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Antitumor and apoptotic effects of quercetin on human melanoma cells involving JNK/P38 MAPK signaling activation

https://doi.org/10.1016/j.ejphar.2019.172568 Get rights and content

Abstract

In this study, we investigated whether Quercetin has anti-cancer effects on A375SM and A375P human melanoma cells. Cell viability was assessed using an MTT assay. The proliferation of melanoma cells was measured by a wound-healing assay. Quercetin significantly decreased viability and proliferation of A375SM cells in a concentration-dependent manner. However, quercetin had no effect on A375P cells. DAPI staining showed increased chromatin condensation in a concentration-dependent manner, indicating apoptosis. Flow cytometric analysis indicated that quercetin suppressed the viability of A375SM cells by inducing apoptosis. Expression of quercetin-induced apoptosis proteins was investigated by Western blot analysis. Quercetin increased the expression of Bax, phospho-JNK, phospho-p38 and phospho-ERK1/2, cleaved poly-ADP ribose polymerase and decreased Bcl-2 in a concentration-dependent manner. We also investigated the in vivo tumor-growth inhibitory effect of quercetin. Quercetin (at 50 and 100 mg/kg) significantly decreased the A375SM tumor volume compared to the control group and increased apoptosis as assessed by the TUNEL assay. Immunohistochemistry staining revealed that the level of phosphor-JNK and phosphor-p38 increased in the quercetin-treated mice. These results indicate that quercetin inhibited the growth of A375SM melanoma cells through apoptosis and thus can be regarded as a new and effective chemo-preventive or therapeutic agent.

Introduction

Melanoma is the most malignant type of skin cancer caused by UV rays from the sun. It is easily metastasized to other organs. It accounts for ~4% of all skin cancers, but its mortality rate is as high as 80%. Although melanoma may be treated by surgery, radiation therapy and chemotherapy, the treatments are often ineffective. Despite recent improvements in immunotherapy and targeted therapy, it is difficult to treat because of the side effects caused by drug resistance and high toxicity (Menaa, 2013; White et al., 2009; Megahed et al., 2002). Therefore, over the past several decades, researchers have investigated potential new anticancer substances derived from natural products obtained from plants (Srivastava and Singh, 2004; Ma et al., 2014; Gao et al., 2011).

Quercetin is commonly found in nature, in a variety of foods such as vegetables, tea, fruit and wine Ishizawa et al., 2011). It is also known to have anti-oxidant (Park et al., 2006; Han et al., 2009), anti-inflammatory (Lee et al., 1981; Parveen et al., 2007), antibiosis (Kataoka et al., 2001) as well as anticancer effects, causing apoptosis of prostate cancer cells (Lee et al., 2008a; Vijayababu et al., 2005), liver cancer cells (Tanigawa et al., 2008; Granado-Serrano et al., 2006), colorectal cancer cells (Lim et al., 2007), breast cancer cells (Choi et al., 2008), pancreatic cancer cells (Lee et al., 2013) and lung cancer cells (Lee et al., 2008b). However, the anti-cancer efficacy of quercetin in A375SM, A375P melanoma cells and the underlying molecular mechanisms remain unknown.

Apoptosis is a potential treatment for cancer. It is a type of defense mechanism that prevents the development and progression of cancer by spontaneously removing unnecessary or malfunctioning cells that have DNA damage (Han et al., 2008). Apoptosis inhibits the survival and proliferation of cancer cells and is caused by the Bcl-2 family, which is composed of a pro-apoptotic protein and an anti-apoptotic protein. The Bcl-2 family of proteins play a key role in regulating apoptosis. (Burz et al., 2009).

The mitogen-activated protein kinase cascade (MAPK), which is a mechanism to transmit the extracellular signals to the nucleus in the cell, plays an important role in cell proliferation and differentiation, or apoptosis and survival. MAPK can be classified into extracellular signal-regulated protein kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In general, p38 and JNK are activated in response to extracellular stress and play an important role in the death of inflammatory cells (Yang et al., 2007). In contrast, ERK1/2 activation plays a role in cell proliferation and differentiation but may have anti-apoptotic or pro-apoptotic effects, depending on the nature of the cell (Ballif and Blenis, 2001; Moos and Fitzpatrick, 1998).

