Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. AIM OF THE STUDY: In this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. MATERIALS AND METHODS: The antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear-cytoplasmic separation assays. RESULTS: EAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR- PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR- PCa cells. CONCLUSIONS: EAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR- PCa cells.

Cordia Dichotoma: A Comprehensive Review of its Phytoconstituents and Endophytic Fungal Metabolites and their Potential Anticancer Effects.

Cordia Dichotoma is a valuable medicinal plant belonging to the family Boraginaceae. It consists of several beneficial secondary metabolite components, including alkaloids, carbohydrates, flavonoids, glycosides, saponins, and tannins. Numerous studies have been conducted to assess the anticancer properties of Cordia Dichotoma on MCF-7, A-549, PC3, and HeLa cancer cell lines, primarily utilizing ethanolic extract, methanolic extract, and chloroform extract. The results of these studies have demonstrated significant effects. Furthermore, several studies have revealed the rich phytoconstituent content of Cordia Dichotoma with some significant components previously utilized by researchers to investigate the anticancer properties of specific compounds. This review discusses several of these components, including ß-sitosterol, α-amyrin, Quercitrin, Robinin, betulin, Taxifolin, and Hesperetin. Additionally, a recent study uncovered that the anticancer effect of metabolites from endophytic fungi residing on the Cordia Dichotoma plant is attributed to a property of the plant itself. This review focuses on the current state of anticancer research related to this plant and its components.

Microwave-Assisted Extraction of Anticancer Flavonoid, 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethyl Chalcone (DMC), Rich Extract from Syzygium nervosum Fruits.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid-liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R2 = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.

Exploring the effects of different processing techniques on the composition and biological activity of Platycodon grandiflorus (Jacq.) A.DC. by metabonomics and pharmacologic design.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. (PG) is a common natural medicine with a history of thousands of years. The processing products were mainly recorded as raw, honey-processed, wine-fried, yellow-fried, and bran-fried PG, which were respectively used for different clinical purposes. Therefore, it is necessary to study the chemical composition and pharmacological activity of PG after processing. AIM OF THE STUDY: To explore the effects of different processing methods on the composition and biological activity of PG using metabonomics and pharmacologic design. MATERIALS AND METHODS: UPLC-QTOF-MS combined with multivariate statistical analysis was used to identify different metabolites before and after the processing of PG. Network pharmacology was used to construct the metabolite-target-disease network. CCK-8 assay, flow cytometry, and western blotting were used to detect cell viability, apoptosis, and the expression of related proteins, respectively. RESULT: A total of 43 differentially expressed metabolites (VIP >10) were detected and identified in the analyzed groups. Based on their chemical nature, these metabolites were divided into five categories, namely, saccharolipids, flavonoid glycosides, alkynes, saponins, and lipids (including fatty acids, phospholipids, fatty aldehydes, and sterols). The content of lipids in the five processed groups (CH, FC, JZ, MZI, and MZG) was found to be higher than that in raw PG. In particular, the processing approaches explored herein increased the contents of many phospholipids, such as, glycerophosphoinositols, phosphatidic acids, and lysophosphatidyle·thanolamines. The 8 metabolites were found by venn diagram to distinguish different processed products (metabolites 2, 6, 19, 20, 21, 26, 28, and 38). The results of network pharmacology analysis showed that the primary anti-cancer targets of 43 metabolites of PG processing products are PIK3CA, Akt, and STAT3, and based on CCK-8 assay, MZI has a significant killing effect on A549 cells, compared to other processing techniques. Moreover, flow cytometry analysis showed that the cells treated with MZI exhibit significantly increased cell apoptosis, and that the effect is dose-dependent. Finally, the western blots performed herein demonstrated that the MZI effectively inhibits the expression of p-Akt and p-STAT3, which is consistent with the network pharmacology results. CONCLUSION: Depending on the processing technique, the contents of 43 different metabolites in PG were varied significantly. Specifically, the contents of phospholipids and fatty acids increase, whereas the contents of large Mw saponins decrease. Compared to the other investigated processing methods, MZI increases the potential of PG in inducing cell apoptosis and inhibiting cell proliferation by affecting the Akt and STAT3 signaling pathways. The increased levels of 3-O-ß-glucopyranosyl polygalacic acid and platycoside F after honey-frying confirm these results.

