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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Anticancer Res. 2021 Dec;41(12):5935–5944. doi: 10.21873/anticanres.15412

Activity in MCF-7 Estrogen-Sensitive Breast Cancer Cells of Capsicodendrin from Cinnamosma fragrans

Ulyana Muñoz Acuña 1,2, Nathan Ezzone 1, L Harinantenaina Rakotondraibe 1, Esperanza J Carcache de Blanco 1,*
PMCID: PMC8750382  NIHMSID: NIHMS1768376  PMID: 34848447

Abstract

In our screening program for new anticancer agents from natural origin, capsicodendrin from Cinnamosma fragrans Baill. (Canellaceae) showed inhibitory activity in a NF-κB p65 assay (IC50=8.6 μM) and cytotoxic activity (IC50=7.5 μM) in a SRB assay against MCF-7 estrogen-dependent breast cancer cells. Previous studies found capsicodendrin can be converted to the toxic cinnamodial and that capsicodendrin inhibited angiogenesis and VEGFR2 mediated AKT signaling in endothelial cells. Herein, the effect of capsicodendrin on the NF-κB pathway was assessed in MCF-7 treated cells using western blot analysis. Both the upstream mediator IκB kinase (IKK) and the downstream intracellular adhesion molecule (ICAM-1) were downregulated in MCF-7 treated cells. The effect on the cell cycle was examined using a fluorescence-activated cell sorter. The sub G1-phase population increased to 81% after 12 h of treatment with capsicodendrin (10 μM). JC-1 monomers were not detected in treated cells and there was no loss of the mitochondrial membrane potential (ΔΨM). Thus, capsicodendrin seems to induce cell death through a mitochondrial-independent pathway. The upstream effect of mitochondrial events in apoptosis together with the effect on both caspase-1 and caspase -7 were investigated. These findings suggest that increased levels of intracellular reactive oxygen species (ROS) promoted activity of caspase-1 to impact levels of caspase-induced cell death in estrogen-sensitive breast cancer cells. This study also showed that capsicodendrin inhibited TNF-α activation in the NF-κB signaling pathway. In conclusion, capsicodendrin may be a future lead molecule in the development of new effective anticancer agents that prevent the progression of metastatic breast cancer.

Keywords: Capsicodendrin, MCF-7, NF-κB, IKKβ, ICAM-1, ROS, Caspase-7, Caspase -1

1. Introduction

Despite new effective anticancer treatments, chemoresistant breast tumor cells spread to other organs and the survival rate (26%) remains low for metastatic breast cancer1, 2, 3. Breast cancer patients have high serum levels of pro-inflammatory cytokinin tumor necrosis factor-α (TNF-α). It has also been shown that transmembrane TNF-α plays a role in the development of chemoresistance and metastasis of breast cancer cells4 5. TNF-α induces the activity on the nuclear factor kappa B (NF-κB) (Fig. 1), which is a heterodimer composed by p65 bound to IκB. IκB kinase (IKK) is responsible for initiation and phosphorylation of IκB. The release from IκB activates NF-κB before it is translocated to the nucleus and the blockage of both IKKβ and nuclear import reduces tumor expansion6, 7, 8. Activation of the NF-κB p65 signal plays an important role in vivo in tumor progression and promotes the resistance to certain tumorigenic agents and chemotherapy9, 10. Previous studies have shown that NF-κB p65 prevents cancer cells from entering apoptosis and contributes to chemoresistance as well as to the progression of the disease11. In addition, it has been shown that MCF-7 estrogen-sensitive breast cancer cells have an overexpression of anti-apoptotic protein Bcl-2, which prevents apoptosis from occurring. Thus, inhibition of NF-κB p65 prevents tumor growth and metastasis of estrogen-sensitive breast cancer12, 13.

Figure 1.

Figure 1.

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Capsicodendrin role in the NF-κB p65 pathway. (A) Structure of capsicodendrin isolated from Cinnamosma fragrans and (B) Proposed pathway for the transcription factor NF-κB p65 in MCF-7 estrogen-dependent cells.

