2-Methoxyestradiol-bis-sulphamate refrains from inducing apoptosis and autophagy in a non-tumorigenic breast cell line
© Visagie and Joubert; licensee BioMed Central Ltd. 2012
Received: 14 March 2012
Accepted: 2 May 2012
Published: 20 August 2012
Anticancer research resulted in the discovery of a promising antimitotic metabolite, 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate, a bis-sulphamoylated analogue exerts antiproliferative- and antimitotic activity. Investigating the anticancer potential of 2-methoxyestradiol-bis-sulphamate requires demonstrating the influence of 2-methoxyestradiol-bis-sulphamate on non-tumorigenic cells. This project focused on the in vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A breast epithelial cell line.
The in vitro influence of 2-methoxyestradiol-bis-sulphamate was investigated on cell cycle progression, possible induction of apoptosis and autophagy and reactive oxygen species generation. Cell cycle progression was done using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Displaying effects on the mitochondrial membrane potential was achieved utilizing flow cytometry and the MitoCapture TM Mitochondrial apoptosis detection kit. Autophagy detection was done by means of flow cytometry and anti-LC3B conjugated to DyLight 488. Reactive oxygen species generation was conducted employing flow cytometry and 2,7-dichlorofluorescein diacetate and hydroethidine.
This study demonstrated that 2-methoxyestradiol-bis-sulphamate did not affect cell cycle progression or reactive oxygen species in a statistically significant manner in the non-tumorigenic MCF-12A cell line. In addition, 2-methoxyestradiol-bis-sulphamate did not statistically significantly induce apoptosis or autophagy.
Reports indicate that 2-methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in several tumorigenic cell lines. The anticancer ability of 2-methoxyestradiol-bis-sulphamate is due to its antimitotic activity. However, this study demonstrates the promising notion that 2-methoxyestradiol-bis-sulphamate does not affect the non-tumorigenic MCF-12A cells. This project contributes to the embedded scientific knowledge regarding the differential death mechanisms used by 2-methoxyestradiol-bis-sulphamate on tumorigenic and non-tumorigenic cell lines.
Researchers have identified a promising anticancer compound, 2-methoxyestradiol (2ME), that exerts antimitotic- and antiproliferative activity in vitro and in vivo [1, 2]. However, 2ME failed to advance to United States Food and Drug Administration approval due to efficacy concerns . 2ME possesses low bioavailability and rapid metabolic breakdown due to being targeted by the 17β-hydroxysteroid dehydrogenase-mediated metabolism pathway where 2ME is swiftly inactivated by conjugation and oxidation of the hydroxyl groups at the C3/C17 positions .
2-Methoxyesradiol-bis-sulphamate is (2MEBM) a bis-sulphamoylated derivative of 2ME that exerts antiproliferative- and anticancer activity with improved bioavailability and a superior pharmacokinetic profile [5–7]. 2MEBM reduced cell growth in a range of cell lines including the estrogen receptor positive breast adenocarcinoma cell line (MCF-7), prostate cancer cell line (LNACaP), estrogen receptor negative breast adenocarcinoma cell line (MDA-MB-231), human cervical adenocarcinoma cell line (HeLa) and human ovarian carcinoma (A2780) [7–12]. In addition, apoptosis was induced by 2MEBM in MCF-7, prostate cancer cells (PC-3), human umbilical vein endothelial cells (HUVEC) and the human breast adenocarcinoma CAL51 cell line [13–15].
Reports have also established that 2MEBM is tenfold more potent than 2ME in its antiproliferative activity [7, 16]. The increased potency of sulphamoylated agents are due to their ability to interact with sulphatase, carbonic anhydrase and proteins such as tubulin . In addition, the estrogen 3-O-sulphamates strongly inhibit carbonic anhydrase II which is responsible for the conversion of carbon dioxide and water to carbonic acid. This interaction is most likely responsible for the high bioavailability of the sulphamoylated analogues as reversible uptake by red blood cells and interaction with carbonic anhydrase II ensures transiting the liver without undergoing first pass metabolism [1, 17, 18]. The superior bioavailability of 2MEBM is demonstrated by a previous study where 2MEBM was still detectable in the plasma after 24 h of exposure . Newman, et al. (2006) reported the increased bioavailability of 2MEBM is due to its resistance to metabolism by 17β-hydroxysteriod dehydrogenase type 1 and type 2 enzymes .
