Cancer Res Treat.  2019 Oct;51(4):1620-1631. 10.4143/crt.2018.340.

Low Doses of Nonylphenol Promote Growth of Colon Cancer Cells through Activation of ERK1/2 via G Protein‒Coupled Receptor 30

• Xie M
• Liang JL
• Huang HD
• Wang MJ
• Zhang T
• Yang XF

• KMID: 2460610
• DOI: http://doi.org/10.4143/crt.2018.340

Abstract

PURPOSE
Nonylphenol (NP) is an endocrine disruptor found in products such as cleaners, plastics, and detergents. It exerts actions similar to endogenous 17β-estradiol (E2) and is reported to influence various cancers. However, its role in colon cancer remains elusive.
MATERIALS AND METHODS
Colon cancer cell lines COLO 205 and SW480 were employed in our study. The cells were treated with NP or E2 followed by measurement of apoptosis and proliferation using flow cytometry and MTT assays, respectively. G protein-coupled estrogen receptor 30 (GPR30) expression was visualized using immunofluorescence and Western blot. To investigate the underlying mechanism, the expression levels of GPR30, p-protein kinase A (PKA), c-myc, cyclin D1, and ERK1/2 were analyzed using Western blot. Meanwhile, the GPR30 antagonist G15 was utilized to validate the role of GPR30 in colon cancer progression. Finally, the effect of a GPR30 inhibitor on tumor growth was determined in vivo using tumor xenograft mouse models.
RESULTS
NP facilitated the proliferation of colon cancer cells and induced apoptosis failure in vitro. Western blot revealed increased GPR30 expression levels in response to NP treatment. Cyclin D1, p-PKA, c-myc, and proliferating cell nuclear antigen, proteins that regulate the cell cycle, were all upregulated by NP, and NP-mediated ERK1/2 activation and subsequent cell proliferation were abrogated by the GPR30 inhibitor G15. Moreover, colon cancer mice that received G15 administration demonstrated impaired tumor growth in vivo.
CONCLUSION
Low dose NP promotes the growth of colon tumors through GPR30-mediated activation of ERK1/2 signaling.

Keyword

Nonylphenol; GPR30; Colon neoplasms; ERK1/2; Cell proliferation; Cyclin D1

MeSH Terms

Animals
Apoptosis
Blotting, Western
Cell Cycle
Cell Line
Cell Proliferation
Colon*
Colonic Neoplasms*
Cyclin D1
Detergents
Estrogens
Flow Cytometry
Fluorescent Antibody Technique
Heterografts
In Vitro Techniques
Mice
Phosphotransferases
Plastics
Proliferating Cell Nuclear Antigen
Cyclin D1
Detergents
Estrogens
Phosphotransferases
Plastics
Proliferating Cell Nuclear Antigen

Figure

• 

  Fig. 1. Nonylphenol (NP) increases cell proliferation and inhibits apoptosis in colon cancer cells. (A) Cell viability detected by MTT assay. (B) Apoptosis analyzed by flow cytometry. n=3. Significant difference compared to the control group (**p < 0.01). E2, 17β-estradiol.

• 

  Fig. 2. Nonylphenol (NP) increases expression of G protein–coupled estrogen receptor 30 (GPR30). (A) Immunocytochemistry staining of GPR30 (red) and nuclei (blue) in COLO 205 and SW480 cells. (B) Western blot of GPR30 levels in cells (**p < 0.01). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 3. Nonylphenol (NP) promotes cell proliferation of colon cancer cells through G protein–coupled estrogen receptor 30 (GPR30). (A) Cell viability detected by MTT assay. (B) Colony formation efficiency analyzed using soft agar colony formation assay. (C) Apoptosis analyzed by flow cytometry. n=3. Significant difference compared to the control group (*p < 0.05, **p < 0.01). G15, GPR30 inhibitor.

• 

  Fig. 4. Nonylphenol (NP) upregulates anti-apoptotic and proliferative protein expression. (A) mRNA levels of cyclin D1, c-myc, and proliferating cell nuclear antigen (PCNA) in cells detected by real-time quantitative reverse transcription polymerase chain reaction. (B) Western blot of cyclin D1, c-myc, and PCNA protein levels in cells. (C) Western blot of G protein–coupled estrogen receptor 30 (GPR30), phosphorylated protein kinase A (pPKA), and protein kinase A (PKA) protein levels in cells. Graphs represent the quantified grey-scale of bands. n=3. Significant difference compared to the control group (*p < 0.05, **p < 0.01). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant.

• 

  Fig. 5. Nonylphenol (NP) activates ERK1/2 signaling in colon cancer cells. (A) Western blot of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 proteins in COLO 205 cells. Graph represents the quantified grey-scale of bands. n=3. (B) Significant difference compared to the control group (**p < 0.01). ns, not significant.

• 

  Fig. 6. Nonylphenol (NP) accelerates tumor growth of colon carcinoma in vivo. (A) Representative images of tumors from xenografts receiving various treatments for 15 days. (B) Tumor volume was calculated every 5 days after injection, and tumors were dissected after 30 days. (C) Weight of tumors harvested from mice at the end of experiments (*p < 0.05, **p < 0.01). (D) Immunohistochemistry staining of Ki67. Scale bar=300 μm.

Reference

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