Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets.

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus-host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.

Antitumor Efficacy of the Herbal Recipe Benja Amarit against Highly Invasive Cholangiocarcinoma by Inducing Apoptosis both In Vitro and In Vivo.

Thailand is the country with highest incidence and prevalence of cholangiocarcinoma (CCA) in the world. Due to the frequently late diagnosis that is associated with this disease, most CCA patients are prescribed chemotherapy as a form of treatment. However, CCA is able to resist the presently available chemotherapy, so to the prognosis of this disease is still very poor. In this study, we investigated the anticancer potential of a Thai herbal recipe, Benja Amarit (BJA) against CCA and the relevant mechanisms of action that are involved. We found that BJA inhibited CCA cell viability in a dose-dependent manner, especially in highly invasive KKU-213 cells. The extract induced mitochondrial- and caspase-dependent apoptosis in CCA cells by regulating the nuclear factor-κB (NF-κB) signaling pathway. BJA also triggered autophagy in CCA cells. Nonetheless, the inhibition of autophagy enhanced BJA-induced CCA cell death via apoptosis. An in vivo xenograft model revealed the growth-inhibiting and death-inducing effects of BJA against CCA by targeting apoptosis. However, general toxicity to blood cells, kidneys and the liver, as well as changes in body weight, did not appear. Our findings suggest that the herbal recipe BJA might be used as a potentially new and effective treatment for cholangiocarcinoma patients.

The Effects of Houttuynia cordata Thunb and Piper ribesioides Wall Extracts on Breast Carcinoma Cell Proliferation, Migration, Invasion and Apoptosis.

Houttuynia cordata Thunb. (HCT) and Piper ribesioides Wall. (PR) are common herbs that are widely distributed throughout East Asia and possess various biological properties including anti-cancer effects. However, in breast cancer, their mechanisms responsible for anti-carcinogenic effects have not been clarified yet. In this study, the inhibitory effects of HCT and PR ethanolic extracts on breast cancer cell proliferation, migration, invasion and apoptosis were examined. In MCF-7 and MDA-MB-231 cells, HCT and PR extracts at low concentrations can inhibit colony formation and induce G1 cell cycle arrest by downregulating cyclinD1 and CDK4 expression. Additionally, HCT and PR extracts also decreased the migration and invasion of both breast cancer cell lines through inhibition of MMP-2 and MMP-9 secretion. Moreover, the induction of apoptosis was observed in breast cancer cells treated with high concentrations of HCT and PR extracts. Not only stimulated caspases activity, but HCT and PR extracts also upregulated the expression of caspases and pro-apoptotic Bcl-2 family proteins in breast cancer cells. Altogether, these findings provide the rationale to further investigate the potential actions of HCT and PR extracts against breast cancer in vivo.

Potential of Thai Herbal Extracts on Lung Cancer Treatment by Inducing Apoptosis and Synergizing Chemotherapy.

The incidence of lung cancer has increased while the mortality rate has continued to remain high. Effective treatment of this disease is the key to survival. Therefore, this study is a necessity in continuing research into new effective treatments. In this study we determined the effects of three different Thai herbs on lung cancer. Bridelia ovata, Croton oblongifolius, and Erythrophleum succirubrum were extracted by ethyl acetate and 50% ethanol. The cytotoxicity was tested with A549 lung cancer cell line. We found four effective extracts that exhibited toxic effects on A549 cells. These extracts included ethyl acetate extracts of B. ovata (BEA), C. oblongifolius (CEA), and E. succirubrum (EEA), and an ethanolic extract of E. succirubrum (EE). Moreover, these effective extracts were tested in combination with chemotherapeutic drugs. An effective synergism of these treatments was found specifically through a combination of BEA with methotrexate, EE with methotrexate, and EE with etoposide. Apoptotic cell death was induced in A549 cells by these effective extracts via the mitochondria-mediated pathway. Additionally, we established primary lung cancer and normal epithelial cells from lung tissue of lung cancer patients. The cytotoxicity results showed that EE had significant potential to be used for lung cancer treatment. In conclusion, the four effective extracts possessed anticancer effects on lung cancer. The most effective extract was found to be E. succirubrum (EE).

Goniothalamin Induces Necroptosis and Anoikis in Human Invasive Breast Cancer MDA-MB-231 Cells.

