Elsevier

Biochemical Pharmacology

Volume 63, Issue 8, 15 April 2002, Pages 1415-1421
Biochemical Pharmacology

The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death

https://doi.org/10.1016/S0006-2952(02)00902-4Get rights and content

Abstract

Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 μM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 μM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.

Introduction

Sanguinarine (Scheme 1) is derived from the plant Sanguinaria canadensis[1]. Its principal pharmacologic use to date is in dental products based on its antibacterial, antifungal, and anti-inflammatory activities, which reduce gingival inflammation and supragingival plaque formation [2], [3], [4]. Sanguinarine also has been reported to have antiviral and tumor-targeting activity [5], [6], [7].

Molecular biological studies indicate that sanguinarine has multiple cellular targets [8]. For example, it can interact with and intercalate DNA [9], [10], inhibit microtubule assembly [11], affect membrane permeability [12], [13], and inhibit a wide variety of enzymes, including Na+/K+ ATPase [14]. Most interestingly, it also is a potent inhibitor of protein kinases [15] and NF-κB [16], which are involved in signal transduction pathways leading to cell proliferation and/or cell death [7].

Cell death is important for normal homeostasis, cell proliferation, and differentiation. The importance of cell death is demonstrated by the observation that dysregulation of cell death can lead to cancer, developmental abnormalities, and autoimmune disorders [17], [18], [19]. Cells undergoing PCD (or apoptosis) are characterized by morphologic changes, including cellular shrinkage, blebbing, and nuclear DNA condensation with or without fragmentation [20], [21], [22], [23], [24]. However, it is stated that apoptosis is rarely observed in vivo and may not be the sole mechanism of cell death [25]. The discovery of intact novel forms of cell death pathways induced by potential anticancer agents may have an important bearing in overcoming chemoresistance.

Of all neoplasms found in females worldwide, cervical cancer has the third highest incidence and is fourth on the list of the leading causes of death by cancer [26], [27]. The available drugs most commonly used for treating cervical malignancies are impeded by frequent progression to chemotherapy resistance. Sanguinarine may be effective against MDR, since the related Sanguinaria canadensis-derived alkaloid, chelerythrine, has been shown to be cytotoxic to cancer cells and MDR cells [28]. In this study, we used our recently established in vitro cervical cancer model system for MDR [29] to investigate whether sanguinarine is effective against MDR in human cervical cells, and to understand the cellular and molecular mechanisms by which it may induce cell death.

Section snippets

Cell culture, cell viability assays, and clonogenic survival assays

Most cell culture protocols, the HPV type 16-immortalized human endocervical cell line (HEN-16-2), the CSC-transformed HEN-16-2 cell line (HEN-16-2T), and the MDR HEN-16-2 cell line (HEN-16-2/CDDP) have been described previously [29], [30], [31]. Cells were cultured in keratinocyte growth medium (KGM). HeLa cervical carcinoma, CEM-VLB leukemia, CEM-T4 leukemia, K562 erythroleukemia, and JM1 pre-B cell lymphoblastic cells were obtained from the American Type Culture Collection. HeLa cells were

Evasion of MDR of human cervical HEN-16-2/CDDP cells by sanguinarine

We examined the chemotherapeutic potential of sanguinarine for MDR cervical cancer cells in a human cervical in vitro system, which is composed of MDR HEN-16-2/CDDP cells and their drug-sensitive parental HEN-16-2 cells [29]. Cell viability, measured by the trypan blue exclusion assay, was similar in both types of cells treated with 0, 0.13, 0.26, 0.53, 1.06, 2.12, and 4.24 μM sanguinarine for 4 or 48 hr (Table 1; Fig. 1). The propidium iodide exclusion assay also showed no significant difference

Discussion

We established the in vitro MDR cervical cell system used in this report by treating HPV16-immortalized human endocervical HEN-16-2 cells with cisplatin [29]. Cell viability was significantly higher in the MDR HEN-16-2/CDDP cells than in the parental cells after treatment with cisplatin, actinomycin D, doxorubicin, etoposide, paclitaxel, 5-fluorouracil, staurosporine, heat shock, or UV radiation [29], [39]. However, this study found no significant difference in the effect of sanguinarine on

Acknowledgements

We thank Mr. G. Chernenko, Ms. Y. Hao, and Ms. L. Lee for excellent technical assistance. The work was supported by a National Cancer Institute of Canada grant (2734 to A.P.) with funds from the Canadian Cancer Society; Medical Research Council of Canada grants (MT-9782 and MT-10140 to A.P. and MT-13178 to A.L.); and a Canadian Institutes of Health Research (CIHR) grant (ROP-40859 to A.P.).

