Accepted Manuscript Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells
Yi Feng, Yonghua Wang, Chengrui Jiang, Zishui Fang, Zhiqiang Zhang, Xiaoying Lin, Liwei Sun, Weiying Jiang PII: DOI: Reference:
S0024-3205(17)30277-1 doi: 10.1016/j.lfs.2017.06.003 LFS 15229
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
13 March 2017 19 May 2017 3 June 2017
Please cite this article as: Yi Feng, Yonghua Wang, Chengrui Jiang, Zishui Fang, Zhiqiang Zhang, Xiaoying Lin, Liwei Sun, Weiying Jiang , Nicotinamide induces mitochondrialmediated apoptosis through oxidative stress in human cervical cancer HeLa cells, Life Sciences (2017), doi: 10.1016/j.lfs.2017.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells
Yi Feng a, Yonghua Wang a, Chengrui Jiang a, Zishui Fang a, Zhiqiang Zhang a,
RI
PT
Xiaoying Lin a, Liwei Sun a, Weiying Jiang a, ⁎
a
SC
Department of Medical Genetics, Zhongshan School of Medicine, Sun Yat-sen
University, University and Key Laboratory of Tropical Disease Control (Sun Yat-sen
MA
NU
University), Ministry of Education Guangzhou, 510080, China
⁎
Corresponding Author: Weiying Jiang, Prof.
PT E
Guangdong, 510080, China
D
Mailing address: No. 74, 2nd Zhongshan Road, Yuexiu District, Guangzhou,
Tel: +86-20-87331928
CE
Fax: +86-20-87331928
AC
E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Aims: Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both celluar survival and death. Recent
PT
studies have shown that it induced proliferation inhibition and apoptosis in many
RI
cancer cells. However, little is known about the effects of nicotinamide on human
SC
cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa
NU
cells and investigated the apoptotic mechanism.
MA
Materials and Methods:
After the treatment of the indicated concentrations nicotinamide, HeLa cell
D
proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive
PT E
oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with
CE
fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by
AC
flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Key findings: Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa
2
cell apoptosis via the intrinsic
ACCEPTED MANUSCRIPT mitochondrial apoptotic pathway. Significance: Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that
PT
nicotinamid may be an inexpensive, safe and promising therapeutic agent or a
RI
neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new
SC
drugs for cervical cancer therapy.
NU
Keywords
AC
CE
PT E
D
MA
Nicotinamide; Oxidative stress; Mitochondrial apoptotic pathway; Cervical cancer
3
ACCEPTED MANUSCRIPT 1. Introduction Cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer death among females [1]. Currently, surgery, radiotherapy and cisplatin-based chemotherapy are the primary methods for treating cervical cancer [2].
PT
However, drug resistance of cervical cancer leading to the low efficacy of the clinical
RI
therapeutics and the high cost of the treatment became the major cause of
SC
the high incidence and the high mortality rate in developing parts of the world. Hence, it is urgent to find the safe, effective, and affordable treatment for cervical cancer.
NU
Human cervical cancer HeLa cell line, derived from cervical cancer cells taken on
MA
February from a patient named Henrietta Lacks who died of her cancer on October in 1951 [3], was selected as the experimental model in the present study.
D
Nicotinamide, the amide form of niacin (vitamin B3), is the necessary nutrient
PT E
that provided by dietary source and supplement [4]. It also has been used to treat with pellagra clinically for a long time and lack of reported side effects [5]. Nicotinamide
CE
is a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD+),
AC
participating in the cellular energy metabolism in the mitochondrial electron transport chain [6]. In addition, nicotinamide also is essential for the synthesis of nicotinamide adenine dinucleotide phosphate (NADP+) [7], which is involved in the synthesis of fatty acids and cell survival under oxidative stress condition [8]. And the reduced pyridine nucleotides, NAD(P)H, depending upon an adequate supply of NAD(P)+ [8], also play a pivotal role in the regulation of cellular redox status. So far, various studies have demonstrated that nicotinamide could modify redox
4
ACCEPTED MANUSCRIPT balance in vivo and in vitro, from human fibroblasts [9] to mouse preadipocyte cells [10]. Additionally, emerging evidences reported that nicotinamide might be a potential candidate as anticancer agent or in combination with other chemotherapeutics in several cancer cell models, including pancreatic cancer [11], chronic lymphocytic
PT
leukemia [12] and prostate carcinoma [13], breast cancers [14]. Furthermore, as a
RI
traditional component of accelerated radiotherapy with carbogen and nicotinamide
SC
(ARCON), nicotinamide has been extensively studied for its radiosensitising properties, which is a therapeutic strategy used in head, neck and bladder cancer [15].
MA
disrupt homeostasis of redox status.
