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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Arch Oral Biol. 2021 Feb 19;125:105087. doi: 10.1016/j.archoralbio.2021.105087

Propolis Reduces the Stemness of Head and Neck Squamous Cell Carcinoma

Felipe Nör 1,2, Carolina Nör 1,3, Letícia W Bento 1, Zhaocheng Zhang 1, Walter A Bretz 4, Jacques E Nör 1,5,6
PMCID: PMC8532192  NIHMSID: NIHMS1677871  PMID: 33639480

Abstract

Objective:

To evaluate the effect of Brazilian propolis on head and neck cancer stem cells in vitro.

Methods:

Head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-17B and UM-SCC-74A), human keratinocytes (HK), and primary human dermal microvascular endothelial cells (HDMEC) were treated with 0.5, 5.0, or 50 μg/ml green, brown or red Brazilian propolis or vehicle control for 24, 36, and 72 hours. Cell viability was evaluated by Sulforhodamine B assay. Western blots evaluated expression of cancer stem cell (CSC) markers (i.e. ALDH, CD44, Oct-4, Bmi-1) and flow cytometry was performed to determine the impact of propolis in the fraction of CSC, defined as ALDHhighCD44high cells.

Results:

propolis significantly reduced cell viability of HNSCC and HDMEC cells, but not HK. Notably, red propolis caused a significant reduction in the percentage of CSC, reduced the number of orospheres, and downregulated the expression of stem cell markers.

Conclusions:

Collectively, our data demonstrate an anti-CSC effect of propolis, and suggest that propolis (i.e. red propolis) might be beneficial for patients with head and neck cancer.

Keywords: Head and neck squamous cell carcinoma, Cancer stem cell, Stemness, Orospheres, Propolis

Introduction

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent head and neck cancer types worldwide with over 53,000 new cases estimated in the United States in 2019 (Surveillance, Epidemiology, and End Results Program (SEER), 2019). The standard of care for HNSCC patients is surgery combined with radiotherapy and chemotherapy. Although current therapy has improved the local disease control and tissue preservation, the overall survival rates in advanced cases remains low due to frequent recurrence and distant metastasis (León et al., 2005). In this context, novel adjuvant therapies should focus on targeting the tumor cells that are resistant to therapy, thus preventing tumor relapse.

One hypothesis to explain why some tumor cells escape from therapy is postulated by the cancer stem cell (CSC) hypothesis. CSC constitute a small subpopulation of neoplastic cells that are endowed with stem cell properties (e.g. multipotency and self-renewal). This specific population of tumor cells is typically in a quiescent state, making them resistant to cytotoxic agents that are designed to destroy cells that proliferate rapidly (i.e. platinum-based therapies or radiotherapy) (Prince & Allies, 2008). For this reason, CSC may persist and contribute to tumor progression and recurrence (Dean, Fojo, & Bates, 2005). We have demonstrated that treatment with Cisplatin increases the proportion of CSC in HNSCC tumors (Nör et al., 2014). Therefore, it seems reasonable to hypothesize that targeting CSC might be a plausible adjuvant therapy in the treatment of cancer.

Tumors are increasingly viewed as complex organs formed not only by tumor cells, but also by stromal cells that play role in the pathobiology of cancer (e.g. endothelial cells, immune cells, fibroblasts). To efficiently target head and neck CSC, defined as cells with high CD44 expression and aldehyde dehydrogenase (ALDH) activity (i.e. ALDHhighCD44high) (Krishnamurthy et al., 2010), it is necessary to define the factors that sustain the CSC pool of cells inside the tumor microenvironment. Normal stem cells reside in a distinct environment called the “stem cell niche”. The niche, which is composed of diverse stromal cells (i.e. mesenchymal and immune cells, a vascular network, soluble factors, and extracellular matrix components), regulates stemness, proliferation, and apoptosis resistance of stem-like cells (Borovski, De Sousa, Vermeulen, & Medema, 2011). CSC also depends on a specific environment, called the “tumor niche”. The cytokine loops result from the interactions between the tumor cells and the different cells from the microenvironment play essential roles in the maintenance of CSC as well as in tumor growth and development (Podberezin, Wen, & Chang, 2013). A previous work from our group has shown that CSC from HNSCC are found in close proximity to blood vessels (Krishnamurthy et al., 2010). In that work, CSC presented a strong dependency on endothelial cell-derived factors in order to survive and self-renew. Thus, depletion of tumor associated-blood vessels could be a reasonable adjuvant strategy for targeting CSC.

