Reactive oxygen species mediate soft corals-derived sinuleptolide-induced antiproliferation and DNA damage in oral cancer cells
OncoTargets and Therapy
reactive oxygen species mediate soft corals- derived sinuleptolide-induced antiproliferation and Dna damage in oral cancer cells
0 hsueh-Wei chang Department of Biomedical science and environmental Biology, Kaohsiung Medical University , 100, shih-chuan 1st road, Kaohsiung 80708 , Taiwan
1 Department of Medical research, Kaohsiung Medical University hospital , Kaohsiung , Taiwan
2 r esearch c enter for n atural Products and Drug Development, Kaohsiung Medical University , Kaohsiung , Taiwan
3 Department of Marine Biotechnology and resources, n ational sun Yat-sen University , Kaohsiung , Taiwan
4 Doctoral Degree Program in Marine Biotechnology , academia sinica,Taipei , Taiwan
5 Doctoral Degree Program in Marine Biotechnology, n ational sun Yat-sen University , Kaohsiung , Taiwan
6 c ancer c enter, Kaohsiung Medical University hospital; Kaohsiung Medical University , Kaohsiung , Taiwan
7 Department of Medical r esearch, c hina Medical University hospital, china Medical University , Taichung , Taiwan
8 Department of Biomedical science and environmental Biology, Kaohsiung Medical University , Kaohsiung , Taiwan
9 Department of radiation Oncology, Kaohsiung Municipal Ta-Tung hospital , Kaohsiung , Taiwan
10 Department of radiation Oncology, Faculty of Medicine, college of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
11 institute of Medical science and Technology, n ational sun Yat-sen University , Kaohsiung , Taiwan
12 Frontier c enter for Ocean science and Technology, n ational sun Yat-sen University , Kaohsiung , Taiwan
We previously reported that the soft coral-derived bioactive substance, sinuleptolide, can inhibit the proliferation of oral cancer cells in association with oxidative stress. The functional role of oxidative stress in the cell-killing effect of sinuleptolide on oral cancer cells was not investigated as yet. To address this question, we introduced the reactive oxygen species (ROS) scavenger (N-acetylcysteine [NAC]) in a pretreatment to evaluate the sinuleptolide-induced changes to cell viability, morphology, intracellular ROS, mitochondrial superoxide, apoptosis, and DNA damage of oral cancer cells (Ca9-22). After sinuleptolide treatment, antiproliferation, apoptosis-like morphology, ROS/mitochondrial superoxide generation, annexin V-based apoptosis, and γH2AX-based DNA damage were induced. All these changes were blocked by NAC pretreatment at 4 mM for 1 h. This showed that the cell-killing mechanism of oral cancer cells of sinuleptolide is ROS dependent.
soft corals; oral cancer; N-acetylcysteine; oxidative stress; γH2AX
open access to scientific and medical research
O r i g i n a l r e s e a r c h
Yung-Ting chang, 1,2,*
chiungn ian li, 6 Jing-ru liu, 6
Jyhhorng sheu, 1,3,7,8 hsueh-Wei
*These authors contributed equally
to this work
Oral cancer is the sixth most prevalent form of cancer worldwide.1,2 Treatment
options for oral cancer include surgery and chemotherapy. Several clinically approved
drugs such as cisplatin are getting ineffective due to drug resistance in oral cancer
therapy.3 Therefore, the discovery of new anti-oral cancer drugs becomes a
Marine microbes, flora, and fauna provide promising sources of bioactive natural
products, and they are used to develop well-received anticancer drugs.4–6 For example,
peptides and roe protein hydrolysates derived from marine fish have been reported
to inhibit the proliferation of oral cancer cells.7 The methanolic extract of red alga
Gracilaria tenuistipitata was found to inhibit oral cancer cell proliferation.8
Luminacin, a marine microbial extract, was reported to induce autophagy and cell death in
head and neck cancer cells.9 Accordingly, marine resources feature abundant natural
marine products with potential anticancer effects.
Recently, many soft coral-derived compounds have been reported as having
potential applications as anticancer drugs.10,11 Studies have investigated Sinularia
lochmodes-derived sinuleptolide for marine natural product identification12 and for use
in treating inflammation13 and skin cancer.14 The structure of sinuleptolide was first
derived from the soft coral Sinularia sp.15 Alternatively, sinuleptolide was extracted
from the soft corals Sinularia leptoclados and S. lochmodes in our laboratory.16,17
However, few studies have investigated the effects of sinuleptolide in the treatment
of oral cancer.