Cytotoxic terpenoids from Tripterygium hypoglaucum against human pancreatic cancer cells SW1990 by increasing the expression of Bax protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium hypoglaucum (Kunmingshanhaitang in Chinese) is a plant of the genus Tripterygium which have been used as anti-tumor folk medicines in Yi and Bai ethnic groups in Yunnan province, China for hundreds of years. Terpenoids from T. hypoglaucum presented therapeutic effects on multiple tumors. But there were few studies about pancreatic cancer treatment of these terpenoids. Pancreatic cancer is an aggressive malignancy and lacked of specific drugs. Currently, anti-tumor drugs have poor therapeutic effect and prognosis for pancreatic cancer. AIM OF THE STUDY: This study aimed to elucidate the terpenoids from T. hypoglaucum and illuminate their anti-pancreatic cancer bioactivities. MATERIAL AND METHODS: Terpenoids were obtained through sequential chromatographic methods including silica gel, MCI gel, Sephadex LH-20, and preparative HPLC. Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configurations of some new diterpenoids were assigned through comparison of experimental and calculated circular dichroism spectra. The cytotoxicity of isolates was measured using the MTT method on human pancreatic cancer cells SW1990. The effects on expressions of AKT, Erk1/2, p-AKT, p-Erk1/2, and Bax proteins in human pancreatic cancer cells SW1990 of these compounds were determined by western blotting assays. RESULTS: Eleven new (compounds 1∼11) and fourteen known terpenoids (compounds 12∼25) were isolated from the underground parts of T. hypoglaucum. These compounds were belonged to abietane diterpenoids, isoprimara diterpenoids, ent-kaurane diterpenoids, oleanane triterpenoids, and friedelane triterpenoids. Compounds 5, 7, 8, 9, 16, 18, 22, 24, and 25 possessed significant cytotoxicity against SW1990 cells with IC50 values of 19.28 ± 4.39, 9.91 ± 2.23, 27.32 ± 5.89, 56.43 ± 6.92, 0.16 ± 0.05, 0.58 ± 0.15, 0.81 ± 0.04, 0.48 ± 0.11, and 10.01 ± 1.39 µM respectively. After compounds 16, 22, and 24 been treated with the pancreatic cancer cells in medium and high doses, the protein expressions of AKT, p-AKT, Erk, and p-Erk were not remarkably reduced and the expressions of Bax protein were significantly increased. CONCLUSION: This study indicated that terpenoids from T. hypoglaucum could inhibit human pancreatic cancer cells SW1990. Especially, compounds 16, 22, and 24 possessed significant cytotoxicity against SW1990 cells with low IC50 values and could increase the expressions of Bax protein. These compounds shared a wide variety of structural characteristics which provided us more candidate molecules for the development of anti-pancreatic cancer drugs and further prompted us to investigate their anti-pancreatic mechanisms.

Brusatol inhibits the growth of renal cell carcinoma by regulating the PTEN/PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Brucea javanica (L.) Merr. is a medicinal herb used in China for the prevention and treatment of diseases such as cancer and malaria. Brusatol was isolated from the seeds of Brucea javanica (L.) Merr, brusatol has a wide range of pharmacological effects, including anti-inflammation and anti-cancer effects. AIM OF THE STUDY: Renal cell carcinoma is one of the most common urinary system tumours and seriously threatens the lives of patients. We aimed to study the mechanism by which brusatol regulates the growth of renal cancer cells through the PTEN/PI3K/AKT signalling pathway. MATERIALS AND METHODS: We chose the A498, ACHN, and OSRC-2 cell lines as experimental models. After intervention with brusatol, CCK-8 experiments and plate cloning experiments were used to detect the cell proliferation ability; flow cytometry was used to detect the cell apoptosis rate; scratch and transwell invasion assays were used to detect the cell migration and invasion ability; qRT-PCR and Western blotting was used to detect PTEN, p-PI3K/PI3K, p-AKT/AKT, Bax, Bcl2, E-cadherin, N-cadherin, and vimentin relative expression. Then, we knocked down the PTEN gene in the three cell lines and again tested the proliferation, apoptosis, migration, and invasion capabilities of each group of cells. RESULTS: Brusatol significantly inhibited the proliferation, migration and invasion and increased the rate of apoptosis of the A498, ACHN, and OSRC-2 cell lines, and brusatol significantly increased the expression of PTEN mRNA and protein, and inhibited the expression of p-PI3K and p-AKT. Moreover, knockdown of PTEN significantly reduced the inhibitory effect of brusatol on the growth of renal cancer cells. CONCLUSION: Our research results show that brusatol has an effective inhibitory effect on the growth of A498, ACHN, and OSRC-2 renal cancer cell lines, and this effect is likely to be produced by regulating the PTEN/PI3K/AKT signalling pathway.