Anticancer agents ideally induce cell death in tumorigenic cells leaving non-tumorigenic cells unaffected. Thus, determining the influence of 2MEBM on non-tumorigenic cell lines is essential. During this study the in vitro effects of 2MEBM on reactive oxygen species generation and the induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line was determined. This study contributes toward the unravelling of differential action mechanisms on tumorigenic and non-tumorigenic cell lines.
Methods and materials
The MCF-12A cell line is a non-tumorigenic spontaneously immortalized adherent human breast epithelial cell line and forms domes in confluent cultures. The MCF-12A cells were a gift from Professor Parker (Department of Medical Biochemistry, University of Cape Town, South Africa).
All required reagents of cell culture analytical grade were purchased from Sigma (St. Louis, United States of America) unless otherwise specified. Heat-inactivated fetal calf serum (FCS), sterile cell culture flasks and plates were purchased from Sterilab Services (Kempton Park, Johannesburg, South Africa). Penicillin, streptomycin and fungizone were obtained from Highveld Biological Pty (Ltd) (Sandringham, South Africa). The Annexin V fluorescein isothiocyanate (FITC) kit, MitoCapture TM Mitochondrial apoptosis detection kit and a rabbit polyclonal anti-LC3B conjugated to DyLight 488 were purchased from BIOCOM biotech (Pty) Ltd. (Clubview, South Africa). 2,7-Dichlorofluorescein diacetate and hydroethidine was acquired from Sigma (St. Louis, United States of America). Since 2MEBM is not commercially available, it was synthesized by Professor Vleggaar from the Department of Chemistry (University of Pretoria, Pretoria, South Africa).
Cells were grown in sterile 25 cm2 tissue culture flasks at a humidified atmosphere at 37°C and 5% CO2. MCF-12A cells were cultured in medium consisting of a 1:1 mixture of Dulbecco’s minimum essential medium eagle (DMEM) and Ham’s-F12 medium, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 μg/ml insulin and 500 ng/ml hydrocortisone, supplemented with 10% heat-inactivated fetal calf serum (56°C, 30 min), 100U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
A stock solution of 2x10-3 M 2MEBM dissolved in dimethyl sulphoxide (DMSO) was prepared and diluted with medium to the desired concentrations prior to exposure of the cells. Media of all control experiments were supplemented with an equal volume of DMSO (vehicle) with the DMSO content of the final dilutions never exceeding 0.05% (v/v). Experiments entailed seeding 500 000 exponentially growing MCF-12A cells per 25 cm2 cell culture flasks containing a final volume of 5 ml of maintenance medium. A 24 h incubation period at 37°C was allowed for cell adherence, the medium was discarded and cells were exposed to 0.4 μM 2MEBM and incubated for 48 h at 37°C. These conditions were selected since previous studies in our laboratory have demonstrated successful antiproliferative activity in tumorigenic cell lines [5, 6].
Cell cycle progression
Flow cytometry was utilized to investigate the DNA content to determine cell cycle distribution, G2/M block and the presence of a sub-G1 apoptotic peak. After 48 h of exposure to 0.4 μM 2MEBM, cells were trypsinized and resuspended in 1 ml growth medium. 1x106 cells were centrifuged for 5 min at 300xg. The pellet resuspended twice in ice-cold phosphate buffer solution (PBS). The supernatant was discarded and the cells were resuspended in 200 μl of ice-cold PBS containing 0.1% FCS. Ice-cold 70% ethanol (4 ml) was added in a drop wise manner and cells were stored at 4°C for 24 h. After 24 h, cells were pelleted by centrifugation for 5 min. The supernatant was removed and cells were resuspended in 1 ml of PBS containing propidium iodide (40 μg/ml) and incubated at 37°C, 5% CO2 for 45 min. Subsequently, cells were analysed by means of FACS FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd) equipped with an air-cooled argon laser excited at 488 nm. Data from at least 10 000 cells were captured with CXP software [Beckman Coulter South Africa (Pty) Ltd, Johannesburg, Gauteng, South Africa] and analyzed with Cyflogic (CyFlo Ltd., Turku, Finland).
