Dammarane-Type 3,4-seco-Triterpenoid from Silver Birch (Betula pendula Roth) Buds Induces Melanoma Cell Death by Promotion of Apoptosis and Autophagy.

Despite unquestionable advances in therapy, melanoma is still characterized by a high mortality rate. For years, high expectations have been raised by compounds of natural origin as a component of pharmacotherapy, particularly by triterpenes found in the bark of birch trees. In this study, 3,4-seco-dammara-4(29),20(21),24(25)-trien-3-oic acid (SDT) was isolated from buds of silver birch and its mechanisms of cell death induction, including apoptosis and autophagy, were determined. Cytotoxicity of SDT was evaluated by the cell viability test and clonogenic assay, whereas induction of apoptosis and autophagy was determined by annexin V staining and Western blot. The results revealed dose- and time-dependent reductions in viability of melanoma cells. Treatment of cells for 48 h led to an increase in the percentage of annexin V-positive cells, activation of caspase-8, caspase-9, and caspase-3, and cleavage of PARP, confirming apoptosis. Simultaneously, it was found that SDT increased the level of autophagy marker LC3-II and initiator of autophagy beclin-1. Pretreatment of cells with caspase-3 inhibitor or autophagy inhibitor significantly reduced the cytotoxicity of SDT and revealed that both apoptosis and autophagy contribute to a decrease in cell viability. These findings suggest that 3,4-seco-dammaranes may become a promising group of natural compounds for searching for anti-melanoma agents.

Accumulation ability of trace metals by silver birch leaves in areas contaminated by Zn-Pb ore processing: Effects of excessive trace metal accumulation on specialized metabolism.

The study was carried out in the most industrialised region of southern Poland. Four areas were selected for the study: the vicinity of the Miasteczko Slaskie Zn-Pb-works (ZW; with three transects selected: A, B, and C), and the metalliferous waste heaps (H1, H2, H3). Samples were taken from the surface soil layer, while only mature silver birch (Betula pendula Roth) trees (3-4 m high) growing spontaneously in the study area were selected for the collection of leaves. The analysis of metals including trace metals (TMs; Ag, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, Pb, Tl, and Zn) in the soil and leaves, as well as the contents of specialized leaf compounds belonging to polyphenols were performed. In general, the B. pendula trees in transects A and B located within 500 m of the furnaces exhibited 3-9 times higher content of TMs in their leaves compared to the plants located further away from the Zn smelter. In contrast, the quantity of TM in trees from transect C was independent of distance. The accumulation of TMs in the leaves was significantly lower in the metalliferous waste heap sites compared to the ZW area. It was hypothesised that the accumulation of TMs in B. pendula leaves would depend on the location and distance from the polluting source. It was demonstrated that TMs content in soil affects the accumulation of phenolic acids and flavonoids.

Biotechnological potential of red yeast isolated from birch forests in Poland.

OBJECTIVES: This study aimed to isolate red yeast from sap, bark and slime exudates collected from Polish birch forests and then assessment of their biotechnological potential. RESULTS: 24 strains of red yeast were isolated from the bark, sap and spring slime fluxes of birch (Betula pendula). Strains belonging to Rhodotorula mucilaginosa (6), Rhodosporidiobolus colostri (4), Cystrofilobasidium capitaum (3), Phaffia rhodozyma (3) and Cystobasidium psychroaquaticum (3) were dominant. The highest efficiency of carotenoid biosynthesis (5.04 mg L-1) was obtained by R. mucilaginosa CMIFS 004, while lipids were most efficiently produced by two strains of P. rhodozyma (5.40 and 5.33 g L-1). The highest amount of exopolysaccharides (3.75 g L-1) was produced by the R. glutinis CMIFS 103. Eleven strains showed lipolytic activity, nine amylolytic activity, and only two proteolytic activity. The presence of biosurfactants was not found. The growth of most species of pathogenic moulds was best inhibited by Rhodotorula yeasts. CONCLUSION: Silver birch is a good natural source for the isolation of new strains of red yeast with wide biotechnological potential.

