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Integrative Medicine: A Clinician's Journal logoLink to Integrative Medicine: A Clinician's Journal
. 2018 Feb;17(1):32–37.

Apoptotic Activities of Sanguinaria canadensis: Primary Human Keratinocytes, C-33A, and Human Papillomavirus HeLa Cervical Cancer Lines

Cynthia Ann Leaver , Hang Yuan, Gwenyth R Wallen
PMCID: PMC6380990  PMID: 30962774

Abstract

Background

Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis. Multiple published, preclinical studies have demonstrated that sanguinarine causes apoptosis in human cancer cells. An expert panel of naturopathic doctors identified sanguinarine as a component for consideration in cervical atypia management protocols. Clinical studies have identified positive outcomes when Sanguinaria is a constituent of escharotic treatment in cervical atypia management protocols. Therefore, further investigation and development of sanguinarine as a potential cancer treatment is recommended. Thus, the aim of this study is to further elucidate the signaling pathways of sanguinarine-induced cell death.

Methods

Cell cultures of cervical cancer line C-33A (human papillomavirus [HPV] negative), HeLa (HPV positive), and normal primary human keratinocytes were established. Microscopic plate inspection, cell viability, initiator and effector caspase assays were completed in triplicate on C-33A (HPV negative), HeLa (HPV positive), and normal primary human keratinocytes cells exposed to incremental doses of sanguinarine.

Results

Sanguinarine induced dose-dependent cell death in cervical cancer lines as well as in normal human keratinocytes. Microscopic plate inspection confirmed morphologic changes of cell death in normal keratinocytes, C-33A, and HeLa including cell shrinkage, round up, and detachment from plate surface. Caspase assays demonstrated that sanguinarine treatment of C-33A cells resulted in an induction of caspase-3/7 when compared with vehicle-treated controls.

Conclusions

Sanguinarine demonstrates potential as an agent with therapeutic impact because of the capacity to eliminate cancerous cells through apoptosis, the process of programed cell death. Further elucidation of signaling pathways of sanguinarine-induced cell death is necessary when considering sanguinarine as a treatment for cervical cancer.


Sanguinaria canadensis is a perennial, herbaceous flowering plant native to the North America continent. Sanguinarine (13-methyl-[1,3]benzodioxole[5,6-c]-1,3-dioxolo [4-5i] phenanthridinium chloride) is a benzophenanthridine alkaloid derivative present in the rhizome of S canadensis and is identified as the active constituent of S canadensis (Figure 1). In laboratory studies sanguinarine has been known to have antimicrobial,1 anti-inflammatory,2 antioxidant,3 and immunomodulatory properties.4,5 Sanguinarine has been shown to induce cell death in prostate cancer cell lines DU-145,6 epidermoid carcinoma cell lines A431,7 and immortalized HaCaT keratinocytes.8 Most recently, sanguinarine inhibited cell proliferation and metabolism and induced cell death by apoptosis in DHD/K12/TRb colorectal adenocarcinoma cells.9

Figure 1.

Figure 1.

A. Sanguinaria canadensis Canada Pucoon (by Sydneham Edwards from The Botanical Magazine, 1791)

B. Sanguinarine (C20H14NO4)

Uncontrolled clinical studies have demonstrated efficacy of topical application of S canadensis to dysplastic cervical tissue.10-12 In addition, several in vitro and animal studies suggest that cell cycle arrest and stimulation of apoptosis are possible mechanisms of action of S canadensis.13-19 The cell biology of sanguinarine at micromolar concentrations has defined sanguinarine as a marker substance, the active constituent of S canadensis, that can inhibit the growth of cancer cells via cell cycle arrest and stimulation of apoptosis.13-19 Sanguinarine has been identified as a potent inhibitor of the activation of nuclear transcription factor nuclear factor kappa B (NF-κB).18 When multidrug resistance (MDR) is a significant obstacle to the success of chemotherapeutic agents, sanguinarine has been effective against MDR in immortalized cervical cancer cells.20 In MDR cervical cancer cells, sanguinarine-induced apoptosis is characterized by cell surface blebbing and activation of caspase-3.20 Only a few agents are known to maintain the capacity for selective and preferential elimination of cancer cells without affecting the normal cells.21-22 There is evidence that sanguinarine exhibits a cell growth-inhibitory response in human squamous carcinoma (A431) by apoptotic pathway, and human foreskin keratinocyte cells (HFKs) do not show any evidence of apoptosis but undergo necrotic cell death with treatment of higher concentrations of sanguinarine.23