Mitochondrial membrane potential
Mitochondrial integrity was investigated using the MitoCapture TM mitochondrial kit. The mitochondrial (intrinsic) apoptosis pathway is activated by stress signals that results in the permeabilization of the mitochondrial outer membrane. This will be demonstrated by the MitoCapture TM mitochondrial kit that supplies quantitive apoptosis data . After 48 h of exposure to 0.4 μM 2MEBM, cells were trypsinized and centrifuged at 13 000 x g. Cells were resuspended in 1 ml MitoCapture solution (1 μl MitoCapture: 1 ml pre-warmed incubation buffer provided by the MitoCaptureTM mitochondrial kit), incubated at a humidified atmosphere (37°C, 5% CO2) for 20 min and subsequently centrifuged at 500 x g. After the supernatant was discarded, cells were resuspended in 1 ml prewarmed incubation buffer (37°C). Afterwards, cells were analyzed immediately using fluorescence activated cell sorting (FACS) FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd). Apoptotic cells were detected in the FITC channel (usually FL1) showing diffused green fluorescence. Data from at least 10 000 cells were analysed using CXP software [Beckman Coulter South Africa (Pty) Ltd, Johannesburg, Gauteng, South Africa] and analyzed with Cyflogic (CyFlo Ltd., Turku, Finland).
Flow cytometry utilizing the rabbit polyclonal anti-LC3B conjugated to DyLight 488 demonstrated possible autophagy induction. LC3-I is converted to LC3-II by a series of reactions . Enhancement of LC3-I conversion to LC3-II results in the upregulation of autophagy. Thus, detection of LC3-II is a useful indication for the presence of autophagy . After 48 h of exposure to 0.4 μM 2MEBM cells were trypsinised and centrifuged. Cells were washed with cold PBS, pelleted and fixed with 3 ml 0.01% formaldehyde in PBS for 10 min at 4°C. Cells were centrifuged and resuspended in 200 μl PBS, followed by 1 ml ice-cold methanol (−20°C) for 15 min at 4°C. Afterwards cells were washed twice with cold PBS. Cells were stained with 0.5 ml of the antibody cocktail (0.05% Triton X-100, 1% bovine serum albumin (BSA), 40 μg/ml propidium iodide and 0.5 μg/ml conjugated rabbit polyclonal anti-LC3B antibody) prepared in PBS for 2 h at 4°C. Cells were washed trice with PBS/0.05% Triton/1% BSA and analyzed by means of flow cytometry. Data from at least 10 000 cells were analyzed employing Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).
Reactive oxygen species generation
Hydrogen peroxide (H2O2) generation was assessed using 2,7-dichlorofluorescein diacetate (DCFDA), a non-fluorescent probe, which, upon oxidation by ROS and peroxides is converted to the highly fluorescent derivative 2,7-dichlorofluorescein (DCF). Superoxide generation was assessed using hydroethidine (HE) that is oxidized by superoxide, to a red fluorescent compound. After 0.4 μM 2MEBM exposure for 48 h exposure, cells were trypsinized and 1x106 cells were resuspended in 1 ml PBS and washed with PBS. Cells were incubated with 20 μM DCFDA for 25 min and 10 μM HE for 15 min at 37°C. DCF (FL1) and HE fluorescent product fluorescence (FL2) were measured with a FACS FC500 System flow cytometer equipped with an air-cooled argon laser excited at 488 nm. The information generated from at least 10 000 cells were analyzed by means of Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).
Measurement of FITC-, HE- and DCF-derived fluorescence was expressed as a ratio of the value measured for the 2MEBM-treated cells compared to vehicle-treated exposed cells (mean relative fluorescence). Flow cytometry analysis involved data from at least 10 000 events that was repeated thrice where after a representative figure was chosen for each experiment. Flow cytometry data were analyzed by means of Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).