Pharmacological studies of rhizomes of extract of Cyperus tegetum, emphasized on anticancer, anti-inflammatory and analgesic activity.

ETHNOPHARMACOLOGICAL RELEVANCE: With over 950 species, Cyperus is one of the most promising health boosting genera in the Cyperaceae family. Traditional uses of Cyperus sp. have been described for gastrointestinal blood abnormalities, menstrual irregularities, and inflammatory diseases, among others. Cyperus tegetum Roxb belonging to Cyperaceae family, is used in traditional medicine to treat skin cancers. AIM OF THE STUDY: The present study was carried out to explore the potential effect of the extract of the plant Cyperus tegetum against different pharmacological activity namely inflammatory, analgesic activity as well as skin cancer activity in mice. MATERIALS AND METHODS: Cytotoxicity of the extract was measured by MTT and Live/death assay on HeLa cell line. Skin cancer was induced by 7,12-dimethylbenz(a) anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice to measure its effects. RESULT: Stigmasterol and some poly phenolic compounds are identified using HPTLC process from the methanol extract of the rhizome of the plant Cyperus tegetum (CT-II). After confirmation of the presence of different polyphenolic compound and triterpenoids in the extract, it was subject to MTT and Live/death assay on HeLa cell line. From the observation it could be concluded that the IC50 of the extract is 300 µg/ml. Thus, the CTII was evaluated further for its in vivo anticancer property. In the tumorigenesis study, the number of tumor growths, the area and weight of the tumor significantly decreases with increment in the dose of CT-II extract and some elevated enzyme release in renal (creatinine, urea) as well as hepatic (AST, ALT, ALP) enzymes are also controlled with the increased dose of the same extract. The elevated enzyme release may be due to cancer induced rupture of the plasma and cellular damage. This CT-II extract also exhibits some other pharmacological activity like anti-inflammatory and analgesic activity. CONCLUSION: As metabolic activation via carcinogens and inflammation response plays important role in development of cancer, antioxidant, anti-inflammatory and analgesic properties can be correlated with anti-cancer properties. Taken all the above studies, it was illustrated that the extract of Cyperus tegetum might be a promising compound to reduce skin cancer risk.

Preparation and structural properties of selenium modified heteropolysaccharide from the fruits of Akebia quinata and in vitro and in vivo antitumor activity.

Cancer is a complex disease, and blocking tumor angiogenesis has become one of the most promising approaches in cancer therapy. Here, an exopoly heteropolysaccharide (AQP70-2B) was firstly isolated from Akebia quinata. Monosaccharide composition indicated that the AQP70-2B was composed of rhamnose, glucose, galactose, and arabinose. The backbone of AQP70-2B consisted of →1)-l-Araf, →3)-l-Araf-(1→, →5)-l-Araf-(1→, →3,5)-l-Araf-(1→, →2,5)-l-Araf-(1→, →4)-d-Glcp-(1→, →6)-d-Galp-(1→, and →1)-d-Rhap residues. Based on the close relationship between selenium and anti-tumor activity, AQP70-2B was modified with selenium to obtain selenized polysaccharide Se-AQP70-2B. Then, a series of methods for analysis and characterization, especially scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS), indicated that Se-AQP70-2B was successfully synthesized. Furthermore, zebrafish xenografts and anti-angiogenesis experiments indicated that selenization could improve the antitumor activity by inhibiting tumor cell proliferation and migration and blocking angiogenesis.

Paeoniflorigenone regulates apoptosis, autophagy, and necroptosis to induce anti-cancer bioactivities in human head and neck squamous cell carcinomas.