Differences in birch tar composition are explained by adhesive function in the central European Iron Age.

Birch bark tar is the most widely documented adhesive in prehistoric Europe. More recent periods attest to a diversification in terms of the materials used as adhesives and their application. Some studies have shown that conifer resins and beeswax were added to produce compound adhesives. For the Iron Age, no comparative large-scale studies have been conducted to provide a wider perspective on adhesive technologies. To address this issue, we identify adhesive substances from the Iron Age in north-eastern France. We applied organic residue analysis to 65 samples from 16 archaeological sites. This included residues adhering to ceramics, from vessel surface coatings, repaired ceramics, vessel contents, and adhesive lumps. Our findings show that, even during the Iron Age in north-eastern France, birch bark tar is one of the best-preserved adhesive substances, used for at least 400 years. To a lesser extent, Pinaceae resin and beeswax were also identified. Through statistical analyses, we show that molecular composition differs in samples, correlating with adhesive function. This has implications for our understanding of birch bark tar production, processing and mode of use during the Iron Age in France and beyond.

An Indicating Role of Antioxidant System Enzymes at the Stage of Active Structural Anomalies Formation in Karelian Birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti).

INTRODUCTION: A complex study of the antioxidant system enzymes (AOS) is an important subject of biochemical research; changes in the activity of these enzymes can be used as a biochemical marker of various processes in plants. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions in different wood formation processes, such as xylogenesis. This article discusses the outcomes of different behaviors of AOS enzymes, which are involved in both the redistribution of the ROS balance and phenolic compounds at the early stages of wood formation in young plants of silver birch (Betula pendula Roth) with straight-grained wood and Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet-Ahti) with non-figured and figured parts within the single trunk. BACKGROUND: Spectrophotometric determination of AOS enzymes' activity can be used as a biochemical marker in the different wood formation processes, including xylogenesis. In this study, we studied structural anomalies of the woody plant trunk of Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet- Ahti). OBJECTIVE: This study aimed to study AOS enzymes' activity in 12-year-old plants of silver birch (Betula pendula Roth) with straight-grained wood and Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet-Ahti) with non-figured and figured parts within the single trunk. METHODS: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4°C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 20 min extraction, the homogenate was centrifuged at 10000 g for 20 min (MPW-351R, Poland). The sediment was washed in the buffer thrice. The pooled supernatant and sediment were dialyzed at 4°C for 18-20 h against a tenfold diluted homogenization buffer. The enzymes' activity was determined spectrophotometrically (Spectrophotometer SF-2000, OKB Spectr, Russia). Proteins in the extracts were quantified by the method of Bradford. RESULTS: We observed different behaviors of the studied enzymes involved in both the redistribution of the ROS balance and phenolic compounds with subsequent lignification even at the early stages of wood formation in young plants and even in different trunk parts within a tree, which was consistent with results obtained earlier on adult plants. High SOD activity in the phloem compared to the activity in the xylem was accompanied by higher CAT activity. The POD/SOD ratio was significantly higher in the figured trunk parts in Karelian birch compared to other variants in the xylem and higher in Karelian birch plants compared to plants of common birch in the phloem. The CAT/POD ratio was significantly higher in plants with no signs of anomalies. The high POD and PPO activity in the xylem of figured trunk parts and in the phloem of figured and non-figured trunk parts of B. pendula var. carelica can be associated with the high activity of apoplast invertase. CONCLUSION: The study showed that at the stage of active formation of structural anomalies in the figured trunk parts in young plants of Karelian birch, hydrogen peroxide utilization occurred mainly due to increased POD activity. An increase in PPO activity in the trunk of figured plants could also be considered an indicator of the formation of structural anomalies. At the same time, in areas with developing abnormal wood, the POD/SOD ratio increased, and the CAT/POD ratio decreased, indicating a fine-tuning of the balance between superoxide radical and hydrogen peroxide, which, when changed, might regulate the rearrangement of xylogenesis towards proliferation in relation to differentiation.