In our recent study,18 we reported that oxidative stress
was associated with the sinuleptolide-induced killing of oral
cancer cells. However, the dependence of oxidative stress
in the cell-killing effect of sinuleptolide on oral cancer cells
was not investigated. N-acetylcysteine (NAC), a glutathione
precursor for replenishing cellular glutathione storage, is
a well-known reactive oxygen species (ROS) scavenger.19
NAC pretreatment can be used to investigate the role of
oxidative stress dependence in drug and natural
productmediated cancer cell death.20–23 Therefore, the purpose of this
study is to evaluate the role of oxidative stress in the
cellkilling effects of sinuleptolide against oral cancer cells.
Materials and methods
cell cultures and chemicals
Human oral cancer cells (Ca9-22), purchased from the Health
Science Research Resources Bank (HSRRB) (Osaka, Japan),
were incubated with DMEM medium (Gibco, Grand Island,
NY, USA) and fetal bovine serum.24 Human normal
gingi.dow lsue val fibroblast cells (HGF-1), purchased from the American
/w a Type Culture Collection (ATCC; Manassas, VA, USA),
tt:sp rspe were incubated with DMEM-F12 medium (Gibco, Grand
h ro Island, NY, USA).25 These cells were maintained at 37°C
from F in a humidified 5% CO2 atmosphere. The structure and
aedd preparation of soft corals Sinularia-derived sinuleptolide
lno was isolated from S. lochmodes as described in our
previdow ous study.17 It was freshly prepared in dimethyl sulfoxide
ryap (DMSO) for cell studies. All the DMSO concentrations of
ehT sinuleptolide treatments were unified at 0.24%. NAC (Sigma,
and St Louis, MO, USA) was pretreated with 4 mM for 1 h before
tsge sinuleptolide treatment.
CellTiter 96® AQueous One Solution Cell Proliferation Assay
(MTS) (Promega Corporation, Madison, WI, USA) was
chosen to measure cell proliferation.24 After plating overnight,
Ca9-22 cells were incubated with sinuleptolide for 24 h with
or without NAC pretreatment. Finally, the MTS response
was measured by an ELISA reader (EZ Read 400 Research
Reader; BioChrom, Cambridge, UK).
intracellular r Os production
Intracellular hydrogen peroxide or other oxidizing ROS
can react with 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH-DA) and generate fluorescence.26,27 The ROS level can
be detected using flow cytometry.8 In brief, after plating
overnight, Ca9-22 cells were incubated with sinuleptolide for 3 h
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with or without NAC pretreatment. After washing with PBS,
cells were incubated with 100 nM DCFH-DA in PBS at 37°C
for 30 min. After harvesting, cells were resuspended in 1 mL
PBS for flow cytometry analysis (BD Accuri™ C6; Becton,
Dickinson and Company, Franklin Lakes, NJ, USA). Mean
intensity of ROS was collected from 1×104 cell counts.
Mitochondrial superoxide production
The mitochondrial superoxide was reacted with MitoSOX™
Red (Molecular Probes; Invitrogen, Eugene, OR, USA) and
generated fluorescence.28 MitoSOX Red was also applied to
flow cytometry.18 In brief, after plating overnight, Ca9-22
cells were incubated with sinuleptolide for 1 h with or without
NAC pretreatment. Subsequently, cells were incubated with
5 µM MitoSOX 37°C for 30 min. After harvesting, cells were
resuspended in 1 mL PBS for flow cytometer analysis (BD
Accuri C6). Mean intensity of mitochondrial superoxide was
collected from 1×104 cell counts.
Dna damage by γh2aX/propidium
γH2AX is the marker of DNA double-strand breaks, and it
can be detected by flow cytometry.24 In brief,
sinuleptolidetreated cells were fixed in 70% ethanol. After washing with
BSA-T-PBS solution (1% bovine serum albumin and 0.2%
Triton X-100 in PBS; Sigma), cells were incubated with
p-Histone H2A.X (Ser 139) monoclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) and BSA-T-PBS
buffer in 1:50 dilution at 4°C for 1 h. After washing, Alexa Fluor
488-tagged secondary antibody (Jackson Laboratory, Bar
Harbor, ME, USA) with BSA-T-PBS buffer in a 1:50 dilution
was added for 30 min at room temperature. After the addition
of 20 µg/mL of propidium iodide (PI), cells were resuspended
for flow cytometry analysis (BD Accuri C6). Mean intensity
of γH2AX was collected from 1×104 cell counts.