Goniothalamin (GTN) is toxic to several types of cancer cells in vitro. However, its effects on non-apoptotic cell death induction of human cancer cells have been poorly documented. Here, an investigation of the anti-cancer activity of GTN and the molecular signaling pathways of non-apoptotic cell death in the invasive human breast cancer MDA-MB-231 cell line were undertaken. Apoptotic cell death was suppressed by using a pan-caspase inhibitor (Benzyloxycarbonyl-Val-Ala-Asp-[O-methyl]-fluoromethylketone), z-VAD-fmk) as a model to study whether GTN induced caspase-independent cell death. In the anoikis study, MDA-MB-231 cells were cultured on poly-(2-hydroxyethyl methacrylate)- or poly-HEMA- coated plates to mimic anoikis-resistance growth and determine whether GTN induced cell death and the mechanisms involved. GTN and z-VAD-fmk induced human breast cancer MDA-MB-231 cells to undergo necroptosis via endoplasmic reticulum (ER) and oxidative stresses, with increased expressions of necroptotic genes such as rip1, rip3, and mlkl. GTN induced MDA-MB-231 cells to undergo anoikis via reversed epithelial-mesenchymal transition (EMT) protein expressions, inhibited the EGFR/FAK/Src survival signaling pathway, and decreased matrix metalloproteinase secretion.

Effect of Pseuderanthemum palatiferum (Nees) Radlk fresh leaf ethanolic extract on human breast cancer MDA-MB-231 regulated cell death.

Leaf extracts of Pseuderanthemum palatiferum (Nees) Radlk were investigated for their effects on human breast cancer MDA-MB-231 cell growth inhibition. Pseuderanthemum palatiferum (Nees) Radlk extracts were prepared using fresh or dried leaves and extracted by either water or 95% ethanol, respectively. Fresh leaf ethanolic extract was the most toxic to MDA-MB-231 cells measured by 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide assay. Fresh leaf ethanolic extract-treated MDA-MB-231 cell death was stained with propidium iodide and examined under fluorescence microscopy. Cell death was confirmed by annexin V-fluorescein isothiocyanate/propidium iodide and propidium iodide-stained cells employing flow cytometry. The mitochondrial transmembrane potential was disrupted in fresh leaf ethanolic extract-treated MDA-MB-231 cells and the percentage of cells with reduced mitochondrial transmembrane potential increased according to concentrations. Mitochondrial transmembrane potential-driven regulated cell deaths were in the form of both apoptosis and necrosis. Oxidative stress probe, 2',7'-dichlorodihydrofluorescein diacetate, was used to indicate the redox status. Dichlorofluorescein level was significantly lower at high fresh leaf ethanolic extract concentrations. Total phenolic contents were found in all Pseuderanthemum palatiferum (Nees) Radlk extracts, whereas Ca2+ level in the cytosol increased, indicating Ca2+ overload and endoplasmic reticulum stress involvement with the activation of caspase-3, -8, and -9. In conclusion, fresh leaf ethanolic extract induced human breast cancer MDA-MB-231 programmed cell death via endoplasmic reticulum and oxidative stress by activating both extrinsic and intrinsic signaling pathways.

Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

Cytotoxic aporphine alkaloids from leaves and twigs of Pseuduvaria trimera (Craib).

From ethyl acetate-methanol extracts of leaves and twigs of Pseuduvaria trimera a new aporphine alkaloid; 8-hydroxy-1,4,5-trimethoxy-7-oxoaporphine or 8-hydroxyartabonatine C (1) was isolated, together with the known 1,2,3-trimethoxy-4,5-dioxo-6a,7-dehydroaporphine (ouregidione, 2). Their structures were elucidated by a combination of spectral methods; mainly 2D NMR; IR and MS. Compounds 1 and 2 exhibited cytotoxic activity with IC50 values of 26.36±5.18 µM and 12.88±2.49 µM, respectively, for human hepatocellular carcinoma HepG2 cells, and 64.75±4.45 and 67.06±3.5 µM, respectively, for human breast cancer MDA-MB231 cells. Both compounds displayed anti-cancer activity but less than that of doxorubicin; a conventional chemotherapeutic drug, the IC50 levels of which were 2.21±1.72 and 1.83±0.09 µM for HepG2 and MDA-MB231 cells, respectively.

Anti-cancer Effect and Active Phytochemicals of Houttuynia cordata Thunb. against Human Breast Cancer Cells.