ETHNOPHARMACOLOGICAL RELEVANCE: Paonia suffruticosa Andr. belonging to the family Paeoniaceae and has been used as a medicinal plant in Asian countries including China, Korea, and Japan. The roots of P. suffruticosa has been used in traditional medicine in various diseases including cancer and cardiovascular, female genital, and inflammatory diseases. AIM OF THE STUDY: Head and neck squamous cell carcinomas (HNSCCs) pathologically account for 90% of all head and neck cancers. However, effective targeted therapies for HNSCCs are insufficient and the prognosis is very poor, especially in patients with metastatic HNSCCs. To overcome the current limitations of available therapies for HNSCCs, pathological approaches using natural compounds are attracting attention. Our study aimed to demonstrate the anti-cancer effects of paeoniflorigenone (Paeo, 98.9% purity) isolated from the root bark of P. suffruticosa. MATERIALS AND METHODS: Our scientific methodology was performed as follows: cytotoxicity, morphological changes, and apototic DNA fragmentation were analyzed using MTT, light microscopy, and TUNEL assays. Protein expression, apoptosis, necroptosis, and autophagy were analyzed using Western blot and immunofluorescence assays. Cell migration and invasion were analyzed using wound healing and Boyden chamber assays. RESULTS: We demonstrated that Paeo significantly reduced cell proliferation and cell division, leading to caspase-dependent apoptotic cell death in human YD-10B HNSCC cells. This result was associated with PI3K/AKT/mTOR/p70S6K signaling in these cells. In addition, we investigated other programmed cell death mechanisms associated with apoptosis and found that Paeo inhibited necroptosis via dephosphorylation of key necroptotic proteins (RIP and MLKL), whereas Paeo induced autophagy via increased LC3I/II expression and autophagosome formation in human YD-10B HNSCC cells. The anti-metastatic effects of Paeo significantly suppressed cell migration and invasion in human YD-10B HNSCC cells. CONCLUSION: Overall, our results demonstrated that the bioactive compound, Paeo, exhibited anti-cancer bioactivities in human YD-10B HNSCC cells, suggesting that Paeo may be an attractive pathological approach for patients with human HNSCCs.

Radioprotective Effects of Plants from the Lamiaceae Family.

BACKGROUND: Edible and medicinal plants are still an interesting source of promising biologically active substances for drug discovery and development. At a time of increasing cancer incidence in the world, alleviating the bothersome side effects of radiotherapy in debilitated cancer patients is becoming an important challenge. OBJECTIVE: The aim of the study was to overview the literature data concerning the radioprotective activity of extracts, essential oils, and some chemical compounds obtained from 12 species belonging to the Lamiaceae family, gathering of numerous spice and medicinal plants rich in valuable phytochemicals. RESULTS: The analysis of available publications showed radioprotective effectiveness of essential oils and complex extracts containing phenolic acids and flavonoids in various in vitro and in vivo models. Relatively welldocumented preventive properties exhibited the following species: Mentha × piperita, Ocimum tenuiflorum, Origanum vulgare, and Rosmarinus officinalis. However, few plants such as Lavandula angustifolia, Mentha arvensis, M. spicata, Plectranthus amboinicus, Salvia miltiorrhiza, S. officinalis, Scutellaria baicalensis, and Zataria multiflora should be more investigated in the future. Among the mechanisms of radioprotective effects of well-studied extracts and phytochemicals, it can be mentioned mainly the protection against chromosomal damage, scavenging free radicals, decreasing of lipid peroxidation and elevating of glutathione, superoxide dismutase, catalase, and alkaline phosphatase enzyme levels as well as the reduction of the cell death. The plant substances protected the gastrointestinal tract, bone marrow and lung fibroblasts. CONCLUSION: The studied species of Lamiaceae family and their active chemical compounds are potent in alleviating the side effects of radiotherapy and should be considered as a complementary therapy.

Antitumour effect of odoroside A and its derivative on human leukaemia cells through the ROS/JNK pathway.

Oleandrigenin-3-O-ß-D-diginoside (a derivative of odoroside A), isolated and purified by our group, has seldom been explored for its pharmacological activity. This study aimed at clarifying the mechanisms towards the leukaemia-suppressive role of odoroside A (compound #1) and its derivative, oleandrigenin-3-O-ß-D-diginoside (compound #2) isolated from Nerium oleander. Viability and nuclear morphology change were assessed by CCK-8 assay and fluorescence microscope, respectively. Then, the cell apoptosis and autophagy induced by the compounds were detected by flow cytometry and Western blot. Xenograft model of nude mice was also applied to measure the leukaemia-suppressive effects of compound #2 in vivo. The result displayed that compound #1 and compound #2 inhibited the proliferation of HL60 and K562 cells and stronger effects were found in HL60 than K562 cells. Both of the compounds induced a dose-dependent apoptosis and autophagy in HL60 cells, where compound #2 was more potent than compound #1. Compound #2 also demonstrated a time-dependent apoptosis and autophagy in HL60 cells. Furthermore, ROS generation and JNK phosphorylation occurred in a dose-dependent manner in the cells treated with compound #2. Mitochondria also played critical role, proved by the decrease of Bcl-2, the release of cyto c to cytosol and the activation of caspase-3 and caspase-9. Moreover, the antitumour effects of compound #2 were validated in the nude mouse xenograft model in vivo. Odoroside A and its derivative inhibited the growth of leukaemia by inducing apoptosis and autophagy through the activation of ROS/JNK pathway. These results suggest that the compounds can serve as potential antitumour agents against leukaemia, especially acute myeloid leukaemia (AML).