Propolis is a mixture of pollen from plants and vegetation collected and metabolized by honeybees to assemble and protect the honeycomb structure (Park, Alencar, & Aguiar, 2002). The variability of propolis composition is dependent on the geographical location and plants available for the bees (Toreti, Sato, Pastore, & Park, 2013; Paulino et al., 2008; Machado et al., 2016). Propolis has been used in folk medicine and cosmetic preparations due to its remarkable wound healing properties, among several other therapeutic attributes (Thomsom, 1990). Given the extensive list of biological properties of propolis already documented, it has scientifically shown potent antitumor properties in several cancers (e.g. glioblastoma, melanomas, lung and prostate) (Markiewicz-Zukowska et al., 2013; Ozturk et al., 2012), including HNSCC (Hehlgans, Lange, Eke, Kammerer, & Cordes, 2011; Ribeiro et al., 2015; Kuo et al., 2015; Celińska-Janowicz et al., 2018). Propolis was also effective in reducing the formation of new blood vessels (i.e. angiogenesis) in vitro and in vivo (Song, Park, Jung, & Jin, 2002; de Moura et al., 2011). Interestingly, propolis was also effective decreasing the number of CSC in breast cancer (Omene, Wu, & Frenkel, 2012) and neuroblastoma (Díaz-Carballo et al., 2014). However, to date the effect of propolis on CSC from HNSCC has not been addressed.

The aim of this study was to evaluate the effect of Brazilian propolis on CSC from HNSCC. Our results demonstrate that propolis not only reduces the proportion of CSC, but also downregulates the expression of key functional regulators of cancer stemness. Moreover, this natural compound is cytotoxic to HNSCC cells, suggesting a potential role of propolis as adjuvant agent for treatment of head and neck cancer.

Material & Methods

Cell Culture

Head and neck squamous cell carcinoma cell lines, UM-SCC-17B – laryngeal cancer (metastasis), UM-SCC-74A – base of tongue (primary tumor), and UM-SCC-81B – tonsillar pillar (metastasis), were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen; Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and with 100 U/ml penicillin-streptomycin (Invitrogen). The identity of all tumor cell lines was confirmed by genotyping at the University of Michigan DNA sequencing core facility. Human dermal microvascular endothelial cells (HDMEC; Lonza, Walkersville, MD, USA) were cultured in endothelial cell growth medium-2 (EBM2; Lonza), supplemented with human epidermal growth factor (hEGF), Hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), 5% FBS, vascular endothelial growth factor (VEGF), basic human fibroblast growth factor (hFGF-B), recombinant 3 insulin-like growth factor-1 (R3-IGF-1), and Ascorbic Acid (EGM2-MV Bulletkit). Human telomerase-immortalized gingival keratinocytes (HK) were cultured in keratinocyte basal medium (KBM; CC-3101, Lonza) supplemented with keratinocyte growth medium SingleQuot (KGM; CC-4131, Lonza). Mycoplasma Detection Kit (PlasmoTest®, InvivoGen, San Diego, CA) was performed to ensure that all cell lines were Mycoplasma free. This study was approved by the University of Michigan Institutional Biosafety Committee (protocol #IBCA00001111).

Propolis Composition and Extraction

The Brazilian propolis was previously typified by high-performance liquid chromatography (HPLC) in which prenylated compounds, cinnamic acid derivatives and flavonoids were detected as the main constituents for brown and green propolis (Moncla et al., 2012), whereas the main constituents for red propolis were antioxidants (Machado et al., 2016). The Brazilian different color types of propolis were collected in distinct geographical areas across the country (green – southeastern; brown – southern; red – northeastern). Natural raw propolis was extracted in dimethyl sulfoxide (DMSO) by shaking for 3 days at 37° C. The resulting solution was centrifuged at 13,000 rpm for 5 min at 25°C and filtered through a 0.22 μM strainer. The propolis solution was named Extract of Propolis in DMSO (EPDMSO). The final concentration of DMSO was set at 0.1%. Experimental groups were, as follows: Vehicle control (DMSO); 0.5 μg/ml, 5 μg/ml, or 50 μg/ml (EPDMSO).