References (58)

  • P. Weerasinghe et al.

    Bax, bcl-2, and NF-κB expression in sanguinarine induced bimodal cell death

    Exp. Mol. Pathol.

    (2001)
  • A. Sapirstein et al.

    Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts

    Biochim. Biophys. Acta

    (2000)
  • L. Xue et al.

    Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution

    Mol. Cell Neurosci.

    (1999)
  • M. Shamma et al.

    Aporphinoid alkaloids

    Nat. Prod. Rep.

    (1986)
  • M.M. Kuftinec et al.

    Sanguinaria toothpaste and oral rinse regimen clinical efficacy in short- and long-term trials

    J. Can. Dent. Assoc.

    (1990)
  • L.L. Laster et al.

    New perspectives on Sanguinaria clinicals: individual toothpaste and oral rinse testing

    J. Can. Dent. Assoc.

    (1990)
  • K.C. Godowski et al.

    Whole mouth microbiota effects following subgingival delivery of sanguinarium

    J. Periodontol.

    (1995)
  • M.D. Faddeeva et al.

    Sanguinarine and ellipticine cytotoxic alkaloids isolated from well-known antitumor plants. Intracellular targets of their action

    Tsitologiia.

    (1997)
  • N. Ahmad et al.

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

    Clin. Cancer Res.

    (2000)
  • D. Walterova et al.

    Benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine: biological activities and dental care applications

    Acta Univ. Palacki. Olomuc. Fac. Med.

    (1995)
  • A. Saran et al.

    1H NMR investigation of the interaction of berberine and sanguinarine with DNA

    Indian J. Biochem. Biophys.

    (1995)
  • J. Wolff et al.

    Antimicrotubule properties of benzophenanthridine alkaloids

    Biochemistry

    (1993)
  • H. Babich et al.

    Cytotoxicity of sanguinarine chloride to cultured human cells from oral tissue

    Pharmacol. Toxicol.

    (1996)
  • M. Das et al.

    Clinicoepidemiological, toxicological, and safety evaluation studies on argemone oil

    Crit. Rev. Toxicol.

    (1997)
  • B.H. Wang et al.

    Inhibition of eukaryote protein kinases by isoquinoline and oxazine alkaloids

    Planta Med.

    (1997)
  • S. Orrenius

    Apoptosis: molecular mechanisms and implications for human disease

    J. Intern. Med.

    (1995)
  • C.B. Thompson

    Apoptosis in the pathogenesis and treatment of disease

    Science

    (1995)
  • E. White

    Life, death, and the pursuit of apoptosis

    Genes Dev.

    (1996)
  • A. Liepins

    Morphological, physiological and biochemical parameters associated with cell injury: a review

    Immunopharmacol. Immunotoxicol.

    (1989)
  • Cited by (105)

    • Alkaloids as potential anticancer agent

      2023, Recent Frontiers of Phytochemicals: Applications in Food, Pharmacy, Cosmetics, and Biotechnology
    • Natural compound library screening identifies Sanguinarine chloride for the treatment of SCLC by upregulating CDKN1A

      2022, Translational Oncology
      Citation Excerpt :

      In purpose of searching for effective pharmaceutical of SCLC, we performed high-throughput screening with a natural compound library, and identified Sanguinarine chloride (Sg) with excellent anti-SCLC activity. Sg is a natural compound derived from Macleaya cordata and recent studies have highlighted that Sg have different degrees of anti-tumor effects on prostate cancer [7,8], cervical cancer [9], breast cancer [10–12], colorectal cancer [13], gastric cancer [14], melanoma [15,16], pancreatic cancer [17] and non-small cell lung cancer (NSCLC) [18]. But the role of Sg in SCLC has not been elucidated.

    View all citing articles on Scopus
    View full text