Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma

  • Authors:
    • Xiaoran Yin
    • Rong Zhang
    • Cheng Feng
    • Jun Zhang
    • Dong Liu
    • Kun Xu
    • Xijing Wang
    • Shuqun Zhang
    • Zongfang Li
    • Xinlian Liu
    • Hongbing Ma
  • View Affiliations

  • Published online on: July 25, 2014     https://doi.org/10.3892/or.2014.3361
  • Pages:1748-1756
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with high incidence and mortality worldwide. Diallyl disulfide (DADS) is a natural organosulfur compound, isolated from garlic. In this study, MTT assay showed that DADS significantly reduced cell viability in a dose- and time-dependent manner in ESCC cells, with lower toxicity in normal liver cells. Cell cycle analysis revealed that DADS made G2/M phase arrest. Molecular analysis suggested that this cell cycle arrest was likely made by the decrease of cyclin B1, cdc2, p-cdc2, cdc25c in concomitance with activation of the p53/p21 pathway. Apoptosis was detected by Annexin V/PI staining. The molecule markers showed that DADS induced apoptosis through activating caspases, altering the Bax/Bcl-2 balance and suppressing the MEK-ERK pathway. Our data indicated that DADS has the potential to be an effective and safe anticancer agent for ESCC therapy in the near future.

Introduction

Esophageal squamous cell carcinoma (ESCC) is one of the most refractory malignant diseases worldwide, and more than 50% of cases occur in China. Increasing rates of morbidity and mortality of ESCC have been reported in recent years (1). Current treatment generally employs surgical resection combined with chemotherapy and radiotherapy for advanced ESCC. However, both the high recurrence rate and strong systemic toxicity of the anticancer treatments led us to investigate new agents to improve the therapeutic effect (2).

In recent years, plant food-derived phytochemical anticancer strategies have been proposed to control various types of carcinoma (3). Diallyl disulfide (DADS), CH2=CH-CH2-SS-CH2CH=CH2, is one of the fat-soluble sulfur compounds obtained from crushed garlic and represents 40–60% of garlic essential oil (4). DADS has a wide variety of biological activities including antifungal (5), antibacterial (6), antiviral (7), antioxidant (8), antiplatelet and antithrombotic properties (9). Moreover, DADS has gained increasing attention due to its protective effects of cancer development and anticancer effects against different types of malignancies (1013). Notably, the apoptotic effects of DADS were even superior to those of chemotherapy agents such as 5-Fu and CTX in human breast cancer cells (14).

Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions, which plays an important role in the treatment of cancer (1517). The exact mechanism of DADS on ESCC is still unclear. The ability of DADS to control tumor growth may be due to its capacity to interfere different pathways presented in the cell.

In the present study, we characterized the effects of DADS on ESCC cell line ECA109. Our results support the use of DADS as a potent anticancer drug by disrupting cell viability, arresting G2/M cell cycle arrest and inducing apoptosis of ECA109 cells. These data suggest that DADS may be a suitable candidate for the treatment of ESCC in the future.

Materials and methods

Materials

ECA109 human ESCC cell line and L02 human normal liver cell line were purchased from the Chinese Academy of Shanghai Institute of Cell Biology. DADS, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), propidium iodide (PI), RNase A and phosphate-buffered saline (PBS) were purchased from Sigma Aldrich (USA). Fetal bovine serum (FBS), RPMI-1640 medium and 0.25% trypsin digest were purchased from Hyclone Co. (USA). TRIzol reagent was purchased from Invitrogen (USA). Annexin V conjugated to FITC (Annexin V-FITC) apoptosis kit was purchased from Roche Technology Co. (USA). PrimeScript™ RT Master Mix kit, SYBR® Premix Ex Taq II were purchased from Takara Technology Company (Japan). Enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Sciences (UK). Antibodies to ERK1/2, phospho-ERK1/2 (p-ERK1/2), MEK1, phospho-MEK1 (p-MEK1), and caspase-3, cleaved caspase-3, cyclin B1, cdc2, phospho-cdc2 (p-cdc2), cdc25c, and PCNA were purchased from Cell Signaling Technology Co. (USA). β-actin antibody was purchased from Santa Cruz Biotechnology (USA). Horseradish peroxidase (HRP)-coupled goat anti-mouse IgG and anti-rabbit IgG (secondary antibody) were purchased from Santa Cruz Biotechnology (USA).