NU
Thus, treatment with nicotinamide in cervical cancer cells would be expected to
However, to the best of our knowledge, few data are available on the effects of
D
nicotinamide on human cervical cancer cells. Therefore, our present study
PT E
investigated the in vitro effect of nicotinamide on cell proliferation, apoptosis, production of ROS and redox-related parameters in human cervical cancer HeLa cells
CE
to explore its potential mechanism. And further identified the underlying pathway of
AC
induction of apoptosis. We found that nicotinamide effectively eliminated HeLa cells through oxidative stress-induced pathway. We expect our study can provide more evidences for nicotinamide to be a safe and inexpensive potential anticancer agent or for combined therapy for cervical cancer patients.
2. Material and Methods 2.1. Cell culture and Reagents
5
ACCEPTED MANUSCRIPT HeLa cells, the human cervical carcinoma cell line [3], were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) according to the instruction provided by the American Type Culture Collection, supplemented with 10% fetal calf serum (FBS, GEMINI) and 1% penicillin-streptomycin (Gibco) at 37 °C and 5% CO2
PT
in a humidified incubator. Nicotinamide, N-acetylcysteine (NAC) and all other
SC
culture were purchased from Corning Incorporated.
RI
chemicals were purchased from Sigma Aldrich. All sterile plastic materials for cell
2.2. Cell proliferation assay
NU
Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8, Dojindo,
MA
Molecular Technologies, Japan) as the manufacturer's instruction. Briefly, after treatment with the indicated concentrations and time points, cells were incubated with
D
50 μl of CCK-8 solution for 2 h. Optical density (450 nm) was measured using a
PT E
microplate reader (Sunrise Tecan). The growth curves were calculated as the mean OD values of each group at the different time points.
CE
2.3. Measurement of intracellular ROS
AC
Intracellular ROS was measured using 2',7'-Dichlorofluorescin diacetate (DCFH-DA, Molecular Probes). Because the groups of 0.25 and 0.5 mg/ml did not induce apparent effect, thus these two groups were excluded. Cells were plated in six-well plates and incubated for 24 h to allow exponential growth. After pretreatment with 5mM NAC for 1 h and treatment with the indicated concentrations of nicotinamide for 24 h, the cells were trypsinised and incubated with 10mM DCFH-DA for 20 min at 37 °C in the dark. Then the cells were washed twice with
6
ACCEPTED MANUSCRIPT PBS and analysed the mean fluorescence intensity immediately using flow cytometry (CytoFLEX). 2.4. Measurement of lipid peroxidation and GSH Intracellular lipid peroxidation and GSH were measured using the Lipid
PT
Peroxidation Malondialdehyde (MDA) assay kit and the Reduced glutathione assay
RI
kit (Nanjing Jiancheng, China), respectively. MDA is the biomarker of plasma
SC
membrane lipid peroxidation [16]. Briefly, after pretreatment with 5mM NAC and treatment with the indicated concentrations of nicotinamide for 24 h, the cells were
NU
harvested and then homogenized and sonicated in RIPA buffer on ice. Cells lysates
MA
were centrifuged at 12,000 g for 15 min at 4℃ to collect the supernatant, then measured their concentrations and subjected to MDA assay and GSH assay as
D
described in the manufacturer's instructions. Thereafter, the levels of MDA and GSH
PT E
were detected using microplate readers at 532 nm and 405 nm, respectively. 2.5. Morphologic analysis of Hela cells
washed
with
PBS,
examined
directly
or
stained
with
AC
were
CE
After treatment with the indicated concentrations of nicotinamide for 24 h, cells
4',6-diamidino-2-phenylindole, (DAPI, Sigma) for 5 min at 37 °C and then imaged on fluorescence microscope at 200× (Axio Observer Z1, Carl Zeiss) in order for the cellular morphology and nuclear morphology determination. 2.6. Cell apoptosis assay Annexin V-FITC/PI Apoptosis Detection Kit (BD biosciences, USA) was used to evaluate cellular apoptosis. After treatment with the indicated concentrations of
7
ACCEPTED MANUSCRIPT nicotinamide for 24 h in the absence or presence of pretreatment of 5mM NAC for 1 h, the cells were trypsinized, washed, collected and resuspended in 200 μl binding buffer, labeled with 5 μl annexin V-FITC and 5 μl PI for 15 min in the dark at room temperature. Then 300 μl binding buffer was added to each sample and the cells were
PT
evaluated by flow cytometry (CytoFLEX, Beckman Coulter).
RI
2.7. Measurement of mitochondrial membrane potential (Δψm)
SC
Δψm was measured using the mitochondrial membrane potential assay kit with JC-1 (Beyotime, China). HeLa cells were seeded at a density of 5×10^5 per well in
NU
six-well plates and incubated for 24 h. After pretreatment with NAC and treatment
MA
with the indicated concentrations of nicotinamide for 24 h, the cells were trypsinised, washed and incubated with 0.5 ml JC-1 working solution at 37 °C for 20 min.
D
Subsequently, removed the staining solution at 600 g centrifugation for 3 min, washed
PT E
the cells twice with buffer. Then the cells were resuspended in 0.3 ml buffer and analyzed using the flow cytometry (CytoFLEX).