Three clinical case studies, which were published as a series in a peer-reviewed naturopathic medical journal, specifically identified sanguinarine incorporated into the management of cervical dysplasia.10 In the first study, 7 consecutive patients were seen for the diagnosis and treatment of carcinoma in situ of the cervix, class IV, S canadensis was implemented as an escharotic treatment within the context of a naturopathic protocol that also included an oral protocol consisting of vitamins and botanical medicine, as well as a vaginal suppository treatment.10 At the 1-year follow-up, 4 were disease free, 1 improved to atypia and then reverted back to mild dysplasia, 1 had resolution to the cells of the endocervix and not the ectocervix, and 1 had resolution to the cells of the ectocervix and not to the endocervix.10 The second study was a 2-year follow-up study reporting outcomes in 43 subjects treated with S canadensis, again within the context of a naturopathic protocol that also included an oral protocol consisting of vitamins and botanical medicine, for cervical atypia and dysplasia of all degrees.11 Outcomes were identified as regression to normal (n = 38), partial regression to normal (n = 3), persistence of the condition (n = 2), and progression of the condition (n = 0).11 The third single case study presents the outcomes for 1 woman with recurrent high-grade neoplasm—CNII, CNIII (moderate to severe dysplasia)—managed with S canadensis as 1 component of an escharotic treatment protocol, again as a component of an oral protocol of vitamins and botanicals, and a vaginal suppository protocol.12 Findings indicated complete remission of evidence for her cervical dysplasia, with negative reports of cervical intraepithelial neoplasm (CIN) on follow-up colposcopy and Papanicolaou (Pap) for 5 years after the initial visits.12

An expert panel of naturopathic doctors participated in a Delphi method study to confirm that current, typical naturopathic management of cervical atypia includes escharotic treatment with Sanguinaria preparations.24

Further investigation of the cell biology of cervical cancer lines exposed to sanguinarine is justified to elucidate mechanisms of action suggested by other preclinical and clinical case study outcomes of sanguinarine in cervical dysplasia. This study specifically explores the cellular response and signaling pathways of apoptosis in HeLa, C-33A immortalized cell lines, and primary HFKs when exposed to sanguinarine by measuring cell viability and apoptosis, initiator caspase-2, -8, and -9; and effector caspase-3, -6, and -7 activation.

Materials and Methods

Cell Culture and Viability Assay

The human cervical carcinoma cell lines HeLa, human papillomavirus (HPV)-18 immortalized cell line (ATCC, Manassas, VA, USA) and C-33A, non-HPV cervical cancer immortalized cell line, (ATCC) were cultured in a T-175 flask with DMEM (Sigma-Aldrich, St Louis, MO, USA) with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), 2 mM Glutamine, and 1% penicillin-streptomycin at 37°C in a humid environment incubator with 5% CO2. Healthy HFK control cells were prepared from human foreskins donated by Georgetown University Hospital Department of Pathology (Washington, DC, USA). These human foreskin tissues were collected with the informed consent according to Georgetown University Institutional Review Board (Washington, DC, USA) protocols. The HFKs are commonly used in HPV studies instead of primary normal cervical cells because they are readily available, and because their sustainability beyond 4 or 5 passages with higher and faster cell growth (similar to the rates of cervical cancer cell lines) is desirable in contrast to the more limited life span of normal cervical cell lines. The HFK were cultured in a T-175 flask with KGM (Sigma-Aldrich). Sanguinarine (Sigma Chemical Co, St Louis, MO, USA) was dissolved in H2O as a stock solution at a 2.72 mM concentration and stored in aliquots at -20°C. This allowed for very minimal micromolar concentrations for cell line exposure. The sanguinarine used in this study, also known as S5890_SIGMA, was confirmed and classified by the National Center for Biology Information as PubChem Substance 24278199. C-33A, HeLa, and HFK cells were grown to 80% confluence then seeded in triplicate into 96-well plates. HeLa and C-33A were seeded to grow to 24 000 in 24 hours. HFK was seeded to grow to 12 500 in 24 hours. Cells were treated in triplicate with serial dilutions of sanguinarine (16.32 μM, 8.16 μM, 4.08 μM, 2.04 μM, 1.02 μM, 0.51 μM, 0.255 μM, and control of 0 μM) and incubated again for 24 hours. Cell viability was determined by Cell Titer-glo Luminescent, Cell Viability Assay (Promega Corporation, Madison, WI, USA) and Veritas Microplate Luminometer (Turner Bio Systems, Madison, WI, USA).