Cytotoxicity Assay

Sulforhodamine B (SRB) assay was performed to evaluate the effect of propolis on HNSCC cell density. Briefly, UM-SCC-17B, UM-SCC-74A, HDMEC, and HK cells were seeded at 2x104 cells per well in 96-well plates, allowed to attach overnight, and treated with vehicle control (DMSO); 0.5 μg/ml, 5 μg/ml, or 50 μg/ml EPDMSO (Red, Brown and Green) for 24, 48 and 72 hours. Cells were fixed using 10% trichloroacetic acid, stained with 0.4% SRB (Sigma-Aldrich) in 1% acetic acid, and plates were read in a microplate reader at 560 nm (GENios; Tecan, Männedorf, Switzerland). Data were obtained from triplicate wells/condition and represent 3 independent experiments.

Flow Cytometry – Cell Sorting for CD44/ALDH

Single cell suspensions were obtained from trypsinization of UM-SCC-17B cells after treatment for 24 hours and re-suspended at 1x106 cells/ml phosphate buffered saline (PBS). Aldefluor kit (Stem Cell Technologies; Vancouver, Canada) was used to identify ALDH activity, as described (Podberezin, Wen, & Chang, 2013). Cells were incubated with activated Aldefluor substrate BODIPY-aminoacetaldehyde (BAAA) or the ALDH inhibitor diethylaminobenzaldehyde (DEAB) for 45 minutes at 37°C. Then, cells were exposed to anti-CD44 antibody (clone G44-26BD; BD Pharmingen; Franklin Lakes, NJ, USA). 7-Aminoactinomycin (7-AAD, BD Pharmingen) was used to select viable cells and flow cytometry assay was carried out to sort the CSC (i.e. ALDHhighCD44high cells) and non-CSC (i.e. ALDHlowCD44low cells).

Orosphere Culture

Orospheres (i.e. non-adherent spheroids formed by ≥25 HNSCC cells) (Krishnamurthy & Nör, 2013) were generated from 1,500 cells in 6-wells ultra-low attachment plates (Corning, NY, USA). Cells (ALDHhighCD44high or ALDHlowCD44low) were plated immediately after sorting and maintained in low glucose DMEM, 10% FBS (Invitrogen), and 100 U/ml Penicillin-streptomycin (Invitrogen) overnight before treatment. ALDHhighCD44high cells were treated with increasing concentrations of red propolis (0.5, 5, 50 μg/ml) for 10 days. Alternatively, ALDHhighCD44high or ALDHlowCD44low sorted cells were plated as single cells in 96-well plates and exposed to 5 μg/ml of red propolis or vehicle control for 10 days. This experimental design (i.e. single cell assay) avoids cell aggregation, once CD44 is a molecule involved in cell adhesion. New media and treatment were added every 4 days. At the completion of the experimental period, orospheres were counted under light microscope. Data were obtained from triplicate wells/condition and represent 3 independent experiments.

Western Blot Analyses

UM-SCC-17B cells were treated with increasing concentrations of red propolis (0.5-50 μg/ml) for 24 hours. Alternatively, cells were exposed to 5 μg/ml red propolis or vehicle control for 24, 48, or 72 hours. Primary antibodies were, as follows: rabbit anti-human Bmi-1 or Oct-4 (Cell Signaling Technology); mouse anti-human CD44 (Cell Signaling Technology), ALDH1/2 (Santa Cruz Biotechnology) or β-actin (Chemicon/Millipore). Immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).

Statistical Analyses

Parametric data was evaluated by one-way ANOVA followed by post-hoc tests (Tukey). Resultant means ± standard deviations (SD) of three independent experiments are depicted on Fig. 1–3 as well as Fig. 5 (A and B). Non-parametric data was analyzed by Mann Whitney test. Resultant medians of three independent experiments are depicted on Fig. 5 (D). Statistical analysis was carried out using the SigmaStat 16.0 software (SPSS, Chicago, IL). Statistical significance was determined at P<0.05.

Figure 1 –

Figure 1 –

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Propolis is cytotoxic to head and neck tumor cells. UM-SCC-17B cells were treated with increasing concentrations of red, brown or green propolis (0.5, 5, 50 μg/ml) for 72 hours (A-C). Cell viability was accessed through Sulforhodamine B (SRB) assay. Asterisks indicate statistical difference (P<0.05), as compared to control group (vehicle) for each time point. The bars represent the means while the error bars indicate the standard deviations.

