Summary

METHODS
After the development of an animal model of breast cancer, a heterogeneous population of breast cancer cells were isolated from the tumor mass. Spheroid formation as a reliable in vitro assay to assess the presence of BCSCs was conducted among these cells. The cytotoxic activity of curcumin on multicellular breast cancer spheroids was assessed by MTT assays. Induction of apoptosis was measured by Annexin V-propidium iodide (pi) flow cytometric analysis.

RESULTS
The curcumin has potent cytotoxic and apoptotic effects on breast cancer spheroids. Although, compared with monolayer, breast cancer cells are more resistant to apoptosis when cultured as multicellular spheroids.

CONCLUSION
This is the first report of the anticancer effects of curcumin on breast cancer stem like cells. Compared to many anti-cancer drugs and compounds, which have very limited ability to fight cancer stem cells, curcumin is a good candidate to combat BCSCs.

Introduction

Herbal extracts and their active components are a promising candidate for new treatment strategies against a variety of diseases included malignancies. [7] Curcuma genus has been extensively used in traditional medicine for treating several diseases.[8] One of the constituents of Curcuma species is curcuminoids including curcumin.[9] Antioxidant, anti-inflammatory, antimicrobial, antiviral, and anticancer effects are the main biological activities of these phytochemicals. Among all, its anticancer potential has been the most described and remains under investigation.[10] Curcumin is believed to show its impact on cell growth and invasion of breast cancer through inducing apoptosis by regulating the expression of apoptosis-related genes, cell cycle arrest at the G2M phase and late S phase.[11-13] Studies have demonstrated that curcumin alone or combined with other drugs presents anticancer activities against malignant cell lines.[14]

Several studies have been focused on anticancer activities of curcumin but there is a lack of enough information on the effects of curcumin on multicellular breast cancer spheroids. In the present study, we are aimed to elucidate the anticancer properties such as cytotoxicity and apoptotic effects of curcumin on breast cancer multicellular tumor spheroids which are a great candidate on representing the native tumor microenvironment.

Methods

Induction of Syngeneic Animal Model of Breast Cancer
As described in previous works[15] for tumor induction in female BALB/c mice, 4T1 cells were subcutaneously injected to the flank (or the right hind limb) of the mice (105 cells suspended in 100 µL phosphatebuffered saline [PBS]) using an insulin syringe. All animal experiments were in compliance with the relevant laws, and this study was approved by the Ethics Committee of Shahrood University of Medical Sciences (registration number: IR.SHMU.REC.1398.109).

Preparation of Heterogeneous Population of Breast Cancer Cells
CSCs are a small subset of the cancer cells among a heterogeneous population of cancer cells. Accordingly, for the first step, it is necessary to dissociate a tumor tissue sample into a single cell suspension to be able to isolate CSCs from the rest of the cancer cells. In the present research for isolation of heterogeneous population of tumor cells, primary tumor of cancerous mice was excised after 20 days of tumor induction in mice, and surface blood was removed by rinsing it in PBS. After mincing with scissors, fragments were placed to 50 ml conical tube. For enzymatic digestion, primary tumor was digested in 10 mg/ml collagenase type IV at 37°C for 75 min on a platform rocker. All enzymes were purchased from Sigma (St. Louis, MO, USA). The digested tumor filtered through 70 um cell strainers and washed with PBS. In the next step, washed cells were resuspended in medium containing 10% FBS, 100 U/ml penicillin, and 100 ug/ml streptomycin (all from Gibco, USA). Ultimately, the cells were cultured at 37°C in 5% CO₂ and passaged 2 times.

Cytotoxic Effect of Curcumin on Breast Cancer Spheroids
Suspension of heterogeneous population of cancer cells (isolated in previous steps) was prepared and cultured as described below:

1. For 2D monolayer, cells were seeded at a density of 1×104 cells/well in a 96-well culture plates and cultured in high glucose DMEM containing 10% FBS and 2% penicillin-streptomycin (all from Gibco, USA) in humidified atmosphere of 5% CO₂ at 37ºC. After 24 h incubation, cell culture medium was exchanged with complete medium supplemented with different concentrations of curcumin (5, 10, 15, 20, and 30 µM). Following 48 h incubation at 37ºC, medium was removed and 50 ml of MTT solution at 5 mg/ml (Sigma) was added to the cultures and the incubation continued for a further 4 h period, after which 150 ml of dimethyl sulfoxide (DMSO) was added. Formed formazan crystals were allowed to dissolve for 30 min before measuring the optical density at 570 nm using CYTATION/5 imaging reader (Bio-Tek Instrument, USA). Finally, cell viability was expressed as percent compared to control wells according to the following equation:

In this equation, blank means culture medium without cells and control means culture medium with cells. This experiment was performed in triplicate.