CE
2.8. Identification of the apoptotic pathways of HeLa cells induced by
AC
nicotinamide with western blotting assay To make sure whether the apoptotic effect of HeLa cells induced by nicotinamide is mediated through activation of the intrinsic or extrinsic apoptotic pathways, HeLa cells were seeded in six-well plates, incubated for 24 h and treated with nicotinamide at the indicated concentrations for 24 h. Then total protein was extracted using the RIPA Lysis Buffer Kit (Beyotime, China). The protein concentration was measured using a BCA Kit (Beyotime, China) and equal amounts of protein was separated by a
8
ACCEPTED MANUSCRIPT 12% SDS-polyacrylamide gel and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Then the membranes were blocked by 5% non-fat milk at room temperature for 2 h and incubated with various primary antibodies against caspase-3 (Abcam, ab32351), caspase-9 (ab185719), caspase-8 (ab32379) and
PT
GAPDH (Jetway Biotech, E021060). After washing three times with TBST, the
RI
membranes were incubated with horseradish peroxidase (HRP)-conjugated goat
SC
anti-rabbit antibody (Jetway, E030120) for 1 h at room temperature. The immunoblots were visualized by the Enhanced Chemiluminescence method using ChemiDoc Touch
NU
Imaging System (Bio-Rad). Densitometric analyses were done on scanned the
MA
membranes by using the public domain ImageJ software. 2.9. Identification of redox-related genes by quantitative real-time PCR
D
(qRT-PCR)
PT E
To evaluate the expression of redox- and apoptosis-related genes, Hela cells were seeded in six-well plates, incubated for 24 h and treated with nicotinamide at the
CE
indicated concentrations for 24 h. Total RNA was isolated from the treated cells using
AC
RNAprep pure Cell Kit (Tiangen Biotech) and then was reverse transcribed to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo) according to the manufacturer's instructions. The concentration and purity of the total RNA from each sample was analyzed by the OD260/OD280 ratio using the Nanodrop 2000 (Thermo Fisher Scientific, Massachusetts, USA). Quantitative real-time PCR was carried out by applying real-time SYBR Green PCR technology with the use of the iQ5 Real Time PCR (Bio-Rad, California, USA). Each well (25μl reaction volume) contained 11 μl
9
ACCEPTED MANUSCRIPT of 2×RealStar Power SYBR Mixture (Genstar, Beijing, China), 1 μl of template, 1 μl of each primer, and 11 μl H2O. The amplification reaction conditions included an initial denaturation for 10 min at 95℃ followed by 40 cycles each of denaturation for 15 sec at 95℃, annealing for 25 sec at 60℃, extension for 35 sec at 72℃, and a final
PT
extension for 5 sec at 72℃. All the primers are listed in the Table 1. All Threshold
RI
cycle (Ct) values were repeated in triplicate. The internal standard (β-actin) was used
method, as previously reported [17].
NU
Table 1. PCR Primers used in this study
SC
to normalize the relative gene expression levels, which were calculated with the 2–ΔΔCt
Primers
GPx F
5’-CAGTCGGTGTATGCCTTCTCGG-3’
GPx R
5’-GCGTTCTCCTGATGCCCAAA-3’
Catalase F
5’-GTTGAAGATGCGGCGAGAC-3’
D
MA
Human Gene
5’-CCTGTGGCAATGGCGTTA-3’
PT E
Catalase R
5’-GGTCCTCACTTTAATCCTCTATC-3’
SOD1 F
5’-TTCTTCATTTCCACCTTTGC-3’
SOD1 R
5’-GGACAAACCTCAGCCCTAAC-3’
SOD2 R Bcl-2 F
CE
SOD2 F
5’-TTGAAACCAAGCCAACCC-3’ 5’-GGTGAACTGGGGGAGGATTG-3’ 5’-GACAGCCAGGAGAAATCAAACAG-5’
Bak F
5’-AGGAACAGGAGGCTGAAGGG-3’
Bak R
5’-CATAGCGTCGGTTGATGTCG-3’
Cytochrome c F
5’ -GCCCCTGGATACTCTTACAC-3’
Cytochrome c R
5’ -TCTGCCCTTTCTTCCTTCT-3’
β-actin F
5’-CATTAAGGAGAAGCTGTGCT-3’
β-actin R
5’ -GTTGAAGGTAGTTTCGTGGA-3’
AC
Bcl-2 R
F, forward; R,reverse.
10
ACCEPTED MANUSCRIPT 2.10. Statistical analysis All experiments were performed in triplicate in three independent trials and all data are presented as means ± SD. Statistical analysis was performed with one-way ANOVA followed by Dunnett’s or Student’s t-tests. *p < 0.05 was considered
RI
PT
statistical differences and * indicates p < 0.05 and ** indicates p < 0.01.
