FM, El-Taweel MA, Abu-Bedir FA.In vitro
challenge using thymoquinone on hepatocellular carcinoma [HepG2] cell line. Iran J Pharm Res 2008;7:283-90.
Rooney S, Ryan MF. Modes of action of alpha-hederin and thymoquinone, active constituents of Nigella sativa
, against HEp-2 cancer cells. Anticancer Res 2005;25:4255-9.
Asfour W, Almadi S, Haffar L. Thymoquinone suppresses cellular proliferation, inhibits VEGF production and obstructs tumor progression and invasion in the rat model of DMH-induced colon carcinogenesis. Pharmacol Pharm 2013;4:7-14.
Banerjee S, Kaseb AO, Wang Z, Kong D, Mohammad M, Padhye S, et al.
Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Res 2009;69:5575-83.
Dixon RA, Ferreira D. Genistein. Phytochemistry 2002;60:205-11.
Bektic J, Guggenberger R, Eder IE, Pelzer AE, Berger AP, Bartsch G, et al.
Molecular effects of the isoflavonoid genistein in prostate cancer. Clin Prostate Cancer 2005;4:124-9.
Dave B, Eason RR, Till SR, Geng Y, Velarde MC, Badger TM, et al.
The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN. Carcinogenesis 2005;26:1793-803.
Li Y, Ahmed F, Ali S, Philip PA, Kucuk O, Sarkar FH. Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells. Cancer Res 2005;65:6934-42.
Khaw AK, Yong JW, Kalthur G, Hande MP. Genistein induces growth arrest and suppresses telomerase activity in brain tumor cells. Genes Chromosomes Cancer 2012;51:961-74.
Alhasan SA, Ensley JF, Sarkar FH. Genistein induced molecular changes in a squamous cell carcinoma of the head and neck cell line. Int J Oncol 2000;16:333-8.
Casagrande F, Darbon JM. p21CIP1 is dispensable for the G2 arrest caused by genistein in human melanoma cells. Exp Cell Res 2000;258:101-8.
Yin F, Giuliano AE, Van Herle AJ. Growth inhibitory effects of flavonoids in human thyroid cancer cell lines. Thyroid 1999;9:369-76.
Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002;30:e36.
Ouchi H, Ishiguro H, Ikeda N, Hori M, Kubota Y, Uemura H. Genistein induces cell growth inhibition in prostate cancer through the suppression of telomerase activity. Int J Urol 2005;12:73-80.
Li Y, Liu L, Andrews LG, Tollefsbol TO. Genistein depletes telomerase activity through cross-talk between genetic and epigenetic mechanisms. Int J Cancer 2009;125:286-96.
Guo Y, Wang S, Hoot DR, Clinton SK. Suppression of VEGF-mediated autocrine and paracrine interactions between prostate cancer cells and vascular endothelial cells by soy isoflavones. J Nutr Biochem 2007;18:408-17.
Yu X, Zhu J, Mi M, Chen W, Pan Q, Wei M. Anti-angiogenic genistein inhibits VEGF-induced endothelial cell activation by decreasing PTK activity and MAPK activation. Med Oncol 2012;29:349-57.
Li Z, Li J, Mo B, Hu C, Liu H, Qi H, et al.
Genistein induces cell apoptosis in MDA-MB-231 breast cancer cells via the mitogen-activated protein kinase pathway. Toxicol In Vitro
Kumi-Diaka J, Sanderson NA, Hall A. The mediating role of caspase-3 protease in the intracellular mechanism of genistein-induced apoptosis in human prostatic carcinoma cell lines, DU145 and LNCaP. Biol Cell 2000;92:595-604.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]