A new discovery: Total Bupleurum saponin extracts can inhibit the proliferation and induce apoptosis of colon cancer cells by regulating the PI3K/Akt/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC has a history of using herb in China for more than 2000 years, which can be traced back to the Classic of Shennong Materia Medica in the Han Dynasty. Although Saikosaponin, the main active ingredient of Bupleurum, has the effects of anti-tumor, yet we still do not know the mechanism by total Bupleurum saponin extracts (TBSE) produces this effect on colon cancer. AIM OF THE STUDY: It is predicted by network pharmacology that TBSE may play an anti-colon cancer role by regulating the PI3K-Akt-mTOR pathway. The purpose of this study is to investigate whether TBSE inhibits proliferation and promote apoptosis of colon cancer cells by regulating PI3K/Akt/mTOR pathway. MATERIALS AND METHODS: The effect of saikosaponins on the proliferation of SW480 and SW620 cells was detected by CCK-8, apoptosis was determined by flow cytometry, morphological changes of cells were observed by microscope, nuclear morphological changes were observed after immunofluorescence staining, the expression of apoptosis-related proteins Bax, Bcl2, Caspase3, Caspase9, Cleaved Caspase3 and Cleaved Caspase9 were detected by Western Blot, and the expression of apoptosis-related genes Bax, Bcl2, Caspase3 and Caspase9 were detected by RT-PCR. According to the theory of network pharmacology, the potential targets of saikosaponins and colon cancer were predicted by database Pharmmapper and Genecards database respectively. The intersection of saikosaponins and colon cancer was enriched and analyzed on the Metascape platform. Then, the expression of PI3K/Akt/mTOR pathway related protein PI3K, Akt, Mtor, p-PI3K, p-Akt, p-mTOR were detected by Western Blot, and the corresponding amount of RNA expressions in the pathway was confirmed by RT-PCR. RESULTS: The results of CCK-8 demonstrated that the survival rate of SW480 and SW620 cells decreased significantly when the concentration of TBSE was in the range of 25-200 µg/ml. The morphological observation showed that the cells lost normal cell morphology, cytoplasmic condensation, and partial loss of adhesion after treatment with TBSE. Flow cytometry indicated that the apoptosis rates of SW480 cells and SW620 cells treated with TBSE (50 µg/ml) were 48.47% ± 1.20% and 36.13% ± 1.76%, respectively. Western Blot firstly confirmed that TBSE significantly up-regulated the expression of pro-apoptotic proteins Bax, Caspase3, Caspase9, Cleaved Caspase3 and Cleaved Caspase9, and down-regulated the expression of anti-apoptotic protein Bcl2. And RT-PCR results implied that TBSE significantly up-regulated the gene expression of apoptotic factors Bax, Caspase3 and Caspase9, and significantly decreased the gene expression of Bcl2. It was predicted that the PI3K/Akt/mTOR pathway may be the main regulatory object of the antitumor effect of TBSE by network pharmacology. Subsequent WB experiment also revealed that TBSE could significantly down-regulate (P < 0.01) the expressions of PI3K, Akt, mTOR and phosphorylated proteins P-PI3K, P-Akt, P-MTOR. Meanwhile, RT-PCR results also indicated that TBSE could significantly down-regulate (P < 0.01) the gene expression levels of PI3K, Akt and mTOR. CONCLUSIONS: TBSE activated Bax/Bcl2 and caspase-9/caspase-3 cascade to induced apoptosis of human colon cancer SW480 and SW60 cells in a dose-dependent manner, which was obviously related to the inhibition of PI3K/Akt/mTOR signaling pathway.

The modulation of PI3K/Akt pathway by 3ß hydroxylup-12-en-28-oic acid isolated from Thymus linearis induces cell death in HCT-116 cells.

