OROncology Reports1021-335X1791-2431D.A. Spandidos10.3892/or.2019.6958or-41-03-2060ArticlesAnticancer effects of methotrexate in combination with α-tocopherol and α-tocopherol succinate on triple-negative breast cancerWeiChyou-Wei12*YuYung-Luen3456*ChenYu-Hsun1HungYu-Ting13YiangGiou-Teng78Department of Nutrition, Master Program of Biomedical Nutrition, Hungkuang University, Taichung 433, Taiwan, R.O.C.Department of Nursing, Hungkuang University, Taichung 433, Taiwan, R.O.C.Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan, R.O.C.Drug Development Center, China Medical University, Taichung 404, Taiwan, R.O.C.Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan, R.O.C.Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.Department of Emergency Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City 231, Taiwan, R.O.C.Department of Emergency Medicine, School of Medicine, Tzu Chi University, Hualien 970, Taiwan, R.O.C.Correspondence to: Dr Giou-Teng Yiang, Department of Emergency Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, 289 Jianguo Road, Xindian, New Taipei City 231, Taiwan, R.O.C., E-mail: gioutengyiang@gmail.com; jtyiang@tzuchi.com.tw
Triple-negative breast cancers (TNBCs) lack the estrogen receptor, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Therefore, hormone or targeted therapies are not effective in the treatment of TNBC and thus the development of novel therapeutic strategies is crucial. Methotrexate (MTX), a folate antagonist, has been used in the treatment of various types of cancer; however, the anticancer effects of MTX treatment on breast cancer have thus far been ineffective. Vitamin E variants and derivatives have been applied for cancer therapy. Previous studies have indicated that vitamin E variants and derivatives exert distinct anticancer effects on different types of cancer. However, whether MTX plus vitamin E variants or its derivatives can inhibit TNBC remains unclear. The aim of the present study was to examine the anticancer effects and mechanisms of action of MTX in combination with vitamin E variants (α-tocopherol) and derivatives (α-tocopherol succinate) on TNBC. In the present study, MTT assay and western blot analysis were used to determine the cell survival rates and protein levels. The results demonstrated that combination treatment with MTX and α-tocopherol suppressed TNBC cell proliferation. In addition, various concentrations of MTX exerted distinct cytotoxic effects on α-tocopherol succinate-treated cells. Furthermore, high-dose MTX enhanced α-tocopherol succinate-induced anticancer activity; however, low-dose MTX inhibited α-tocopherol succinate-induced anticancer activity. The present study also demonstrated that caspase-3 activation and poly(adenosine diphosphate-ribose) polymerase cleavage were observed in the α-tocopherol succinate/MTX-treated cells. In conclusion, the findings of the present study demonstrated that high-dose MTX enhanced anticancer activity in α-TOS-treated TNBC, while low-dose MTX reduced anticancer activity in α-TOS-treated TNBC.
Breast cancer is a common type of non-skin malignant tumor affecting women (1). A previous study revealed that ~1.67 million breast cancer cases are diagnosed each year worldwide (2). Breast cancers with or without estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) can be divided into hormone-dependent and -independent breast cancers (3,4). It is considered that ~15–20% of breast cancer cells lack ER, PR and EGFR2, and thus are classed as triple-negative breast cancers (TNBCs) (5,6). As TNBCs lack hormone receptors, hormone or targeted therapies are not effective in the treatment of TNBCs in clinical practice (7,8). Thus, the development of novel treatment strategies for TNBCs is of utmost importance.
Methotrexate (MTX) is known as a folate antagonist used widely in the treatment of rheumatoid arthritis and cancer (9,10). Low-dose MTX has been shown to exert anti-inflammatory effects when used in the treatment of rheumatoid arthritis; however, high-dose MTX has been shown to have anti-proliferative cytotoxic activities with a number of side-effects, such as renal damage (9,11,12). Previous studies have suggested that MTX can induce cell cytotoxicity which is related to increases in reactive oxygen species (ROS) production (13,14). Furthermore, a number of studies have demonstrated that MTX can inhibit cell proliferation in various types of cancers including lung cancer, lymphoma, leukemia and hepatoma (15–19). However, to date, to the best of our knowledge, no previous study has demonstrated that MTX alone can exert sufficient anticancer effects on breast cancer. In order to enhance the anticancer effects of MTX on breast cancer, combination treatment with MTX with other agents has been considered (4,20,21).
