Santin and cirsimaritin from Betula pubescens and Betula pendula buds induce apoptosis in human digestive system cancer cells

Abstract Flavonoids are bioactive secondary metabolites of plants, which exert anti‐cancer effects. However, metabolism in enterocytes and the liver can influence the biological activity of flavonoids contained in the diet. Therefore, results from in vitro studies on cancer cells from the digestive tract and liver may reflect the real effects in the human body. Previously, we have found that the extract from birch buds exerts antiproliferative activity in a panel of cancer cells. In the present study, the anti‐cancer activity of ten flavonoids isolated from the buds of Betula pubescens and Betula pendula was characterized. Among them, santin and cirsimaritin significantly reduced viability, proliferation and clonogenicity of gastric (AGS), colon (DLD‐1) and liver (HepG2) cancer cells. Both flavonoids induced apoptosis, accompanied by activation of caspase‐3, caspase‐7, caspase‐8 and caspase‐9. Moreover, upregulation of p53 was detected only in wild‐type p53 harbouring cells. Together, our results suggest that santin and cirsimaritin exhibit promising anti‐cancer activity in cultures of digestive system cancer cells.

quercetin glycosides, as well as glycosides of kaempferol and myricetin, which together comprise up to 3% of the mass of birch leaves. 7,8 Birch buds are also used as a diuretic, mainly in Russian traditional medicine and to a lesser extent in Poland. 9 The composition and form of flavonoids in buds and leaves are different. In the leaves, flavonoids exist as glycosides while the buds are characterized by high content of aglycones, mainly various methyl ethers of naringenin, apigenin and kaempferol. 10 In vitro studies show higher cytotoxicity of flavonoid aglycones when compared with glycosides. 11 In addition indeed, alcohol extracts from silver birch leaves were shown to exert low cytotoxicity in glioma, breast cancer and epidermoid carcinoma cells. 12 However, flavonoids contained in birch extracts were reported to efficiently induce cancer cell death by various mechanisms. For instance, isoquercitrin promotes autophagy and subsequently activates mitochondrial-dependent pathway leading to increase in the level of active caspase-3 in liver cancer. 13 Mitochondrial pathway of apoptosis induced by hyperoside in colorectal cancer is triggered by p53. 14 Apigenin suppress cell cycle progression through G2/M arrest and promotes p53-dependent mitochondrial pathway of apoptosis in gastrointestinal cancers. [15][16][17] However, decrease in viability of apigenintreated liver cancer cells through induction of autophagy was also reported. 18 Both mitochondrial and extrinsic pathways are involved in induction of apoptosis in colorectal cancer cells treated with kaempferol. 19 Our recent report has shown that extracts of birch buds exhibit anti-cancer activity. 9 Therefore, it is necessary to investigate the effect of birch bud-derived flavonoid aglycones on cancer cell death.
The aim of this study was to quantify the content of selected flavonoids in birch buds, assess their antitumor effect and elucidate the molecular mechanism of flavonoid-mediated anti-cancer activity. The cytotoxic activity was investigated for ten flavonoids previously isolated from birch buds.

| Quantification of the flavonoid content in birch bud extracts
Carbon dioxide supercritical extraction (SFE) of birch buds was described in our previous publication. 9 The later optimisation of the procedure presented in our further study provided an increase in the yield of the extract from the buds of B. pubescens and B. pendula to 19.7 ± 1.7% and 25.3 ± 1.3%, respectively. All flavonoids studied in this work were isolated from the obtained extracts by column chromatography, identified by mass spectrometry and NMR as described in our recent work. 20 The content of selected flavonoids in the obtained extracts was determined from the results of gas chromatography-mass spectrometric (GC-MS) analysis. For this purpose, the mass spectrometric detector was calibrated by analysing a series of six calibration solutions prepared by the weight-volumetric method. The concentration range of the calibration solutions of flavonoids, isolated in sufficient quantities for the purposes of the work, was 0.06 to 2 mg/mL. Before GC-MS analysis, both birch bud extracts and flavonoid aglycones were submitted to a silylation procedure. The birch bud extracts (20 mg) were diluted with 1 mL of pyridine, and 100 μL of N,O-bis(trimethylsilyl)-trifluoroacetamide containing 1% trimethylchlorosilane was added. For each calibration solution (1 mL), 100 μL of silylating agent was added. The obtained mixtures were heated for 30 min at 60°C.
Quantitative analysis of flavonoids in birch bud extracts was conducted using an Agilent 7890A gas chromatograph with an Agilent 5975C mass spectrometer (Agilent, USA). A chromatographic separation was performed on a HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) at a helium flow rate of 1 mL/min. The initial column temperature was 50°C, rising to 325°C at3°C/min, and the final temperature was held for 10 min. The injector worked in a split mode (1:10) at a temperature of 300°C. The ion source and quadrupole temperatures were 230°C and 150°C, respectively. Electron ionization mass spectrums (EIMS) were obtained at ionization energy 70 eV.
To ensure selectivity in the separation of birch bud extracts, which are complex multicomponent mixtures, GC-MS analysis was carried out in the monitoring mode of selected ions (SIM), having the highest intensity in the mass spectrum of each of the compounds being determined. In the case of silanized flavonoids containing a TMS group at the C5 position, the most intense peaks are [M -15] + , which are formed as a result of the loss of the CH 3 group by the molecular ion. 21 The detection of the TMS derivatives of 7,4'-dimethylnaringenin, sakuranetin and santin was carried out using ions at m/z 357, 415 and 473, respectively, while in the mass spectra of kumatakenin, cirsimaritin and ermanin, the ion at m/z 473 has the highest intensity.
The GC-MS analyses were performed in triplicate for both birch bud extracts, as well as for each calibration sample. Table 1 shows the results of the quantitative determination of six flavonoids, as well as the regression equations and values of the determination coefficients R 2 . The mass spectra of all quantified flavonoids, as well as the resulting calibration plots, are provided in Supplemental Information (Figure S1, Figure S2, Table S1). Medium (PAN-Biotech, Germany) supplemented with 10% foetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) in 95% air and 5% carbon dioxide at 37°C.