The presence of intricate carbon skeletons in natural compounds enhances their bioactivity spectrum with unique modes of action at several targets in various dreadful diseases like cancer. The present study was designed to purify the molecules from Thymus linearis and elucidate their antiproliferative activity. The compounds were isolated from the active methanolic extract of Thymus linearis through column chromatography and characterized by various spectroscopic techniques. Antiproliferative activity of isolated compounds was evaluated using MTT assay on cancer and normal cell lines. Mechanism of cell death was elucidated using flow cytometric, microscopic, and Western blot analysis. Four compounds, Sitosterol, Chrysin, 3ß-hydroxylup-12-en-28-oic acid (3BH), and ß-Sitosterol glycoside, were isolated. Among these, 3BH was most potent antiproliferative agent across all cell lines under study, HCT-116 being the most affected one. 3BH was demonstrated to downregulate PI3Ksubunits (p110α and p85α), downstream pAktSer473 and prompted G1 phase cell cycle arrest. The cell cycle CDK inhibitor p27 and p21 were upregulated with simultaneous downregulation of cyclin D1 and cyclin E in HCT-116 cells. This was accompanied by apoptosis, as depicted by decrease in Bcl-2/Bax ratio, with increase in active caspases-3 and caspase-9, cleavage of PARP-1, the generation of reactive oxygen species (ROS), and the loss of mitochondrial membrane potential. The findings established that 3BH induced cell death in HCT-116 cells by modulating PI3K/Akt signaling axis, impeding cell cycle, and instigating apoptosis.

The anti-tumor effect of OP-B on ovarian cancer in vitro and in vivo, and its mechanism: An investigation using network pharmacology-based analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Maidong (Liliaceae) is used as a yin-nourishing medication for the treatment of cardiovascular disease, inflammation, and assistant cancer chemotherapy in the clinic. Ophiopogonin B (OP-B), a major saponin extracted from Maidong, is reported to have potential antitumor activities against various human cancers. However, the effects of OP-B on human ovarian cancer (OC) and the potential mechanisms of action are yet elusive. AIM OF THE STUDY: In this study, we aimed to explore the potential molecular mechanisms of OP-B in the treatment of OC using network pharmacology. In vivo and in vitro experiments were conducted to further verify the therapeutic effects of OP-B on OC. MATERIALS AND METHODS: To investigate the functions of OP-B against OC holistically, the related targets of OP-B and OC were each predicted based on four public databases. Subsequently, the identified PPI network was constructed to detect the hub potential targets. In addition, GO and KEGG enrichment analysis were applied by Metascape database. Furthermore, we simultaneously investigated the anticancer effects of OP-B on SKOV3 and A2780 human ovarian cancer cells using a cell viability assay, transwell assay, and an image-based cytometric assay. The quantitative real-time PCR and western-blot assay were used to validate the RNA and protein levels of target genes in OP-B treated OC cells. At last, SKOV3-bearing BALB/c nude mice were applied to observe the effectiveness and toxicity of OP-B. RESULTS: Through network pharmacological analysis, OP-B was found to play a critical role in OC via multiple targets and pathways, especially the STAT3 signaling pathways. In addition, in vitro experiments found OP-B suppressed SKOV3 and A2780 cells proliferation in a time and concentration dependent manner, and markedly impaired cancer cell migration. Flow cytometry analysis revealed that OP-B significantly increased early and late apoptosis, induced G2/M phase cell cycle arrest in SKOV3 cells and G0/G1 phase cell cycle arrest in A2780 cells. Moreover, OP-B administration down-regulated the expression of p-STAT3 protein, whereas the RNA expression and total protein levels of STAT3 were not altered. Finally, in vivo experiments confirmed the therapeutic effects of OP-B on OC in nude mice with low toxicity in heart, liver, lung, and kidney. CONCLUSION: OP-B could efficiently suppress OC cellular proliferation, migration and induce apoptosis, cell cycle arrest mainly via the regulation of STAT3 signaling pathway. This study provides a promising potential application for an alternative to chemotherapy in ovarian cancer.