SC
3. Results
3.1. Nicotinamide treatment inhibits proliferation of HeLa cells
NU
The proliferation of HeLa cells treating with nicotinamide was measured using
MA
CCK-8 assay. As shown in Figure 1, different concentrations of nicotinamide did not obviously change HeLa cell viability (all p > 0.05) after treatment for 18 h. However,
D
the groups of 2 mg/ml and 4 mg/ml started to reduce the proliferation of the cells after
PT E
24 h comparing with the control group (p < 0.05). As time went on, cell viability was dramatically inhibited showing statistically significance at the groups of 2 mg/ml and
CE
4 mg/ml at 56 h and 72 h (p < 0.05). What’s more, there were no significant
AC
differences in cells proliferation at the groups below 1mg/ml (all p > 0.05). While the concentration of nicotinamide exceeded 1mg/ml, cell viability shown statistically significant difference after 18 h, suggesting nicotinamide could inhibit proliferation of HeLa cells in time- and dose-dependent manners.
11
RI
PT
ACCEPTED MANUSCRIPT
SC
3.2. Nicotinamide treatment induces oxidative stress in HeLa cells
NU
Subsequently, to test if nicotinamide altered the intracellular redox situation of HeLa cells, we investigated the production of intracellular ROS, MDA and other
MA
redox-related parameters after treatment with the rising concentrations of nicotinamide. As shown in Figure 2A-2B, the levels of ROS and MDA culminated in
PT E
D
the group of 4 mg/ml nicotinamide with the maximum production with about 3-fold and 1-fold increase, respectively, indicating nicotinamide induced oxidative damage
CE
in a dose-dependent manner in HeLa cells. Moreover, the reduced GSH levels and the expression of GPx gradually
AC
decreased with the increasing concentrations of nicotinamide and significantly declined at the group of 4 mg/ml (Figure 2C-2D). However, the mRNA level of Catalase increased about fourfold at 1 mg/ml and sevenfold at 2mg/ml group (p < 0.05), while showing no statistical difference at 4 mg/ml group comparing with the control group (Figure 2E). Likewise, SOD1/2 showed a tendency similar with Catalase (Figure 2F-2G). Taken together, these results demonstrated that treatment with nicotinamide induced oxidative stress and imbalance of redox status in HeLa 12
ACCEPTED MANUSCRIPT
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NU
SC
RI
PT
cells, mediating by the GSH depletion and severe ROS accumulation.
3.3. Nicotinamide treatment induces apoptosis of HeLa cells
D
To investigate the apoptotic effect of nicotinamide on HeLa cells, we first
PT E
observed the morphological changes of the cells and nucleus. As shown in Figure 3A, the quantity of cells altering its morphology increased with the rising of
CE
concentrations of nicotinamide, exhibiting cell shrinkage, loss of cell-to-cell contact
AC
and detachment from the plate and suggesting the manifestations of apoptosis [18]. And the proportion of chromatin condensation containing nuclear fragments was increased in a dose-dependent manner, especially at the concentration of nicotinamide exceeding 1 mg/ml (Figure 3B). We next measured the apoptotic rate using Annexin V method, the quantity of FITC+/PI− cells corresponded to early apoptosis, while FITC+/PI+ cells corresponded to late apoptosis (Figure 3C). Our results demonstrated that the higher the
13
ACCEPTED MANUSCRIPT concentration of nicotinamide, the more the cells entered apoptosis. As shown in Figure 3D-3E, the early apoptosis rate significantly increased when the concentration of nicotinamide exceeding 1 mg/ml while the late apoptosis rate was more pronounced at the groups of 4 mg/ml (p < 0.01), indicating nicotinamide-induced
PT
apoptosis began in 2 mg/ml groups and peaked in 4 mg/ml groups in this assay.
RI
To further evaluate whether the induction of oxidative stress was the primary
SC
event that triggered apoptosis in HeLa cells or not, cells were pretreated with an antioxidant NAC, the GSH precursor and ROS scavenger [19], before treatment with
NU
nicotinamide. Our results demonstrated that pretreatment of NAC consistently
MA
prevented the nicotinamide-induced ROS accumulation (Figure 2A), MDA production (Figure 2B) and completely reversed the level of GSH (Figure 2C) after exposure of 2
D
mg/ml nicotinamide in HeLa cells. Moreover, the apoptotic rate was partly reversed in
PT E
the presence of NAC (Figure 3D). These results suggested that relatively high concentration nicotinamide induced apoptosis in HeLa cells through oxidative stress,
AC
CE
at least partly.