Caspase Assay

Consistent with the cell culture and viability assays, the C-33A, HeLa, and HFK cells were seeded in triplicate into 96-well plates. HeLa and C-33A were seeded to grow to 24 000 in 24 hours. HFK was seeded to grow to 12 500 in 24 hours. Cells were treated in triplicate with serial dilutions of sanguinarine (4.08 μM, 2.04 μM, 1.02 μM, 0.51 μM, 0.255 μM, and control of 0.0 μM), and incubated for 24 hours. Then, initiator caspase-2, -8, and -9 and effector caspase-3, -6, and -7 activation were measured using caspase-Glo Assays (Promega Corporation).

Results

Sanguinarine Induces Cell Death in Cancer Cell Line and in Normal Primary Cells

In C-33A, HeLa, and HFK, microscopic plate inspection revealed typical morphologic signs of cell death including cell shrinkage, round up, and detachment from plate surface (Figure 2A). Cell viability assays of HeLa, C-33A, and primary HFK exposed to high dose sanguinarine 16.32 μM for 24 hours revealed close to 100% death (Figure 2B). With exposure to lower doses of sanguinarine, 0.255 μM and 0.51 μM, loss of cell viability was slightly more pronounced in the C-33A cell line than in the HeLa or the HFK (Figure 2B). This finding is in keeping with previous observations showing that sanguinarine resulted in dose-dependent decrease in viability of both cancer cells and normal cells.7 The finding unique to this study is the increased cytotoxicity of sanguinarine exhibited by the C-33A cell line.

Figure 2.

Figure 2.

A. Representative Images of HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

B. HFK, C-33A, and HeLa Cell Viability After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 0.51 μM, 1.02 μM, 2.04 μM, 4.08 μM, 8.16 μM, 16.32 μM) of Sanguinarine

C-33A, HeLa, and HFK exposed to incremental doses of sanguinarine (4.08 μM, 2.04 μM, 1.02 μM, 0.51 μM, 0.255 μM, and control of 0.0 μM) and incubated for 24 hours revealed no difference in the activation of initiator caspase-2 and -6 (Figure 3A, 3B). There was no observed difference in activation of caspase-8 observed between HeLa and HFK at any dose and incubated for 24 hours (Figure 3C). Initiator caspase-8 revealed an increase in activation in C-33A cells exposed to low dose sanguinarine 1.02 μM and incubated for 24 hours. The slight increase in the activation of caspase-8 unique to C-33A cells is distinctive and a unique finding of this study.

Figure 3.

Figure 3.

A. Initiator Caspase-2 Activation Assay in HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

B. Initiator Caspase-8 Activation Assay in HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

C. Initiator Caspase-9 Activation Assay in HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

Effector caspase-9 revealed an observable increased activation in C-33A cells exposed to sanguinarine at the low dose 1.02 μM and incubated for 24 hours (Figure 4B). There was no difference in caspase-9 activation observed among HFK, HeLa, or C-33A with exposure to sanguinarine at the low dose 0.255 μM and incubated for 24 hours. Effector caspase-3/7 revealed an observable increase in activation between C-33A and HFK, with exposure to an intermediate dose of sanguinarine 2.04 μM, and continued increase of activation was observed in C-33A at high dose 4.08 μM when incubated for 24 hours (Figure 4A).

Figure 4.

Figure 4.

A. Effector Caspase-3/7 Activation Assay in HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

B. Effector Caspase-6 Activation Assay in HFK, C-33A, and HeLa Cells After 24 Hours of Incubation With Incremental Doses (Control, 0.225 μM, 1.02 μM, 2.04 μM, 4.08 μM) of Sanguinarine

Discussion

Escharotic treatment is an ablative therapy that has been implemented to treat cervical dysplasia as an adjunct therapy to oral protocols of vitamins and botanicals, as well as vaginal suppository protocols. Criticism of escharotic treatments in the literature identified cases where escharotics were self-prescribed, independently purchased, and unsupervised by the care of licensed health care professionals with expertise in areas of gynecology and integrative medicine.27

In this study, we initially analyzed the effects of sanguinarine, the active constituent of S canadensis, treatment of HeLa, C-33A, and primary HFKs. The study design including HeLa, C-33A, and primary HFK allowed for side-by-side analysis of HPV and non-HPV transformed cervical cancer cell lines and primary HPV-naive cells, as well as the exploration of sanguinarine as a potential agent for selective and preferential elimination of cancer cells without affecting normal/healthy cells. At lower doses of 0.255 μM and 0.51 μM, the loss of viability was slightly more pronounced in C-33A cell line than in either HeLa or HFK. These data suggest a differential response of non-HPV immortalized human cervical cancer cells to sanguinarine when compared with HPV-18 positive immortalized cervical cancer cells or even normal human foreskin keratinocytes.