Figure 3 –

Figure 3 –

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Propolis is cytotoxic to human endothelial cells. Human dermal microvascular endothelial cells (HDMEC) were treated with increasing concentrations of red, brown or green propolis (0.5, 5, 50 μg/ml) for 72 hours (A-C). Cell viability was accessed through Sulforhodamine B (SRB) assay. Asterisks indicate statistical difference (P<0.05), as compared to control group (vehicle) for each time point. The bars represent the means while the error bars indicate the standard deviations.

Figure 5 –

Figure 5 –

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Propolis inhibits orosphere formation and reduces the proportion of UM-SCC-17B cancer stem cells. A, Graph depicting the effect of red propolis treatment (0.5, 5, or 50 μg/ml) or vehicle control on the number of orospheres formed by UM-SCC-17B sorted cells (ALDHhighCD44high). B, Graph depicting the number of orospheres formed by ALDHhighCD44high or ALDHlowCD44low cells upon 5 μg/ml propolis treatment or vehicle control. The bars represent the means while the error bars indicate the standard deviations (A and B). C, Representative flow cytometry graphs depicting the percentage of UM-SCC-17B ALDHhighCD44high cells (p10) upon 5 μg/ml red propolis treatment or vehicle control. D, Graph depicting the percentage of UM-SCC-17B ALDHhighCD44high cells upon 5 μg/ml red propolis treatment or vehicle control. The bars represent the medians (D). Asterisks indicate statistical difference (P<0.05), as compared to control group (vehicle).

Results

Propolis is cytotoxic to HNSCC cells

SRB assay was performed to evaluate the effect of propolis on cell viability. Treatment with 50 μg/ml green, brown or red propolis caused a marked reduction in cell density of UM-SCC-17B (Fig. 1) or UM-SCC-74A (Fig. 2 A–C) after 72 hours of treatment when compared to lower concentrations or vehicle control. Importantly, the three propolis extracts did not affect the cell proliferation of human keratinocytes (Fig. 2 D–F). We also observed an inhibitory effect of propolis on the density of human endothelial cells, suggesting a possible anti-tumor angiogenesis effect (Fig. 3). Among all extracts evaluated here, red propolis induced the lower trend in cell viability during the experimental period (Fig. 1A, 2A, and 3A), when compared to green or brown samples. For this reason, red propolis was chosen to perform the subsequent experiments.

Figure 2 –

Figure 2 –

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Propolis is cytotoxic to head and neck tumor cells. UM-SCC-74A cells were treated with increasing concentrations of red, brown or green propolis (0.5, 5, 50 μg/ml) for 72 hours (A-C). Cell viability was accessed through Sulforhodamine B (SRB) assay. D-F, cell proliferation of human keratinocytes is not affected by propolis. Primary human keratinocytes were treated with increasing concentrations of red, brown or green propolis (0.5, 5, 50 μg/ml) for 72 hours. Cell viability was accessed through Sulforhodamine B (SRB) assay. Asterisks indicate statistical difference (P<0.05), as compared to control group (vehicle) for each time point. The bars represent the means while the error bars indicate the standard deviations.

Red propolis reduces the expression of stem cell markers

UM-SCC-17B cells were chosen for cell sorting, orosphere assay and western blot analysis due to the fact that this cell line is derived from a metastatic site of HNSCC, thus posing an additional challenge to each propolis extract since those cells theoretically present with a more aggressive behavior compared to UM-SCC-74A that is derived from a primary site. To evaluate the effect of propolis on the stemness of head and neck cancer cells, we performed western blot assays to determine the expression of key cancer stem cell (i.e. ALDH, CD44) and stem cell/self-renewal (i.e. Oct-4, Bmi-1) markers (Prince et al., 2007). Treatment with red propolis down-regulates the expression of ALDH, CD44, Oct-4 and Bmi-1 in a time- (Fig. 4A and C) and dose-dependent manner (Fig. 4B and D). Notably, propolis (5 μg/ml) was capable to shut down the expression of CD44 and Bmi-1 after 48 hours of treatment (Fig. 4A and C). UM-SCC-81B, a HNSSC cell line known to express high constitutive levels of ALDH, Oct-4 and CD44 (data not shown), was used as positive control (Fig. 4B). These results unveiled the first evidence indicating a role of red propolis in the regulation of critical stemness regulators in HNSCC cells.

