Glossogin, a novel phenylpropanoid from Glossogyne tenuifolia, induced apoptosis in A549 lung cancer cells

Glossogyne tenuifolia has been shown to exhibit good antioxidant and anticancer activity. In this study, a new phenylpropanoid compound, glossogin (1′-acetoxy-4-O-isovalyryleugenol), was isolated from ethyl acetate extract of G. tenuifolia by using column chromatography and HPLC. Its chemical structure was determined by ¹H and ¹³C NMR, MS and IR spectroscopic evidence. This compound showed the cytotoxicity against A549 human lung cancer cell line and it induced the progressing apoptosis on A549 cells. This apoptosis was verified as A549 cells were arrested at the sub-G₁ phase. The apoptosis was accompanied by release of cytochrome C and activation of caspase-9 and -3. It was also associated with the decrease in Bcl-2 and Bcl-xL protein levels, and the increase in Bad protein expression. Data analysis suggests glossogin exerted significant apoptotic effect on A549 cells through the mitochondrial pathway. Hence, our findings showed that glossogin exhibited potential anticancer activity against lung cancer through proliferating inhibition and apoptosis induction of cancer cells.     

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G ₀/G₁‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)?1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.     

Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways

Autophagy is a response of cancer cells to various anticancer therapies. It is designated as programmed cell death type II and characterized by the formation of autophagic vacuoles in the cytoplasm. The Akt/mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) and the extracellular signal-regulated kinases 1/2 (ERK1/2) pathways are two major pathways that regulate autophagy induced by nutrient starvation. These pathways are also frequently associated with oncogenesis in a variety of cancer cell types, including malignant gliomas. However, few studies have examined both of these signal pathways in the context of anticancer therapy-induced autophagy in cancer cells, and the effect of autophagy on cell death remains unclear. Here, we examined the anticancer efficacy and mechanisms of curcumin, a natural compound with low toxicity in normal cells, in U87-MG and U373-MG malignant glioma cells. Curcumin induced G(2)/M arrest and nonapoptotic autophagic cell death in both cell types. It inhibited the Akt/mTOR/p70S6K pathway and activated the ERK1/2 pathway, resulting in induction of autophagy. It is interesting that activation of the Akt pathway inhibited curcumin-induced autophagy and cytotoxicity, whereas inhibition of the ERK1/2 pathway inhibited curcumin-induced autophagy and induced apoptosis, thus resulting in enhanced cytotoxicity. These results imply that the effect of autophagy on cell death may be pathway-specific. In the subcutaneous xenograft model of U87-MG cells, curcumin inhibited tumor growth significantly (P < 0.05) and induced autophagy. These results suggest that curcumin has high anticancer efficacy in vitro and in vivo by inducing autophagy and warrant further investigation toward possible clinical application in patients with malignant glioma.     

B1, a novel topoisomerase II inhibitor, induces apoptosis and cell cycle G1 arrest in lung adenocarcinoma A549 cells

In our previous studies, we demonstrated that 2,6-bis-(2-chloroacetamido) anthraquinone (B1) showed a highly significant cytotoxic effect. However, its influence in the cell cycle and apoptotic induction effects has not been investigated yet. Here we report the antiproliferative effect of B1, for which IC50 values were 0.57 μmol/l for lung cancer A549 cells, 0.63 μmol/l for colon cancer HT-29 cells, and 0.53 μmol/l for breast cancer MCF-7 cells. DNA topoisomerase II (Topo II), an essential enzyme in DNA synthesis and meiotic division, is highly expressed in cancer cells. Some currently used clinical anticancer drugs (doxorubicin and mitoxantrone) targeting Topo II are very effective antineoplastic agents. B1, sharing the basic structure of known Topo II inhibitors, demonstrated a significant inhibitory effect on Topo II bioactivity. In A549 cells, B1 increased apoptotic cell population with induction of Fas, Bax, and cleaved poly(ADP-ribose) polymerase and by reduction of Bcl-2 expression. Moreover, cell cycle analysis indicated that B1 induced G1 phase arrest through modulation of G1 cell cycle regulatory proteins, such as the downregulation of cyclin D1 and upregulation of Cip/p21, Kip1/p27, and p53. Thus, our study suggests that B1, with the ability to inhibit Topo II activity and cause cell cycle G1 arrest and apoptosis, has potential as a novel anticancer agent.     