2. To form 3D spheroids, cells were seeded at a density of 1×10⁴ cells/well in a 96-well Ultra-Low Attachment microplate and cultured in spheroid forming media comprised high glucose DMEM containing 0.5% FBS and 2% penicillin-streptomycin (all from Gibco, USA) in humidified atmosphere of 5% CO₂ at 37°C and incubating them for 6 days. The resulting tumor spheroids were treated for 48 h with different concentrations of curcumin (10, 25, 50, 75, and 100 µM) and MTT assay was performed as explained above.

Apoptosis Assay
For apoptosis assay, similar to previous step, 2D monolayer and 3D spheroids of breast cancer cells were prepared and treated for 48 h with IC50 concentrations of curcumin. Cell apoptosis assays were carried out with the use of MabTag's Annexin-V Apoptosis Detection Kit, according to the manufacturer protocol.

Statistical Analysis
Results are expressed as the mean ± standard deviation. Data were analyzed with GraphPad Prism statistical software 6.0 (GraphPad Software, La Jolla, CA, USA) using paired samples t-test. P<0.05 was considered statistically significant.

Results

Fig. 1. Primary tumor cells isolation and multicellular breast cancer spheroids generation. (a) Animal model of triple-negative breast cancer was generated after 20 days of tumor induction in Balb/c mice. (b) Primary tumor isolation and H&E staining of tumor tissue were performed. (c) Primary tumor cell extraction was performed on primary tumor tissues. (d) Multicellular breast cancer spheroids generated from heterogeneous population of tumor cells in special culture condition after 6 days.

Multicellular Breast Cancer Spheroids Formation
For spheroid formation among heterogeneous population of tumor cells, we used non-adherent 96 well plates. As shown in Figure 1, after 6 days, the spheroids formed in the well (Fig. 1d). At this stage, the spheroids were ready for treatment with the curcumin.

Cytotoxic Effects of Curcumin against Multicellular Breast Cancer Spheroids
To determine the growth inhibitory activity of curcumin on breast cancer cells, heterogeneous population of tumor cells were treated with different concentrations of curcumin for 24 h, 48 h, and 72 h and cells viability was measured by MTT assay. The initial results indicated that cytotoxic effect was better analyzable qualitatively and statistically after 48 h. Therefore, the MTT assay was only done in dose-dependent manner. Exposing heterogeneous population of primary tumor cells to curcumin resulted in a significant decrease in cells viability in a dose-dependent manner (p<0.05), (Fig. 2). The IC50 value was considered as the concentration of the curcumin that caused a 50% decrease in cell viability relative to the negative control which was constituted by cell culture and DMSO without the curcumin. For 2D monolayer of tumor cells, IC50 value was found to be 10 uM by MTT assay. Compared with 2D monolayer, breast cancer cells are more resistant to cytotoxic effects of curcumin when cultured as multicellular 3D spheroids. IC50 value was found to be 60 uM by MTT assay for tumor cells presented in multicellular 3D spheroids. These IC50 concentrations were selected for all further mechanistic studies.

Fig. 2. Breast cancer cells viability in 2D monolayer and 3D multicellular spheroids under different concentration of curcumin. MTT assay results quantifying the viability of cells under different treatment conditions. Untreated cells were used as the control. Exposing heterogeneous population of primary tumor cells to curcumin resulted in a significant decrease in cells viability in a dose-dependent manner. IC₅₀ value was found to be 10 µM for these cells in 2D monolayer format. Breast cancer cells in 3D culture were are more resistance to curcumin and IC₅₀ value was found to be 50 µM for these cells in 3D multicellular spheroid format.

Apoptotic Effects of Curcumin
To determine the apoptotic effects of the curcumin, we used annexin test. For this purpose, both the heterogeneous population of tumor cells in 2D conditions and the population of cancer stem cells located in spheroids were treated with a concentration of IC50 concentrations of curcumin. The results of Annexin V/PI staining for curcumin after 48 h are shown in Figure 3. The curcumin could be induced apoptosis in breast cancer cells both in 2D and 3D condition but result indicated that breast tumor cells are significantly more resistant to apoptosis in 3D multicellular spheroids.