Novel compounds discovery approach based on UPLC-QTOF-MS/MS chemical profile reveals birch bark extract anti-inflammatory, -oxidative, and -proliferative effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Betula pendula subsp. Mandshurica (Regel) Ashburner & McAll. Cortex (birch bark) is a globally traditional medicine for treating multiple inflammatory diseases. Its records are included in the Compendium of Materia Medica and other ancient medical literatures. However, uncovering its chemical profile and exploring novel biologically active compounds from birch bark remains a significant challenge. AIM OF THE STUDY: To uncover the anti-inflammatory, -oxidative, and -proliferative mechanisms and potentially effective compounds of birch bark extract by combing chemical profiling, isolation, identification, together with in vivo, in vitro, and silico evaluation. MATERIALS AND METHODS: Ultra-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) was used to obtain the chemical profile of birch bark extract. The new compounds were obtained via column chromatography and analyzed using X-ray diffraction and electronic circular dichroism for absolute configuration confirmation. The zebrafish caudal fin inflammation-induced model, qPCR, and Western blot analysis were used to explore the effects and underlying mechanisms of birch bark extract. In vitro cytotoxicity assays and kinases screening conducted to gain preliminary insight into the anti-proliferative effects of birch bark extract and its isolated compounds. In addition, in-silico molecular docking was performed to investigate the putative mechanism. RESULTS: UPLC-QTOF-MS/MS chemical profiles revealed 105 compounds in birch bark extract, with 80 of these were first reported in B. pendula subsp. Mandshurica cortex. We selected five compounds speculated as novel and isolated three ones (one triterpenoid derivative and two lupine series triterpenoids) for further analysis. Birch bark extract exerted antioxidative and anti-inflammatory effects on zebrafish, as shown by the downregulated reactive oxygen species levels and COX-2α, IL-1ß, and TNF-α expression, which occurred through NF-ĸB signaling pathway activation. The in vitro anti-proliferative effects of birch bark extract and compound 44 were also unveiled. Moreover, the putative anti-tumor mechanism of compound 44 was revealed using kinase screening and in-silico molecular docking. CONCLUSIONS: This study provided a predictable chemical profile and demonstrated the pharmacological effects of birch bark extract, elucidated the mechanism of this traditional Chinese medicine and suggested it as a novel anti-cancer candidate.

Comparative Analyses of Bioactive Compounds in Inonotus obliquus Conks Growing on Alnus and Betula.

Inonotus obliquus grows in the Northern Hemisphere on some living broadleaved tree species as a pathogen, causing stem rot. In Estonia, the fungus is well known in the Betula species but can also be found on Alnus. Sterile conks of I. obliquus contain different bioactive compounds, but the quantitative and comparative research of these compounds in conks on different host species is limited. In the current work, I. obliquus was isolated and, evidently, determined from Alnus incana (L.) Moench., Alnus glutinosa (L.) Gaertn., and Betula pendula Roth, and the content of bioactive compounds in conks on these hosts were analysed. All the analysed conks sampled from A. incana and B. pendula contained betulin that varied from 111 to 159 µg/g. A significantly (p < 0.05) higher betulinic acid content was found in conks sampled from A. incana when compared with B. pendula: 474−635 and 20−132 µg/g, respectively. However, the conks from Betula were richer in total polyphenols, flavonols, and glucans. The content of inotodiol was quite similar in the conks from A. incana (7455−8961 µg/g) and B. pendula (7881−9057 µg/g). Also, no significant differences in the lanosterol content were found between the samples from these two tree species. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on Alnus. The results demonstrate that the bioactive compounds are promising in conks of I. obliquus growing not only on Betula but also on the Alnus species. It supports the opportunity to cultivate I. obliquus, also on the Alnus species, thus increasing the economic value of growing this tree species in forestry.