Riccardin D induces cell death by activation of apoptosis and autophagy in osteosarcoma cells

Macrocyclic bisbibenzyls, characteristic components derived from liverworts, have various biological activities. Riccardin D (RD), a liverwort-derived naturally occurring macrocyclic bisbibenzyl, has been found to exert anticancer effects in multiple cancer cell types through apoptosis induction. However, the underlying mechanisms of such effects remain undefined. In addition, whether RD induces other forms of cell death such as autophagy is unknown. In this study, we found that the arrest of RD-caused U2OS (p53 wild) and Saos-2 (p53 null) cells in G1 phase was associated with the induction of p53 and p21^WAF1 in U2OS cells. RD-mediated cell cycle arrest was accompanied with apoptosis promotion as indicated by changes in nuclear morphology and expression of apoptosis-related proteins. Further studies revealed that the antiproliferation of RD was unaffected in the presence of p53 inhibitor but was partially reversed by a pan-inhibitor of caspases, suggesting that p53 was not required in RD-mediated apoptosis and that caspase-independent mechanisms were involved in RD-mediated cell death. Except for apoptosis, RD-induced autophagy occurred as evidenced by the accumulation of microtubule-associated protein-1 light chain-3B-II, formation of AVOs, punctate dots, and increased autophagic flux. Pharmacological blockade of autophagy activation markedly attenuated RD-mediated cell death. RD-induced cell death was significantly restored by the combination of autophagy and caspase inhibitors in osteosarcoma cells. Overall, our study revealed RD-induced caspase-dependent apoptosis and autophagy in cancer cells, as well as highlighted the importance of continued investigation on the use of RD as a potential anticancer candidate.     

Plumbagin induces apoptosis,cell cycle arrest,and inhibits protein synthesis in LoVo colon cancer cells: A proteomic analysis

Extracted from the roots of Plumbago zeylanica L., plumbagin is a natural naphthoquinone with potential as an anticancer compound. However, no studies have investigated its impact on LoVo (colon cancer) cells, and the specific mechanisms by which plumbagin exerts its anticancer effects remain to be established. The anticancer potential of plumbagin against LoVo cells was evaluated using a battery of assays, including MTT assay, clone formation assay, transwell chamber invasion assay, and wound-curing assay. Cell cycle analysis and cell apoptosis analysis were conducted to break down the anticancer impact of plumbagin on LoVo cells. A label-free proteomics technology was employed to investigate alterations in protein expression in LoVo cells treated with plumbagin. Our investigation indicated that plumbagin markedly inhibited the LoVo cells proliferation, and induced the apoptosis in LoVo cells, simultaneously induced G0/G1 phase cell cycle arrest. The LC-MS/MS proteomics assay revealed 78 proteins that were differentially expressed upon treatment with plumbagin. Bioinformatics and functional analyses indicated that these proteins were predominantly involved in protein synthesis and translation. Our findings revealed that multiple mechanisms are involved in the anticancer activity of plumbagin against LoVo cells, resulting in decreased cell viability. Proteomic analysis suggests that plumbagin may impede protein synthesis by reducing the expression of eukaryotic initiation factors. Our findings demonstrate that plumbagin exerts its anticancer activity against LoVo cells through multiple mechanisms, including inhibition of cell proliferation, induction of apoptosis, cell cycle arrest, and disruption of protein synthesis. These results provide new insights into the therapeutic potential of plumbagin for colon cancer treatment.     

Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro

Aim:

Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro.

Methods:

Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay.

Results:

Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L).

Conclusion:

Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.     