BACKGROUND: Houttuynia cordata Thunb (HCT) is a medicinal herb used in Southeast Asia. Aim of this work: This study aimed at investigating the cytotoxicity of this plant extract and fractions towards human breast cancer MDA-MB-231 and MCF-7 cells. HCT's phytoactive compounds are determined. MATERIALS AND METHODS: Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of cell death was measured by staining with annexin V-FITC and propidium iodide (PI) employing flow cytometry technique. The oxidative stress was measured by using 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE+) fluorescent probes and using a fluorescence microplate reader. HCT phytochemicals were characterized by high performance liquid chromatography (HPLC). RESULTS: The proliferation of MDA-MB-231 and MCF-7 cells was dramatically decreased by the crude extract and individual fraction of HCT. Ethyl acetate was the solvent fraction with the highest toxicity against MCF-7 cells, followed by dichloromethane, crude, and hexane fractions, respectively, whereas in MDA-MB231 cells, dichloromethane, crude, hexane, and ethyl acetate fractions each had the strongest impact, respectively. The methanol fraction had no effect on either cell line up to 200 µg/ml. The extract and fractions were less harmful to the NIH3T3 normal murine fibroblast cell line. The mode of both cell death was apoptosis evidenced by the increase of cell population stained with annexin V-FITC and PI. The fluorescence probes of both DCFH-DA and DHE in MDA-MB-231 cell line were enhanced. Phenolic acids included chlorogenic acid (CA), gallic acid (GA), transcoumaric acid (TCA), vanillic acid (VA), and syringic acid (SA), as well as flavonoids like quercetin and rutin, were identified as the active phytochemicals in the crude and fractions by using HPLC method. CONCLUSION: MDA-MB-231cells underwent apoptosis via oxidative stress when induced with HCT hexane fraction. Phenolic acids and flavonoids were identified in HCT's extract and fractions.

Houttuynia cordata Thunb. Hexane fraction induces MDA-MB-231 cell apoptosis via caspases, ER stress, cell cycle arrest and attenuated Akt/ERK signaling.

Houttuynia cordata Thunb. (HCT) is a perennial plant used in traditional Thai medicine for many centuries. This study aimed to investigate the antiproliferative effect of the hexane fraction, which has not been explored before. HCT ethanol extract (crude extract) was sequentially fractionated to obtain a hexane (H) fraction. GC-MS was used to determine the phytochemicals. The H fraction consisted of lipids, mainly α-linolenic acid and some terpenoids. MTT assay was used to determine the cytotoxic effects of H fraction in MCF-7, MDA-MB-231, NIH3T3 and PBMCs. The mode of cell death and cell cycle analysis were determined by flow cytometry. The mechanisms of cell death were defined by mitochondrial transmembrane potential (MTP) reduction and activation of caspase-3, -8 and -9. The expression levels of the Bcl-2 family, cell cycle-related, endoplasmic reticulum (ER) stress-associated proteins; and Akt/ERK signaling molecules were investigated by immunoblotting. The H fraction was toxic to MDA-MB-231 more than MCF-7 cells but not to NIH3T3 and PBMCs. The growth of MDA-MB-231 cells was inhibited through apoptosis. MTP was disrupted whereas caspase-3, -8 and -9 were activated. The expression of pro-apoptotic Bax and Bak was upregulated, while Bid and anti-apoptotic Bcl-xL proteins were downregulated. Cyclin D1 and CDK4 levels were downregulated. The cell cycle was arrested at G1. Moreover, GRP78 and CHOP elevation indicated ER stress-mediated pathway. The expression ratio of pAkt/Akt and pERK/ERK were reduced. Taken together, the molecular mechanisms of MDA-MB-231 cell apoptosis were via intrinsic/extrinsic pathways, cell cycle arrest, ER stress and abrogation of Akt/ERK survival pathways. According to the most current research, the H fraction may be used as an adjuvant in the BC treatment; however, before the anticancer strategy can be applied to patients, it is important to determine each active compound's effects in cell lines and in vivo when compared with a combined mixture.

Gambogic Acid and Piperine Synergistically Induce Apoptosis in Human Cholangiocarcinoma Cell via Caspase and Mitochondria-Mediated Pathway.

