Evaluation of anticancer effects of frankincense on breast cancer stem‐like cells

Abstract Background Relapse and metastasis in breast cancer are linked to cancer stem cells (CSCs) resistant to anticancer therapies. The presence of cancer stem‐like cells (CSLCs) and their ability to self‐renew is determined by in vitro spheroid formation. Aims Many studies have found that frankincense has anticancer impacts, although these effects on breast CSLCs have never been evaluated. Methods and results A population of heterogeneous breast tumor cells was extracted from the tumor mass after generating an animal model of triple‐negative breast cancer (TNBC). Spheroid formation was used as an in vitro assay to determine the existence of CSLCs in these cells. MTT assay was used to determine frankincense's cytotoxic activity. An annexin V‐ propidium iodide (PI) staining and scratch test were used to assess the induction of apoptosis and antimetastatic effects of frankincense. The frankincense extract has significant cytotoxic and apoptotic effects on breast CSLCs. Although, the breast CSLCs are more resistant to these impacts than other breast cancer cells. Conclusion Our study is the first report that indicates that frankincense extract has anticancer properties in breast CSLCs. Compared to many anticancer chemicals, which have limited potential to battle cancer stem cells, frankincense is an appropriate option to combat breast CSCs.


| INTRODUCTION
The most frequent malignancy among women worldwide is breast carcinoma. 1 Triple-negative breast cancer (TNBC) is the most aggressive and invasive type, with a dismal prognosis among all types of breast cancer. Patients with TNBC are currently treated with chemotherapeutic drugs. 2 For some kinds of breast cancer, innovative tailored medicines are improving patient outcomes.
However, in the case of TNBC, this sort of treatment poses significant and fundamental difficulties. Chemoresistance, recurrence, and metastasis are the most common complications among TNBC patients. 3 The presence of a small minority of stem cells within heterogeneous populations of cancer cells has been explained by decades of cancer cell research. Cancer stem cells (CSCs) also known as cancer-initiating cells, are special cells that initiate cancer. Surface indicators, multidrug resistance pumps, and altered self-renewal pathways differentiate these cells. They have an essential role in cancer carcinogenesis and metastasis. 4 Several investigations in breast cancer have revealed the existence of breast CSCs. They stressed the importance of these cells in tumor development, metastasis, and resistance to current cancer treatments. 5 CSCs have been shown to form multicellular spheroid forms when cultivated under specific conditions. 6 These sphere-forming cells, which allow CSCs to proliferate and multiply, are used as a standard experimental test for determining the potential of stemness in cancer cells and are the greatest instrument for determining cancer stemness. 7 Traditional medicine products, which have been used for many years, are still popular among the majority of people around the world.
These items are usually affordable and have no adverse side effects.
Frankincense, an aromatic resin derived from Boswellia trees (Burseraceae family), is used to treat various ailments, including cancer. 8 Boswellic acid, as the primary component of frankincense, has been found to induce apoptosis in different cancer cells, including prostate, 9 colon, 10 melanoma, 11 hepatocellular, 12 leukemia, and brain. 13 Several authors have underlined the anticancer capabilities of frankincense in preclinical and clinical trials in breast cancer, 14-17 but these effects on breast CSCs have not been previously documented.
The goal of this study was to assess these effects.

| Cell culture
The murine mammary cancer cell line 4T1 was obtained from the Pasteur Institute of Iran's cell bank (C604) and cultured in high glucose cell culture media [DMEM (Dulbecco's Modified Eagle's Medium) with 10% FBS and 2% Penicillin-Streptomycin (both from Gibco, USA)]. The cells were incubated at 37 C with 95% air and 5% carbon dioxide (CO 2 ).

| Breast tumor induction and isolation of heterogeneous population of cancer cells
Induction of mammary tumors was performed as described in our previous study. 18 Briefly, female BALB/c mice weighing 20-25 g were obtained from the Royan Institute (Iran). The animals were kept in cages at 12 h photoperiod while they had free access to food and water. The ethics committee of Shahroud University of Medical Sciences approved this study for ethics in animal research (registration number: IR.SHMU.REC.1398.109). Subcutaneous injection of 4T1 cells (1 Â 10 5 ) was performed into the mice's flank (or the right hind limb). For isolation of a heterogeneous population of tumor cells, as described in our previous work, 19 tumors were excised from mice after 35 days of tumor induction. Mincing and enzymatical digestion of tumor tissue were performed in aseptic conditions. The digested tumor was filtered through 70 μm cell strainers and cultured in a DMEM with 10% FBS and 2% Penicillin-Streptomycin (all from Gibco, USA). The cells were ultimately incubated at 37 C in 5% CO 2 and passaged twice.