A new diaryl urea compound, D181, induces cell cycle arrest in the G1 and M phases by targeting receptor tyrosine kinases and the microtubule skeleton

Receptor tyrosine kinases (RTKs) modulate a variety of cellular events, including cell proliferation, differentiation, mobility and apoptosis. In addition, RTKs have been validated as targets for cancer therapies. Microtubules are another class of proven targets for many clinical anticancer drugs. Here, we report that 1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-cyano-4-hydroxyphenyl)urea (D181) functions as both a receptor tyrosine kinase inhibitor and a tubulin polymerization enhancer. D181 displayed potent inhibitory activities against a panel of RTKs, including Flt3, VEGFR, cKit, FGFR1 and PDGFRβ. D181 also enhanced tubulin polymerization and modified the secondary structure of tubulin proteins to disrupt their dynamic instability. Because of synergistic cooperation, D181 strongly inhibited the proliferation of various cancer cell lines, induced LoVo cell cycle arrest in the G1 and M phases and suppressed tumor growth in nude mice bearing human LoVo and HT29 xenografts. Our studies have provided a new, promising lead compound and novel clues for multi-target anticancer drug design and development.     

A novel Diels–Alder adduct of mulberry leaves exerts anticancer effect through autophagy-mediated cell death

Guangsangon E (GSE) is a novel Diels–Alder adduct isolated from leaves of Morus alba L, a traditional Chinese medicine widely applied in respiratory diseases. It is reported that GSE has cytotoxic effect on cancer cells. In our research, we investigated its anticancer effect on respiratory cancer and revealed that GSE induces autophagy and apoptosis in lung and nasopharyngeal cancer cells. We first observed that GSE inhibits cell proliferation and induces apoptosis in A549 and CNE1 cells. Meanwhile, the upregulation of autophagosome marker LC3 and increased formation of GFP–LC3 puncta demonstrates the induction of autophagy in GSE-treated cells. Moreover, GSE increases the autophagy flux by enhancing lysosomal activity and the fusion of autophagosomes and lysosomes. Next, we investigated that endoplasmic reticulum (ER) stress is involved in autophagy induction by GSE. GSE activates the ER stress through reactive oxygen species (ROS) accumulation, which can be blocked by ROS scavenger NAC. Finally, inhibition of autophagy attenuates GSE-caused cell death, termed as “autophagy-mediated cell death.” Taken together, we revealed the molecular mechanism of GSE against respiratory cancer, which demonstrates great potential of GSE in the treatment of representative cancer.     

Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines

Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4′-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Meanwhile, autophagy was detected as early as 12 h after an exposure to low-dose dioscin, as indicated by an up-regulated expression of LC3-II and beclin-1 proteins. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR. Taken together, this study demonstrated for the first time that autophagy occurred earlier than apoptosis during dioscin-induced human lung cancer cell line apoptosis. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways may provide a protective mechanism for cell survival against dioscin-induced apoptosis to act as a cytoprotective reaction.     

NF-κB activation was involved in reactive oxygen species-mediated apoptosis and autophagy in 1-oxoeudesm-11(13)-eno-12,8α-lactone-treated human lung cancer cells

1-oxoeudesm-11(13)-eno-12,8α-lactone (OEL), a novel eudesmane-type sesquiterpene compound, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. Here, we investigated the anti-cancer activities of OEL in human lung cancer cells. Our studies demonstrated that OEL induced both apoptosis and autophagy in A549 and H460 cells. OEL-induced autophagy was assessed by appearance of autophagic vacuoles, formation of acidic vesicular organelles, conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes, and activation of autophagy genes. Furthermore, administration of autophagic inhibitor 3-methyladenine augments OEL-induced apoptotic cell death. The induction of autophagy and apoptosis by OEL links to NF-κB activation and the generation of reactive oxygen species (ROS). Interruption of NF-κB activation by specific inhibitor promotes apoptosis, but decreases autophagy. ROS antioxidants (N-acetylcysteine) attenuated both OEL-induced autophagy and apoptosis. Further experiments confirmed that OEL-induced activation of ROS was increased by NF-κB inhibitor whereas NF-κB activation was not affected by ROS inhibition. These findings suggest that OEL-elicited autophagic response plays a protective role that impedes cell death, and inhibition of autophagy could be an adjunctive strategy for enhancing the chemotherapeutic effect of OEL as an antitumor agent.     

