Elsevier

Biochemical Pharmacology

Volume 78, Issue 11, 1 December 2009, Pages 1374-1381
Biochemical Pharmacology

Antiproliferative and antiangiogenic effects of the benzophenanthridine alkaloid sanguinarine in melanoma

https://doi.org/10.1016/j.bcp.2009.07.011Get rights and content

Abstract

This study was aimed at evaluating the potential application of benzophenanthridine alkaloids, sanguinarine and cheleritrine, in the therapy of melanoma cancer. In vitro antiproliferative activity of sanguinarine was higher than that of cheleritrine against the B16 melanoma 4A5 cells. Both agents were able to produce DNA breaks, and the DNA unwinding assay showed that they act as DNA intercalating agents. Sanguinarine was selected for determination of its in vivo preclinical efficacy. Oral treatment with sanguinarine reduced the tumor burden in a transplantable murine tumor grown in a syngeneic host (B16 melanoma 4A5 in C57BL/6 mice), and in a human tumor xenograft grown in immunodeficient mice (A375 human melanoma in athymic nude mice). In A375 tumors a significant decrease in the proliferation marker Ki67, and a reduction in the activated mitogen-activated protein kinases (p-p44/42 MAPK), and in protein kinase B (pAKT) were also observed. Three out of eleven A375-bearing treated mice were tumor-free at the end of treatment, and did not develop any tumor after a further, treatment-free, observation period of 60 days. Sanguinarine also showed a striking antiangiogenic activity in mice. Data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer.

Graphical abstract

Preclinical in vivo antiproliferative and antiangiogenic effects of sanguinarine in melanoma.

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Introduction

Malignant melanoma is an aggressive, therapy-resistant malignancy of melanocytes. The incidence of melanoma has been steadily increasing worldwide, resulting in an increasing public health problem. In Europe, 26 368 males and 35 909 females are diagnosed each year with melanoma, and around 8500 males and 8000 females die because of it [1]. In the United States, about 62 480 cases of melanoma were expected to be diagnosed in 2008, with about 8420 Americans dead from this cancer [2]. As with most malignant disorders, prognosis is highly dependent on the clinical stage (extent of tumor burden) at the time of diagnosis, and the patient's clinical stage at diagnosis dictates selection of therapy. If melanoma is diagnosed early it can be cured by surgical resection, and about 80% of cases undergo this treatment. However, metastatic malignant melanoma is largely refractory to existing therapies, and has a poor prognosis, with a median survival rate of 6 months, and a 5-year survival rate of less than 5% [3]. There are several approved postoperative adjuvant therapies for malignant melanoma [3, and references therein]. Interferon-α (IFN-α) is the most commonly used adjuvant immunotherapy for advanced melanoma, although its efficacy is still a matter of debate. High-dose interleukin-2 (IL-2) has also been approved, but response rates are low, and toxicity is a problem. Dacarbazine (DTIC) is the reference approved chemotherapeutic agent for the treatment of advanced melanoma, and drugs such as carmustine (BiCNU), paclitaxel (Taxol), temozolomide and cisplatin have shown single-agent activity in metastatic disease [4]. In addition, many different immunotherapies have been tested, but so far none of these approaches has reached regulatory approval [5]. Overall, despite decades of investigation, no systemic treatment that improves overall survival in patients with advanced metastatic melanoma has been developed, and new treatment options are urgently needed.

Natural products have long been a fertile source of cure for cancer, with plant-derived drugs becoming increasingly explored and integrated into chemotherapy strategies; there are at least 250 000 species of plants, out of which more than 1000 have been found to possess significant anticancer properties [6]. Recently, the benzophenanthridine alkaloids, which are mainly distributed in Papaveraceae (Chelidonium majus, Macleaya cordata, and Sanguinaria canadensis L.), have been the focus of increasing attention for their anticancer activity [7], [8], [9]. Specifically, several studies have indicated that sanguinarine [13-methyl-(1,3)benzodioxolo[5,6-c]-1,3-dioxolo[4,5-i] phenanthridinium], a compound endowed with antimicrobial, antioxidant, and anti-inflammatory properties [10, and the references therein], is able to inhibit the growth of various human cancer cell lines, at micromolar concentration, inducing a selective apoptotic response in cancer cells vs normal cells [7], [9], [11]. Notably, a recent study has also shown that sanguinarine reduces in vitro cell proliferation of K1375-M2, a very invasive melanoma cell line; Authors reported that sanguinarine acts as a DNA damaging agent, showing also collateral damage to mitochondrial bioenergetics, in a fashion similar to doxorubicin [12].

In the present study, we have explored the potential application of benzophenanthridine alkaloids sanguinarine and cheleritrine in the therapy of melanoma cancer. To this end, in vitro antiproliferative activity of the compounds was investigated, along with their mechanism of action; sanguinarine was selected for preclinical in vivo evaluations. Results obtained demonstrated that the antitumoral effect of sanguinarine relies on its activity on several critical steps in primary tumor progression, including cell proliferation and angiogenesis.

Section snippets

Drugs and reagents

For in vitro studies, sanguinarine chloride and cheleritrine chloride (donated by INDENA Milan, Italy) were diluted in DMSO. Solutions were further diluted at each experimental day in order to achieve a 0.1% final DMSO concentration. All reagents were purchased from Sigma (Sigma-Chemical Co., St. Louis, MO, USA), unless indicated.

Cell lines

B16 melanoma 4A5 cells, a cell line derived from subcutaneously inoculated B16F0 tumors in C57BL/6 strain mouse, and A375 cells, derived from a 54-year-old female with

In vitro studies

The benzophenanthridine alkaloids sanguinarine and cheleritrine, tested in B16 melanoma 4A5 cell line, yielded IC50 values of 1.96 ± 0.22 and 2.9 ± 0.22 μM, respectively, following 72 h of drug exposure; these results clearly pointed out that sanguinarine was more active than cheleritrine against melanoma cancer cells, and, for this reason, it was selected for subsequent in vivo investigations. A potential effect of drugs on the nuclear structure was assessed by Comet assay and results are shown in

Discussion

Experimental data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer. During a cellular screening of benzophenanthridine alkaloids we found that the drug resulted more active than the analogous compound cheleritrine against the murine cell line B16 melanoma 4A5, and that it mainly acts as an intercalating agent producing DNA breaks. This finding is in keeping with earlier data from Maiti et al. [21], showing that sanguinarine strongly interacts

Acknowledgements

This study was supported in part by the Ministero Istruzione Università e Ricerca (Decreto No. 1587). Sanguinarine and Cheleritrine were donated by INDENA s.p.a.

References (31)

  • V. Gray-Schopfer et al.

    Melanoma biology and new targeted therapy

    Nature

    (2007)
  • A.A. Tarhini et al.

    Cutaneous melanoma: available therapy for metastatic disease

    Dermatol Ther

    (2006)
  • J.M. Kirkwood et al.

    Strategies for the development of more effective adjuvant therapy of melanoma: current and future explorations of antibodies, cytokines vaccines, and combinations

    Clin Cancer Res

    (2006)
  • A.K. Mukherjee et al.

    Advances in cancer therapy with plant based natural products

    Curr Med Chem

    (2001)
  • N. Ahmad et al.

    Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells

    Clin Cancer Res

    (2000)
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    These Authors equally contributed to this work.

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