Acetylenic Synthetic Betulin Derivatives Inhibit Akt and Erk Kinases Activity, Trigger Apoptosis and Suppress Proliferation of Neuroblastoma and Rhabdomyosarcoma Cell Lines.

Neuroblastoma (NB) and rhabdomyosarcoma (RMS), the most common pediatric extracranial solid tumors, still represent an important clinical challenge since no effective treatment is available for metastatic and recurrent disease. Hence, there is an urgent need for the development of new chemotherapeutics to improve the outcome of patients. Betulin (Bet), a triterpenoid from the bark of birches, demonstrated interesting anti-cancer potential. The modification of natural phytochemicals with evidenced anti-tumor activity, including Bet, is one of the methods of receiving new compounds for potential implementation in oncological treatment. Here, we showed that two acetylenic synthetic Bet derivatives (ASBDs), EB5 and EB25/1, reduced the viability and proliferation of SK-N-AS and TE671 cells, as measured by MTT and BrdU tests, respectively. Moreover, ASBDs were also more cytotoxic than temozolomide (TMZ) and cisplatin (cis-diaminedichloroplatinum [II], CDDP) in vitro, and the combination of EB5 with CDDP enhanced anti-cancer effects. We also showed the slowdown of cell cycle progression at S/G2 phases mediated by EB5 using FACS flow cytometry. The decreased viability and proliferation of pediatric cancers cells after treatment with ASBDs was linked to the reduced activity of kinases Akt, Erk1/2 and p38 and the induction of apoptosis, as investigated using Western blotting and FACS. In addition, in silico analyses of the ADMET profile found EB5 to be a promising anti-cancer drug candidate that would benefit from further investigation.

Dosing intact birch pollen grains at the air-liquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B.

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.

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

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.

Evaluation of Antioxidative Mechanisms In Vitro and Triterpenes Composition of Extracts from Silver Birch (Betula pendula Roth) and Black Birch (Betula obscura Kotula) Barks by FT-IR and HPLC-PDA.

Silver birch, Betula pendula Roth, is one of the most common trees in Europe. Due to its content of many biologically active substances, it has long been used in medicine and cosmetics, unlike the rare black birch, Betula obscura Kotula. The aim of the study was therefore to compare the antioxidant properties of extracts from the inner and outer bark layers of both birch trees towards the L929 line treated with acetaldehyde. Based on the lactate dehydrogenase test and the MTT test, 10 and 25% concentrations of extracts were selected for the antioxidant evaluation. All extracts at tested concentrations reduced the production of hydrogen peroxide, superoxide anion radical, and 25% extract decreased malonic aldehyde formation in acetaldehyde-treated cells. The chemical composition of bark extracts was accessed by IR and HPLC-PDA methods and surprisingly, revealed a high content of betulin and lupeol in the inner bark extract of B. obscura. Furthermore, IR analysis revealed differences in the chemical composition of the outer bark between black and silver birch extracts, indicating that black birch may be a valuable source of numerous biologically active substances. Further experiments are required to evaluate their potential against neuroinflammation, cancer, viral infections, as well as their usefulness in cosmetology.

Betulinic acid in the treatment of tumour diseases: Application and research progress.

Betulinic acid (BA) is a pentacyclic triterpene compound that can be obtained by separation, chemical synthesis and biotransformation from birch. BA has antitumour activity, and its mechanisms of action mainly include the induction of mitochondrial oxidative stress; the regulation of specificity protein transcription factors, and the inhibition of signal transducer and activator of transcription 3 and nuclear factor-κB signalling pathways. In addition, BA can increase the sensitivity of cancer cells to other chemotherapy drugs. Recent studies have shown that BA plays an anticancer role in several kinds of tumour diseases. In this article, the anticancer mechanism of BA and its application in the treatment of tumour diseases are reviewed.

Benzophenones from Betula alnoides with Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Cell Line.