Fig. 3. Curcumin regulates apoptosis in breast cancer cells both in 2D monolayer and 3D multicellular spheroids. Breast cancer cells in 2D and 3D culture were treated with IC50 concentrations of curcumin (10 um and 50 um) for 48 h. Cell apoptosis was analyzed by flow cytometry. Compared with 2D monolayer, breast cancer cells are more resistant to apoptosis when cultured as multicellular spheroids. The level of apoptosis was 28% in 10 um concentration of curcumin in 2D conditions but this level was 5% in spheroids containing cancer stem cells (**p<0.005).

Discussion

Curcumin showed cytotoxic and apoptotic potential against breast cancer in other studies as well. Several mechanisms such as inhibition of oncogene protein expression, stem-like properties, regulating the EMT process, cell cycle arrest, and interaction with oncogenic and tumor-suppressive miRNAs underlie curcumin cytotoxic effects.[13,17-19]

Most cytotoxicity assays are designed to evaluate anticancer drug effects on classic 2D cultures. Consequently, many cell characteristics and dynamic nature of tumor microenvironments are usually lost so 3D models are more predictive than monolayers in 2D cultures.[20,21] Similar to our findings, Abuelba et al.[22] also reported slight cytotoxic reduction in 3D breast cancer model compared to 2D condition. They worked on metastatic breast adenocarcinoma cell line (MDA-MB-231) and concluded that 3D tumor cell culture systems appear to be the ideal environment for in vitro assays regarding anticancer drug effects on cell viability.

Most 3D culture models are supposing tumor cell seeding on polymer scaffolds or cell embedding in hydrogels while some models are speculating adherent cell ability to cluster in suspension or on low adherence surface. In a recent work, for achieving a 3D environment, researcher used encapsulation of cells in alginate hydrogel. The results of this study showed that curcumin in 3D culture conditions causes mortality in breast cancer cells (MCF-7).[23] Because of cell behavior in cell clusters is essential to further development of the tumor microenvironment, in our work, we used spheroids for 3D culture of breast cancer cells.

Evaluation of human MCF-7 breast cancer cells seeded in 2 and 3D culture systems confirmed that curcumin significantly decreased MCF-7 cells viability in dose? and time?dependent manners in 2 and 3D systems. However, cell viability in 2D cultures was significantly lower compared to 3D cultures; most probably due to cell clustering effect that may prevent curcumin penetration to inner cells in spheroids.[24] In this work, researchers used human MCF-7 breast cancer cell line. Most breast cancer cell lines have undergone changes in their function and genome due to multiple passages and manipulations.[16] Accordingly, in our work, we use the heterogeneous population of primary tumor cells to create spheroid.

It should be noted that curcumin effectiveness has been limited due to low bioavailability.[25] Several studies have been conducted to increase curcumin effectiveness.[26] To boost the bioavailability of this chemical component, various approaches have been undertaken like using adjuvant, liposomal curcumin, nanoparticles, phospholipid complex, and structural analogs of curcumin.[25]

In case of clinical use, since cancer is still one of the leading causes of death in the world, there is an increasing demand of new therapeutic interventions. [27] According to Sen et al.[28] study, curcumin is a potent chemosensitizer that improves the therapeutic index of widely used anti-cancer drugs. Therefore, it can be developed into an adjuvant chemotherapeutic drug. Xiong et al.[29] designed a dual-drug codelivery system which consisted of PTX (a US Food and Drug Administration approved chemotherapeutic drug for the treatment of breast cancer) and curcumin. The results of this in vivo study suggest that dual drugs could be a potential system for the treatment of breast cancers. Ferguson and Orlando also reported that addition of curcumin during 5-fluorouracil (an antimetabolite that inhibits cell proliferation) therapy enhanced the chemotherapeutic effectiveness by protecting normal cells from reduced viability and consequently permitting higher dosing or longer treatment times.[30]

Conclusion

Acknowledgments: We would like to thank the research assistant of Shahrood University of Medical Sciences and all the participants who helped us in this project.

Peer-review: Externally peer-reviewed.

Conflict of Interest: The author declares that they have no competing interest.

Ethics Committee Approval: This study was approved by the Shahrood University of Medical Sciences Ethics Committee (No: IR.SHMU.REC.1400.027, Date: 08/05/2021).

Financial Support: This work was supported by a grant from the Shahrood University of Medical Sciences (SHMU) grant no. 99130.

Authorship contributions: Concept - M.K.F.; Design - M.K.F.; Supervision - M.K.F.; Funding - M.K.F., A.Atashi; Materials - M.K.F., A.Atashi; Data collection and/or processing - M.K.F., A.Asadi; Data analysis and/or interpretation - M.K.F., A.Atashi; Literature search - M.K.F., A.Asadi; Writing - M.K.F., A.Asadi; Critical review - M.K.F., A.Atashi

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