Figure 4 –

Figure 4 –

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Red propolis inhibits the expression of stem cell markers. A and C, Western blots for ALDH, Oct-4, CD44 and Bmi-1 in UM-SCC-17B cells exposed to red propolis (5 μg/ml) or vehicle control for 24, 48, and 72 hours. B and D, Western blots for ALDH, Oct-4, CD44 and Bmi-1 in UM-SCC-17B cells exposed to increasing concentrations of red propolis (0.5 or 50 μg/ml) or vehicle control for 24 hours. UM-SCC-81B cells were used as positive control.

Propolis inhibits orosphere formation

Our group has developed and characterized the orosphere assay, an in vitro method for the study of head and neck cancer stem cells (Krishnamurthy & Nör, 2012). This method allows for the propagation of CSC that retain stemness and self-renewal properties. As described, orospheres were generated from sorted cells (ALDHhighCD44high and ALDHlowCD44low) and treated with increasing concentrations of red propolis (0.5, 5, 50 μg/ml) for 10 days. Propolis (5 or 50 μg/ml) caused a significant reduction on the number of orospheres when compared to low propolis concentrations or vehicle control (P<0.05). Remarkably, no orospheres were observed with the highest dose of propolis (Fig. 5A).

In a separate set of experiments, single ALDHhighCD44high or ALDHlowCD44low sorted cells were plated in 96-well ultra-low attachment plates to form orospheres. As previously noticed, ALDHhighCD44high cells have a higher capacity to form orospheres when compared to ALDHlowCD44low cells. Cells were treated with an intermediate dose of propolis (5 μg/ml) or vehicle control. After 10 days, red propolis significantly reduced the number of orospheres compared to vehicle control (P<0.05). As expected, ALDHlowCD44low cells formed less orospheres when compared to ALDHhighCD44high cells (Fig. 5B). Additionally, we treated UM-SCC-17B cells with 5 μg/ml red propolis or vehicle control for 24 hours and analyzed the percentage of cancer stem cells (ALDHhighCD44high) by flow cytometry. Red propolis significantly reduced the percentage of cancer stem cells when compared to vehicle control (Fig. 5C and D). Taken together, these results indicate a role of red propolis reducing the viability of head and neck cancer stem cells.

Discussion

Head and neck cancer is one of the most common tumor types. The prognosis and survival rate of patients with head and neck cancer are generally poor. A variety of treatments such as surgery, radiotherapy, and chemotherapy have been employed with modest results. Therefore, there has been an increasing interest to research natural alternative products available in nature for the treatment of this malignancy. Propolis, a natural compound metabolized by bees, has received significant attention for its ability to combat cancer cells (Markiewicz-Zukowska et al., 2013; Ozturk et al., 2012; Hehlgans, Lange, Eke, Kammerer, & Cordes, 2011; Ribeiro et al., 2015; Kuo et al., 2015; Celińska-Janowicz et al., 2018). Here, we studied the impact of propolis on the stemness of head and neck cancer cells.

Cancer stem cells have been considered important targets for cancer treatment due to their capacity to escape from conventional therapy, thus contributing to tumor recurrence and metastasis (Dean, Fojo, & Bates, 2005; Diehn, Cho, & Clarke, 2009). Caffeic acid phenyl ester (CAPE), a major component of propolis, was shown to be effective against CSC in breast cancer cell lines via interaction with the mitochondrial metabolism (Omene, Wu, & Frenkel, 2012; Bonuccelli, De Francesco, de Boer, Tanowitz, & Lisanti, 2017). Here, for the first time, we showed that Brazilian red propolis causes a significant reduction on the number of orospheres. This a method that allows for the propagation of cells that present with stem cell properties (also defined as ALDHhighCD44high by flow cytometry) due to their capability to form spheroid aggregates in low-attachment cell culture conditions. On the other hand, ALDHlowCD44low cells do not show the same properties which is reflected on their limited capability to form the cell clusters. The effect of red propolis reducing the formation of spheres was more robust in ALDHhighCD44high compared to ALDHlowCD44low cells, indicating a role of propolis reducing the stemness of HNSCC cells. Additionally, we observed a marked reduction of stem cell markers (i.e. CD44, ALDH, Oct-4 and Bmi-1) (Krishnamurthy & Nör, 2012) in HNSCC cells exposed to propolis, indicating a potential role of this compound as adjuvant agent for treatment of head and neck squamous cell carcinoma.