Cell cycle progression
Mitochondrial membrane potential
Reactive oxygen species generation
During the investigation of a compound’s potential anticancer activity it is crucial to scrutinize the effects on non-tumorigenic cell lines as it is essential that the compound leaves non-tumorigenic cells less-or unaffected. As previously mentioned 2MEBM exerts antiproliferative activity and demonstrates potential as an anticancer agent [5, 6]. This in vitro study explored the effects of 2MEBM on cell cycle progression and cell death induction in a non-tumorigenic breast epithelial cell line.
Various reports have indicated that 2MEBM exerts an antimitotic effects and induced a G2/M block in several cell lines including drug resistant human breast adenocarcinoma cell line (MCF-7 DOX40), mitoxantrone resistant breast adenocarcinoma cell line (MCF-7 MR) and MDA-MB-231 [9, 12]. Cell cycle progression analysis from the current study revealed that 2MEBM did not affect the G2/M fraction. In addition, the statistically insignificant sub-G1 fraction in the current study demonstrates that 2MEBM does affect non-tumorigenic cell lines less as 0.4 μM 2MEBM exposure for 48 hours resulted in apoptosis induction in 50% of MCF-7 cells [5, 6]. The notion that 2MEBM affects non-tumorigenic cell lines less with regard to apoptosis induction is verified by the MitoCapture TM mitochondrial results that indicate 2MEBM has no significant effects the mitochondrial membrane potential.
Previously published novel findings indicate that 2MEBM induces autophagy in MCF-7 cells and the oesophageal tumorigenic cell line (SNO) [6, 23]. However, Vorster, et al. (2010) reported that 2MEBM did not induce autophagy in the non-tumorigenic cell line . The latter is supported in this study where 2MEBM failed to induce autophagy in the MCF-12A cells.
The increased reactive oxygen species production after exposure to 2MEBM in MCF-7 cells was recently reported . The current study revealed the novel finding that 2MEBM exposure did not result in a significantly significant increase in hydrogen peroxide or hydrogen peroxide production. Studies indicate that reactive oxygen species is associated with apoptosis and autophagy . Reactive oxygen species may be involved in the direct activation of death receptors and reactive oxygen species-induced receptor clustering and signaling by means of the lipid raft platforms; however, this suggestion is novel and requires research . In addition, increased reactive oxygen species production results in permeabilization of the mitochondrial outer membrane and subsequent cytochrome c release leading to apoptosis . In addition, Shrotriya, et al. (2012) reposted that reactive oxygen species generation resulted in apoptosis induction in neck sqamous carcinoma cells . Recent reports indicate that reactive oxygen species involved with autophagy induction using several mechanisms including Atg4, catalase and the mitochondrial electron transport chain resulting in both cell survival and cell death . Reactive oxygen species production results in Beclin-1 overexpression resulting in autophagy induction. Hydrogen peroxide is necessary for starvation-induced autophagy and directly targets Atg4 for oxidation and inactivates Atg4 inducing autophagy .
In conclusion, this promising study revealed evidence that 2MEBM does not affect the non-tumorigenic MCF-12A cells in a statistically significant manner. Thus, the latter indicates that 2MEBM influences tumorigenic and non-tumorigenic cell lines differentially as 2MEBM is a well-reported compound with antiproliferative activity that induces cell death [5, 6]. This study contributes to elucidating the differential mechanism of action used by 2MEBM in tumorigenic and non-tumorigenic cell lines.