Cell culture

Human ECA109 cell line and human L02 cell line were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate, at 37°C with 5% CO2. The medium was replaced every other day. After reaching 70–80% confluency, cells were digested with 0.25% trypsin and subcultured. Exponentially growing cells were used for assays.

Cell viability assay

Cell viability was determined by using MTT assay. DADS was dissolved in PBS with less than 0.01% DMSO and prepared at a concentration of 1,000 μg/ml. PBS with less than 0.01% DMSO was used as the negative control. ECA109 and L02 cells were seeded in 96-well plates at 1×104 cells/well and incubated with DADS (10–60 μg/ml). After incubation, 20 μl MTT (5 mg/ml) was added to each well. Four hours later, the blue crystals were solubilized with 150 μl DMSO. The absorbance was measured at 570 nm. The following formula was used for the calculation: Cell viability ratio = 1 - [(A value of the control - A value of the experimental samples)/A value of the control] × 100%. Half maximal inhibitory concentration (IC50) values were calculated by SPSS® software. Each assay was performed in 5 replicates.

Cell cycle assay

A total of 5×105 ECA109 cells/plate in 6 cm plates were incubated with different concentrations of DADS (20, 40, 60 μg/ml). PBS with less than 0.01% DMSO was used as control. Following 24 h treatment, cells were fixed with ice-cold 70% ethanol overnight at −20°C, then stained with 5 μl PI at room temperature for 30 min in the dark. Finally, cell cycle analysis was performed using Flow Cytometer, and data were analyzed with Cell Quest Pro® software. The experiments were repeated three times.

Apoptosis assay

The apoptotic ratios of cells were determined with the Annexin V-FITC apoptosis detection kit. A total of 5×105 ECA109 cells/plate in 6 cm plates were incubated with different concentrations of DADS (20, 40, 60 μg/ml). Cells treated with PBS with less than 0.01% DMSO were used as control. The cells were trypsinized, washed twice with PBS, resuspended in 100 μl of binding buffer at a concentration of 1×106 cells/ml, incubated with 5 μl Annexin V-FITC and 10 μl PI for 15 min at room temperature in the dark, and added with 400 μl binding buffer. Apoptosis analysis was performed by Flow Cytometer, and data were analyzed with Cell Quest Pro® software. The experiments were repeated at least three times.

Real-time polymerase chain reaction (PCR)

Cells were treated with serially diluted concentrations of DADS (20, 40, 60 μg/ml) and harvested for 24 h. PBS with less than 0.01% DMSO was used as control. Total RNA was extracted by the TRIzol method and the quantity of RNA was assessed by spectrophotometry. cDNA was obtained by reverse transcription with 1 μg total RNA using PrimeScript™ RT Master Mix kit. Real-time PCR was performed with the SYBR-Premix Ex Taq™ II Perfect Real Time kit. All reactions were performed on an ABI QPCR System. The individual values were normalized to the β-actin control, and the ratio of the relative expression levels was calculated. The specific primers are shown in Table I. The samples were run in triplicate.

Table I

Primers for real-time PCR analysis.

Table I

Primers for real-time PCR analysis.

GeneForward sequenceReverse sequence
β-actin 5′-TGGCACCCAGCACAATGAA-3′ 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′
p21 5′-AACATGTTGAGCTCTGGCATAGAAG-3′ 5′-GCATGGGTTCTGACGGACA-3′
p53 5′-TCAGCATCTTATCCGAGTGGAA-3′ 5′-TGTAGTGGATGGTGGTACAGTCA-3′
Bcl-2 5′-GTGTGGAGAGCGTCAACC-3′ 5′-CTTCAGAGACAGCCAGGAG-3′
Bax 5′-ATGGGCTGGACATTGGACTTC-3′ 5′-TGGTGAGTGAGGCGGTGAG-3′
Western blot assay

ECA109 cells were prepared by serially diluted concentrations of DADS (20, 40, 60 μg/ml). Cells treated with PBS with less than 0.01% DMSO were used as control. After incubation for 24 h, cells were harvested to obtain protein concentrations by RIPA lysis buffer. Proteins (50 μg) were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, then probed with appropriate primary antibodies. HRP-conjugated goat anti-mouse antibody or anti-rabbit antibody (1:10,000) was used as the secondary antibody. Antigen-antibody complex signals were visualized with BeyoECL Plus. The densitometric analysis was performed by Image J® software. The experiments were repeated three times.