Vitamin E is known as a fat-soluble vitamin with anti-oxidative function (22). Vitamin E has 8 variants, including 4 tocopherols (α-, β-, γ- and δ-tocopherols) and 4 tocotrienols (α-, β-, γ- and δ-tocotrienols); it has also been demonstrated to lower cancer risk (23–25). α-tocopherol is a major variant found in vitamin E and has anti-oxidative functions (26,27). The majority of studies have suggested that γ- and δ-tocopherol and γ- and δ-tocotrienol can inhibit breast cancer growth, while α-tocopherol does not exert obvious anticancer activity in breast cancer (25,28). Studies have demonstrated that γ- and δ-tocopherol only inhibit ER-positive (ER+) breast cancer, while γ- and δ-tocotrienol can inhibit both ER+ breast cancer and TNBC (23,28). In addition, a previous study also revealed that high-dose (100 µM) α-tocopherol inhibited cell proliferation in ER+ breast cancer, including MCF-7 and T47D cells in a dose-dependent manner (29). Previous studies have also demonstrated that only α-tocopherol succinate (α-TOS) has potent antioxidant activities, while γ- and δ-tocotrienol do not (22–27). Clinical MTX treatment can induce oxidative stress resulting in side-effects (9,11,12); therefore, perhaps MTX-induced oxidative stress can be decreased by the use of α-TOS. Therefore, whether α-tocopherol has potent anticancer activities in breast cancer warrants further investigation.
α-TOS is an analogue of α-tocopherol with anticancer activity (30–32). Previous studies have revealed that α-TOS has anticancer activities in various hormone-dependent breast cancers, such as MCF-7, MDA-MB-435, 4T1 and MDA-MB-453 cells (33–36). Another previous study demonstrated that α-TOS induced the apoptosis of TNBC cells, such as MDA-MB-231 and SKBR-3 cells (37). However, that study only demonstrated that apoptotic characteristics and Fas signals were induced in α-TOS-treated cells. The other mechanisms associated with α-TOS treatment in TNBC warrant further investigation.
Based on the above-mentioned studies, MTX, vitamin E and its analogue have been previously administered in the treatment of breast cancers. The present study aimed to determine whether combination treatment with MTX and vitamin E or its analogue may have more potential for use in the treatment of TNBCs. The anticancer effects on TNBC were determined following treatment with MTX, α-tocopherol, α-TOS, MTX/α-tocopherol and MTX/α-TOS.
Materials and methodsMaterials, reagents and antibodies
Fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), non-essential amino acids, L-glutamine and penicillin/streptomycin were obtained from Gibco/Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The MTT assay kit was obtained from Bio Basic Canada Inc. (Markham, OT, Canada). Luminol, lucigenin, α-tocopherol, α-TOS and Hoechst 33342 were purchased from Sigma-Aldrich/Merck KGaA (Darmstadt, Germany). Anti-tubulin (1:1,000; cat. no. BS1699) primary rabbit polyclonal antibody was acquired from Bioworld Technology, Inc. (Louis Park, MN, USA). Anti-cleaved poly(adenosine diphosphate-ribose) polymerase (PARP; 1:2000; cat. no. 9544) and anti-caspase-3 (1:1000; cat. no. 9665) primary rabbit polyclonal antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. 7074) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Cells and cell culture
The TNBC cell lines, MDA-MB-231 and MDA-MB-468, were purchased from the Bioresource Collection and Research Center (Shin Chu, Taiwan). The cells were cultured with DMEM supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine and 100 IU/ml penicillin/streptomycin and maintained at 37°C with a humidified atmosphere containing 5% CO2.
Cell survival rate assay
The cell survival rate was determined using an MTT assay. The cells were cultured in 96-well dish (3×103 cells/well). Every 24 h, the control and experimental groups (MTX, vitamin E, MTX/vitamin E treatments) were treated with the MTT kit. The cells were incubated with MTT solution for 3 h at 37°C and the formazan product was produced. The formazan product was dissolved and the absorbance was determined at 570 nm (A570) using a Multiskan™ FC Microplate Photometer (Molecular Devices LLC, Sunnyvale, CA, USA). The survival rate (%) was calculated as [(A570 experimental group)/(A570 control group)] × 100%.
Measurements of intracellular H<sub>2</sub>O<sub>2</sub>
The production of cellular H2O2 was determined using the lucigenin-amplified chemiluminescence method (38,39). The control and experimental samples (200 µl) were treated with 0.2 mmol/ml of luminol solution (100 µl) to determine the H2O2 levels. All samples were analyzed and observed for 5 min using a chemiluminescence analyzing system (CLA-FSI; Tohoko Electronic Industrial Co., Ltd., Sendai, Japan).
SDS electrophoresis and western blot analysis
The control and experimental cells were treated with lysis buffer (radio-immunoprecipitation assay buffer; cat. no. 20–188; EMD Millipore, Billerica, MA, USA). Following centrifugation (16,000 × g; 4°C) for 30 min, proteins were obtained from the supernatant layer. The protein concentration was determined using a protein assay kit (cat. no. 23200; Thermo Fischer Scientific, Inc.). A total of 40 µg protein was loaded and separated on 13.3% SDS-PAGE under 80 volts. Separated proteins were transferred onto PVDF membranes (EMD Millipore). The membranes were firstly blocked with 5% non-fat milk at room temperature for 2 h. After washing with phosphate-buffered saline (PBS) for 15 min (3 times), the membranes were treated with primary antibodies for 4 h at room temperature, and the membranes were then washed with PBS for 15 min (3 times). The membranes were subsequently incubated with anti-rabbit HRP-conjugated secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. Finally, immunolabeled proteins were added and incubated with Western Lightning® Chemiluminescence Plus reagent (PerkinElmer, Inc., Waltham, MA, USA). The protein band was observed and analyzed with a Luminescence Image Analysis system (LAS-4000; FUJIFILM Electronic Materials Taiwan Co., Ltd., Taiwan) and ImageJ 1.51j8 by Wayne Rasband (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
All data were obtained and calculated from 3 or 4 independent experiments. Values are expressed as the means ± SD and analyzed using one-way ANOVA (SPSS for Windows, version 10; SPSS, Inc., Chicago, IL, USA) followed by Tukey's test for the comparisons of group means. All statistical analyses were performed using SAS for Windows, version 9.4. The level of significance was set at a P-value <0.05.