| Preparation of frankincense extract
The dried powder of gum resin (5 g) from Boswellia sacra (gifted from Dr. Fatemeh Jamshidi-Addgene-University of Nizwa-Oman) was extracted in 50 ml of methanol in a mechanical shaker at room temperature for 48 h. The mixture was then filtered with Whatman no. 1 filter paper. The filtrates obtained from methanol extraction were evaporated to dryness at 45 C in an oven. The dried extract sample was dissolved in sterile dimethyl sulfoxide (DMSO) and stored at À20 C until use. 150, and 200 μg/ml), and an MTT test was carried out as described.

| Apoptosis assay
For the apoptosis assay, 2D monolayer and 3D spheroids of breast

| Multicellular breast cancer spheroids formation
We used non-adherent 96 well plates for spheroid formation among the heterogeneous population of tumor cells. The spheroids formed in the well after 6 days, as seen in Figure 2. The spheroids were ready to be treated with the extract at this point.

| Cytotoxic effects of frankincense extract against monolayer and multicellular breast cancer spheroids
A diverse population of tumor cells was treated with different extract concentrations for 48 h to assess the growth inhibitory activity of frankincense extract on breast cancer cells. Cell viability was determined using the MTT method. The preliminary findings showed that the cytotoxic effects were more qualitative and statistically analyzable after 48 h. As a result, only a dose-dependent MTT assay was performed.
The vitality of primary tumor cells was significantly reduced dosedependent after exposure to extract (p < .05, Figure 3A). The

| Apoptotic effects of Frankincense extract
The annexin test was utilized to detect the extract's apoptotic effects.
The population of tumor cells in two-dimensional circumstances and the population of CSLCs in spheroids were treated with a

| Frankincense extract significantly reduces cells migration
This stage involved examining the effect of frankincense extract on the migration of a heterogeneous population of tumor cells. Figure 5 depicts the findings of the wound healing assay. Cell migration was extremely rapid in the control group, reaching 40% after only 24 h. . After frankincense extract treatment, apoptosis was 28% in 2D conditions, but this level was 16% in spheroids containing cancer stem cells (**p < .001).
F I G U R E 5 Frankincense suppresses the migration and invasion of a heterogeneous population of tumor cells. The migration ability was compared in treated and control groups based on wound healing.
apoptosis and have potent cytotoxic effects on malignant glioma cells in glioma. 28 Acetyl-11-keto-beta-boswellic acid (AKBA) at 10 mg/kg/day has also been found to suppress tumor growth in animals with prostate tumors. It was discovered that AKBA reduces tumor angiogenesis in this kind of malignancy. 29 Regarding safety issues, past reports have found no major, long-term, or irreversible side effects associated with B. Serrata. As a result, it can be used to treat various of disorders, including cancer, with minimal risk. 30 The antitumor effects of frankincense have been studied extensively, particularly in breast cancer. The antitumor properties of an alcoholic extract of B. Serrata gum resin were studied in vitro and in vivo. It was discovered that the extract causes cell-specific cytotoxicity in vitro and reduces cell proliferation, angiogenesis, and metastatic rate in vivo. 15  Cancer stem cells have been proven resistant to standard chemotherapy treatments in many lab investigations. 34,35 Several natural substances, such as curcumin, 24 piperine, 24 and sulforaphane 36 have recently been discovered to have anti-CSCs properties. However, toxicity and poor dose-response limit their application to a large extent. In our work, we proposed frankincense as a new natural substance with anti-CSCs properties whose clinical applications must be further analyzed.

| CONCLUSIONS
For the first time, the anticancer effects of frankincense extract on breast cancer stem-like cells were investigated in our study. Frankincense has great anticancer effects on breast cancer stem cells compared to many anticancer medications and chemicals. By focusing on cancer stem cells and establishing a therapy targeting this population, we can overcome metastatic breast cancer in the foreseeable future.

ACKNOWLEDGMENTS
We would like to thank the research assistant of Shahroud University of medical sciences and all the participants who helped us in this project.

FUNDING INFORMATION
This study was supported by Grant No. 9886 from Shahroud University of Medical Sciences.

CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ETHICS STATEMENT
The ethics committee of Shahroud University of medical sciences approved this study for ethics in animal research (registration number: IR.SHMU.REC.1398.109). All studies were carried out in compliance with the ARRIVE guidelines (https://arrive.guidelines.org) for the reporting of animal experiments. All methods were performed in accordance with the relevant guidelines and regulations.