Experimental data were analyzed and expressed as mean ±
SD. Data were analyzed using two-sample Student’s t-test
with Bonferroni correction. The P-values ,0.01 (=0.05/5)
are considered as statistically significant.
nac effect on sinuleptolide-induced cell
In the MTS assay (Figure 1A and B), sinuleptolide
concentration responsively decreased the cell viability (%) of
oral cancer cells (Ca9-22) and oral normal cells (HGF-1),
cells was significantly reduced by a pretreatment with NAC
but sinuleptolide selectively killed Ca9-22 cells and was
(P,0.002) (Figure 1A and B).
less harmful to HGF-1 cells, which was consistent with our
previous study.18 The IC
values of sinuleptolide in Ca9-22
and HGF-1 cells were 11.76 and 22.3 µg/mL, respectively.
As NAC is a common ROS scavenger,29,30 this NAC effect
was used in the present study to address the dependence of
oxidative stress for the sinuleptolide effect on oral cancer
compared with normal cells. We found that the sinuleptolide
nac effect on sinuleptolide-induced
The cell morphology was changed and became more
abnormal in sinuleptolide-treated oral cancer cells
(Ca9-22) (Figure 2A) than in HGF-1 cells (Figure 2B). At
higher concentrations of sinuleptolide (12 and 24 µg/mL),
(3–24 µg/mL)-induced cell killing in Ca9-22 and HGF-1
apoptosis-like morphological changes, such as apoptotic
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bodies and cell shrinkages, were observed in Ca9-22
cells. However, these sinuleptolide-induced
apoptosislike or abnormal morphologies were reduced by NAC
nac effect on the sinuleptolide-induced
r Os generation
81 Figure 3A and B shows the relative ROS intensity patterns
l-02 of sinuleptolide-induced ROS generation of Ca9-22
-J2u and HGF-1 cells with or without NAC pretreatment.
o1n At higher concentrations of sinuleptolide (12 and 24 µg/mL)
172 (Figure 3C), the ROS generation of Ca9-22 cells was
upregu.136 lated, which was consistent with the results of our previous
..73 study.18 After NAC pretreatment, the sinuleptolide-induced
y45 ROS generation of Ca9-22 cells was significantly reduced by
/bm NAC pretreatment (P,0.002) (Figure 3C). In contrast, the
.sco ROS generation of HGF-1 cells was maintained at a basal
rvpee l.yno level with or without NAC pretreatment (Figure 3D).
nac effect on the generation of
Mitochondria-specific ROS staining dye (MitoSOX Red) was
used to evaluate mitochondrial superoxide by flow cytometry.
Figure 4A and B shows the relative mitochondrial
superoxide intensity patterns of NAC pretreatment effects against
sinuleptolide-treated oral cancer and normal cells. Higher
concentrations of sinuleptolide (12 and 24 µg/mL) (Figure 4C
and D) induced the mitochondrial superoxide generation of
Ca9-22 and HGF-1 cells. NAC pretreatment significantly
reduced the sinuleptolide-induced mitochondrial superoxide
generation of Ca9-22 and HGF-1 cells (P,0.002).
nac effect on sinuleptolide-induced
γh2aX/Pi-based Dna damage
Figure 5A and B shows the relative γH2AX intensity
patterns of sinuleptolide-induced DNA damage in Ca9-22 and
HGF-1 cells with or without NAC pretreatment. The higher
concentrations of sinuleptolide (12 and 24 µg/mL) (Figure 5C)
dramatically induced the γH2AX expression of Ca9-22 cells,
which was consistent with our previous study.18 After NAC
pretreatment, the sinuleptolide-induced γH2AX expression
in Ca9-22 cells was significantly reduced by NAC
pretreatment (P,0.002). In contrast, sinuleptolide-induced γH2AX
expression of HGF-1 cells was maintained at a basal level
with or without NAC pretreatment (Figure 5D).
We could show here that NAC pretreatment inhibited
sinuleptolide-induced cell killing, apoptosis-like morphology,
and apoptosis of oral cancer cells. This indicated the role of
oxidative stress in the cytotoxicity provided by sinuleptolide
for oral cancer cells. Moreover, oxidative stress is involved
in early apoptosis31 and mitochondrial dysfunction.32–34 NAC
can interact with ROS, such as hydrogen peroxide, hydroxyl
radical, superoxide, and hypochlorous acid.35 Consistently,
we also found that NAC pretreatment inhibited
sinuleptolideinduced ROS and the generation of mitochondrial superoxide.