ResultsCombined treatment with MTX and α-tocopherol suppresses the proliferation of TNBC cells
MTX and α-tocopherol were used to treat the TNBC MDA-MB-231 cells. The survival rates were ~90% following 72 h of treatment with 0.1 µM MTX, 10 µM MTX and 5 µM α-tocopherol in the MDA-MB231 cells (Fig. 1). These results indicated that MTX alone and α-tocopherol alone did not effectively suppress the proliferation of the MDA-MB-231 cells. Combined treatment with MTX and α-tocopherol was then applied in the MDA-MB-231 cells. The survival rate was ~75% following treatment of the MDA-MB231 cells with 0.1 µM MTX plus 5 µM α-tocopherol (Fig. 2A). In addition, the survival rate was below 70% following treatment of the MDA-MB231 cells with 10 µM MTX plus 5 µM α-tocopherol (Fig. 2B). As shown in Fig. 2, when compared with the group treated with MTX alone, the group treated with MTX plus α-tocopherol (MTX/α-tocopherol) exhibited significantly lower survival rates at 48 and 72 h. These data suggested that MTX/α-tocopherol treatment suppressed the proliferation of the MDA-MB-231 cells. However, the survival rate was above 60% in the MTX/α-tocopherol-treated group following 72 h of treatment. Thus, it was considered that MTX/α-tocopherol may only attenuate cell proliferation and thus MTX/α-tocopherol treatment may be not be an effective strategy for clinical treatment.
α-tocopherol decreases the H<sub>2</sub>O<sub>2</sub> levels in MTX-treated cells
Previous studies have demonstrated that MTX can induce an increase in ROS in cells, particularly H2O2 levels, causing cell cytotoxicity (14,19). In the present study, we wished to determine whether MTX also induces an increase in H2O2 levels in MDA-MB-231 cells. Compared to the control group, treatment with 10 µM MTX significantly increased the H2O2 levels in the MDA-MB-231 cells, while treatment with 0.1 µM MTX did not markedly increase the H2O2 levels (Fig. 3A). These results indicated that MTX increases the H2O2 levels in a dose-dependent manner. α-tocopherol is known to have anti-oxidative activities (26,27). In the present study, whether α-tocopherol can inhibit the MTX-induced increase in H2O2 levels was investigated. The results revealed that α-tocopherol decreased the H2O2 levels in the MTX-treated cells (Fig. 3B). Notably, as shown in Figs. 2 and 3, the H2O2 levels were not associated with cell proliferation in the MTX-treated and MTX/α-tocopherol-treated MDA-MB-231 cells.
α-TOS induces cell cytotoxicity in TNBC in a time- and concentration-dependent manner
A previous study indicated that α-TOS induces the apoptosis of and Fas activation in breast cancer cells (37). In the present study, the concentration of α-TOS and the incubation time were further investigated in the α-TOS-treated MDA-MB-231 cells. As shown in Fig. 4, the cell survival rate was ~90% following treatment of the MDA-MB-231 cells with 5 and 20 µM α-TOS for 72 h; however, the cell survival rate at 72 h was ~50% following treatment with 40 µM α-TOS and below 50% following treatment of the MDA-MB-231 cells with 60 and 80 µM α-TOS. These results indicated that α-TOS induced cytotoxicity in a concentration-dependent manner. Furthermore, following treatment with 40, 60 and 80 µM α-TOS, the survival rates of the MDA-MB-231 cells gradually decreased during the 72-h treatment period. Thus, α-TOS induced cytotoxicity in a time-dependent manner.
High- and low-dose MTX induces distinct cytotoxicity in α-TOS-treated TNBC cells
Our data demonstrated that MTX/α-tocopherol treatment suppressed TNBC cell proliferation, although MTX/α-tocopherol induced-anticancer activity was ineffective (Fig. 2). The present study further investigated the anticancer effects on MDA-MB-231 cells following treatment with MTX plus α-TOS, or α-tocopherol derivatives. The 72-h cell survival rate was ~50% following treatment of the MDA-MB-231 cell with 40 µM α-TOS alone (Fig. 5). Notably, the 72-h MDA-MB-231 cell survival rate was ~35% in the group treated with 40 µM α-TOS plus 10 µM MTX; however, it was ~60% in the 40 µM α-TOS plus 0.1 µM MTX group (Fig. 5). During the 72-h treatment period, the survival rate curve of the group treated with 40 µM α-TOS plus 10 µM MTX was below that of the group treated with 40 µM α-TOS only. However, the survival rate curve of the group treated with 40 µM α-TOS plus 0.1 µM MTX was above that of the group treated with 40 µM α-TOS alone. Therefore, these results indicated that high-dose MTX enhanced anticancer activity in α-TOS-treated MDA-MB-231 cells, while low-dose MTX reduced anticancer activity in α-TOS-treated MDA-MB-231 cells.