Propolis has well characterized anticancer and anti-angiogenic properties in vitro and in vivo (Markiewicz-Zukowska et al., 2013; Ozturk et al., 2012; Hehlgans, Lange, Eke, Kammerer, & Cordes, 2011; Ribeiro et al., 2015; Kuo et al., 2015; Celińska-Janowicz et al., 2018; Song, Park, Jung, & Jin, 2002; de Moura et al., 2011). Here, we show that three different extracts of propolis evaluated side by side (i.e. green, brown and red) caused significant reduction on cell viability of two HNSCC lineages. Importantly, the three extracts of propolis were not cytotoxic to normal human gingival keratinocytes highlighting their specific anti-tumoral effect. As previously mentioned, propolis samples were collected in distinct areas across Brazil, where botanical sources are different. In fact, Park et al. (2002) have shown that Brazilian propolis can be classified into 3 groups, based on the geographical origin (i.e. southern, southeastern, and northeastern Brazil). In this same work, they conclude that differences in plant ecology from each location define the chemical composition of propolis. Those specificities on chemical composition may explain the robust effect of red propolis (northeastern) when compared to green (southeastern) and brown (southern) samples observed in the present study.

To better understand the cytotoxic mechanism of propolis, Hehlgans et al. (2011) have demonstrated that propolis increased apoptosis and induced Caspase 3 cleavage in HNSCC cell lines. Propolis also caused important modifications in proteins involved in tumor progression, such as AKT, ERK1/2, FAK, E-cadherin and Cyclin D. Moreover, pre-treatment of propolis radiosensitized cancer cells to ionizing radiation therapy (Hehlgans, Lange, Eke, Kammerer, & Cordes, 2011). In the same way, Kuo et al. (2013) have verified that propolis caused down-regulation of AKT and Cyclin D, as well as FOXO and NF-κB, in HNSCC cells. Additionally, they showed that combination of propolis with 5-fluorouracil led to an additive effect on cell proliferation inhibition. These results show that propolis not only regulates different pathways associated with the progression of cancer, but also appears to facilitate radiotherapy and chemotherapy.

Here, we also observed a reduction of endothelial cell viability, when HDMEC cells were exposed to green, brown or red propolis extracts. Endothelial cells can regulate diverse aspects of cancer cell function, including proliferation, tumor growth, invasiveness and resistance (Song, Park, Jung, & Jin, 2002; de Moura et al., 2011). Borovski et al. (2011) showed that application of antiangiogenic therapy to gliomas and eradication of tumor vasculature results in a higher susceptibility of the CSC to cytotoxic agents. Furthermore, inhibition of angiogenesis and depletion of blood vessels by the VEGF neutralizing antibody bevacizumab reduced the CSC pool and, subsequently, inhibited tumor growth. This evidence supports the role of angiogenesis on CSC, and consequently in tumor progression, making the regulation of the perivascular niche by propolis a potential therapeutic strategy in cancer.

Conclusion

We demonstrated that propolis inhibits head and neck cancer stemness and survival. Besides the well-known established bioavailability by the oral administration and the long-term safety profile of propolis, data from in vitro experiments provide a rationale for further investigations evaluating the use of propolis as an adjuvant agent for patients with head and neck squamous cell carcinoma. Preclinical studies and further randomized clinical trials are appropriate study designs to test this potential new therapeutic application of propolis.

Highlights:

  • Cancer stem cells are associated with tumor progression and resistance to therapy

  • Propolis is cytotoxic to head and neck squamous cell carcinoma cells

  • Propolis reduces the fraction of head and neck cancer stem cells

Acknowledgements

We would like to thank the University of Michigan Angiogenesis Research Lab team for all support with the protocols and cell culture techniques. We thank Dr. Carey for kindly providing the HNSCC cell lines used in this study. We also thank the University of Michigan Flow Cytometry Core for their expert support to this project funded in part by the National Institutes of Health (NIH) Grant # R01-DE021139 (JEN), and by the National Council for Scientific and Technological Development (PIBIC/CNPq/Brazil) (FN).

Footnotes

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Conflict of Interest

The authors of the manuscript entitled “Propolis reduces the stemness of head and neck squamous cell carcinoma” have no conflict of interest to disclose.

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