Most cholangiocarcinoma (CCA) patients undergo chemotherapy as a therapeutic approach due to the disease's frequently late diagnosis. However, because CCA is resistant to currently available treatments, the prognosis for this cancer is still quite poor. Combination therapy has emerged as a novel and promising strategy in cancer treatment, as monotherapy frequently results in tumor recurrence and drug resistance. Gambogic acid has been shown to have a synergism with other compounds in combating certain cancer cells. Moreover, piperine has been shown to improve the efficacy of numerous chemotherapy drugs and other anticancer natural substances. However, no research has been done on the combination of these two compounds in the treatment of bile duct cancer. In this study, the cytotoxic activity was determined by using the MTT assay, and then, the combined effect was assessed by using the combination index (CI). We found that the combination of gambogic acid and piperine inhibited cell viability more effectively than either treatment alone, and it also demonstrated a synergistically cytotoxic effect against CCA cells. Interestingly, the findings allowed the use of lower concentrations of gambogic acid in cancer treatment when combined with piperine, which could reduce its adverse effect on normal cholangiocytes. Furthermore, the combination of the two compounds increased CCA cell death by inducing apoptosis via both the extrinsic and intrinsic or mitochondria-mediated pathways, as determined by caspase-3, -8, and -9 activity and the reduction of mitochondrial transmembrane potential (ΔΨm). It is possible that the use of these two natural compounds together could be a promising strategy for the treatment of bile duct cancer.

Three-Dimensional RAW264.7 Cell Model on Electrohydrodynamic Printed Poly(ε-Caprolactone) Scaffolds for In Vitro Study of Anti-Inflammatory Compounds.

Inflammation plays an essential role in the human immune system, and anti-inflammatory compounds are important to promote health. However, the in vitro screening of these compounds is largely dependent on flat biology. Herein, we report our efforts in establishing a 3D inflammation murine macrophage model. Murine macrophage RAW 264.7 cells were cultured on poly(ε-caprolactone) (PCL) scaffolds fabricated through an electrohydrodynamic jetting 3D printer and their behavior were examined. Cells on PCL scaffolds showed a 3D shape and morphology with multilayers and a lower proliferation rate. Moreover, macrophages were not activated by scaffold material PCL and 3D microenvironment. The 3D cells showed greater sensitivity to lipopolysaccharide stimulation with higher production activity of nitric oxide (NO), nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). Additionally, the 3D macrophage model showed lower drug sensitivity to commercial anti-inflammatory drugs including aspirin, ibuprofen, and dexamethasone, and natural flavones apigenin and luteolin with higher IC50 for NO production and lower iNOS and COX-2 inhibition efficacy. Overall, the 3D macrophage model showed promise for higher accurate screening of anti-inflammatory compounds. We developed, for the first time, a 3D macrophage model based on a 3D-printed PCL scaffold that provides an extracellular matrix environment for cells to grow in the 3D dimension. 3D-grown RAW 264.7 cells showed different sensitivities and responses to anti-inflammatory compounds from its 2D model. The 3D cells have lower sensitivity to both commercial and natural anti-inflammatory compounds. Consequently, our 3D macrophage model could be applied to screen anti-inflammatory compounds more accurately and thus holds great potential in next-generation drug screening applications.

Suppressive Effect and Molecular Mechanism of Houttuynia cordata Thunb. Extract against Prostate Carcinogenesis and Castration-Resistant Prostate Cancer.

Houttuynia cordata Thunb. (HCT) is a well-known Asian medicinal plant with biological activities used in the treatment of many diseases including cancer. This study investigated the effects of HCT extract and its ethyl acetate fraction (EA) on prostate carcinogenesis and castration-resistant prostate cancer (CRPC). HCT and EA induced apoptosis in androgen-sensitive prostate cancer cells (LNCaP) and CRPC cells (PCai1) through activation of caspases, down-regulation of androgen receptor, and inactivation of AKT/ERK/MAPK signaling. Rutin was found to be a major component in HCT (44.00 ± 5.61 mg/g) and EA (81.34 ± 5.21 mg/g) in a previous study. Rutin had similar effects to HCT/EA on LNCaP cells and was considered to be one of the active compounds. Moreover, HCT/EA inhibited cell migration and epithelial-mesenchymal transition phenotypes via STAT3/Snail/Twist pathways in LNCaP cells. The consumption of 1% HCT-mixed diet significantly decreased the incidence of adenocarcinoma in the lateral prostate lobe of the Transgenic rat for adenocarcinoma of prostate model. Similarly, tumor growth of PCai1 xenografts was significantly suppressed by 1% HCT treatment. HCT also induced caspase-dependent apoptosis via AKT inactivation in both in vivo models. Together, the results of in vitro and in vivo studies indicate that HCT has inhibitory effects against prostate carcinogenesis and CRPC. This plant therefore should receive more attention as a source for the future development of non-toxic chemopreventive agents against various cancers.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2',6'-dihydroxy-4,4'-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2',7'-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Extracts of Bridelia ovata and Croton oblongifolius induce apoptosis in human MDA­MB­231 breast cancer cells via oxidative stress and mitochondrial pathways.

