Elsevier

World Neurosurgery

Volume 114, June 2018, Pages e182-e190
World Neurosurgery

Original Article
Thymoquinone Induces Apoptosis in B16-F10 Melanoma Cell Through Inhibition of p-STAT3 and Inhibits Tumor Growth in a Murine Intracerebral Melanoma Model

https://doi.org/10.1016/j.wneu.2018.02.136Get rights and content

Highlights

  • •

    Thymoquinone exerts effective treatment against melanoma both in vitro and in vivo.

  • •

    Survival increased by thymoquinone in a murine intracerebral melanoma model.

  • •

    Thymoquinone enhanced apoptosis in melanoma cells through inhibition of p-STAT3.

Background

Prognosis of patients with melanoma brain metastasis is poor despite various chemotherapeutic agents. Researchers focus on finding effective treatment with a low risk of toxicity. Thymoquinone (TQ) has been found to be effective on different types of cancer. However, no data exist regarding the effect of TQ in intracerebral melanoma. The purpose of this study was to assess the effect of TQ in B16-F10 melanoma cell in vitro and intracerebral melanoma in vivo.

Methods

The mechanisms of efficacy were investigated using adenosine triphosphate assay for cytotoxicity, flow cytometry, and acridine orange staining for apoptosis, comet assay for genotoxicity, CM-H2DCF-DA (2,7-dichlorodihydrofluorescein) for intracellular reactive oxygen species (ROS) generation and ELISA methods for inflammatory cytokines. Western blotting was performed to assess the expressions of p-JAK2, p-STAT3, caspase-3, Bax, Bcl-2, and survivin. In addition, the effect of TQ was investigated in a model system of intracerebral melanoma in syngeneic mice.

Results

The median survival was improved by TQ in mice with intracerebral melanoma compared with the control group (16 days vs 9 days; P = 0.008). Cytotoxicity was enhanced by TQ in B16-F10 cells in a dose-dependent manner. TQ also induced apoptosis, DNA damage, and increased intracellular ROS. TQ inhibited p-STAT3, resulting in apoptosis through regulation of proapoptotic and antiapoptotic proteins.

Conclusions

Our findings suggest that TQ would be an effective treatment in intracerebral metastatic lesions. This warrants further investigation.

Introduction

Expected median survival of patients with metastatic brain disease from melanoma is less than 5 months, and those patients have a particularly poor prognosis.1, 2 The treatment for patients with a stage IV melanoma is challenging because of the aggressiveness of the disease and lack of effective treatment. Various chemotherapy regimens and clinical trials have provided insufficient response and only a modest improvement of survival in patients with metastatic brain tumor from melanoma.3 In addition, cancer treatment regimens usually consist of cytotoxic chemotherapeutic agents, which induce severe toxic effects and require prolonged breaks to avoid adverse effects. Therefore, new strategies, including possible targeted therapies, are necessary.

Thymoquinone (TQ), a major bioactive constituent of the volatile oil of black seed (Nigella sativa), has been known to have antioxidant, anti-inflammatory and anti-neoplastic effects.4 TQ shows inhibitory effects in various types of cancer cells including breast cancer, ovarian cancer,5 colon cancer,6 pancreas cancer,7 uterine cancer,8 human osteosarcoma,9 fibrosarcoma and lung carcinoma,10 multiple myeloma,11 and melanoma.12, 13 TQ has been shown to exert anti-cancer effects through various mechanisms including inhibition of cell proliferation, inducing apoptosis, generating reactive oxygen species (ROS) and inhibiting metastases and angiogenesis.12, 14 Although in vivo studies have shown the efficacy of TQ in animal models,12 the use of TQ in an animal model with central nervous system (CNS) metastasis has been neglected.

Signal transducer and activator of transcription 3 (STAT3) has been shown to be a key regulator of tumor growth, metastases, and tumor-associated immune suppression in patients with various types of malignancies, including multiple myeloma, leukemia, lymphoma, squamous cell carcinoma, prostate cancer, breast cancer, and melanoma.15, 16, 17 Upon phosphorylation, phosphorylated STAT3 (p-STAT3) becomes activated and results in nuclear translocation and the upregulation of various gene products in tumorigenesis, metastases, angiogenesis, and tumor immune evasion.18, 19 Constitutively active p-STAT3 was reported in most of the melanoma cell lines and primary tumors.20, 21 Conversely, inhibition of p-STAT3 remarkably induces cytotoxic effects in melanoma cells, inhibits tumor growth, and prevents metastasis in melanoma murine models.21, 22 TQ has been studied in various types of malignancies, including melanoma in murine models7, 12, 23; however, the efficacy of TQ in a murine model with CNS metastases has not been studied. Therefore, we hypothesized that TQ would be an effective treatment for melanoma with CNS disease through inducing apoptosis secondary to inhibition of p-STAT3.

Section snippets

Cell Culture and Reagents

The B16-F10 murine melanoma cell line was obtained from the American Type Culture Collection (Rockville, Maryland) and cultured in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin from Life Technologies (Carlsbad, California, USA). The cells were maintained in 5% CO2 in a humidified incubator at 37°C. TQ was obtained from Sigma (St. Louis, Missouri, USA).

Murine Intracerebral Melanoma Model

The in vivo experiments used 4–6-week-old female

TQ Exerts Cytotoxic Effect on B16-F10 Melanoma Cell Line

To assess the ability of TQ to inhibit the proliferation of the B16-F10 melanoma cell line, these cells were treated with varying concentrations of TQ (5–200 μM) at various times (24 and 48 hours after incubation), and the cytotoxic effect of TQ was measured using the ATP method. TQ suppressed cell proliferation in a dose-dependent manner, with an IC50 value of 60 μM (Figure 1).

TQ Is Efficacious in CNS Melanoma Metastasis In Vivo

To determine whether TQ treatment is efficacious against established CNS tumors, C57BL/6J mice with intracerebral

Discussion

In the present study, we have shown that TQ effectively suppressed tumor growth in a CNS tumor model in vivo, exerted cytotoxic effect, enhanced apoptosis and DNA damage, and inhibited ROS and cytokine-related immune response in vitro. TQ did exert an antitumor effect with suppressing p-STAT3, resulting in direct cytotoxicity and apoptosis and inhibition of downstream proteins, including Bcl-2 and survivin. To our knowledge, this report is the first to show the antitumor effect of TQ in an

Conclusion

We have shown that TQ exerts a significant antitumor effect in an established murine intracerebral melanoma model. In vitro results suggest that this effect is evident through the induction of apoptosis by inhibition of p-STAT3. Further testing is warranted to assess the efficacy of TQ in treatment of patients with metastatic brain disease before clinical use.

Acknowledgment

We thank Omer Uysal for doing the statistical analysis.

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    Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

    This work was supported by the Bezmialem Vakif University Fund (grant number 2013/227, 2013).

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