Moreover, intracellular adhesion molecule ICAM-1 has been shown to be induced by TNF-α in MCF-7 cells14 (Fig. 1). Herein, the effects on ICAM-1 and the anti-proliferative effect of capsicodendrin in MCF-7 estrogen-dependent breast cancer cells was investigated. Also, the effect of capsicodendrin in the NF-κB pathway and the viability of treated cells was further analyzed. The apoptotic effect and the effect on cellular oxidative stress additionally was investigated in vitro. The aim of this study was to assess the possible mechanism of action through which capsicodendrin (Fig. 1 A) induces cell-cycle arrest in breast cancer cells.

2. Materials and Methods

Cell culture

The MCF-7 cancer cell line was obtained from American Type Culture Collection (ATCC# HTB-22). Cells were cultured in Dubelcco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute medium (RPMI-1640), containing 10% fetal bovine calf serum and 10% Antibiotic-Antimycotic (Gibco, Rockville, MD). Cells were kept at 37°C and in an atmosphere with 5% CO2.

SRB assay

MCF-7 cells were plated in a 96-well plate and treated with capsicodendrin for 72 h at 37°C and incubated in an atmosphere with 5% CO2. Cells were fixed using 20% trichloroacetic acid for 30 min, followed by staining with sulforhodamine (SRB) (0.4%) for 30 min at room temperature. SRB was removed by washing with acetic acid three times. After adding 200 μM Tris base solution to each well, the 96-well plates were placed on a shaker for 5 min. Absorbance reading was performed at a wavelength of 515 nm using a Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Paclitaxel (Tocris Bioscience, Bristol, UK) was used as a positive control.

NF-κB assay

The NF-κB p65 assay was performed according to a previously established protocol15. Nuclear extracts were prepared from HeLa cells and the Transcription Assay System (Pierce Biotechnology, Rockford, IL) was used to evaluate the binding affinity of the NF-κB subunit p65 to the biotinylated consensus sequence. Luminescence was detected using a Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Rocaglamide (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used as a positive control.

Immunoblotting

Cells were treated with capsicodendrin at different concentrations (0.008, 0.016, 0.4, 2.0 and 10 μM) for 3 h. Briefly, cells were lysed using PhosphoSafe Lysis Buffer (Novagen). Protein concentration was determined by using a Bradford protein assay kit and albumin standard (Thermo Scientific). Absorbance was measured using a Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Lysates were analyzed by western blot analysis with primary (1:1000) and secondary antibodies (1:2000). Equal amounts of protein (20 μg) were loaded together with a LDS sample loading buffer (Invitrogen) and resolved using Nu-PAGE 10% SDS-PAGE Bis-Tris gels together with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen). Proteins were transferred to a polyvinyldiene fluoride (PVDF) membrane using transfer buffer, TBS-T. The blots were then blocked at room temperature using non-fat milk and probed using primary antibodies against each target protein including NF-κB p65, IKKβ, ICAM-1 and caspase-7, using BSA in TBS-T overnight. Conjugated antibodies were detected using chemiluminescent substrates with a Supersignal Femto kit from Thermo Scientific.

Reactive Oxygen Species

A Reactive Oxygen Species (ROS) assay was performed following a previously described procedure16. Generated intracellular levels of ROS were measured using the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). MCF-7 cells were seeded in a 96-well plate, treated with capsicodendrin, daunomycin, vitamin C or DMSO and followed by 5 h incubation at 37°C with 5% CO2. Subsequently, cells were incubated with H2O2 (1.25 mM) and FeSO4 for 30 min at 37 °C. Later, the fluorescent probe DCFH-DA was added to determine intracellular ROS. Fluorescence was measured using FLUOstar Optima fluorescence plate reader (BMG Lab technologies GmbH Inc. Durham. NC, USA) at an excitation wavelength of 485 nm and emission wavelength of 530 nm. All treatments performed in triplicate are representative of at least two different experiments.