| Colony formation assay
The method description is available as Supplemental Information.

| Apoptosis assay
The method description is available as Supplemental Information.

| Western immunoblot
The method description is available as Supplemental Information.

| Immunofluorescence microscopy
The method description is available as Supplemental Information.

| Statistical analysis
The results were analysed in GraphPad Prism software using a one-way ANOVA followed by Tukey's test, accepting p < 0.05 as significant.

| Quantitative content of selected flavonoids in SFE extracts of birch buds
The results of the quantitative analysis of the selected flavonoids in the extracts of both birch species, carried out using GC-MS, are presented in Table 1. The total flavonoid content in the extract of downy birch (121.78 ± 7.8 mg/g) was 4.7 times higher than in the extract from silver birch buds (25.85 ± 1.63 mg/g). It was found that the content of santin in the downy birch buds (17.71 ± 0.85 mg/g) is over twice as high as in the silver birch buds (7.35 ± 0.22 mg/g).

| Flavonoids isolated from birch buds exert differential effects on viability of digestive system cancer cells and fibroblasts
The cytotoxic effect of ten flavonoids isolated from birch bud extract:   Figure S3). For the most cytotoxic compounds, half maximal inhibitory concentration (IC 50 ) values were determined (Table 2).
Flavonoids with IC 50 values below 50 µM were considered to be active.
Among the tested compounds, santin was more cytotoxic than apigenin in all cancer cell lines; however, upon 24 h treatment,

| Santin and cirsimaritin induce apoptosis of digestive system cancer cells
The above findings indicate that treatment of cancer cells with santin and cirsimaritin results in a significant decrease in cell viability and proliferation. To further explore the mechanism by which these flavonoids decrease cell viability, an annexin V/propidium iodide staining assay was used, followed by fluorescence microscopy.   These data show that santin and cirsimaritin act as apoptosis inducers and santin has a prolonged proliferation-suppressing effect.