However, NAC is known to show other properties as well.
For example, NAC also has anti-inflammatory effects.36–38
Since our study lacks experiments to exclude this possibility,
sinuleptolide-induced cytotoxicity of oral cancer cells may
also include other than an “ROS-dependent” mechanism.
DCFH-DA is used to detect intracellular ROS such as
hydrogen peroxide, but does not specifically detect
mitochondrial ROS. In contrast, MitoSOX Red dye has been reported to
selectively detect superoxide in the mitochondria of live cells
rather than other ROS.39 The role of mitochondrial superoxide
was first reported as being involved in sinuleptolide-induced
cytotoxicity in the present study. However, sinuleptolide
induces cell death in both kinds of cells with different IC50
values (Ca9-22=11.76 and HGF-1=22.3 µg/mL). The reason for
some differences only observed with ROS and DNA damage
in sinuleptolide-treated cells remains unclear as yet. One
possiinvestigated in the current study. Furthermore, the status of p53
bility is that mitochondrial and cytoplasmic ROS have diverse
in Ca9-22 cells is a mutant form,43,44 but it is wild-type (wt)
functions. For example, accumulating evidence suggests that
in the HGF-1 cells. Cells with mutant or wt p53 may display
mitochondrial ROS are important for normal cell
functiondifferent responses. For example, hyperthermia induced
apoping.40 Mitochondrial and cytoplasmic ROS may play opposing
tosis in oral squamous cell carcinoma (OSCC) cells (wt p53)
effects throughout the life cycle. For Caenorhabditis elegans,
and decreased IL-12 expression, but it increased IL-12Rβ1 in
the increase of mitochondrial ROS increases the lifespan of this
OSCC (mutated p53).45 Accordingly, the role of p53 in
sinuinvertebrate, whereas the increase of cytoplasm ROS decreases
leptolide-induced antiproliferation and DNA damage effects of
its lifespan.41 However, the detailed function of mitochondrial
oral cancer cells warrants further investigation in the future.
and cytoplasmic ROS in sinuleptolide-induced cytotoxicity
Mitochondria are commonly assumed to have a tubular
against cancer cells warrants further investigation.
form in healthy cells, but donut or blob forms increased with
p53 is reported to highly regulate redox homeostasis and to
mitochondrial superoxide,46 suggesting that mitochondrial
modulate several ROS-regulating genes.42 In contrast, ROS can
shape may change at different conditions of mitochondrial
modify p53 conformation to adjust the transcription of p53.42
ROS generation. The mitochondrial superoxide intensity
In the current study, ROS was validated to play an important
increased upon oxidative stress with inhibitors of the
role in sinuleptolide-induced cell death of oral cancer cells.
mitochondrial complex I (rotenone) and mitochondrial
However, the role of p53 in sinuleptolide-treated cells was not
complex II (antimycin).46 NAC pretreatment has been reported
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to reduce mitochondrial superoxide levels and mitochondrial
donut or blob formations.46 This suggests further investigation
of sinuleptolide-induced mitochondrial shape change.
Moreover, oxidative stress commonly induces DNA
damage,47 but we found that sinuleptolide-induced DNA
damage was reduced by NAC pretreatment, suggesting that
oxidative stress plays an important role in the DNA damaging
effect of sinuleptolide in oral cancer cells. In addition, the
sinuleptolide-induced DNA damage effect may have led to
apoptosis in our previous study.18 Our preliminary result also
found that sinuleptolide-induced apoptosis may be reduced
by NAC pretreatment as indicated by the cleaved Poly
(ADP-ribose) polymerase (PARP) assay (data not shown).
Some oxidative stress modulating drugs have been
reported to modulate the endoplasmic reticulum (ER) stress
provided by oxidative stress.48,49 ROS has also been reported
to induce autophagy when exposed to nonmarine drugs
and marine drugs.50 Because sinuleptolide-induced
killing of oral cancer cells was shown to depend on oxidative
stress, the possible responses of ER stress and autophagy in
sinuleptolide-treated oral cancer cells warrant further
investigation. Moreover, NAC was reported to inactivate c-Jun
N-terminal kinase, p38 mitogen-activated protein kinase,
activating protein-1, and nuclear factor kappa B.51 These
kinase signaling proteins need to be further investigated in
terms of the sinuleptolide mediation in the future.