Cell cycle

Cells were plated and treated using five different concentrations of capsicodendrin. After 12 h of incubation, cells were harvested and pelleted by centrifugation, washed with PBS and fixed in ice-cold 70% ethanol. The DNA was stained with 10 μg/mL propidium iodine (PI) in a reaction solution containing 1 mM EDTA and 100 μg/mL RNase A. Fluorescence emitted from the propidium iodine-DNA complex was quantified using BD FACS Canto II (Biosciences, San Jose, CA) at 488 nm.

Mitochondrial transmembrane potential (ΨΔm) assay

The mitochondrial transmembrane potential (MTP) fluorescence-activated cell sorting (FACS) assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) was used to assess MTP (ΨΔm) in MCF-7 cells after treatment, using FACS analysis15. Cells were seeded on 10 cm plates and treated with capsicodendrin for 24 h. Next, cells were harvested using Trypsin-EDTA (Gibco), washed in phosphate-buffered saline (PBS) and re-suspended in assay buffer. The potentiometric dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbensimidazoylcarbocyanine iodide (JC-1) was used to stain MCF-7 cells. A volume of 50 μl of JC-1 stain was added to the cells and incubated for 15 min at 37 °C in 5% CO2. At high ΨΔm, red fluorescence J-aggregates are formed in healthy cells; however, in apoptotic cells with low ΨΔm, JC-1 remains in the monomeric form, which only exhibits green fluorescence. Analysis was performed using FACS Canto II (BD Bioscience, San Jose, CA, USA). The mitochondrial function was assessed, and the J-aggregates were detected using an excitation wavelength of 520–570 nm, and emission of 570–610 nm, respectively.

Caspase-Glo 1 inflammasome assay

Caspase-1 activity was determined using a modified protocol from Promega Caspase-Glo® 1 Inflammasome Assay (G9951). MCF-7 cells were plated overnight in white 96-well plates in 100 μL medium at a density of 20×104 cells/mL, and at 37°C in a humidified 5% CO2 incubator. Next, cells were treated with test sample, capsicodendrin or doxorubicin (control), for 24 h. Four concentrations of both capsicodendrin and the control were tested, using the IC50 value as the second concentration and one concentration 5x higher than IC50 as well as two concentrations 5x and 10x lower than the IC50 value. Reagents used in assay were prepared according to the specifications detailed by the manufacturer for full well plate. After treatment, 70 μL of medium were then discarded and the appropriate volume of Caspase-Glo®1 Reagent was added while Caspase-Glo®1 YVAD-CHO Reagent was added at a ratio of 1:1 sample volume to reagent volume. The plate was then incubated at room temperature for 1 h and the luminescence was measured up to 120 min using a Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC).

3. Results and Discussion

Capsicodendrin was isolated from the bark of Cinnamosma fragrans Baill. (Canellaceae) (Fig. 1 A)17. C. fragrans is a medicinal plant endemic to Madagascar and it is used in the treatment of malaria symptoms, fatigue, intestinal parasite infections, intoxication and headaches17, 18.

The results obtained from the SRB assay showed that capsicodendrin displayed cytotoxic activity in the MCF-7 hormone-dependent breast cancer cell line (IC50=7.5 μM), suggesting the potential for further analysis as breast cancer is a leading cause of death in women. The cytotoxic activity was compared to the inhibitory activity of paclitaxel (IC50=0.0021 μM). Capsicodendrin has previously displayed cytotoxic activity against other cancer cell lines: HeLa cervical cells (IC50=2.97 μM), HT-29 colon cancer cells (IC50=1.04 μM) and murine leukemia cell line L1210/0 (IC50= 0.58 μM), human T-lymphocyte cell lines Molt4/C8 (IC50=1.51 μM) and CEM/0 (IC50=1.61 μM)17, 19, 20.

Capsicodendrin also displayed NF-κB inhibitory activity (IC50=8.6 μM) when tested in the NF-κB p65 ELISA assay (Fig. 1 B). The NF-κB p65 inhibitory effect in HeLa cells was compared to rocaglamide (IC50=0.075 μM). Subsequently, western blot analysis was performed at five different concentrations to assess the expression levels of NF-κB p65 specifically in treated hormone-dependent breast cancer MCF-7 cells. At a concentration of 0.04 μM, the levels of NF-κB p65 were downregulated in treated MCF-7 cells when compared to untreated cells (Fig. 2A).