Alkaloid-rich fraction of Ervatamia coronaria sensitizes colorectal cancer through modulating AMPK and mTOR signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ervatamia coronaria, a popular garden plant in India and some other parts of the world is known traditionally for its anti-inflammatory and anti-cancer properties. The molecular bases of these functions remain poorly understood. AIM OF THE STUDY: Efficacies of the existing therapies for colorectal cancer (CRC) are limited by their life-threatening side effects and unaffordability. Therefore, identifying a safer, efficient, and affordable therapeutic is urgent. We studied the anti-CRC activity of an alkaloid-rich fraction of E. coronaria leaf extracts (AFE) and associated underlying mechanism. MATERIALS AND METHODS: Activity guided solvant fractionation was adopted to identify the activity in AFE. Different cell lines, and tumor grown in syngeneic mice were used to understand the anti-CRC effect. Methodologies such as LCMS, MTT, RT-qPCR, immunoblot, immunohistochemistry were employed to understand the molecular basis of its activity. RESULTS: We showed that AFE, which carries about six major compounds, is highly toxic to colorectal cancer (CRC) cells. AFE induced cell cycle arrest at G1 phase and p21 and p27 genes, while those of CDK2, CDK-4, cyclin-D, and cyclin-E genes were downregulated in HCT116 cells. It predominantly induced apoptosis in HCT116p53+/+ cells while the HCT116p53-/- cells under the same treatment condition died by autophagy. Notably, AFE induced upregulation of AMPK phosphorylation, and inhibition of both of the mTOR complexes as indicated by inhibition of phosphorylation of S6K1, 4EBP1, and AKT. Furthermore, AFE inhibited mTOR-driven conversion of cells from reversible cell cycle arrest to senescence (geroconversion) as well as ERK activity. AFE activity was independent of ROS produced, and did not primarily target the cellular DNA or cytoskeleton. AFE also efficiently regressed CT26-derived solid tumor in Balb/c mice acting alone or in synergy with 5FU through inducing autophagy as a major mechanism of action as indicated by upregulation of Beclin 1 and phospho-AMPK, and inhibition of phospho-S6K1 levels in the tumor tissue lysates. CONCLUSION: AFE induced CRC death through activation of both apoptotic and autophagy pathways without affecting the normal cells. This study provided a logical basis for consideration of AFE in future therapy regimen to overcome the limitations associated with existing anti-CRC chemotherapy.

Matrine injection inhibits pancreatic cancer growth via modulating carbonic anhydrases- a network pharmacology-based study with in vitro validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Matrine injection is a complex mixture of plant bioactive substances extracted from Sophora flavescens Aiton and Smilax glabra Roxb. Since its approval by the Chinese Food and Drug Administration (CFDA) in 1995, Matrine injection has been clinically used as a complementary and alternative treatment for various cancers; however, the underlying mechanism of pancreatic cancer treatment is yet to be elucidated. AIM OF THE STUDY: The present study explores the potential mechanism of matrine injection on pancreatic cancer through network pharmacology technique and in vitro experimental validation. MATERIALS AND METHODS: Genes differentially expressed in pancreatic cancer were obtained from the Gene Expression Omnibus (GEO) database (GSE101448). The potential active components of matrine injection were selected following a literature search, and target prediction was performed by the SwissTarget Prediction database. Overlapping genes associated with survival were screened by the Gene Expression Profiling Interactive Analysis (GEPIA) database. In vitro experimental validation was performed with cell counting kit-8 (CCK-8) assay, apoptosis detection, cell cycle analysis, immunoblotting, and co-immunoprecipitation of the identified proteins. RESULTS: One thousand seven hundred genes differentially expressed among pancreatic tumor and non-tumor tissues were screened out. Sixteen active components and 226 predicted target genes were identified in matrine injection. A total of 25 potential target genes of matrine injection for the treatment of pancreatic cancer were obtained. Among them, the prognostic target genes carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) based on the GEPIA database are differently expressed in tumors compared to adjacent normal tissue. In vitro experiments, the results of CCK-8 assay, apoptosis and cell cycle analysis, immunoblotting, and co-immunoprecipitation showed that matrine injection inhibited Capan-1 and Mia paca-2 proliferation, arrested the cell cycle at the S phase, and induced apoptosis through up-regulated CA12 and down-regulated CA9. CONCLUSIONS: In this study, bioinformatics and network pharmacology were applied to explore the treatment mechanism on pancreatic cancer with matrine injection. This study demonstrated that matrine injection inhibited proliferation, arrested the cell cycle, and induced apoptosis of pancreatic cancer cells. The mechanism may be related to the induction of CA12 over-expression, and CA9 reduced expression. As novel targets for pancreatic cancer treatment, Carbonic anhydrases require further study.

Propolis Extract Regulates microRNA Expression in Glioblastoma and Brain Cancer Stem Cells.