14
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SC
RI
PT
ACCEPTED MANUSCRIPT
potential (Δψm) in HeLa cells
MA
3.4. Nicotinamide treatment triggers the collapse of mitochondrial membrane
D
Considering the pivotal role of mitochondria in orchestrating the apoptotic
PT E
pathway, we measured the Δψm using the JC-1 method. JC-1, a Δψm-sensitive dye, accumulates in the matrix of mitochondria by forming J-aggregates with high
CE
orange-red fluorescence when Δψm is high and becomes monomer with green
AC
fluorescence when Δψm is low [20]. The ratio of green and red fluorescence indicates depolarization percentage of mitochondria [21]. As shown in Figure 4A, our results showed that the ratio of green and red fluorescence increased with rising concentrations of nicotinamide, suggesting that it increased the percentage of depolarized mitochondria and triggered the collapse of Δψm in HeLa cells. Moreover, pretreatment of 5mM NAC partly restored the Δψm after exposure of 2 mg/ml nicotinamide (p
Yi Feng, Yonghua Wang, Chengrui Jiang, Zishui Fang, Zhiqiang Zhang, Xiaoying Lin, Liwei Sun, Weiying Jiang PII: DOI: Reference:
S0024-3205(17)30277-1 doi: 10.1016/j.lfs.2017.06.003 LFS 15229
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
13 March 2017 19 May 2017 3 June 2017
Please cite this article as: Yi Feng, Yonghua Wang, Chengrui Jiang, Zishui Fang, Zhiqiang Zhang, Xiaoying Lin, Liwei Sun, Weiying Jiang , Nicotinamide induces mitochondrialmediated apoptosis through oxidative stress in human cervical cancer HeLa cells, Life Sciences (2017), doi: 10.1016/j.lfs.2017.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells
Yi Feng a, Yonghua Wang a, Chengrui Jiang a, Zishui Fang a, Zhiqiang Zhang a,
RI
PT
Xiaoying Lin a, Liwei Sun a, Weiying Jiang a, ⁎
a
SC
Department of Medical Genetics, Zhongshan School of Medicine, Sun Yat-sen
University, University and Key Laboratory of Tropical Disease Control (Sun Yat-sen
MA
NU
University), Ministry of Education Guangzhou, 510080, China
⁎
Corresponding Author: Weiying Jiang, Prof.
PT E
Guangdong, 510080, China
D
Mailing address: No. 74, 2nd Zhongshan Road, Yuexiu District, Guangzhou,
Tel: +86-20-87331928
CE
Fax: +86-20-87331928
AC
E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Aims: Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both celluar survival and death. Recent
PT
studies have shown that it induced proliferation inhibition and apoptosis in many
RI
cancer cells. However, little is known about the effects of nicotinamide on human
SC
cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa
NU
cells and investigated the apoptotic mechanism.
MA
Materials and Methods:
After the treatment of the indicated concentrations nicotinamide, HeLa cell
D
proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive
PT E
oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with
CE
fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by
AC
flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Key findings: Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa
2
cell apoptosis via the intrinsic
ACCEPTED MANUSCRIPT mitochondrial apoptotic pathway. Significance: Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that
PT
nicotinamid may be an inexpensive, safe and promising therapeutic agent or a
RI
neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new
SC
drugs for cervical cancer therapy.
NU
Keywords
AC
CE
PT E
D
MA
Nicotinamide; Oxidative stress; Mitochondrial apoptotic pathway; Cervical cancer
3
ACCEPTED MANUSCRIPT 1. Introduction Cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer death among females [1]. Currently, surgery, radiotherapy and cisplatin-based chemotherapy are the primary methods for treating cervical cancer [2].
PT
However, drug resistance of cervical cancer leading to the low efficacy of the clinical
RI
therapeutics and the high cost of the treatment became the major cause of
SC
the high incidence and the high mortality rate in developing parts of the world. Hence, it is urgent to find the safe, effective, and affordable treatment for cervical cancer.
NU
Human cervical cancer HeLa cell line, derived from cervical cancer cells taken on
MA
February from a patient named Henrietta Lacks who died of her cancer on October in 1951 [3], was selected as the experimental model in the present study.
D
Nicotinamide, the amide form of niacin (vitamin B3), is the necessary nutrient
PT E
that provided by dietary source and supplement [4]. It also has been used to treat with pellagra clinically for a long time and lack of reported side effects [5]. Nicotinamide
CE
is a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD+),
AC
participating in the cellular energy metabolism in the mitochondrial electron transport chain [6]. In addition, nicotinamide also is essential for the synthesis of nicotinamide adenine dinucleotide phosphate (NADP+) [7], which is involved in the synthesis of fatty acids and cell survival under oxidative stress condition [8]. And the reduced pyridine nucleotides, NAD(P)H, depending upon an adequate supply of NAD(P)+ [8], also play a pivotal role in the regulation of cellular redox status. So far, various studies have demonstrated that nicotinamide could modify redox
4
ACCEPTED MANUSCRIPT balance in vivo and in vitro, from human fibroblasts [9] to mouse preadipocyte cells [10]. Additionally, emerging evidences reported that nicotinamide might be a potential candidate as anticancer agent or in combination with other chemotherapeutics in several cancer cell models, including pancreatic cancer [11], chronic lymphocytic
PT
leukemia [12] and prostate carcinoma [13], breast cancers [14]. Furthermore, as a
RI
traditional component of accelerated radiotherapy with carbogen and nicotinamide
SC
(ARCON), nicotinamide has been extensively studied for its radiosensitising properties, which is a therapeutic strategy used in head, neck and bladder cancer [15].