Our study investigated the in vitro effect of quercetin on the viability and proliferation of A375SM and A375P melanoma cells, the induction of apoptosis and its mechanism, and quercetin's in vivo effect on inhibiting the proliferation of tumors.

Section snippets

Cell lines and materials

The human melanoma cell lines (A375SM and A375P) for this experiment were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). MEM and DMEM were purchased from Welgene (Gyeonsan, Korea). Penicillin-streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Anti-rabbit IgG, anti-Bax, anti-Bcl-2, anti-β-actin, anti-PARP, anti-JNK, anti-phospho-JNK, anti-ERK1/2, anti-phospho-ERK1/2, anti-p38 and anti-phospho-p38 were purchased from Cell

The effect of quercetin on the viability of melanoma cells

We conducted the MTT assay with quercetin at concentrations of 0, 20, 40, 60, 80, 100, 150 and 200 μM to confirm the effect of quercetin on the survival of A375SM and A375P melanoma cells. When treated with quercetin at different concentrations for 24 h, the survival rate of A375SM melanoma cells was 109% at 20 μM, 89% at 40 μM, 74% at 60 μM, 61% at 80 μM, 59% at 100 μM, 41% at 150 μM, and 32% at 200 μM. When compared to the control group, there was a significant decrease at 40 μM (Fig. 2A).

Discussion

To examine the inhibitory effects of quercetin on the growth and proliferation of A375SM and A375P melanoma cells, each cell was treated with quercetin at concentrations of 0, 20, 40, 60, 80, 100, 150 and 200 μM for 24 h. The MTT assay results showed that the cell viability of the A375SM melanoma cells was 108, 85, 57, 43, 38, 39 and 37% at quercetin concentrations of 0, 20, 40, 60, 80, 100, 150 and 200 μM, respectively. Cell viability decreased significantly at concentrations of 40 μM and

Conclusion

Quercetin significantly decreased the viability and proliferative activity of A375SM cells in a concentration-dependent manner. However, it had no effect on A375P cells. Quercetin showed a significant increase in the stained cells with condensed chromatin due to apoptosis, and the showed a significant increase in apoptosis cells in concentration-dependent manner. Quercetin increased the expression of Bax, cleaved poly-ADP ribose polymerase and phospho-JNK, phospho-p38 and phospho-ERK1/2 while

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF, 2017R1A2B4005516).

Conflicts of interest

The authors declare no conflict of interest.

CRediT authorship contribution statement

Sung-Hyun Kim: Conceptualization, Data curation, Project administration, Formal analysis, Writing - original draft, Writing - review & editing. Eun-Seon Yoo: Conceptualization, Validation, Formal analysis. Joong-Seok Woo: Conceptualization, Validation, Formal analysis. So-Hee Han: Investigation, Visualization. Jae-Han Lee: Investigation, Visualization. Soo-Hyun Jung: Investigation, Software. Hyeong-Jin Kim: Conceptualization, Methodology. Ji-Youn Jung: Supervision, Conceptualization,

References (34)

  • S.I. Han et al.

    Role of apoptotic and necrotic cell death under physiologic conditions

    BMB Rep.

    (2008)
  • D.S. Han et al.

    Study on the antioxidant and anticancer effects of extract of stamens of Nelumbo nucifera and kaempferol

    Kor. J. Herbology

    (2009)
  • M. Hashemzaei et al.

    Anticancer and apoptosis-inducing effects of quercetin in vitro and in vivo

    Oncol. Rep.

    (2017)
  • M. Kataoka et al.

    Antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (Polygonum tinctorium Lour.), against Helicobacter pylori-infected Mongolian gerbils

    J. Gastroenterol.

    (2001)
  • H.J. Kim et al.

    Apoptotic effect of quercetin on HT-29 colon cancer cells via the AMPK signaling pathway

    J. Agric. Food Chem.

    (2010)
  • K.H. Lee et al.

    Antitumor agents. 49. Tricin, kaempferol-3-0-β-d-glucopyranoside and (+)-nortrachelogenin, antileukemic principles from Wikstroemia indica

    J. Nat. Prod.

    (1981)
  • J.H. Lee et al.

    Apoptosis by quercetin through induction of Bax and activation of caspase in human lung carcinoma cells

    Cancer Prev. Res.

    (2008)
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