The following primary antibodies were used: anti-cyclin B1 (1:1,000), anti-cdc2 (1:1,000), anti-p-cdc2 (1:500), anti-cdc25c (1:1,000), anti-ERK1/2 (1:1,000), anti-p-ERK1/2 (1:300), anti-caspase-3 (1:1,000), anti-cleaved caspase-3 (1:500), anti-MEK1 (1:1,000), anti-p-MEK1 (1:500) and anti-β-actin (1:1,000) antibodies.

Quantification and statistical analysis

Quantitative data are expressed as the mean ± SD from at least three independent experiments. The two-tailed Student’s t-test was performed for paired samples, one-way analysis of variance (ANOVA) or two-factor factorial ANOVA was used for multiple groups. Results were considered to indicate a statistically significant difference at the P<0.05 level. Statistical analyses were performed by SPSS 17.0 statistical software.

Results

DADS inhibits cell viability

The MTT assay was used to detect the viability effects of DADS at various concentrations (10–60 μg/ml) on ECA109 and L02 cells. Our data showed that DADS clearly inhibited cell viability at the concentrations of 10–60 μg/ml following exposure for 24 h (P<0.05, Fig. 1), 20–60 μg/ml for 48 and 72 h (P<0.01, Fig. 1) compared with the control group. The IC50 was 49.02±4.78, 33.14±5.02 and 22.74±4.05 μg/ml for 24, 48 and 72 h DADS treatment respectively. The 72-h treatment group had an apparent difference compared with the 24-h treatment group (P<0.05, Fig. 1). DADS had less of an influence on L02 normal cells than on ECA109 esophageal carcinoma cells (P<0.05, Fig. 2). These results indicate that DADS significantly inhibited ECA109 cell viability in a dose- and time-dependent manner, and had a lower effect on normal cells.

DADS induces G2/M phase cell cycle arrest

Cell cycle distribution of ECA109 cells treated with different doses of DADS for 24 h was analyzed by flow cytometry. Results showed that the percentage of cells in the DADS groups, particularly at the 20, 40, 60 μg/ml concentrations, significantly increased at the G2/M phase in a dose-dependent manner compared with the control group. The percentage of cells at the G2/M phase increased from 13.43±1.25 to 58.65±3.79%, and decreased from 60.01±3.13 to 18.97±1.37% and from 26.56±2.02 to 22.38±2.56%, at the G0/G1 and S phase, respectively (Table II). These results suggest that DADS induced cell cycle arrest at the G2/M phase (Fig. 3), which may be one of the reasons for the viability inhibition and apoptosis induction of ECA109 cells.

Table II

Effect of DADS on the cell cycle of ECA109 cells.

Table II

Effect of DADS on the cell cycle of ECA109 cells.

GroupsNG0/G1G2/MS
Control360.01±3.1313.43±1.2526.56±2.02
20 μg/ml348.51±2.65a28.43±2.56a23.06±2.45b
40 μg/ml342.08±1.12a39.90±2.06a18.02±1.24b
60 μg/ml318.97±1.37a58.65±3.79a22.38±2.56b

{ label (or @symbol) needed for fn[@id='tfn1-or-32-04-1748'] } Data are expressed as the mean ± SD from three independent experiments.

a P<0.01,

b P<0.05 vs. control group.

DADS induces cell apoptosis

ECA109 cells were examined by phase contrast microscopy after incubation with DADS at different concentrations (20, 40, 60 μg/ml) for 24 h. Cells treated with PBS were used as control. The control group cells showed a typical polygonal and intact appearance, whereas the DADS-treated cells displayed dose-dependent changes in cell shape, such as membrane blebbing, formation of apoptotic bodies, cellular shrinkage, poor adherence and floating shapes (Fig. 4).