In conclusion, we demonstrated that sinuleptolide induces
cell killing, apoptosis, and DNA damage in oral cancer
cells by allowing for relatively high cellular ROS levels.
This effect was significantly reduced by NAC pretreatment
that allows ROS scavengers to reduce the actual ROS
content (Figure 6). This suggests that the marine bioactive
compound sinuleptolide kills oral cancer cells by mediating
This work was supported by funds of the Ministry of Science
and Technology (MOST 104-2320-B-037-013-MY3, MOST
104-2320-B-110-001-MY2, and MOST
105-2314-B-037036), the National Sun Yat-sen University-KMU Joint
Research Project (#NSYSU-KMU 106-p001), the Kaohsiung
Municipal Ta-Tung Hospital (KMUH105-5R61), and the
Health and welfare surcharge of tobacco products, the
Ministry of Health and Welfare, Taiwan, Republic of China
(MOHW105-TDU-B-212-134007). The authors thank
Dr Hans-Uwe Dahms for help with English editing.
The authors report no conflicts of interest in this work.
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1. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer . Oral Oncol . 2009 ; 45 ( 4-5 ): 309 - 316 .
2. Petersen PE . Oral cancer prevention and control - the approach of the World Health Organization . Oral Oncol . 2009 ; 45 ( 4-5 ): 454 - 460 .
3. Hager S , Ackermann CJ , Joerger M , Gillessen S , Omlin A . Anti-tumour activity of platinum compounds in advanced prostate cancer-a systematic literature review . Ann Oncol . 2016 ; 27 ( 6 ): 975 - 984 .
4. Sithranga Boopathy N , Kathiresan K. Anticancer drugs from marine flora: an overview . J Oncol . 2010 ; 2010 : 214186 .
5. Lee JC , Hou MF , Huang HW , et al. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties . Cancer Cell Int . 2013 ; 13 ( 1 ): 55 .
6. Kim JK , Kang KA , Piao MJ , et al. Generation of reactive oxygen species and endoplasmic reticulum stress by Dictyopteris undulata extract leads to apoptosis in human melanoma cells . J Environ Pathol Toxicol Oncol . 2015 ; 34 ( 3 ): 191 - 200 .
7. Han Y , Cui Z , Li YH , Hsu WH , Lee BH . In vitro and in vivo anticancer activity of pardaxin against proliferation and growth of oral squamous cell carcinoma . Mar Drugs . 2016 ; 14 ( 1 ): 2 .
8. Yeh CC , Yang JI , Lee JC , et al. Anti-proliferative effect of methanolic extract of Gracilaria tenuistipitata on oral cancer cells involves apoptosis, DNA damage, and oxidative stress . BMC Complement Altern Med . 2012 ; 12 ( 1 ): 142 .
9. Shin YS , Cha HY , Lee BS , et al. Anti-cancer effect of luminacin, a marine microbial extract, in head and neck squamous cell carcinoma progression via autophagic cell death . Cancer Res Treat . 2016 ; 48 ( 2 ): 738 - 752 .
10. Yen WH , Hu LC , Su JH , et al. Norcembranoidal diterpenes from a Formosan soft coral Sinularia sp . Molecules . 2012 ; 17 ( 12 ): 14058 - 14066 .
11. Lin YY , Lin SC , Feng CW , et al. Anti-inflammatory and analgesic effects of the marine-derived compound excavatolide B isolated from the culture-type Formosan Gorgonian Briareum excavatum . Mar Drugs . 2015 ; 13 ( 5 ): 2559 - 2579 .
12. el Sayed KA , Hamann MT. A new norcembranoid dimer from the red sea soft coral Sinularia gardineri . J Nat Prod . 1996 ; 59 ( 7 ): 687 - 689 .
13. Takaki H , Koganemaru R , Iwakawa Y , Higuchi R , Miyamoto T. Inhibitory effect of norditerpenes on LPS-induced TNF-alpha production from the Okinawan soft coral, Sinularia sp . Biol Pharm Bull . 2003 ; 26 ( 3 ): 380 - 382 .
14. Liang CH , Wang GH , Chou TH , et al. 5 -epi-Sinuleptolide induces cell cycle arrest and apoptosis through tumor necrosis factor/ mitochondria-mediated caspase signaling pathway in human skin cancer cells . Biochim Biophys Acta . 2012 ; 1820 (7): 1149 - 1157 .