| DISCUSS ION
Parts of plants from the genus Betula, for instance, the leaves, bark, buds and flowers, have been widely used for centuries in traditional medicine. 5 In addition, Betula bark is also a source of triterpenoids, mainly betulinic acid and betulin, which have been extensively studied, both for anti-cancer properties and for obtaining derivatives. 22 Results of the present study demonstrated that the santin and cirsimaritin contained in the buds of both investigated birch species exhibit anti-cancer activity in gastric, colon and liver cell lines.
Moreover, the toxic effect of these flavonoids in normal fibroblasts was less pronounced when compared with cancer cells and these re- This effect of santin treatment might be of great importance, since COX-2 activity positively correlates with the occurrence and progression of cancer. 27,28 Presumably, the anti-inflammatory activity of santin is connected to its ability to attenuate the activity of the transcription factor NF-κB. 29 This protein is known to stimulate transcription of the genes encoding COX-2 and iNOS. 30 Importantly, NF-κB is also able to inhibit apoptosis. 31,32 However, there is no literature indicating the anti-cancer activity of santin, besides a description showing low activity of this compound as mitotic blocker. 33 Our results revealed that the viability of cancer cells is affected more strongly by santin than by any other flavonoid isolated from birch buds. Cytotoxic effect of santin was also stronger than in cells treated with apigenin which beneficial activity on digestive system cancer cells was proven in many reports. [15][16][17][18] In contrast, cirsimaritin belongs to the flavones class, and several studies have shown that cirsimaritin induces the death of various cancer cells, including colon cancer 34,35 and gastric cancer. 35 Interestingly, like santin, cirsimaritin inhibits the NF-κB pathway and down-regulates COX-2 and iNOS 36,37 ; however, its role in cancer needs to be elucidated. Our study confirms the significant anti-cancer activity of cirsimaritin. In addition, the obtained results reveal the mechanism of cell death induced by cirsimaritin and santin.
Our study demonstrated that apoptosis is primarily responsible for flavonoid-induced cell death. In the liver and digestive tract, the apoptotic process triggered by flavones and flavonols is associated with increased or decreased reactive oxygen species (ROS) production, 19,38,39 increase in p53 expression, 19 decrease in the mitochondrial membrane potential, 39,40 elevated Bax/Bcl-2 ratio, 18,39 release of cytochrome c from the mitochondria to the cytosol, 38 activation of caspase-9, 18,19,38,39 activation of caspase-8 39 and activation of caspase-3. 18,19,38,39,41 Consistent with these data, our study has demonstrated that santin and cirsimaritin trigger the intrinsic pathway of apoptosis (activation of caspase-9). These results are consistent with the finding that cirsimaritin activates caspase-9 and caspase-3. 23 Moreover, the detected induction of  [44][45][46] Up-regulation of p53 in wild-type p53-containing AGS and HepG2 cells suggests a partial contribution of p53 to the promotion of apoptosis. P53 is a well-characterized tumour suppressor transcription factor that gets activated by cellular stress such as DNA damage, oxidative stress, oncogene activation, hypoxia and telomere shortening. 40 Importantly, p53 has been reported to play a pivotal role in flavone-and flavonol-induced apoptosis 47,48 ; however, p53independent cell death was also described. 49 Research on the impact of the methoxyl group at C7 in flavones on cell death provides conflicting results depending on the cell line. 54,55 Cirsimaritin, which contains a free hydroxyl group at C4' and an additional methoxyl group at C6, exhibits strong apoptosisinducing activity. It has been shown that the introduction of a methoxyl group to flavonoids increases their lipophilicity and membrane permeability, leading to the accumulation of molecules in the cell. 56 Flavonols differ from flavones, with a hydroxyl group at C3 that is associated with reduced cytotoxicity. 53,57 Consistent with these findings, three flavonols with a free hydroxyl group at C3 (kaempferol, quercetin and rhamnocitrin) showed less pronounced toxicity than apigenin. However, three flavonols with a methylated hydroxyl group at C3 (ermanin, santin and rhamnocitrin) showed a differential effect on cell viability. Kumatakenin, which has a methylated hydroxyl group at C7, exhibits low toxicity, whereas free hydroxyl group-containing ermanin and santin significantly decreased cancer cell viability. This difference in activity may also be related to the methylation of the hydroxyl group at C4', since this modification was shown to have a different effect depending on cell type. 53,55,58 To conclude, the negative influence of the hydroxyl group at C3 is, at least in part, removed by methylation. Additionally, like cirsimaritin, santin is C6-methoxylated. Flavonoids exhibiting this type of F I G U R E 4 Santin and cirsimaritin activate intrinsic and extrinsic pathways of apoptosis. Western blot analysis for PARP, caspase-8, p53, PUMA, caspase-9, caspase-3 and caspase-7 in AGS, DLD-1 and HepG2 cancer cells treated with different concentrations of santin, cirsimaritin and apigenin for 48 h. Actin served as a control for protein loading structure, such as 3,6-dimethoxyapigenin, were found to have significant antiproliferative activity in a variety of cancer cell lines. 59 Therefore, it can be concluded that the methoxyl group at C6 probably leads to an increase in the cytotoxicity of flavonoids; however, this finding should be supported by further research.
It has been previously described that triterpene seco-acids isolated from the SFE extract of Betula pubescens buds induce apoptosis in gastric and colon cancer cells. 60 Our present study indicates that flavonoids, at least in part, may contribute to the previously reported cytotoxic activity of the SFE extract. 9 F I G U R E 5 Santin and cirsimaritin induce nuclear accumulation of p53 and activate effector caspases. Immunofluorescence analysis for p53, cleaved caspase-3 and cleaved caspase-7 in AGS, DLD-1 and HepG2 cancer cells treated with different concentrations of santin, cirsimaritin and apigenin for 48 h In conclusion, our studies indicate that santin and cirsimaritin induce apoptotic cell death via the intrinsic and extrinsic pathways of apoptosis in cancer cells. Further investigation using in vivo assays such as tumour xenografts to test santin and cirsimaritin efficacy would be a natural extension to our current study.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Writing-review & editing (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.