The suppression of cancer cell growth correlates with apoptosis. We explored the apoptosis rate of ECA109 cells impacted with different concentrations of DADS for 24 h with Annexin V-FITC and PI staining observed by flow cytometry. The rate of apoptosis in the control and DADS groups (20, 40 and 60 μg/ml) for 24 h was 10.26±1.05, 14.48±0.99, 42.68±2.08 and 72.96±3.43% respectively (P<0.05, Fig. 5). These results demonstrate that DADS induced apoptosis of ECA109 cells in a dose-dependent manner.

DADS inhibits G2/M phase-associated protein expression of cyclin B1, cdc2, p-cdc2 and cdc25c

Since DADS induced G2/M phase arrest in ECA109 cells, we evaluated the expression of proteins that regulate the G2/M phase transition by western blot assay. Cyclin B1, cdc2, and cdc25c are important proteins related to the G2/M phase. G2/M phase is controlled by a complex formed cyclin B1 and cdc2, and the complex is regulated by cdc25c. Our data showed that cdc2 protein levels had no apparent change (P>0.05, Fig. 6), and the protein levels of cyclin B1 and cdc25c decreased in a dose-dependent manner with significant inhibition occurring at DADS concentrations of 40 and 60 μg/ml (P<0.01, Fig. 6). Moreover, the protein levels of p-cdc2 decreased in a dose-dependent manner, with significant inhibition occurring at DADS concentrations of 20, 40 and 60 μg/ml (P<0.01, Fig. 6). These results indicate that DADS reduced the expression of cyclin B1, p-cdc2, and cdc25c to induce G2/M phase arrest of ECA109 cells.

Effects of DADS on the p53/p21 pathway

The p53/p21 pathway is involved in the cell apoptosis progression and plays an important role in the cell cycle arrest of G2/M phase. Therefore, we used real-time PCR assay to investigate the G2/M phase relative molecular pathway. Our data showed that DADS (20, 40 and 60 μg/ml) upregulated mRNA levels of p21 (P<0.01, Fig. 7), p53 (P<0.05, Fig. 7) compared with the relative control. The upregulated levels of p21 mRNA expression were accompanied by the increase of G2/M arrest. Based on these results, we suggest that DADS-increased G2/M phase arrest and apoptosis rate of ECA109 cells might be partly affected by the p53/p21 signaling pathway.

Protein expression of caspase-3 and cleaved caspase-3 by western blot analysis

As the imbalance of apoptotic signals is closely related to the initiation of the apoptotic program, we analyzed the protein expression levels of caspase-3 and cleaved caspase-3 proteins by western blotting. These proteins were blotted against the corresponding antibodies as mentioned above in the ECA109 cells, following 24 h of treatment with DADS (20, 40 and 60 μg/ml). Our results showed that the protein levels of caspase-3 increased at DADS doses of 40 and 60 μg/ml (P<0.01, Fig. 8), and the expression levels of cleaved caspase-3 fragment (17 kDa, 19 kDa) were clearly upregulated in a dose-dependent manner, particularly at 20, 40 and 60 μg/ml doses (P<0.01, Fig. 8). Therefore, this study suggests that DADS-induced apoptosis of ECA109 cells is mediated by effector caspase-3.

Effects of DADS on Bax, Bcl-2 expression levels and Bax/Bcl-2 ratio

DADS-induced apoptosis of ECA109 cells prompted an examination of certain apoptosis regulatory proteins, such as Bax and Bcl-2. The expression levels of Bax mRNA significantly increased in 40 and 60 μg/ml (P<0.05, Fig. 9), whereas the expression levels of Bcl-2 mRNA were clearly decreased in 20, 40 and 60 μg/ml (P<0.01, Fig. 9). The Bax/Bcl-2 ratio was significantly increased in the presence of DADS at concentrations of 40 and 60 μg/ml (P<0.05, Fig. 9). Collectively, the data suggest that DADS induced apoptosis through upregulation of Bax mRNA, downregulation of Bcl-2 mRNA and a shift of Bax/Bcl-2 ratio in a dose-dependent manner.