MA
disrupt homeostasis of redox status.
NU
Thus, treatment with nicotinamide in cervical cancer cells would be expected to
However, to the best of our knowledge, few data are available on the effects of
D
nicotinamide on human cervical cancer cells. Therefore, our present study
PT E
investigated the in vitro effect of nicotinamide on cell proliferation, apoptosis, production of ROS and redox-related parameters in human cervical cancer HeLa cells
CE
to explore its potential mechanism. And further identified the underlying pathway of
AC
induction of apoptosis. We found that nicotinamide effectively eliminated HeLa cells through oxidative stress-induced pathway. We expect our study can provide more evidences for nicotinamide to be a safe and inexpensive potential anticancer agent or for combined therapy for cervical cancer patients.
2. Material and Methods 2.1. Cell culture and Reagents
5
ACCEPTED MANUSCRIPT HeLa cells, the human cervical carcinoma cell line [3], were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) according to the instruction provided by the American Type Culture Collection, supplemented with 10% fetal calf serum (FBS, GEMINI) and 1% penicillin-streptomycin (Gibco) at 37 °C and 5% CO2
PT
in a humidified incubator. Nicotinamide, N-acetylcysteine (NAC) and all other
SC
culture were purchased from Corning Incorporated.
RI
chemicals were purchased from Sigma Aldrich. All sterile plastic materials for cell
2.2. Cell proliferation assay
NU
Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8, Dojindo,
MA
Molecular Technologies, Japan) as the manufacturer's instruction. Briefly, after treatment with the indicated concentrations and time points, cells were incubated with
D
50 μl of CCK-8 solution for 2 h. Optical density (450 nm) was measured using a
PT E
microplate reader (Sunrise Tecan). The growth curves were calculated as the mean OD values of each group at the different time points.
CE
2.3. Measurement of intracellular ROS
AC
Intracellular ROS was measured using 2',7'-Dichlorofluorescin diacetate (DCFH-DA, Molecular Probes). Because the groups of 0.25 and 0.5 mg/ml did not induce apparent effect, thus these two groups were excluded. Cells were plated in six-well plates and incubated for 24 h to allow exponential growth. After pretreatment with 5mM NAC for 1 h and treatment with the indicated concentrations of nicotinamide for 24 h, the cells were trypsinised and incubated with 10mM DCFH-DA for 20 min at 37 °C in the dark. Then the cells were washed twice with
6
ACCEPTED MANUSCRIPT PBS and analysed the mean fluorescence intensity immediately using flow cytometry (CytoFLEX). 2.4. Measurement of lipid peroxidation and GSH Intracellular lipid peroxidation and GSH were measured using the Lipid
PT
Peroxidation Malondialdehyde (MDA) assay kit and the Reduced glutathione assay
RI
kit (Nanjing Jiancheng, China), respectively. MDA is the biomarker of plasma
SC
membrane lipid peroxidation [16]. Briefly, after pretreatment with 5mM NAC and treatment with the indicated concentrations of nicotinamide for 24 h, the cells were
NU
harvested and then homogenized and sonicated in RIPA buffer on ice. Cells lysates
MA
were centrifuged at 12,000 g for 15 min at 4℃ to collect the supernatant, then measured their concentrations and subjected to MDA assay and GSH assay as
D
described in the manufacturer's instructions. Thereafter, the levels of MDA and GSH
PT E
were detected using microplate readers at 532 nm and 405 nm, respectively. 2.5. Morphologic analysis of Hela cells
washed
with
PBS,
examined
directly
or
stained
with
AC
were
CE
After treatment with the indicated concentrations of nicotinamide for 24 h, cells
4',6-diamidino-2-phenylindole, (DAPI, Sigma) for 5 min at 37 °C and then imaged on fluorescence microscope at 200× (Axio Observer Z1, Carl Zeiss) in order for the cellular morphology and nuclear morphology determination. 2.6. Cell apoptosis assay Annexin V-FITC/PI Apoptosis Detection Kit (BD biosciences, USA) was used to evaluate cellular apoptosis. After treatment with the indicated concentrations of
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ACCEPTED MANUSCRIPT nicotinamide for 24 h in the absence or presence of pretreatment of 5mM NAC for 1 h, the cells were trypsinized, washed, collected and resuspended in 200 μl binding buffer, labeled with 5 μl annexin V-FITC and 5 μl PI for 15 min in the dark at room temperature. Then 300 μl binding buffer was added to each sample and the cells were
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evaluated by flow cytometry (CytoFLEX, Beckman Coulter).
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2.7. Measurement of mitochondrial membrane potential (Δψm)
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Δψm was measured using the mitochondrial membrane potential assay kit with JC-1 (Beyotime, China). HeLa cells were seeded at a density of 5×10^5 per well in
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six-well plates and incubated for 24 h. After pretreatment with NAC and treatment
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with the indicated concentrations of nicotinamide for 24 h, the cells were trypsinised, washed and incubated with 0.5 ml JC-1 working solution at 37 °C for 20 min.