α-TOS- and MTX/α-TOS-induced cytotoxicity is associated with caspase-3 activation and PARP cleavage
Previous studies have demonstrated that both α-TOS and MTX can induce cell cytotoxicity via caspase-3 activation (19,40,41). In the present study, as shown in Fig. 5, treatment with 40 µM α-TOS, 40 µM α-TOS/0.1 µM MTX and 40 µM α-TOS/10 µM MTX induced cell cytotoxicity. Thus, caspase-3 activation and PARP, downstream of caspase-3, were investigated in the present study. As determined by western blot analysis, the ratio of cleaved caspase-3/pro-caspase-3 was increased in the α-TOS- and α-TOS/MTX-treated cells (Fig. 6). In addition, PARP cleavage was also observed in the α-TOS- and α-TOS/MTX-treated cells (Fig. 6). Therefore, these results indicate that α-TOS and α-TOS/MTX may induce cell cytotoxicity via caspase-3 activation in MDA-MB-231 cells.
Discussion
Previous studies have indicated that vitamin E has anti-oxidative functions in cancer chemotherapy, reducing chemical agent-induced side-effects (42,43). Furthermore, vitamin E can prolong the survival of patients with gastric cancer (44) and can attenuate prostate cancer metastasis (45). α-tocopherol is the most common form found abundantly in vitamin E and displays anti-oxidative activity (42,46). α-tocopherol is also used to decrease chemical agent-induced side-effects in cancer therapy (47). The low dose of MTX (0.1 µM) used in the present study, is the concentration that is used in clinical practice for the treatment of rheumatoid arthritis. The high dose of MTX (10 µM) used in the present study is the dose used in clinical practice for cancer therapy. Currently, a dose of ≥10 µM MTX is used in clinical practice for cancer therapy. On the other hand, vitamin E is an antioxidant nutrient, and the dose range of vitamin E used in clinical practice varies widely. Generally, the dose of 5 to 80 µM vitamin E is used for combination treatment in clinical practice for various diseases. Therefore, the dose range of 5 to 80 µM vitamin E was used in the present study. A previous study revealed that α-tocopherol enhances the anti-proliferative effects of gefitinib in cells (48). However, α-tocopherol may attenuate the anticancer activity of some chemical agents, such as tamoxifen and crizotinib (49,50). The present study demonstrated that treatment with α-tocopherol plus MTX decreased the proliferation of MDA-MB-231 cells (Fig. 2). Therefore, it was suggested that α-tocopherol may have different anticancer effects when used in combination with different chemical agents. However, the mechanisms through which α-tocopherol promotes MTX- and gefitinib-induced cell proliferation and inhibits tamoxifen- and crizotinib-induced anticancer activities remain unclear.
A recent study demonstrated that treatment with vitamin C/MTX increased H2O2 levels, resulting in cell cytotoxicity (4). A number of studies have also revealed that high H2O2 levels are associated with cell cytotoxicity (51–53). However, the present study demonstrated that α-tocopherol attenuated the MTX-induced H2O2 levels (Fig. 3B), while treatment with α-tocopherol plus MTX exerted anti-proliferative effects on MDA-MB-231 cells (Fig. 2). Therefore, it was suggested that the anti-proliferative effects of treatment with α-tocopherol/MTX may not be related to the H2O2 levels. The mechanisms underlying the anti-proliferative activity of α-tocopherol/MTX require further investigation in the future.
Previous studies have demonstrated that cyclopentenone prostaglandins/α-TOS treatment and MTX/α-TOS treatment have synergistic anticancer effects on oral squamous carcinoma cells (54) and osteosarcoma cells (55). Similar to these studies, the data from the present study also demonstrated that high-dose MTX/α-TOS had a synergistic anticancer effect on MDA-MB-231 cells. The survival rate was below 50% following treatment with high-dose MTX/α-TOS for 48 h (Fig. 5). Compared with the survival rate of the high-dose MTX/α-TOS treatment group, the survival rate was still above 60% following treatment with high-dose MTX/α-tocopherol for 72 h (Fig. 2B). Therefore, it was suggested that high-dose MTX/α-TOS may be a potential treatment for TNBC, while high-dose MTX/α-tocopherol treatment may not be an effective strategy for TNBC treatment.