With apoptosis as the major mode of cell death in cancer, several mechanisms by which apoptosis is induced in cancer cells have been reported.21-22 Caspase assays offer a novel approach to understanding the cascade of cysteine proteases that play a role in apoptosis, necrosis, and inflammation.25 Where, caspase-8 revealed activation in C-33A with exposure to a low dose of sanguinarine, it is possible that initiation of programmed cell death is through activation of caspase-8 pathways. Interestingly, in C-33A effector caspase-9 activation is noted at low doses as well as caspase-3/7 and sanguinarine-induced apoptosis of C-33A immortalized cervical cancer cells at higher doses 2.04 µM and 4.08 µM through the caspase-3/7-dependent pathway as well.

A distinct and precisely localized control over the fate of specific cells is dependent on many variables including cell cycle, duration of exposure, and dose of agent. It is expected that cells exposed to sanguinarine will not respond simultaneously. Therefore, caspase assays provide a “snapshot” of cellular response. Furthermore, persistent exploration of sanguinarine, time of incubation, and incremental dose exposure is indicated.

Inclusion of HFKs in this study design allowed for evaluation of the effect of dose-related response to sanguinarine of both cancer cells and normal cells. Cell death of HFKs was observed at low dose exposure to sanguinarine with activation of caspase activation at 2.04 µM, indicating potential programing of cell death and participation of Bcl-2, Bcl-xL, or even Bax inactivation.13-17 Sanguinarine-associated leukoplakia profiles of both normal and dysplastic cells identified potential preneoplastic nature of sanguinarine.23,26 Staining profiles for cyclin D1 and p53 proteins were significant between normal and dysplastic specimens.25 The finding from our study is consistent with this finding and theoretically supports a carcinogenic potential for sanguinarine in some normal specimens.

The methods of cell culture applied in this study allowed for amalgamation of primary cells and immortalized cell lines in the preliminary study of the compound sanguinarine that potentially exhibits anticancer properties. Agents that can eradicate cancerous cells via programmed cell death but preserve normal cells may have a therapeutic advantage for the elimination of cancer cells. It is accepted that cell numbers are regulated by factors that influence cell survival, as well as control the proliferation and differentiation of cells.21

Conclusions

Sanguinarine is an agent used by naturopathic doctors in the escharotic treatment of cervical dysplasia atypia.24 This in vitro study elucidates the potential role of sanguinarine as an anticancer agent for management of cervical atypia. Sanguinarine demonstrates potential as an agent with therapeutic advantage because of its capacity to eliminate cancerous cells through apoptosis, the process of programed cell death. Further investigation of sanguinarine on primary cervical cancer cells is required to determine the signaling pathways and gene mediation of sanguinarine-induced apoptosis. Investigation that further differentiates between necrosis and apoptosis of affected cells is necessary to establish dose requirements and optimal duration of exposure of cervical cancer cells to sanguinarine as part of an escharotic treatment protocol.

Acknowledgements

We would like to acknowledge Lena St. John, MSN, research fellow, National Institutes for Health, National Institute of Nursing Research, for her participation in design development of this project.

Biographies

Cynthia Ann Leaver, PhD, APRN, FNP-BC, is a family nurse practitioner, nurse research consultant, and the associate dean of faculty and research and assistant professor at United States University, College of Nursing, in San Diego, California.

Hang Yuan, PhD, is an assistant professor in the Department of Pathology at Georgetown University Medical Center in Washington, DC.

Gwenyth R. Wallen, PhD, is the deputy chief of research and practice development, National Institutes of Health, Clinical Center, in Bethesda, Maryland.

Footnotes

Author Disclosure Statement

The authors declare that they have no financial or nonfinancial competing interests to report. This project has been funded in whole or in part with federal funds from the National Institutes of Health, Clinical Center Intramural Research Program. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services. Additional funding for this work was provided by the Bravewell Collaborative; National Institutes of Health, National Institute of Nursing Research, Intramural Research Program, and Georgetown University Hospital, Department of Pathology.

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