Effects of DADS on the MEK-ERK pathway

Western blot analysis was used to evaluate the effect of DADS on MEK1, ERK1/2 and their phosphorylation levels in ECA109 cells. Our data showed that the protein levels of MEK1 and ERK1/2 had no obvious change, whereas the expression levels of p-MEK1 and p-ERK1/2 were clearly decreased in the 20, 40 and 60 μg/ml DADS groups compared with the control (P<0.01, Fig. 10). As shown in Fig. 10, DADS inhibited p-MEK1 and p-ERK1/2 in a dose-dependent manner. Based on these results, we suggest that DADS-related apoptosis in ECA109 cells might be influenced by the MEK-ERK signaling pathway.

Discussion

Diallyl disulfide (DADS) is a lipid-soluble organic compound from garlic. Scientific investigations have shown that DADS can reduce the risk of cardiovascular disease and diabetes, stimulate the immune system, protect against infections, and show significant protection against different types of malignancies (4,18).

By MTT assay, DADS showed dose- and time-dependent anti-viability effects on ESCC cell lines (Figs. 1 and 2). Moreover, we further demonstrated that DADS had much lower cytotoxicity in L02 normal liver cells compared with ECA109 carcinoma cells (P<0.01). The viability inhibitory properties of DADS may be attributable to its induction of cell cycle arrest and apoptosis cell death, which is consistent with previous findings (1013).

G2/M phase cell cycle arrest can reduce proliferation and induce apoptosis by inhibiting the segregation of damaged chromosomes during mitosis (15). DADS has been demonstrated to induce G2/M cell cycle arrest in several cancer cell lines such as colon cancer (11), breast cancer (12) and human gastric cancer cells (13). In the present study, we demonstrated that the percentage of ECA109 cells in DADS treatment groups significantly increased at the G2/M phase in a dose-dependent manner (Fig. 3). The G2/M phase arrest induced by DADS showed an increase in the number of ECA109 cells from 13.43 to 58.65%, as well as apoptotic cells from 10.2 to 72.96% (Table II), suggesting that the block in the G2/M phase results in triggering the apoptotic program.

Apoptosis is a morphologically and biochemically distinct mode of cell death that plays major roles during carcinogenesis, cancer treatment and toxic cell killing (19). Apoptosis induction involves different apoptotic genes and enzymes depending on the cell type of different tumors (16). DADS has the ability to induce apoptosis of human tumor cells including those of colon, gastric, prostate and breast origin (10,11,18,2022). Our study confirmed the apoptotic effects of DADS on ECA109 cells by using several methods. First, under light microscope, we observed that morphological changes of apoptosis were induced by DADS. Typical morphological characteristics of apoptotic ECA109 cells, such as membrane blebbing, cell shrinkage and formation of apoptotic bodies, were observed in the 40, 60 μg/ml DADS dose group (Fig. 4). Second, we detected the apoptosis rate by the double-staining of Annexin V-FITC and PI. Our flow cytometry analysis showed that DADS promoted ECA109 apoptosis in a dose-dependent manner (Fig. 5).

DADS controlled tumor growth and death. Although the mechanism for DADS-induced apoptosis has been studied in several cellular systems, it remains a controversial issue. As the balance and interplay of multiple signaling networks involved in various signaling contexts, it is not surprising that DADS regulation is complex. Therefore, the results of the present study indicate the noticeable changes in molecular signals of ECA109 cells affected by DADS.

G2/M phase progression is regulated by cdc2 kinases and cyclin B1. In addition, cdc2 activation depends on the dephosphorylation of Tyr15 by cdc25c. Otherwise, p53 and the p53-responsive gene, p21, can suppress cyclin B1 and cdc2 expression by inhibiting either cdc2 kinase activity or blocking the interaction of cyclin B1-cdc2 complexes with their substrates, leading to G2/M-phase cell cycle arrest (2327). Furthermore, the MEK-ERK pathway has crosstalk with p53/p21, and Bax is a key target of the p53 transcription process in aspects of cell apoptosis (26). In the case of our study, the data showed that DADS downregulated cyclin B1, cdc2, p-cdc2 and cdc25c expression (Fig. 7) and upregulated p21 expression of ECA109 cells in a dose-dependent manner (Fig. 6). Thus, we found that upregulation of the p53/p21 pathway could be due to blocking the interaction of cyclin B1-cdc2 complexes, and downregulation of cdc25c consequently reduced the activity of cdc2. Therefore, we suggest that alterations in G2/M phase-associated protein levels and p53/p21 signaling pathway may be the mechanism underlying the growth inhibition and G2/M phase arrest.