The present study, as well as previous studies have demonstrated that α-tocopherol/MTX and α-tocopherol/gefitinib treatment exert synergistic anti-proliferative effects (48). However, previous studies have also revealed that α-tocopherol/tamoxifen and α-tocopherol/crizotinib treatment exert antagonistic anti-proliferative effects (49,50). These studies indicate that α-tocopherol in combination with different chemical agents has distinct anti-proliferative effects. However, in the present study, high-dose MTX enhanced α-TOS-induced cell cytotoxicity, while low-dose MTX antagonized α-TOS-induced cell cytotoxicity in MDA-MB-231cells (Fig. 5). This result was similar to that of a previous study (34). This previous study demonstrated that high-dose sodium selenite promoted α-TOS-induced cell cytotoxicity, while low-dose sodium selenite attenuated α-TOS-induced cell cytotoxicity in MCF-7 cells. Based on these studies, it was suggested that both synergistic and antagonistic effects can arise in the same drug components in a dose-dependent manner. Although the detailed signaling mechanisms require further investigation, the results of the present study suggested that caspase signaling (Fig. 6) had a similar mechanism when comparing the low- and high-dose effects of MTX with α-TOS treatment. However, oxidative stress (such as H2O2 levels; Fig. 3) may have different signaling pathways when comparing the low- and high-dose effect of MTX with α-TOS treatment. In addition, as shown in Fig. 5, high-dose MTX slightly reduced the cell survival rate, while low-dose MTX slightly increased the cell survival rate. These results may be related to the synergistic or antagonistic anticancer effects caused by α-TOS/MTX treatment. Both MDA-MB-231 and MDA-MB-468 were examined in the present study. Similar results were obtained with the MDA-MB-468 cells (data not shown).
In conclusion, the present study demonstrated that combined treatment with MTX and α-tocopherol or α-TOS attenuated the cell survival rate of TNBC cells. However, MTX/α-TOS exerted a more potent anti-proliferative effect than MTX/α-tocopherol. In addition, high-dose MTX enhanced the α-TOS-induced cytotoxic effects, while low-dose MTX antagonized the α-TOS-induced cytotoxic effects.
Acknowledgements
The authors would like to thank the Ministry of Science and Technology, the Ministry of Health and Welfare, the Taipei Tzu Chi Hospital and Drug Development Center, China Medical University (Taiwan, ROC) for making the present study a possibility.
Funding
The present study was supported by grants from the Ministry of Science and Technology, Taiwan (grant nos. MOST106- 2320-B-039-051-MY3 and MOST107-2320-B-039-004), the Ministry of Health and Welfare, Taiwan (grant no. MOHW107- TDU-B-212-112-015), the Taipei Tzu Chi Hospital, Taiwan (grant nos. TCRD-TPE-106-35, TCRD-TPE-106-36, TCRD- TPE-105-20 and TCRD-TPE-105-02) and the study was also financially supported by the ‘Drug Development Center, China Medical University’ from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, ROC.
Availability of data and materials
All data generated or analyzed during the present study are included in this published article or are available from the corresponding author on reasonable request.
Authors' contributions
CWW and YLY performed the experiments, analyzed the data and wrote the manuscript. YHC and YTH performed the experiments and analyzed the data. GTY designed the experiments and analyzed the data. All authors approved the final version of the manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests
ReferencesFanJWuYYuanMPageDLiuJOngIMPeissigPBurnsideEStructure-leveraged methods in breast cancer risk predictionJ Mac Learn Res17852017FerlayJSoerjomataramIDikshitREserSMathersCRebeloMParkinDMFormanDBrayFCancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012Int J Cancer136E359E386201510.1002/ijc.2921025220842RobinsonSPJordanVCAntiestrogenic action of toremifene on hormone-dependent, -independent, and heterogeneous breast tumor growth in the athymic mouseCancer Res491758176219892522347WuCWLiuHCYuYLHungYTWeiCWYiangGTCombined treatment with vitamin C and methotrexate inhibits triple-negative breast cancer cell growth by increasing H2O2 accumulation and activating caspase-3 and p38 pathwaysOncol Rep3721772184201710.3892/or.2017.543928259996YuYLChouRHLiangJHChangWJSuKJTsengYJHuangWCWangSCHungMCTargeting the EGFR/PCNA signaling suppresses tumor growth