- Stander A, Joubert F, Joubert A: Docking, synthesis, and in vitro evaluation of antimitotic estrone analogues. Chem Biol Drug Des. 2011, 77: 173-181. 10.1111/j.1747-0285.2010.01064.x.View ArticlePubMedGoogle Scholar
- Fotsis T, Zhang Y, Pepper MS, Aldercreutz H, Montesano R, Nawroth PP: Schweigerer: The endogenous oestrogen metabolite 2-methoxyestradiol inhibits angiogenesis and suppresses tumor growth. Nature. 1994, 368: 237-239. 10.1038/368237a0.View ArticlePubMedGoogle Scholar
- Risinger AL, Westbrook CD, Encinas M, Mülbaier M, Schultes CM, Wawro S, Lewis JD, Janssen B, Giles FJ, Moolberry SL: ELR5010444, a novel microtubule disruptor with multiple mechanisms of action. Pharmacol Exp Ther. 2011, 6: 652-660.View ArticleGoogle Scholar
- Chander SK, Foster PA, Leese MP, Newman SP, Potter BVL, Purohit A, Reed MJ: In vivo inhibition of angiogenesis by sulphamoylated derivatives of 2-methoxyestradiol. Br J Cancer. 2007, 96: 1368-1376.PubMed CentralPubMedGoogle Scholar
- Visagie MH, Joubert AM: 2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line. Mol Cell Biochem. 2011, 357: 343-352. 10.1007/s11010-011-0905-3.View ArticlePubMedGoogle Scholar
- Visagie MH, Joubert AM: The in vitro effects of 2-Methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity and cell morphology, and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line. Cell Mol Biol Lett. 2010, 15: 564-581. 10.2478/s11658-010-0030-4.View ArticlePubMedGoogle Scholar
- Ireson CR, Chander SK, Purohit A, Perera S, Newman SP, Parish D, Leese MP, Smith AC, Potter BVL, Reed MJ: Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-methoxyoestradiol-bis-sulphamate in vivo in rodents. Br J Cancer. 2004, 90: 932-937. 10.1038/sj.bjc.6601591.PubMed CentralView ArticlePubMedGoogle Scholar
- Raobaikady B, Purohit A, Chander SK, Woo LWL, Leese MP, Potter BVL, Reed MJ: Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyestradiol-bis-sulphamate. J Steroid Biochem Mol Biol. 2003, 84: 351-358. 10.1016/S0960-0760(03)00049-9.View ArticlePubMedGoogle Scholar
- Suzuki RN, Newman SP, Purohit A, Leese MP, Potter BV, Reed MJ: Growth inhibition of multi-drug-resistant breast cancer cells by 2-methoxyoestradiol-bis-sulphamate and 2-ethyloestradiol-bis-sulphamate. J Steroid Biochem Mol Biol. 2003, 84: 269-278. 10.1016/S0960-0760(03)00035-9.View ArticlePubMedGoogle Scholar
- Foster PA, Stengel C, Ali T, Leese MP, Potter BV, Reed MJ, Purohit A, Newman SP: A comparison of two orally bioavailable anti-cancer angents, IRC-110160 and STX140. Anticancer Res. 2008, 28: 1483-1491.PubMedGoogle Scholar
- Raobaikady B, Reed MJ, Leese MP, Potter BVL, Purohit A: Inhibition of MDA-MB-231 cell cycle progression and cell proliferation by C-2-substituted oestradiol mono- and bis-3-O-sulphamates. Int J Cancer. 2005, 117: 150-159. 10.1002/ijc.21066.View ArticlePubMedGoogle Scholar
- Day JM, Foster PA, Tutill HJ, Newman SP, Ho YT, Leese MP, Potter BV, Reed MJ, Purohit A: BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure. Br J Cancer. 2009, 100: 476-486. 10.1038/sj.bjc.6604873.PubMed CentralView ArticlePubMedGoogle Scholar
- Newman SP, Leese MP, Purohit A, James DRC, Rennie CE, Potter BVL: Inhibition of in vitro angiogenesis by 2-Methoxy- and 2-ethyl-estrogen sulfamates. Int J Cancer. 2004, 109: 533-540. 10.1002/ijc.20045.View ArticlePubMedGoogle Scholar