The antiausterity strategy is a promising approach for the discovery of lead compounds with unprecedented anticancer activities by targeting the tolerance of cancer cells to nutrition starvation. These agents are selectively cytotoxic under the tumor microenvironment-mimicking condition of nutrition starvation, without apparent toxicity in the normal nutrient-rich condition. In this study, an ethanol extract of Betula alnoides showed antiausterity activity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions, with a PC50 value of 13.2 µg/mL. Phytochemical investigation of this active extract led to the isolation of eight benzophenones (1-8), including six new compounds, named betuphenones A-F (2-7), and three known xanthones (9-11). The structure elucidation of the new compounds was achieved by HRFABMS, NMR, and ECD spectroscopic analyses. A plausible biogenetic pathway of the new compounds was proposed. Compounds 1-7 displayed antiausterity activity with PC50 values of 4.9-8.4 µM. Moreover, compounds 2 and 7 induced alterations in PANC-1 cell morphology under nutrient-deprived conditions and also inhibited PANC-1 colony formation under nutrient-rich conditions.

Increased Induction of Apoptosis in ESCC (Esophageal Squamous-Cell Carcinoma) by Betula pendula Roth Stem Cell Extract Containing Triterpenoids Compared to Doxorubicin.

BACKGROUND: Esophageal Squamous-Cell Carcinoma (ESCC) is one of the most life-threatening malignancies worldwide, with a growing incidence in Iran higher than the global average. OBJECTIVE: The present study, for the first time under patent number (97668), introduces a method using in vitro production of activated-Birch stem cells using biotechnological techniques of tissue culture and plant stem cell culture from Betula pendula Roth (Birch) bark. METHODS: In the first step, Birch stem cells were produced in large amounts using tissue culture, and then the amount of triterpenoids of its extract was measured by the HPLC method. In the second step, the cytotoxicity was evaluated by MTT, and the IC50 was calculated. The cellular apoptosis in response to the extract compared to doxorubicin was measured using the Annexin V kit and the flow cytometry method. RESULTS: The optimized method introduced in the current study efficiently produced plant stem cells containing triterpenoids in large quantities over a period of 2-4 months. Our findings indicated that the growth of ESCC cells decreased by induction treatment 3 times (24, 36, 48 hours). IC50 values were obtained in 24 hours for the natural bark extract, Birch stem cell extract, doxorubicin and interactions of two extracts with doxorubicin at 300µg/mL, 1700µg/mL, 0.5µM, 150µg/mL, 1800µg/mL, respectively. In the flow cytometric test, the Birch stem cell extract showed the highest percentage of apoptosis, with 92.5% for total apoptosis. The percentage of total apoptosis in doxorubicin treatment was 85.33%, and the combination of doxorubicin with Birch stem cell extract was 88.33%. Natural bark extract and its combination with a lower percentage (69.33% and 70.33%, respectively) caused apoptosis of esophageal cancer cells. CONCLUSION: Owing to the extinction of Birch in Iran and its inaccessibility and exploitation, Birch stem cells can be cultured as an appropriate alternative source to produce valuable triterpenoids for pharmaceutical purposes. Additionally, according to the results of this study, stem cells can be used to enhance the treatment of esophageal cancer and supplementation with chemotherapy.

Betula utilis extract prolongs life expectancy, protects against amyloid-ß toxicity and reduces Alpha Synuclien in Caenorhabditis elegans via DAF-16 and SKN-1.