of triple-negative breast cancer cells with cell-penetrating PCNA peptidesPLoS One8e61362201310.1371/journal.pone.0061362235934723620387Metzger-FilhoOTuttAde AzambujaESainiKSVialeGLoiSBradburyIBlissJMAzimHAJrEllisPDissecting the heterogeneity of triple-negative breast cancerJ Clin Oncol3018791887201210.1200/JCO.2011.38.201022454417PerouCMSørlieTEisenMBvan de RijnMJeffreySSReesCAPollackJRRossDTJohnsenHAkslenLAMolecular portraits of human breast tumoursNature406747752200010.1038/3502109310963602WilliamsCBSoloffACEthierSPYehESPerspectives on epidermal growth factor receptor regulation in triple-negative breast cancer: Ligand-mediated mechanisms of receptor regulation and potential for clinical targetingAdv Cancer Res127253281201510.1016/bs.acr.2015.04.00826093903MalaviyaANLow-dose methotrexate (LD-MTX) in rheumatology practice-a most widely misunderstood drugCurr Rheumatol Rev12168176201610.2174/157339711266616082415180127964706RobienKBoyntonAUlrichCMPharmacogenetics of folate-related drug targets in cancer treatmentPharmacogenomics6673689200510.2217/14622416.6.7.67316207145MoriSHidakaMKawakitaTHidakaTTsudaHYoshitamaTMigitaKUekiYFactors associated with myelosuppression related to low-dose methotrexate therapy for inflammatory rheumatic diseasesPLoS One11e0154744201610.1371/journal.pone.0154744271286794851368MaejimaHWataraiANakanoTKatayamaCNishiyamaHKatsuokaKAdverse effects of methotrexate in three psoriatic arthritis patientsRheumatol Int34571574201410.1007/s00296-012-2649-023274445KolliVKAbrahamPIsaacBSelvakumarDNeutrophil infiltration and oxidative stress may play a critical role in methotrexate-induced renal damageChemotherapy558390200910.1159/00019239119145077CağlarYÖzgürHMaturIYenilmezEDTuliAGÖnlüşenGPolatSUltrastructural evaluation of the effect of N-acetylcysteine on methotrexate nephrotoxicity in ratsHistol Histopathol28865874201323307329YangWZouYMengFZhangJChengRDengCZhongZEfficient and targeted suppression of human lung tumor xenografts in mice with methotrexate sodium encapsulated in all-function-in-one chimeric polymersomesAdv Mater2882348239201610.1002/adma.20160006527383234LarsenECDevidasMChenSSalzerWLRaetzEALohMLMattanoLAJrColeCEicherAHauganMDexamethasone and high-dose methotrexate improve outcome for children and young adults with high-risk B-acute lymphoblastic leukemia: A report from children's oncology group study AALL0232J Clin Oncol3423802388201610.1200/JCO.2015.62.4544271145874981974KansaraRShenkierTNConnorsJMSehnLHSavageKJGerrieASVillaDRituximab with high-dose methotrexate in primary central nervous system lymphomaAm J Hematol9011491154201510.1002/ajh.2420426414492CipolleschiMGMarziIRovidaEOlivottoMDello SbarbaPLow-dose methotrexate enhances cycling of highly anaplastic cancer cellsCell Cycle16280285201710.1080/15384101.2016.125288327841718YiangGTChouPLHungYTChenJNChangWJYuYLWeiCWVitamin C enhances anticancer activity in methotrexate-treated Hep3B hepatocellular carcinoma cellsOncol Rep3210571063201410.3892/or.2014.3289249695444121419BarrosSMenciaNRodriguezLOleagaCSantosCNoéVCiudadCJThe redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cellsPLoS One8e63276201310.1371/journal.pone.0063276236754693652835TanabeMCombination chemotherapy of mitomycin C and methotrexate was effective on metastatic breast cancer resistant to Eribulin, Vinorelbine, and bevacizumab after anthracycline, Taxane, and CapecitabineCase Rep Oncol9422426201610.1159/000447770277217625043245Velthuis-te WierikEJvan den BergHWeststrateJAvan het HofKHde GraafCConsumption of reduced-fat products: Effects on parameters of anti-oxidative capacityEur J Clin Nutr5021421919968730607SmolarekAKSoJYBurgessBKongANReuhlKLinYShihWJLiGLeeMJChenYKDietary administration of δ- and γ-tocopherol inhibits tumorigenesis in the animal model of estrogen receptor-positive, but not HER-2 breast cancerCancer Prev Res513101320201210.1158/1940-6207.CAPR-12-0263ConstantinouCPapasAConstantinouAIVitamin E and cancer: An insight into the anticancer activities of vitamin E isomers and analogsInt J Cancer123739752200810.1002/ijc.2368918512238SmolarekAKSuhNChemopreventive activity of vitamin E in breast cancer: A focus on γ- and δ-tocopherolNutrients3962986201110.3390/nu3110962222540893257724ZapataGLGuajardoMHTerrasaAMThe in vitro protective effect of alpha-tocopherol on oxidative injury in the dog retinaVet J177266272200810.1016/j.tvjl.2007.04.00517581765Ekstrand-HammarstromBOsterlundCLilliehöökBBuchtAVitamin E