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Subsequently, removed the staining solution at 600 g centrifugation for 3 min, washed
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the cells twice with buffer. Then the cells were resuspended in 0.3 ml buffer and analyzed using the flow cytometry (CytoFLEX).
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2.8. Identification of the apoptotic pathways of HeLa cells induced by
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nicotinamide with western blotting assay To make sure whether the apoptotic effect of HeLa cells induced by nicotinamide is mediated through activation of the intrinsic or extrinsic apoptotic pathways, HeLa cells were seeded in six-well plates, incubated for 24 h and treated with nicotinamide at the indicated concentrations for 24 h. Then total protein was extracted using the RIPA Lysis Buffer Kit (Beyotime, China). The protein concentration was measured using a BCA Kit (Beyotime, China) and equal amounts of protein was separated by a
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GAPDH (Jetway Biotech, E021060). After washing three times with TBST, the
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membranes were incubated with horseradish peroxidase (HRP)-conjugated goat
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anti-rabbit antibody (Jetway, E030120) for 1 h at room temperature. The immunoblots were visualized by the Enhanced Chemiluminescence method using ChemiDoc Touch
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Imaging System (Bio-Rad). Densitometric analyses were done on scanned the
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membranes by using the public domain ImageJ software. 2.9. Identification of redox-related genes by quantitative real-time PCR
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(qRT-PCR)
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To evaluate the expression of redox- and apoptosis-related genes, Hela cells were seeded in six-well plates, incubated for 24 h and treated with nicotinamide at the
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indicated concentrations for 24 h. Total RNA was isolated from the treated cells using
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RNAprep pure Cell Kit (Tiangen Biotech) and then was reverse transcribed to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo) according to the manufacturer's instructions. The concentration and purity of the total RNA from each sample was analyzed by the OD260/OD280 ratio using the Nanodrop 2000 (Thermo Fisher Scientific, Massachusetts, USA). Quantitative real-time PCR was carried out by applying real-time SYBR Green PCR technology with the use of the iQ5 Real Time PCR (Bio-Rad, California, USA). Each well (25μl reaction volume) contained 11 μl
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ACCEPTED MANUSCRIPT of 2×RealStar Power SYBR Mixture (Genstar, Beijing, China), 1 μl of template, 1 μl of each primer, and 11 μl H2O. The amplification reaction conditions included an initial denaturation for 10 min at 95℃ followed by 40 cycles each of denaturation for 15 sec at 95℃, annealing for 25 sec at 60℃, extension for 35 sec at 72℃, and a final
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extension for 5 sec at 72℃. All the primers are listed in the Table 1. All Threshold
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cycle (Ct) values were repeated in triplicate. The internal standard (β-actin) was used
method, as previously reported [17].
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Table 1. PCR Primers used in this study
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to normalize the relative gene expression levels, which were calculated with the 2–ΔΔCt
Primers
GPx F
5’-CAGTCGGTGTATGCCTTCTCGG-3’
GPx R
5’-GCGTTCTCCTGATGCCCAAA-3’
Catalase F
5’-GTTGAAGATGCGGCGAGAC-3’
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Human Gene
5’-CCTGTGGCAATGGCGTTA-3’
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Catalase R
5’-GGTCCTCACTTTAATCCTCTATC-3’
SOD1 F
5’-TTCTTCATTTCCACCTTTGC-3’
SOD1 R
5’-GGACAAACCTCAGCCCTAAC-3’
SOD2 R Bcl-2 F
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SOD2 F
5’-TTGAAACCAAGCCAACCC-3’ 5’-GGTGAACTGGGGGAGGATTG-3’ 5’-GACAGCCAGGAGAAATCAAACAG-5’
Bak F
5’-AGGAACAGGAGGCTGAAGGG-3’
Bak R
5’-CATAGCGTCGGTTGATGTCG-3’
Cytochrome c F
5’ -GCCCCTGGATACTCTTACAC-3’
Cytochrome c R
5’ -TCTGCCCTTTCTTCCTTCT-3’
β-actin F
5’-CATTAAGGAGAAGCTGTGCT-3’
β-actin R
5’ -GTTGAAGGTAGTTTCGTGGA-3’
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Bcl-2 R
F, forward; R,reverse.
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ACCEPTED MANUSCRIPT 2.10. Statistical analysis All experiments were performed in triplicate in three independent trials and all data are presented as means ± SD. Statistical analysis was performed with one-way ANOVA followed by Dunnett’s or Student’s t-tests. *p < 0.05 was considered
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statistical differences and * indicates p < 0.05 and ** indicates p < 0.01.