Breast cancer is the most common type of cancer and is also the second leading cause of cancer­associated death in women worldwide. Thus, there is an urgent requirement for the development of effective treatments for this disease. Bridelia ovata and Croton oblongifolius are herbs used in Thai traditional medicine that have been used to treat various health problems; B. ovata has traditionally been used as a purgative, an antipyretic, a leukorrhea treatment and as a birth control herb. C. oblongifolius has been used to increase breast milk production, for post­partum care (where it is used as a hot bath herb), and as a treatment for flat worms and dysmenorrhea. However, there is little research investigating the anticancer properties of these herbs. The present study aimed to investigate the anticancer properties of crude ethyl acetate extracts of B. ovata (BEA) and C. oblongifolius (CEA) in order to explore their underlying mechanisms in breast cancer cell death. The phytoconstituents of the crude extracts of BEA and CEA were studied using gas chromatography­mass spectrometry (GC­MS). GC­MS analysis showed that the primary compound in BEA is friedelan­3­one, and kaur­16­en­18­oic acid in CEA. Cytotoxicity was investigated using an MTT assay, both BEA and CEA showed greater toxicity against MDA­MB­231 breast cancer cells compared with their effect on MCF10A normal epithelial mammary cells. BEA and CEA exerted various effects, including inducing apoptotic cell death, reducing mitochondrial transmembrane potential, increasing the levels of intracellular ROS, activating caspases, upregulating pro­apoptotic and downregulating anti­apoptotic genes and proteins. BEA and CEA were shown to have anticancer activity against breast cancer cells and induce apoptosis in these cells via a mitochondrial pathway and oxidative stress.

Anticancer effects of a traditional Thai herbal recipe Benja Amarit extracts against human hepatocellular carcinoma and colon cancer cell by targeting apoptosis pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: A preparation of Benja Amarit (BJA) has been effectively used in folk medicine to treat diseases related to the liver and colon and forms of cancer for hundreds of years in Thailand. However, there has not been any research on BJA with regard to its anticancer activity against human hepatocellular carcinoma and colon cancer cells. AIM OF THE STUDY: This study was to obtain the scientific supports for the traditional usage in anticancer potential of BJA extracts on hepatocellular carcinoma and colon cancer. MATERIALS AND METHODS: The cytotoxic effects were determined using MTT assay. Apoptosis was quantitated by annexin V-FITC/PI staining. Caspases activities were measured by using specific substrates and colorimetric analysis. The protein expressions were determined by Western blot analysis. Reactive oxygen species (ROS) generation, mitochondrial transmembrane potential, and calcium ion levels were measured by specific fluorescence probes and flow cytometry. The chick embryo chorioallantoic membrane model has been used to study the in vivo anticancer activity. The phytochemical identification was performed by GC-MS and LC-MS. RESULTS: Notably, 95% (BJA-95) and 50% (BJA-50) ethanolic extract of BJA inhibited hepatocellular carcinoma and colon cancer cell viability in a dose-dependent manner. While, the water extract of BJA (BJA-W) was not found to be toxic to both kinds of cancer cell lines. BJA extract induced both the extrinsic and intrinsic or mitochondria-mediated apoptosis pathways. Moreover, BJA-95 caused ROS generation and endoplasmic reticulum stress-mediated apoptosis. The extract exhibited the growth inhibitory effects on cancer cells in vivo. Phytochemical analysis revealed that the major active compounds were piperine, xanthotoxol and dihydrogambogic acid. CONCLUSION: This study is the first to demonstrate anticancer efficiency of BJA extracts on human cancer cells. We consider BJA extract to be a potentially alternative cancer treatment and to be a promising candidate in the future development of antitumor agents.

Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway.

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4',6'-dihydroxy-2',4-dimethoxy-5'-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2',6'-dihydroxy-4,4'-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3'-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

The Effect of Transfusion-Dependent Thalassemia Patient's Serum on Peripheral Blood Mononuclear Cell Viability.