down-modulates mitogen-activated protein kinases, nuclear factor-kappaB and inflammatory responses in lung epithelial cellsClin Exp Immunol147359369200710.1111/j.1365-2249.2006.03285.x172239791810475ComitatoRNesaretnamKLeoniGAmbraRCanaliRBolliAMarinoMVirgiliFA novel mechanism of natural vitamin E tocotrienol activity: Involvement of ERbeta signal transductionAm J Physiol Endocrinol Metab297E427E437200910.1152/ajpendo.00187.200919491296ChamrasHBarskySHArdashianANavasartianDHeberDGlaspyJANovel interactions of vitamin E and estrogen in breast cancerNutr Cancer524348200510.1207/s15327914nc5201_616091003GaoYQiXZhengYJiHWuLZhengNTangJNanoemulsion enhances α-tocopherol succinate bioavailability in ratsInt J Pharm515506514201610.1016/j.ijpharm.2016.10.02627746330KulikovAVVdovinASZhivotovskyBGogvadzeVTargeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatmentCell Mol Life Sci7123252333201410.1007/s00018-013-1489-824142346Angulo-MolinaAReyes-LeyvaJLópez-MaloAHernándezJThe role of alpha tocopheryl succinate (α-TOS) as a potential anticancer agentNutr Cancer66167176201410.1080/01635581.2014.86336724364743YuWLiaoQYHantashFMSandersBGKlineKActivation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-α-tocopheryl succinate-induced apoptosis of human breast cancer cellsCancer Res6165696576200111522656BadrDMHafezHFAghaAMShoumanSAThe combination of α-tocopheryl succinate and sodium selenite on breast cancer: A merit or a demerit?Oxid Med Cell Longev 201647416942016WangXFXieYWangHGZhangYDuanXCLuZJα-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathwayActa Pharmacol Sin3116041610201010.1038/aps.2010.171211274964002948WangDChuangHCWengSCHuangPHHsiehHYKulpSKChenCSalpha-Tocopheryl succinate as a scaffold to develop potent inhibitors of breast cancer cell adhesionJ Med Chem5256425648200910.1021/jm9002457197086612761948TurleyJMFuTRuscettiFWMikovitsJABertoletteDCIIIBirchenall-RobertsMCVitamin E succinate induces Fas-mediated apoptosis in estrogen receptor-negative human breast cancer cellsCancer Res5788189019979041190LinBRYuCJChenWCLeeHSChangHMLeeYCChienCTChenCFGreen tea extract supplement reduces D-galactosamine-induced acute liver injury by inhibition of apoptotic and proinflammatory signalingJ Biomed Sci1635200910.1186/1423-0127-16-35193179202667169YiangGTYuYLLinKTChenJNChangWJWeiCWAcetaminophen induces JNK/p38 signaling and activates the caspase-9-3-dependent cell death pathway in human mesenchymal stem cellsInt J Mol Med36485492201510.3892/ijmm.2015.2254260966464501662AbeNShimizuTMiyoshiNMurataYNakamuraYα-Tocopherol sensitizes human leukemia HL-60 cells to apoptosis induced by benzyl isothiocyanateBiosci Biotechnol Biochem76381383201210.1271/bbb.11066522313765Abo-HadedHMElkablawyMAAl-JohaniZAl-AhmadiOEl-AgamyDSHepatoprotective effect of sitagliptin against methotrexate induced liver toxicityPLoS One12e0174295201710.1371/journal.pone.0174295283340485363865PehHYTanWSLiaoWWongWSVitamin E therapy beyond cancer: Tocopherol versus tocotrienolPharmacol Ther162152169201610.1016/j.pharmthera.2015.12.00326706242YüncüMBükücüNBayatNSencarLTarakciogluMThe effect of vitamin E and L-carnitine against methotrexate-induced injury in rat testisTurk J Med Sci45517525201510.3906/sag-1409-3926281314PyrhönenSKuitunenTNyandotoPKouriMRandomised comparison of fluorouracil, epidoxorubicin and methotrexate (FEMTX) plus supportive care with supportive care alone in patients with non-resectable gastric cancerBr J Cancer71587591199510.1038/bjc.1995.11475335172033628DragoJRNesbittJABadalamentRASmithJChemotherapy and vitamin E in treatment of Nb rat prostate tumorsIn Vivo239940119882979862FuJYHtarTTDe SilvaLTanDMChuahLHChromatographic separation of vitamin E enantiomersMolecules22piiE233201710.3390/molecules2202023328165404DiasMFSousaECabritaSPatricioJOliveiraCFChemoprevention of DMBA-induced mammary tumors in rats by a combined regimen of alpha-tocopherol, selenium, and ascorbic acidBreast J61419200010.1046/j.1524-4741.2000.98071.x11348329YiangGTChenJNLinPSLiuHCChenSYWeiCWCombined treatment with vitamin E and gefitinib has synergistic effects to inhibit TGF-β1-induced renal fibroblast proliferationMol Med Rep1353725378201610.3892/mmr.2016.515527109695UchiharaYUedaFTagoKNakazawaYOheTMashinoTYokotaSKasaharaTTamuraHFunakoshi-TagoMAlpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALKPLoS One12e0183003201710.1371/journal.pone.0183003288064145555621PeraltaEAViegasMLLouisSEngleDLDunningtonGLEffect of vitamin E on