Korean J Physiol Pharmacol.  2016 Sep;20(5):487-498. 10.4196/kjpp.2016.20.5.487.

p53 signaling is involved in leptin-induced growth of hepatic and breast cancer cells

• Shrestha M
• Park PH

• KMID: 2350505
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.487

Abstract

Leptin, an adipokine predominantly produced from adipose tissue, is well known to induce tumor growth. However, underlying molecular mechanisms are not established yet. While p53 has long been well recognized as a potent tumor suppressor gene, accumulating evidence has also indicated its potential role in growth and survival of cancer cells depending on experimental environments. In the present study, we examined if p53 signaling is implicated in leptin-induced growth of cancer cells. Herein, we demonstrated that leptin treatment significantly increased p53 protein expression in both hepatic (HepG2) and breast (MCF-7) cancer cells without significant effect on mRNA expression. Enhanced p53 expression by leptin was mediated via modulation of ubiquitination, in particular ubiquitin specific protease 2 (USP2)-dependent manner. Furthermore, gene silencing of p53 by small interfering RNA (siRNA) suppressed leptin-induced growth of hepatic and breast cancer cells, indicating the role of p53 signaling in tumor growth by leptin. In addition, we also showed that knockdown of p53 restored suppression of caspase-3 activity by leptin through modulating Bax expression and prevented leptin-induced cell cycle progression, implying the involvement of p53 signaling in the regulation of both apoptosis and cell cycle progression in cancer cells treated with leptin. Taken together, the results in the present study demonstrated the potential role of p53 signaling in leptin-induced tumor growth.

Keyword

Apoptosis; Cancer; Cell cycle; Leptin; p53

MeSH Terms

Adipokines
Adipose Tissue
Apoptosis
Breast Neoplasms*
Breast*
Caspase 3
Cell Cycle
Gene Silencing
Genes, Tumor Suppressor
Leptin
RNA, Messenger
RNA, Small Interfering
Ubiquitin
Ubiquitination
Adipokines
Caspase 3
Leptin
RNA, Messenger
RNA, Small Interfering
Ubiquitin

Figure

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  Fig. 1 Effect of leptin on the expression of p53 in hepatic and breast cancer cells.(A, B, C and D) HepG2 cells were treated with indicated concentration of leptin for 48 h (A and C) or 250 ng/ml of leptin for indicated time periods (B and D). (E, F, G and H) Similarly, MCF-7 cells were treated with indicated concentration of leptin for 48 h (E and G) or 250 ng/ml of leptin for indicated time periods (F and H). Protein expression level of p53 was determined by Western blot analysis as described in materials and methods (A, B, E and F). (Upper panel) Representative images of three independent experiments are shown along with β-actin as an internal loading control. (Lower panel) Quantitative analysis of p53 expression was performed by densitometric analysis and presented as mean±SEM (n=3). *p<0.05 compared with control group. (C, D, G and H) Messenger RNA level of p53 was determined by qRT-PCR analysis normalized to GAPDH. Data are expressed as mean±SEM of three to six independent experiments. *p<0.05 compared to the control group.

• 

  Fig. 2 Effect of leptin on the expression of p53 and USP2 in HepG2 Xenograft tumor model.BALB/c nude mice (4 weeks old male) were subcutaneously injected with HepG2 cells into the rear flanks. After two weeks of initial implantation, mice were randomly divided into two groups (control and leptin-treated group, n=5 per group). Leptin (1 mg/kg) was given intraperitoneally every 36 h for 4 weeks. p53 (A) and USP2 (B) protein expression were measured by Western blot analysis as described in materials and methods. Quantitative analysis of the p53 and USP2 expression was performed by densitometric analysis. Values are presented as mean±SEM (n=5). *p<0.05 compared to the mice not treated with leptin.

• 

  Fig. 3 Role of USP2 in leptin-induced p53 expression in cancer cells.HepG2 cells (A) and MCF-7 cells (B) were transfected with siRNA targeting USP2 or scrambled control siRNA followed by leptin treatment. (Upper panel) Transfection efficiency was determined after 48 h of transfection by Western blot analysis. (Lower panel) p53 protein expression was measured by Western blot analysis as described previously. (C) After treatment with leptin (250 ng/ml, 48 h), ubiquitinated p53 level was analyzed by immunoprecipitation using anti-p53 antibody and further immunoblotting with anti-ubiquitin antibody as described in the materials and methods. (D) HepG2 cells were pretreated with betulinic acid; an activator of proteasome, for 2 h followed by treatment with leptin for additional 48 h. p53 expression level was determined by Western blot analysis. Images are representative of three independent experiments that showed similar results.

• 

  Fig. 4 Involvement of p53 signaling in leptin-induced growth of cancer cells.HepG2 cells (A) and MCF-7 cells (B) were transfected with siRNA targeting p53 or scrambled control siRNA followed by incubation with leptin for 48 h. (Upper panel) Transfection efficiency was determined after 48 h of transfection by Western blot analysis. (Lower panel) Cell viability was assessed by MTS assay as described in materials and methods. Values presented are the results of three independent experiments and are expressed as mean±SEM. *p<0.05 compared to the control group; #p<0.05 compared to the cells treated with leptin, but not transfected.

• 

  Fig. 5 Role of p53 signaling in leptin-induced suppression of apoptosis in HepG2 cells.Cells were transfected with siRNA targeting p53 or scrambled control siRNA followed by incubation with indicated concentration of leptin for 48 h. (A) Caspase-3/7 activity was assessed as described in materials and methods. Values are expressed as mean±SEM (n=3). *p<0.05 compared to the control group; #p<0.05 compared to the cells treated with leptin, but not transfected. (B) Bax protein expression was determined by Western blot analysis as described in materials and methods. Images are the representative of three independent experiments that showed similar results.

• 

  Fig. 6 Role of p53 signaling in leptin-induced cell cycle progression in HepG2 cells.Cells were transfected with siRNA targeting p53 or scrambled control siRNA followed by incubation with indicated concentration of leptin for 48 h. (A) Cell cycle was analyzed using flow cytometer as described in materials and methods. (Upper panel) Values are the percentage of the cells in each of the cell cycle phases and are expressed as mean±SEM (n=5). (Lower panel) Images are the representative image of the five independent experiments which showed similar results. (B) Cyclin D1 protein expression level was determined by Western blot analysis. (Upper panel) Representative images of three independent experiments are shown along with β-actin for internal loading control. (Lower panel) Quantitative analysis of p53 protein expression was performed by densitometric analysis and presented as mean±SEM (n=3). *p<0.05 compared to the control cells; #p<0.05 compared to the cells treated with leptin, but not transfected with siRNA.

• 

  Fig. 7 Role of p53 signaling in leptin-induced increase in the expression of the genes related with autophagy.HepG2 cells were transfected with siRNA targeting p53 or scrambled control followed by incubation with leptin (250 ng/ml) for 48 h. Protein expression level of LC3 II (A), Atg5 (B) and Beclin-1 (C) was determined by Western blot analysis as indicated in the materials and methods. Images are the representative of three separate experiments that showed similar results.

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