Betula utilis (BU), an important medicinal plant that grows in high altitudes of the Himalayan region, has been utilized traditionally due to it's antibacterial, hepatoprotective, and anti-tumor properties. Here, we demonstrated the longevity and amyloid-ß toxicity attenuating activity of B. utilis ethanolic extract (BUE) in Caenorhabditis elegans. Lifespan of the worms was observed under both the standard laboratory and stress (oxidative and thermal) conditions. Effect of BUE was also observed on the attenuation of age-dependent physiological parameters. Further, gene-specific mutants and green fluorescent protein (GFP)-tagged strains were used to investigate the molecular mechanism underlying the beneficial effects mediated by BUE supplementation. Our results showed that BUE (50 µg/ml) extended the mean lifespan of C. elegans by 35.99% and increased its survival under stress conditions. The BUE also reduced the levels of intracellular reactive oxygen species (ROS) by 22.47%. A delayed amyloid-ß induced paralyses was observed in CL4176 transgenic worms. Interestingly, the BUE supplementation was also able to reduce the α-synuclein aggregation in NL5901 transgenic strain. Gene-specific mutant studies suggested that the BUE-mediated lifespan extension was dependent on daf-16, hsf-1, and skn-1 but not on sir-2.1 gene. Furthermore, transgenic reporter gene expression assay showed that BUE treatment enhanced the expression of stress-protective genes such as sod-3 and gst-4. Present findings suggested that ROS scavenging activity, together with multiple longevity mechanisms, were involved in BUE-mediated lifespan extension. Thus, BUE might have potential to increase the lifespan and to attenuate neuro-related disease progression.

Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography-tandem mass spectrometry.

Pentacyclic triterpenoids (PCTs) possess high biological activity, including antitumor, anti-inflammatory, antiviral and hepatoprotective properties and are widespread in a plant biomass. Due to significant differences in polarity and other physicochemical properties, the simultaneous determination of different classes of PCTs by the methods of reversed phase liquid chromatography is difficult. In the present study, we proposed a new approach to chromatographic separation of such compounds based on the use of a stationary phase with a mixed retention mechanism combining hydrophobic, weak anion exchange and hydrophilic interactions. The use of the Acclaim Mixed-Mode WAX-1 column and tuning the selectivity by changing the contributions of different types of analyte-stationary phase interactions allowed the separation of 10 PCTs (betulin, erythrodiol, uvaol, friedelin, lupeol, ß-amyrin, α-amyrin, betulinic, oleanolic and ursolic acids) belonging to four different classes (monools, diols, ketones and triterpenic acids) during 7.5 min in isocratic elution mode. The combination of this approach with atmospheric pressure chemical ionization tandem mass spectrometric detection and pressurized liquid extraction of analytes with methanol allowed to develop a rapid, accurate and highly sensitive method for analyzing PCTs in plant tissues with a total duration of the analytical cycle (including sample preparation steps) of not more than 40 min. It provides the detection limits in plant biomass extracts of 3-12 µg L-1 (44 µg L-1 for friedelin). The developed method was validated and successfully tested in the analyses of real birch bark and lingonberry peels.

1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one from Betula platyphylla induces apoptosis by suppressing autophagy flux and activating the p38 pathway in lung cancer cells.

Betula platyphylla (BP) is frequently administered in the treatment of various human diseases, including cancers. This study was undertaken to investigate the pharmacological function of the active components in BP and the underlying mechanism of its chemotherapeutic effects in human lung cancer cells. We observed that BP extracts and 1,7-bis(4-hydroxyphenyl)-4-hepten-3-one (BE1), one of the components of BP, effectively decreased the cell viability of several lung cancer cell lines. BE1-treated cells exhibited apoptosis induction and cell cycle arrest at the G2/M phase. Further examination demonstrated that BE1 treatment resulted in suppression of autophagy, as evidenced by increased protein expression levels of both LC3 II and p62/SQSTM1. Interestingly, the pharmacological induction of autophagy with rapamycin remarkably reduced the BE1-induced apoptosis, indicating that apoptosis induced by BE1 was associated with autophagy inhibition. Our data also demonstrated that BE1 exposure activated the p38 pathway resulting in regulation of the pro-apoptotic activity. Taken together, we believe that BE1 is a potential anticancer agent for human lung cancer, which exerts its effect by enhancing apoptosis via regulating autophagy and the p38 pathway.

Cytotoxicity of Triterpene Seco-Acids from Betula pubescens Buds.