Bax is a key target of the p53 transcription process in aspects of cell apoptosis (26). Anti-apoptotic protein Bcl-2 could bind to and inactivate Bax and other pro-apoptotic proteins to inhibit apoptosis. Therefore, Bax/Bcl-2 ratio plays a pivotal role in apoptosis (28). Moreover, caspase-3 plays an important role as the central effector for initiation of apoptosis (16). Our results suggest that DADS promoted cell death by downregulating Bcl-2 mRNA expression and upregulating the Bax expression in a dose-dependent manner, thus increasing the Bax/Bcl-2 ratio (Fig. 9), leading to a pro-apoptotic process via caspase-3 in ECA109 cells (Fig. 8). Based on these observations, the data indicate that the increase of Bax/Bcl-2 and activation of caspase-3 are involved in the DADS-induced apoptosis mechanism.

The sequential phosphorylation of MAPK kinase1/2 (MEK1/2) makes the activation of extracellular signal-regulated kinase1/2 (ERK1/2). Then, ERK1/2 activation leads to phosphorylation of a variety of transcription factors and results in proliferation and differentiation, protecting cells against apoptosis (29). MEK-ERK signaling cascade may be affected by the activation of the p53/p21 pathway (26). Although DADS was reported to rapidly and potently inhibit the phosphorylation of ERK1/2 to induce apoptosis in human leukemia cell line HL-60 (21), other studies showed that DADS activated ERK1/2 in human non-small cell lung carcinoma H1299 cell death (20). Our results showed that both p-MEK1 and p-ERK1/2 were inhibited following DADS treatment in a dose-dependent manner (Fig. 10), suggesting that the MEK-ERK pathway plays a role in maintaining the regulation of apoptosis by DADS in ECA109 cells.

In summary, the present study confirmed that DADS inhibits ESCC cell viability with only a slight effect on normal cells. To the best of our knowledge, this is the first report to identify mechanisms of the antitumor properties of DADS in human ESCC cells. DADS has been shown to arrest cancer cells at the G2/M phase via the modulation of cell-cycle related proteins, which is associated with the reduction of cyclin B1, cdc2, p-cdc2, cdc25c expression. Furthermore, DADS controlled cellular apoptosis by activating caspase-3, upregulating Bax/Bcl-2 ratio and downregulating the MEK-ERK signaling pathway. Moreover, activation of the p53/p21 pathway is involved in the process of inhibiting cell differentiation, arresting G2/M phase and inducing apoptosis. In brief, DADS regulates ESCC cells via multiple networks involved in various signaling contexts. Therefore, this study suggests that DADS may be a promising anticancer therapeutic for ESCC in the near future.

Acknowledgements

This study was supported by funds from the Science and Technology Program of Shaanxi Province (nos. 2010K01-141 and 2011K13-02-05), the Important Clinic Project of the Chinese Ministry of Health (no. 2007353). This study was also supported by the Office of Oncology Research (Zong-Fang Li). The authors are grateful to Dr Guleng B for his technical assistance.

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October 2014
Volume 32 Issue 4

Print ISSN: 1021-335X
Online ISSN:1791-2431

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APA
Yin, X., Zhang, R., Feng, C., Zhang, J., Liu, D., Xu, K. ... Ma, H. (2014). Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma. Oncology Reports, 32, 1748-1756. https://doi.org/10.3892/or.2014.3361
MLA
Yin, X., Zhang, R., Feng, C., Zhang, J., Liu, D., Xu, K., Wang, X., Zhang, S., Li, Z., Liu, X., Ma, H."Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma". Oncology Reports 32.4 (2014): 1748-1756.
Chicago
Yin, X., Zhang, R., Feng, C., Zhang, J., Liu, D., Xu, K., Wang, X., Zhang, S., Li, Z., Liu, X., Ma, H."Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma". Oncology Reports 32, no. 4 (2014): 1748-1756. https://doi.org/10.3892/or.2014.3361