- Newman SP, Foster PA, Ho YT, Day JM, Raibaikady B, Kasprzyk PG, Leese MP, Potter BV, Reed MJ, Purohit A: The therapeutic potential of a series of bioavailable anti-angiogenic microtubule disruptors as therapy for hormone-independent prostate and breast cancers. Br J Cancer. 2007, 97: 1673-1682. 10.1038/sj.bjc.6604100.PubMed CentralView ArticlePubMedGoogle Scholar
- Wood L, Leese MP, Mouzakiti A, Purohit A, Potter BV, Reed MJ: Packham Gl: 2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases. Apoptosis. 2004, 9: 323-332.View ArticlePubMedGoogle Scholar
- Utsumi T, Leese MP, Chander SK, Gaukroger K, Purohit A, Newman SP, Potter BV, Reed MJ: The effects of 2-methoxyoestrogen sulphamates on the in vitro and in vivo proliferation of breast cancer cells. J Steroid Biochem Mol Biol. 2005, 94: 219-227. 10.1016/j.jsbmb.2005.01.022.View ArticlePubMedGoogle Scholar
- Leese LP, Jourdan FL, Gaokroger K, Mahon MF, Newman SP, Foster PA, Stengel C, Regis-Lydi S, Di Fiore A, De Simone G, Supuran CT, Purohit A, Reed MJ, Potter BVL: Structure-activity relationship of C-17 cyano-substituted estratriene as anticancer agents. J Med Chem. 2008, 51: 1295-1308. 10.1021/jm701319c.View ArticlePubMedGoogle Scholar
- Jourdan F, Leese MP, Dohle W, Hamel E, Ferrandism E, Newman SP, Purohit A, Rees MJ, Potter BVL: Synthesis, antitubulin, and antiproliferative SAR of analogues of 2-methoxyestradiol-3–17-O, O-bis-sulfamate. J Med Chem. 2010, 53: 2942-2951. 10.1021/jm9018806.View ArticlePubMedGoogle Scholar
- Newman SP, Ireson CR, Tutill HJ: The role of 17β-hydroxysteriod dehydrogenase in modulating the activity of 2-methoxyestradiol in breast cancer cells. Cancer Res. 2006, 66: 324-330. 10.1158/0008-5472.CAN-05-2391.View ArticlePubMedGoogle Scholar
- Landes T, Martinou JC: Mitochondrialouter membrane permeabilization during apoptosis: the role of mitochondrial fission. Biochim Biophys Acta. 2011, 1813: 540-545. 10.1016/j.bbamcr.2011.01.021.View ArticlePubMedGoogle Scholar
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J. 2000, 19: 5720-5728. 10.1093/emboj/19.21.5720.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T: Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol. 2001, 152: 657-667. 10.1083/jcb.152.4.657.PubMed CentralView ArticlePubMedGoogle Scholar
- Mqoco T, Joubert A: Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells. Biocell. 2010, 34:Google Scholar
- Vorster CJJ, Joubert AM: In vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A cell line. Cell Biochem Func. 2010, 28: 412-419. 10.1002/cbf.1671.View ArticleGoogle Scholar
- Visagie MH, Joubert AM: In vitro effects of 2-methoxyestradiol-bis-sulphamate on reactive oxygen species and possible apoptosis induction in a breast adenocarcinoma cell line. Cancer Cell Int. 2011, 11: 1-7. 10.1186/1475-2867-11-1.View ArticleGoogle Scholar
- Yang J, Wu LJ, Tashino SI, Onodera S, Ikejima T: reactive oxygen species and nitric oxide regulate mitochondrial-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells. Free Radic Res. 2008, 42: 492-504. 10.1080/10715760802112791.View ArticlePubMedGoogle Scholar
- Circu ML, Aw TY: reactive oxygen species, cellular redox systems, and apoptosis. Free Rad Biol Med. 2010, 48: 749-762. 10.1016/j.freeradbiomed.2009.12.022.PubMed CentralView ArticlePubMedGoogle Scholar
- Shrotriya S, Deep G, Gu M, Kaur M, Jain AK, Inturi S, Agarwal R, Agarwal C: generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck sqamous cell carcinoma cells. Carcimogenesis. 2012, in pressGoogle Scholar
- Azad MB, Chen Y, Gibson B: Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and cancer. Antioxid Redox Signal. 2009, 11 (4): 1-14.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.