Figure 2.

Figure 2.

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Immunoblotting studies on NF-κB. (A) Western blot analysis shows the expression of the transcription factor NF-κB after 3h of incubation. The NF-κB subunit p65 (Rel A) was downregulated in capsicodendrin-treated MCF-7 cells and the effect on protein expression was concentration-dependent. The positive control was treated with rocaglamide and the negative control was not treated. β-Actin was used as an internal control. (B) Western blot analysis showed the expression of proteins in the NF-κB pathway. The expression of IκB kinase β (IKKβ) was downregulated in capsicodendrin treated MCF-7 cells and the effect was concentration dependent. β-Actin was used as an internal control.

Further, IκB kinase (IKK) is a pivotal upstream regulator of the NF-κB pathway19. Thus, the effect on the upstream IKKβ was assessed by western blot analysis (Fig. 2B). Decreased expression of IKKβ was detected at the concentration of 0.04 μM. It has previously been shown that NF-κB acts as a pro-metastatic factor facilitating adhesion of tumor cells to endothelial cells and it has been reported that capsicodendrin prevented the formation of new blood vessels20. In the present study, it was shown that the expression of intracellular adhesion molecule (ICAM-1) was downregulated in MCF-7 cells that were treated with capsicodendrin at 0.016 μM (Fig. 3A). The glucoprotein ICAM-1 is a member of the immunoglobulin superfamily. It is an essential molecule in cell-to-cell adhesion and mediates interaction between different cells. Silencing the expression of ICAM -1 decreases colony-formation in MCF-7 tumor cells and reduces cell adhesion. Further, it is involved in the transmigration of tumor cells and during metastasis. The downregulation of human ICAM-1 inhibits the invasion and metastatic ability of human breast cancer cell lines21. A previous study has also shown that NF-κB p65 inhibition suppressed expression of ICAM-1 and sensitized cells to anticancer compound treatment22, 23. Additionally, the present findings are in complete agreement with our previous studies performed in order to understand the mechanism of action of capsicodendrin, and demonstrated that it displayed angiostatic activity through selective inhibition of VEGFR2-mediated AKT signaling and dysregulated autophagy in endothelial cells during angiogenesis21. In addition, it has previously been shown that ICAM-1 is under the control of NF-κB in MCF-7 cells during the metastatic phase when tumor cells migrate through the vessels in the lymphatic route23. Thus, ICAM-1 is a key player in cancer and the effect of capsicodendrin on this target is worthy of further study.

Figure 3.

Figure 3.

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Expression of ICAM-1 and caspase-7. (A) ICAM-1 expression in capsicodendrin treated MCF-7 cells after 3 h of treatment. Decreased levels of adhesion molecule ICAM-1 were detected in TNF-α treated cells. The effect was concentration-dependent. (B) The effect of capsicodendrin on caspase-7 expression after 3 h of treatment in MCF-7 cells breast cancer cells was analyzed. Immunoblot analysis showed increasing concentrations of caspsicodendrin led to decreased levels of caspase-7. The effect was concentration-dependent. The effect was compared with rocaglamide, the positive control.

Moreover, treatment with capsicodendrin induced oxidative stress in treated estrogen–sensitive MCF-7 cells. The levels of ROS increased in capsicodendrin-treated cells compared to the positive control daunomycin (Fig. 4). Similarly, it has been found that capsicodendrin depleted glutathione in human myeloid leukemia cells and reduced the intracellular antioxidant capacity of cancer cells24. This suggests that oxidative stress promoted cell death in treated MCF-7 cells. Studies have shown that reactive oxygen species (ROS) play an important role in multidrug resistance in MCF-7 cells and thus together with the inhibition of NF-κB p65, the mechanism of action of capsicodendrin may prevent chemoresistance in cancer patients25.