The present study investigated the magnitude and mechanism of the cytotoxic effect on selected cancer cell lines of 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) isolated from downy birch (Betula pubescens) buds by carbon dioxide supercritical fluid extraction and gradient column chromatography. Cell viability in six human cancer lines exposed to these compounds was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by annexin V/propidium iodide staining of gastric cancer AGS and colorectal cancer DLD-1 cells. To evaluate the mechanism of apoptosis, the expression of apoptosis-related proteins was analyzed by Western blot. Compound 1 exhibited non-specific toxicity, while compounds 2 and 3 were specifically toxic to colon and stomach cancer cells. The toxicity of compounds 2 and 3 against these two cell lines was greater than for compound 1. Cleavage of caspase-8, -9, and -3 was found in AGS and DLD-1 cells treated with all three seco-acids, indicating the induction of apoptosis via extrinsic and intrinsic pathways. Therefore, triterpene seco-acids (1-3) decreased cell viability by apoptosis induction. AGS and DLD-1 cells were more susceptible to seco-acids with an oxidized C19 than normal fibroblasts. Hence, it made them a new group of triterpenes with potential anticancer activity.

Platyphylloside Isolated from Betula platyphylla is Antiproliferative and Induces Apoptosis in Colon Cancer and Leukemic Cells.

Betula platyphylla bark has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diarylheptanoids are the major constituents of the B. platyphylla bark and possess various pharmacological effects. Our previous study confirmed the selective antiproliferative effect of platyphylloside (BPP) isolated from B. platyphylla on colon cancer and leukemic cells using 60 different cancer cell lines from thr National Cancer Institution (NCI). In line with previous reports, this study focuses on the apoptotic pathway of BPP, a phenolic glycoside composed of two aromatic rings joined by a seven-carbon chain. Cytotoxicity assays in solid tumor and blood cancer cell models demonstrated that BPP possesses potent antiproliferative activity. The level of apoptosis increased with BPP treatment, causing cell cycle arrest at the G1 phase along with the downregulation of IκBα phosphorylation and BCL-2, as well as upregulation of cleaved caspase 3 and BAX proteins. In addition, BPP displayed potent mitochondrial depolarization effects in Jurkat cells. The combined findings revealed that the cytotoxic effects of BPP were mediated by intracellular signaling, possibly through a mechanism involving the upregulation of mitochondrial reactive oxygen species (ROS). Thus, BPP could be a potential multitarget therapeutic agent in leukemia and colon cancer.

Betulin-1,4-quinone hybrids: Synthesis, anticancer activity and molecular docking study with NQO1 enzyme.

Betulin-1,4-quinone hybrids were obtain by connecting two active structures with a linker. This strategy allows for obtaining compounds showing a high biological activity and better bioavailability. In this research, synthesis, anticancer activity and molecular docking study of betulin-1,4-quinone hybrids are presented. Newly synthesized compounds were characterized by 1H, 13C NMR, IR and HR-MS. Hybrids were tested in vitro against a panel of human cell lines including glioblastoma, melanoma, breast and lung cancer. They showed a high cytotoxic activity depending on the type of 1,4-quinone moiety and the applied tumor cell lines. It was found that cytotoxic activities of the studied hybrids were increasing against the cell line with higher NQO1 protein level, like melanoma (C-32), breast (MCF-7) and lung (A-549) cancer. Selected hybrids were tested on the transcriptional activity of the gene encoding a proliferation marker (H3 histone), a cell cycle regulators (p53 and p21) and an apoptosis pathway (BCL-2 and BAX). The obtained results suggested that the tested compounds caused a mitochondrial apoptosis pathway in A549 and MCF-7 cell lines. The molecular docking was used to examine the probable interaction between the hybrids and human NAD[P]H-quinone oxidoreductase (NQO1) protein. The computational studies showed that the type of the 1,4-quinone moiety affected the location of the compound in the active site of the enzyme. Moreover, it was shown that an interaction of 1,4-quinone fragment with the hydrophobic matrix of the active site near Tyr128, Phe178, Trp105 and FAD cofactor could explain the observed increase of TP53 gene expression.