15. Shoji N , Umeyama A , Arihara S. A novel norditerpenoid from the Okinawan soft coral Sinularia sp . J Nat Prod . 1993 ; 56 ( 9 ): 1651 - 1653 .
16. Ahmed AF , Shiue RT , Wang GH , Dai CF , Kuo YH , Sheu JH . Five novel norcembranoids from Sinularia leptoclados and S. parva . Tetrahedron . 2003 ; 59 ( 8 ): 7337 - 7344 .
17. Tseng YJ , Ahmed AF , Dai CF , Chiang MY , Sheu JH . Sinulochmodins A-C, three novel terpenoids from the soft coral Sinularia lochmodes . Org Lett . 2005 ; 7 ( 17 ): 3813 - 3816 .
18. Chang YT , Huang CY , Li KT , et al. Sinuleptolide inhibits proliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress, and DNA damage . Arch Oral Biol . 2016 ; 66 : 147 - 154 .
19. Gibson KR , Neilson IL , Barrett F , et al. Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity . J Cardiovasc Pharmacol . 2009 ; 54 ( 4 ): 319 - 326 .
20. Chang HS , Tang JY , Yen CY , et al. Antiproliferation of Cryptocarya concinna-derived cryptocaryone against oral cancer cells involving apoptosis, oxidative stress, and DNA damage . BMC Complement Altern Med . 2016 ; 16 ( 1 ): 94 .
21. Chen CY , Yen CY , Wang HR , et al. Tenuifolide B from Cinnamomum tenuifolium stem selectively inhibits proliferation of oral cancer cells via apoptosis, ROS generation, mitochondrial depolarization, and DNA damage . Toxins (Basel) . 2016 ; 8 ( 11 ): 319 .
22. Shu CW , Chang HT , Wu CS , et al. RelA-mediated BECN1 expression is required for reactive oxygen species-induced autophagy in oral cancer cells exposed to low-power laser irradiation . PLoS One . 2016 ; 11 ( 9 ): e0160586 .
23. Wu CH , Bai LY , Tsai MH , et al. Pharmacological exploitation of the phenothiazine antipsychotics to develop novel antitumor agents-A drug repurposing strategy . Sci Rep . 2016 ; 6 : 27540 .
24. Chiu CC , Haung JW , Chang FR , et al. Golden berry-derived 4betahydroxywithanolide E for selectively killing oral cancer cells by generating ROS, DNA damage , and apoptotic pathways . PLoS One . 2013 ; 8 ( 5 ): e64739 .
25. Yen YH , Farooqi AA , Li KT , et al. Methanolic extracts of Solieria robusta inhibits proliferation of oral cancer Ca9-22 cells via apoptosis and oxidative stress . Molecules . 2014 ; 19 ( 11 ): 18721 - 18732 .
26. Chan WH , Wu HJ , Hsuuw YD . Curcumin inhibits ROS formation and apoptosis in methylglyoxal-treated human hepatoma G2 cells . Ann N Y Acad Sci . 2005 ; 1042 : 372 - 378 .
27. Carter WO , Narayanan PK , Robinson JP . Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells . J Leukoc Biol . 1994 ; 55 ( 2 ): 253 - 258 .
28. Mukhopadhyay P , Rajesh M , Yoshihiro K , Hasko G , Pacher P . Simple quantitative detection of mitochondrial superoxide production in live cells . Biochem Biophys Res Commun . 2007 ; 358 ( 1 ): 203 - 208 .
29. Hseu YC , Lee MS , Wu CR , et al. The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway . J Agric Food Chem . 2012 ; 60 ( 9 ): 2385 - 2397 .
30. Shih HC , El-Shazly M , Juan YS , et al. Cracking the cytotoxicity code: apoptotic induction of 10-acetylirciformonin B is mediated through ROS generation and mitochondrial dysfunction . Mar Drugs . 2014 ; 12 ( 5 ): 3072 - 3090 .
31. Samhan-Arias AK , Martin-Romero FJ , Gutierrez-Merino C . Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis . Free Radic Biol Med . 2004 ; 37 ( 1 ): 48 - 61 .
32. Oh SH , Lim SC . A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation . Toxicol Appl Pharmacol . 2006 ; 212 ( 3 ): 212 - 223 .