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3. Results
3.1. Nicotinamide treatment inhibits proliferation of HeLa cells
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The proliferation of HeLa cells treating with nicotinamide was measured using
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CCK-8 assay. As shown in Figure 1, different concentrations of nicotinamide did not obviously change HeLa cell viability (all p > 0.05) after treatment for 18 h. However,
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the groups of 2 mg/ml and 4 mg/ml started to reduce the proliferation of the cells after
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24 h comparing with the control group (p < 0.05). As time went on, cell viability was dramatically inhibited showing statistically significance at the groups of 2 mg/ml and
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4 mg/ml at 56 h and 72 h (p < 0.05). What’s more, there were no significant
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differences in cells proliferation at the groups below 1mg/ml (all p > 0.05). While the concentration of nicotinamide exceeded 1mg/ml, cell viability shown statistically significant difference after 18 h, suggesting nicotinamide could inhibit proliferation of HeLa cells in time- and dose-dependent manners.
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3.2. Nicotinamide treatment induces oxidative stress in HeLa cells
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Subsequently, to test if nicotinamide altered the intracellular redox situation of HeLa cells, we investigated the production of intracellular ROS, MDA and other
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redox-related parameters after treatment with the rising concentrations of nicotinamide. As shown in Figure 2A-2B, the levels of ROS and MDA culminated in
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the group of 4 mg/ml nicotinamide with the maximum production with about 3-fold and 1-fold increase, respectively, indicating nicotinamide induced oxidative damage
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in a dose-dependent manner in HeLa cells. Moreover, the reduced GSH levels and the expression of GPx gradually
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decreased with the increasing concentrations of nicotinamide and significantly declined at the group of 4 mg/ml (Figure 2C-2D). However, the mRNA level of Catalase increased about fourfold at 1 mg/ml and sevenfold at 2mg/ml group (p < 0.05), while showing no statistical difference at 4 mg/ml group comparing with the control group (Figure 2E). Likewise, SOD1/2 showed a tendency similar with Catalase (Figure 2F-2G). Taken together, these results demonstrated that treatment with nicotinamide induced oxidative stress and imbalance of redox status in HeLa 12
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cells, mediating by the GSH depletion and severe ROS accumulation.
3.3. Nicotinamide treatment induces apoptosis of HeLa cells
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To investigate the apoptotic effect of nicotinamide on HeLa cells, we first
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observed the morphological changes of the cells and nucleus. As shown in Figure 3A, the quantity of cells altering its morphology increased with the rising of
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concentrations of nicotinamide, exhibiting cell shrinkage, loss of cell-to-cell contact
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and detachment from the plate and suggesting the manifestations of apoptosis [18]. And the proportion of chromatin condensation containing nuclear fragments was increased in a dose-dependent manner, especially at the concentration of nicotinamide exceeding 1 mg/ml (Figure 3B). We next measured the apoptotic rate using Annexin V method, the quantity of FITC+/PI− cells corresponded to early apoptosis, while FITC+/PI+ cells corresponded to late apoptosis (Figure 3C). Our results demonstrated that the higher the
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ACCEPTED MANUSCRIPT concentration of nicotinamide, the more the cells entered apoptosis. As shown in Figure 3D-3E, the early apoptosis rate significantly increased when the concentration of nicotinamide exceeding 1 mg/ml while the late apoptosis rate was more pronounced at the groups of 4 mg/ml (p < 0.01), indicating nicotinamide-induced
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apoptosis began in 2 mg/ml groups and peaked in 4 mg/ml groups in this assay.
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To further evaluate whether the induction of oxidative stress was the primary
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event that triggered apoptosis in HeLa cells or not, cells were pretreated with an antioxidant NAC, the GSH precursor and ROS scavenger [19], before treatment with
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nicotinamide. Our results demonstrated that pretreatment of NAC consistently
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prevented the nicotinamide-induced ROS accumulation (Figure 2A), MDA production (Figure 2B) and completely reversed the level of GSH (Figure 2C) after exposure of 2
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mg/ml nicotinamide in HeLa cells. Moreover, the apoptotic rate was partly reversed in
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the presence of NAC (Figure 3D). These results suggested that relatively high concentration nicotinamide induced apoptosis in HeLa cells through oxidative stress,
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at least partly.
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potential (Δψm) in HeLa cells
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3.4. Nicotinamide treatment triggers the collapse of mitochondrial membrane
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Considering the pivotal role of mitochondria in orchestrating the apoptotic
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pathway, we measured the Δψm using the JC-1 method. JC-1, a Δψm-sensitive dye, accumulates in the matrix of mitochondria by forming J-aggregates with high
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orange-red fluorescence when Δψm is high and becomes monomer with green
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fluorescence when Δψm is low [20]. The ratio of green and red fluorescence indicates depolarization percentage of mitochondria [21]. As shown in Figure 4A, our results showed that the ratio of green and red fluorescence increased with rising concentrations of nicotinamide, suggesting that it increased the percentage of depolarized mitochondria and triggered the collapse of Δψm in HeLa cells. Moreover, pretreatment of 5mM NAC partly restored the Δψm after exposure of 2 mg/ml nicotinamide (p