Figure 4.

Figure 4.

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Intracellular levels of reactive oxygen species (ROS) formed in capsicodendrin treated estrogen-dependent breast cancer MCF-7 cells after 5 h of treatment. The effect was compared to the positive control daunomycin (a topoisomerase inhibitor). The cells were treated at four different concentrations. The formation of ROS was detected with the fluorescent probe DCFH-DA. The highest concentration of capsicodendrin (20 μM) led to a higher ROS formation than the highest concentration of daunomycin (20 μM). The effect of capsicodendrin was concentration-dependent. Vitamin C was used as a negative control.

The effect of capsicodendrin on cell-cycle arrest was examined using a fluorescence-activated cell sorter (FACS). The sub G1-phase population increased to 81% after 12 h of treatment with capsicodendrin (10 μM). This was compared to 46% of cells detected in sub G1-phase in non-treated MCF-7 cells (Fig. 5A–F). There was a significant loss of cells and the results showed that cell cycle arrest had been induced. The effect observed was concentration-dependent in treated cells; however, mitochondrial induced cell-death was not observed in capsicodendrin-treated cells and JC-1 monomers were not detected (Fig. 6). The results showed no loss of the outer mitochondrial membrane potential (ΔΨM) nor mitochondrial dysfunction or cell-death in capsicodendrin-treated MCF-7 cells. Hence, the effect was found to be independent of mitochondrial activity. According to previous studies, MCF-7 cells have a high expression of the anti-apoptotic protein Bcl-2 and lack the cellular response to apoptotic stimuli such as e.g. the activity of caspase-326, 27. To further understand the mechanism and the biochemical interactions through which capsicodendrin promoted cell-cycle arrest in MCF-7 the effect on both caspase-1 and caspase -7 were investigated.

Figure 5.

Figure 5.

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The effect of capsicodendrin on DNA fragmentation was evaluated in MCF-7 cells using fluorescence-activating cell flow cytometry (FACS). The cells were treated with capsicodendrin at five different concentration levels for 12 h: (A) control, (B) 0.0016 μM, (C) 0.008 μM, (D) 0.4 μM, (E) 2.0 μM, (F) 10 μM. The increasing concentration of capsicodendrin led to an increase of cell population detected in sub-G1 phase.

Figure 6.

Figure 6.

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The loss of mitochondrial membrane potential (ΔΨM) is a major event during apoptosis. Mitochondrial membrane potential (ΔΨM) was assessed using the cationic dye JC-1 in MCF-7 cells in untreated cells (A) and treated with capsicodendrin (B). In apoptotic unhealthy cells, JC-1 monomers were detected as indicated by green fluorescent cells when analyzed by cell flow cytometry. There was no increase in the formation of JC-1 monomers (564–606 nm) observed in capsicodendrin-treated cells when compared with untreated cells.

Caspase-1 is part of the inflammasome and is involved in inflammatory response. It has been shown to activate pro-inflammatory cytokine IL-1β28. Increasing evidence suggests that IL-1β might be involved in autoinflammatory diseases of unknown origin29. Recent studies have shown that there are increased levels of IL-1β in the tumor microenvironment of breast cancer tissue. An increase in IL-1β was estrogen-dependent and IL-antagonists inhibited tumor development and angiogenesis30, 31, 32. Herein, we report the effect of capsicodendrin on caspase-1 enzymatic activity in MCF-7 cells (Fig. 7 A). Capsicodendrin (0.12–3.50 μM) increased caspase-1 activity in a caspase-1 GLO assay compared with doxorubicin (0.14–3.5 μM) (Fig. 7 B). In addition, the treatment with increasing concentrations of caspsicodendrin (0.12–3.0 μM) induced caspase-1 activity even in cells treated with inhibitor YVAD-CHO. The effect correlates with increased levels of ROS and suggests that ROS promotes the activity of caspase-1. This effect was both concentration-dependent and time-dependent. It has previously been reported that caspase-1 induce p53 dependent cell-cycle arrest and inflammasome mediated apoptosis in estrogen dependent MCF-7 cells33.