BACKGROUND: Grade IV gliomas are classified as glioblastoma (GBM), which is the most malignant brain cancer type. Various genetic and epigenetic mechanisms play a role in the initiation and progression of GBM. MicroRNAs (miRNAs) are small, non-coding RNA molecules that belong to the main epigenetic regulatory RNA class that plays different roles in either physiological or pathological conditions, including GBM pathogenesis regulating expression levels of the target genes. Brain Cancer Stem Cells (BCSCs) are responsible for poor prognosis, including therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC selfrenewal and differentiation properties. Propolis is a resinous substance collected by honey bees from various plant sources. The flavonoid content of propolis varies depending on the collection region and the extraction method. Although there are studies that include the effects of different originated-propolis on the miRNA expression levels of the glioblastoma cells, the impact on the BCSCs has not been studied yet. OBJECTIVE: This study aims to evaluate the effects of propolis obtained from Aydin, a city in western Turkey, on miRNA expression levels of BCSCs and GBM cells. METHODS: Aydin propolis was dissolved in 60% ethanol, and after evaporation, distilled water was added to prepare the propolis stock solution. The flavonoids content of the Aydin propolis was determined by MS Q-TOF analysis. Commercially obtained U87MG and BCSCs were used as in-vitro brain cancer models. Cytotoxic and apoptotic effects of Aydin propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile was investigated using the real-time qRT-PCR method. The fold changes were calculated by the2-ΔΔCt method. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases. RESULTS: Quercetin 3-methyl ether was the main component of the Aydin propolis. Aydin propolis did not show significant cytotoxic and apoptotic effects on both GBM and BCSCs up to 2mg/ml concentration. Aydin propolis treatment decreased the expression of nine miRNAs in the U87MG and five miRNAs in the BCSCs. Moreover, ten miRNAs have upregulated from 2.22 to 10.56 folds in propolis treated GBM cells compared to the control group significantly (p<0.05). In the study, the potential roles of two new miRNAs, whose regulations in glioma were not previously defined, were identified. One of them was miR-30d-5p, a novel potential oncomiR in GBM, which was 2.46 folds downregulated in Aydin propolis treated GBM cells. The other one is miR-335-5p, which is a potential tumor suppressor miR in GBM, that was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway, its upstream and downstream regulators, and critically neuronal developmental regulators, NOTCH and WNT pathways, were determined as the most deregulated pathways in Aydin propolis treated cells. CONCLUSION: The determination of the anti-cancer effect of Aydin propolis on the miRNA expression of GBM, especially on cancer stem cells, may contribute to the elucidation of brain cancer genetics by supporting further analyses.

Anti-Proliferative and Anti-Telomerase Effects of Blackberry Juice and Berry- Derived Polyphenols on HepG2 Liver Cancer Cells and Normal Human Blood Mononuclear Cells.

BACKGROUND: Previous studies have provided strong evidence for the anticancer activity of berry fruits. OBJECTIVE: In this study, we investigated the effects of blackberry juice and three berry- polyphenolic compounds on cell proliferation and telomerase activity in human hepatoma HepG2 and normal peripheral blood mononuclear cells (PBMCs). METHODS: The cell viability and telomerase activity were measured by MTT and TRAP assay, respectively. Berry effects on the expression of genes were determined by quantitative RT-PCR assay. RESULTS: Blackberry, gallic acid, and resveratrol inhibited proliferation of both HepG2 and PBMC cells in a dosedependent manner. Resveratrol was more effective than gallic acid for reducing the viability of HepG2 cells, but both showed the same level of growth inhibition in PBMC cells. Berry, resveratrol, and gallic acid significantly inhibited telomerase activity in HepG2 cells. The antiproliferative effect of berry was associated with apoptotic DNA fragmentation. Gallic acid was more effective for reducing telomerase activity than resveratrol, but anthocyanin moderately increased telomerase activity in cancer cells. Telomerase activity was induced by all three polyphenols in PBMCs. Overall, Krumanin chloride was more effective to induce telomerase than gallic acid and resveratrol in PBMC cells. There was no significant difference in hTERT, hTR, and Dnmts expressions between berry treated and the control untreated HepG2 cells. But, a significant downregulation of HDAC1 and HDAC2 and upregulation of SIRT1 were observed in berry-treated cells. CONCLUSION: These data indicate that the berry anticancer effect is associated with antitelomerase activity and changes in HDACs expression. The data also suggest that berry antitelomerase activity is mainly related to its gallic acid and resveratrol, but not anthocyanin content.

Stenophyllols A-C, new compounds from Boesenbergia stenophylla.

Three new compounds, i.e. stenophyllols A-C (1-3), were isolated from the rhizome of Boesenbergia stenophylla. The structures were determined by spectroscopic analysis (UV, IR, NMR and HRESIMS). In-vitro neuroblastoma cell viability assay showed stenophyllol A (1) was able to reduce the N2A cell viability to 20% within 24 h.