33. Li JJ , Tang Q , Li Y , et al. Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid . Acta Pharmacol Sin . 2006 ; 27 ( 8 ): 1078 - 1084 .
34. Ehlers RA , Hernandez A , Bloemendal LS , Ethridge RT , Farrow B , Evers BM . Mitochondrial DNA damage and altered membrane potential (delta psi) in pancreatic acinar cells induced by reactive oxygen species . Surgery . 1999 ; 126 ( 2 ): 148 - 155 .
35. Aruoma OI , Halliwell B , Hoey BM , Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid . Free Radic Biol Med . 1989 ; 6 ( 6 ): 593 - 597 .
36. Atalay F , Odabasoglu F , Halici M , et al. N-acetyl cysteine has both gastro-protective and anti-inflammatory effects in experimental rat models: its gastro-protective effect is related to its in vivo and in vitro antioxidant properties . J Cell Biochem . 2016 ; 117 ( 2 ): 308 - 319 .
37. Lasram MM , Lamine AJ , Dhouib IB , et al. Antioxidant and anti-inflammatory effects of N-acetylcysteine against malathioninduced liver damages and immunotoxicity in rats . Life Sci . 2014 ; 107 ( 1-2 ): 50 - 58 .
38. Sadowska AM , Manuel YKB , De Backer WA. Antioxidant and antiinflammatory efficacy of NAC in the treatment of COPD: discordant in vitro and in vivo dose-effects: a review . Pulm Pharmacol Ther . 2007 ; 20 ( 1 ): 9 - 22 .
39. Mukhopadhyay P , Rajesh M , Hasko G , Hawkins BJ , Madesh M , Pacher P . Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy . Nat Protoc . 2007 ; 2 ( 9 ): 2295 - 2301 .
40. Sena LA , Chandel NS . Physiological roles of mitochondrial reactive oxygen species . Mol Cell . 2012 ; 48 ( 2 ): 158 - 167 .
41. Schaar CE , Dues DJ , Spielbauer KK , et al. Mitochondrial and cytoplasmic ROS have opposing effects on lifespan . PLoS Genet . 2015 ; 11 ( 2 ): e1004972 .
42. He Z , Simon HU . A novel link between p53 and ROS . Cell Cycle . 2013 ; 12 ( 2 ): 201 - 202 .
43. Imai Y , Ohnishi K , Yasumoto J , et al. Glycerol enhances radiosensitivity in a human oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 gene via Bax-mediated induction of apoptosis . Oral Oncol . 2005 ; 41 ( 6 ): 631 - 636 .
44. Kaneda Y , Shimamoto H , Matsumura K , et al. Role of caspase 8 as a determinant in chemosensitivity of p53-mutated head and neck squamous cell carcinoma cell lines . J Med Dent Sci . 2006 ; 53 ( 1 ): 57 - 66 .
45. Yasumoto J , Kirita T , Takahashi A , et al. Apoptosis-related gene expression after hyperthermia in human tongue squamous cell carcinoma cells harboring wild-type or mutated-type p53 . Cancer Lett . 2004 ; 204 ( 1 ): 41 - 51 .
46. Ahmad T , Aggarwal K , Pattnaik B , et al. Computational classification of mitochondrial shapes reflects stress and redox state . Cell Death Dis . 2013 ; 4 : e461 .
47. Orrenius S. Mechanisms of Oxidative Cell Damage . Basel, Switzerland: Birkhäuser Verlag; 1993 .
48. Farooqi AA , Li KT , Fayyaz S , et al. Anticancer drugs for the modulation of endoplasmic reticulum stress and oxidative stress . Tumour Biol . 2015 ; 36 ( 8 ): 5743 - 5752 .
49. Kim JK , Kang KA , Ryu YS , et al. Induction of endoplasmic reticulum stress via reactive oxygen species mediated by luteolin in melanoma cells . Anticancer Res . 2016 ; 36 ( 5 ): 2281 - 2289 .
50. Farooqi AA , Fayyaz S , Hou MF , Li KT , Tang JY , Chang HW . Reactive oxygen species and autophagy modulation in non-marine drugs and marine drugs . Mar Drugs . 2014 ; 12 ( 11 ): 5408 - 5424 .
51. Zafarullah M , Li WQ , Sylvester J , Ahmad M. Molecular mechanisms of N-acetylcysteine actions . Cell Mol Life Sci . 2003 ; 60 ( 1 ): 6 - 20 .