tamoxifen-treated breast cancer cellsSurgery140607615200610.1016/j.surg.2006.07.00717011908ChangHLiCHuoKWangQLuLZhangQWangYWangWLuteolin prevents H2O2-induced apoptosis in H9C2 cells through modulating Akt-P53/Mdm2 signaling pathwayBiomed Res Int 201651258362016MiyazatoHTairaJUedaKHydrogen peroxide derived from marine peroxy sesquiterpenoids induces apoptosis in HCT116 human colon cancer cellsBioorg Med Chem Lett2646414644201610.1016/j.bmcl.2016.08.05727575468WangCWangGLiuHHouYLProtective effect of bioactive compounds from Lonicera japonica Thunb. Against H2O2-induced cytotoxicity using neonatal rat cardiomyocytesIran J Basic Med Sci19971052016270960704823622ElAttarTMVirjiASInhibition of growth in oral squamous carcinoma cells by cyclopentenone prostaglandins: comparison with chemotherapeutic agentsProstaglandins Leukot Essent Fatty Acids56461465199710.1016/S0952-3278(97)90600-19223658AllevaRBenassiMSPazzagliaLTomasettiMGellertNBorghiBNeuzilJPicciPAlpha-tocopheryl succinate alters cell cycle distribution sensitising human osteosarcoma cells to methotrexate-induced apoptosisCancer Lett232226235200610.1016/j.canlet.2005.02.01916458119
MTX alone and α-tocopherol alone do not induce cytotoxicity. MDA-MB-231 cells were incubated with 0.1 and 10 µM MTX and 5 µM α-tocopherol for 72 h. Every 24 h, the samples were treated with an MTT kit. Survival rates were calculated as (A570 experimental group/A570 control group) × 100%. Data were analyzed from 4 independent experiments and are presented as the means ± standard deviation. MTX, methotrexate; α-T, α-tocopherol.
Combined treatment with MTX and α-tocopherol exerts a synergistic effect. (A) MDA-MB-231 cells were incubated with 0.1 µM MTX, 5 µM α-tocopherol and 0.1 µM MTX plus 5 µM α-tocopherol, respectively, for 72 h. (B) MDA-MB-231 cells were incubated with 10 µM MTX, 5 µM α-tocopherol and 10 µM MTX plus 5 µM α-tocopherol, respectively, for 72 h. Every 24 h, the samples were treated with an MTT kit. Survival rates were calculated as (A570 experimental group/A570 control group) × 100%. Data were analyzed from 4 independent experiments and are presented as the means ± standard deviation. *P<0.05 vs. the group treated with MTX only. MTX, methotrexate; α-T, α-tocopherol.
α-tocopherol decreases MTX-induced H2O2 levels. (A) Cells were incubated with 0.1 or 10 µM MTX, respectively. (B) Cells were incubated with 10 µM MTX or 10 µM MTX plus 5 µM α-tocopherol, respectively. Following a 4-h incubation, samples were treated with luminol solution. Data were analyzed from 4 independent experiments and are presented as the means ± standard deviation. *P<0.05 vs. the control group; $P<0.05 vs. the group treated with 10 µM MTX only. MTX, methotrexate; α-T, α-tocopherol.
α-TOS exerts a dose-dependent cytotoxicity effect. Cells were incubated with 5, 20, 40, 60 and 80 µM α-TOS, respectively, for 72 h. Every 24 h, the samples were treated with an MTT kit. Survival rates were calculated as (A570 experimental group/A570 control group) × 100%. Data were analyzed from 4 independent experiments and are presented as the means ± standard deviation. *P<0.05 vs. the 5 µM α-TOS group. α-TOS, α-tocopherol succinate.
High- and low-dose MTX induce distinct cytotoxicity in α-TOS-treated cells. Cells were incubated with 0.1 µM MTX, 10 µM MTX, 40 µM α-TOS, 0.1 µM MTX plus 40 µM α-TOS and 10 µM MTX plus 40 µM α-TOS, respectively, for 72 h. Every 24 h, the samples were treated with an MTT kit. Survival rates were calculated as (A570 experimental group/A570 control group) ×100%. Data were analyzed from 4 independent experiments and are presented as the means ± standard deviation. P-values were evaluated among the 40 µM α-TOS only group, 0.1 µM MTX plus 40 µM α-TOS group and 10 µM MTX plus 40 µM α-TOS group. *P<0.05 vs. the 40 µM α-TOS only group. MTX, methotrexate; α-TOS, α-tocopherol succinate.
MTX and α-TOS induce casepase-3 activation and PARP cleavage. (A) Western blot analysis, (B) cleaved caspase-3/caspase-3 relative intensity and (C) cleaved PARP/tubulin were observed and determined at 72 h in the control group (lane 1), 40 µM α-TOS group (lane 2), 0.1 µM MTX plus 40 µM α-TOS group (lane 3) and 10 µM MTX plus 40 µM α-TOS group (lane 4). Data were determined from 3 independent experiments and are presented as the means ± standard deviation. *P<0.05 vs. the control group. MTX, methotrexate; PARP, poly(adenosine diphosphate-ribose) polymerase; α-TOS, α-tocopherol succinate.