Figure 7.

Figure 7.

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Caspase-1 activity was determined using a modified protocol from Promega Caspase-Glo® 1 Inflammasome Assay (G9951). The YVAD-CHO is a specific caspase-1 inhibitor. The MCF-7 cells were treated with doxorubicin (topoisomerase inhibitor) and capsicodendrin. Capsicodendrin (0.12–3.50 μM) (A) increased caspase-1 activity compared with doxorubicin (0.14–3.5 μM) that showed inhibitory activity (B). Increases in concentration levels of caspsicodendrin (0.12–3.0 μM) induced caspase-1 activity. The effect was concentration-dependent. This suggested that capsicodendrin potentiated caspase-1 activity in a concentration-dependent manner. The effect was compared to the negative control.

Earlier studies have shown that the levels of caspase-7 are overexpressed in estrogen-dependent breast cancer cells and thus the levels were evaluated in capsicodendrin wild-type p53 protein expressing MCF-7 cells34, 35. The results of this study showed that the levels of caspase-7 were downregulated in capsicodendrin treated (0.008 – 10 μM) MCF-7 cells. (Fig. 3B). The CASP7 promoter has five estrogen responsive elements and thus could represent potential specific targets in estrogen-dependent cancer cells34. Furthermore, caspase-7 targets various cell-cycle inhibitors such as p21 and DNA repair proteins36, 37. It has been shown that reduction of p21 contribute to proliferation and cell growth. Cyclin-dependent kinases (CDKs) play an important role and promote cell cycle progression38. Thus, CDK inhibitory proteins, such as p21, inhibit CDK activity, which, in turn, causes cell-cycle arrest in G1/S phase and negatively affects cancer cell proliferation39 40. The downregulated levels of caspase-7 correlated with the cell cycle arrest in MCF-7 treated cells. This inhibitory effect may have depended on the inhibitory effect of p21 on cell proliferation34.

Furthermore, in our previous studies, capsicodendrin was shown to be able to be converted to its more reactive monomer cinnamodial in the presence of nucleophilic solvents such as pyridine and DMSO in vitro. We proposed that capsicodendrin is present in plants as a chemical reservoir of the bioactive cinnamodial against predators21. The conversion of capsicodendrin to cinnamodial may qualify the compound as a prodrug; however, this remains to be evaluated in vivo. In this study, we focused on understanding the antiproliferative mechanisms of capsicodendrin against MCF-7, which is a hormone-dependent breast cancer cell line. Diligent efforts were made to use as little DMSO as possible during the assays in order to understand the mechanism of capsicodendrin. In summary, the results from this study showed that capsicodendrin inhibited the NF-kB pathway, increased oxidative stress, induced cell-death through caspase-1 activity, and exerted downregulation of the expression of caspase-7. Targeting the NF-κB pathway could potentiate specific anticancer activity in tumor cells and also potentiate existing anticancer treatments in the management of metastatic cancer34, 41.

4. Conclusion

The findings from the present drug discovery study showed that the NF-κB inhibitory effect of capsicodendrin induced cell death through a mitochondrial-independent pathway in malignant MCF-7 cells. The NF-κB inhibitory effect of capsicodendrin increased oxidative stress, the activity of caspase-1, and decreased the expression of estrogen-dependent caspase-7. In conclusion, inhibitors that target the NF-κB p65 signal pathway may induce additive anti-cancer therapeutic effects. Capsicodendrin and its potential analogues may be future lead molecules that selectively targets metastatic breast cancer cells and improve the prognosis of recurrent breast cancers.

Acknowledgements

We greatly acknowledge the financial support through program project grant P01 CA125066 and its supplement P01 CA125066-10S1 from the National Cancer Institute, NIH, Bethesda, MD to carry out the presented work. The authors are also thankful for partial support provided by the Jack L. Beal Endowment Fund of Dr. A. Douglas Kinghorn for Mr. Nathan Ezzone.

Footnotes

CONFLICT OF INTEREST

The authors declare that no conflict of interest is disclosed.

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