Iron overload is a major complication in transfusion-dependent thalassemia (TDT) patients. Chronic oxidative stress from iron overload may lead to cellular damage and viability. This is a cross-sectional study. Transfusion-dependent thalassemia patients aged ⩾18 years old were enrolled. Transfusion-dependent thalassemia patient's serum and normal volunteer's serum were separately incubated with healthy peripheral blood mononuclear cells (PBMCs). The cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at 24, 48, and 72 hours. Sixty-nine TDT patients and 22 healthy controls were enrolled. The mean of PBMCs viability after incubation with serum from TDT patients was lower than that with the controls (88.65% vs 103.56% at 24 hours, 78.77% vs 112.04%% at 48 hours, and 71.18% vs 132.16%% at 72 hours, respectively). High serum ferritin level (correlation -0.29, P < .05) and white blood cell (WBC) count negatively affected cell viability (correlation -2.86, P = .05). From multivariate analysis, serum ferritin level is the only significant risk factor that is independently associated with cell viability (correlation -11.42, P < .001). Our findings showed that TDT patient's serum causes decreased cell viability. Serum ferritin level was a significant independent factor influencing cell viability.

Alpha-mangostin, an active compound in Garcinia mangostana, abrogates anoikis-resistance in human hepatocellular carcinoma cells.

Anoikis-resistance is a critical step in cancer progression, especially during the process of metastasis. During this phase, the cancer phenotype that causes cell survival in detachment conditions, drug resistance, and epithelial-to-mesenchymal transition (EMT) is altered. Inhibition of anoikis-resistance can potentially be the molecular target in cancer therapy. Alpha-mangostin, an active compound in Garcinia mangostana, has been reported for its cell-death induction and its chemosensitizing and anti-metastatic properties in many cancer cell types, such as ovarian cancer, lung cancer, and hepatocellular carcinoma. We, therefore, have investigated whether alpha-mangostin could sensitize anoikis in human hepatocellular carcinoma (HepG2). The established anoikis-resistant HepG2 displayed more aggressive malignant behaviors, including rapid proliferation, doxorubicin resistance, up-regulated anti-apoptotic protein levels, and EMT phenotype. Alpha-mangostin significantly sensitized anoikis in HepG2 through the inhibition of cell survival by induced caspase-9, caspase-8 and caspase-3 activities, increased pro-apoptotic protein (Bax, Bim, t-Bid) levels, and decreased anti-apoptotic protein (c-FLIP, Mcl-1) levels. Besides, alpha-mangostin significantly reduced cell re-adhesion and migration, matrix metalloproteinases-2 (MMP-2) and MMP-9 secretions, and EMT-involved protein (N-cadherin, αV, ß1 integrin, and vimentin) expressions. AKT and ERK signaling pathways were dramatically suppressed, which indicated that alpha-mangostin inhibited anoikis-resistance via the inhibition of these pathways in HepG2. These findings support the development of alpha-mangostin to be used in the treatment of anoikis-resistant liver cancer.

Apoptosis Induction via ATM Phosphorylation, Cell Cycle Arrest, and ER Stress by Goniothalamin and Chemodrugs Combined Effects on Breast Cancer-Derived MDA-MB-231 Cells.

Goniothalamin (GTN), a styryl-lactone, exhibits inhibitory effects on many kinds of cancer cells in vitro. The objectives of this study were to investigate the anticancer activities of GTN and molecular signaling pathways associated with cell death in human breast cancer MDA-MB-231 cell line. GTN inhibited the growth of MDA-MB-231 cells. Apoptosis was confirmed by annexin V-FITC and PI staining, and apoptotic morphology was observed by microscopy. Reduction of mitochondrial transmembrane potential and enhanced caspases activities were found in GTN-treated MDA-MB-231 cells. GTN significantly altered apoptosis-related protein expressions, including Noxa, PUMA, Bax, Bim, Bad, Bcl-2, Bcl-xL, and DIABLO, which was related to the gene expression levels. Mitochondrial calcium released to the cytosol and ER stress related proteins increased, which correlated with increases in ER stress gene expression levels. GTN induced hydrogen peroxide and superoxide anion radicals in MDA-MB-231 cells associated with cell cycle arrest in G2/M phase, which was induced by phosphorylation and ATM gene expression. Moreover, GTN had synergistic effects when combined with cyclophosphamide, 5-fluorouracil, paclitaxel, and vinblastine, and additive effect with methotrexate through caspases enzyme-acceleration. In conclusion, goniothalamin-induced MDA-MB-231 cell apoptosis occurred via intrinsic and extrinsic pathways, along with ER stress. These pathways provide new targeted drug strategies for advancements in anticancer medicine.