β-Elemene induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549 cells

Objectives β‐Elemene, a novel traditional Chinese medicine, has been shown to be effective against a wide range of tumours. In this study, the antitumour effect of β‐elemene on human non‐small‐cell lung cancer (NSCLC) A549 cells and the mechanism involved have been investigated. Methods Cell viability and apoptosis were measured by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by Western blotting. Autophagy was evaluated under fluorescence microscopy and transmission electron microscopy. Key findings β‐Elemene inhibited the viability of A549 cells in a dose‐dependent manner. This suppression of cell viability was due to the induction of apoptosis. Further study showed that β‐elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway, and at the same time it triggered a robust autophagy. The autophagy was characterized by the accumulation of punctate LC3 dots in the cytoplasm, morphological changes, and the increased levels of LC3‐II as well as Atg5‐Atg12 conjugated proteins. Inhibition of autophagy with chlorochine significantly enhanced the antitumour effect of β‐elemene. Conclusions Our data indicated that β‐elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway in human NSCLC A549 cells, which resulted in apoptosis as well as protective autophagy. A combination of β‐elemene with autophagy inhibitor might be an effective therapeutic option for advanced NSCLC.     

A synthetic curcumin derivative hydrazinobenzoylcurcumin induces autophagy in A549 lung cancer cells

Context: Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability restricts its application in chemotherapeutic trials. Nowadays, a growing number of curcumin derivatives or analogs are known, hoping to replace curcumin and circumvent this problem. Hydrazinobenzoylcurcumin (HBC) has been synthesized and identified as a potent inhibitor of cell proliferation in previous reports.

Objective: This study presents a novel mechanism of cell autophagy induced by HBC in the human non-small lung epithelial carcinoma (A549) cells.

Materials and methods: Cells were cultured and treated with HBC at different concentrations (10–80?μM) and at different time periods (1–24?h). Microscopic analysis was used to detect the morphology changes and autophagolysosomes of A549 cells. An acridine orange staining assay was conducted to evaluate the autophagolysosomes and autophagic vacuoles was analyzed by monodansylcadaverine (MDC) and GFP-LC3 transfection analysis. Western blotting was used to assess the conversion of microtubule-associated protein light chain 3 (LC3).

Results: HBC could induce A549 cells autophagolysosomes formation in a dose and time-dependent manner and the inhibitory rate of HBC (80?μM) on the viability of A549 cells reached 76.68?±?5.81% after 24?h of treatment. Autophagic vacuoles increased in a concentration-dependent manner in HBC-treated cell. Furthermore, conversion of LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles and increased fusion of autophagosomes with lysosomes suggested the occurrence of autophagy.

Conclusion: Our data indicate that HBC induced A549 cell autophagy, which is a novel cell death mechanism induced by curcumin derivatives.     

A phenylacetate derivative,SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.     

Preliminary study on fabrication,characterization and synergistic anti-lung cancer effects of self-assembled micelles of covalently conjugated celastrol–polyethylene glycol–ginsenoside Rh2

The aim of this study was to develop an amphipathic polyethylene glycol (PEG) derivative that was bi-terminally modified with celastrol and ginsenoside Rh2 (Celastrol-PEG-G Rh2). Such derivative was capable of forming novel, celastrol-loaded polymeric micelles (CG-M) for endo/lysosomal delivery and thereby synergistic treatment of lung cancer. Celastrol-PEG-G Rh2 with a yield of 55.6% was first synthesized and characterized. Its critical micellar concentration was 1?×?10^?5?M, determined by pyrene entrapment method. CG-M had a small particle size of 121.53?±?2.35?nm, a narrow polydispersity index of 0.214?±?0.001 and a moderately negative zeta potential of –23.14?±?3.15?mV. Celastrol and G Rh2 were rapidly released from CG-M under acidic and enzymatic conditions, but slowly released in normal physiological environments. In cellular studies, the internalization of celastrol and G Rh2 by human non-small cell lung cancer (A549) cells treated with CG-M was 5.8-fold and 1.8-fold higher than that of non-micelle control. Combinational therapy of celastrol and G Rh2 using CG-M exhibited synergistic anticancer activities in cell apoptosis and proliferation assays via rapid drug release within endo/lysosomes. Most importantly, the celastrol in CG-M exhibited a long elimination half-life of 445.3?±?43.5?min and an improved area under the curve of 645060.8?±?63640.7?ng/mL/h, that were 1.03-fold and 2.44-fold greater than those of non-micelle control, respectively. These findings suggest that CG-M is a promising vector for precisely releasing anticancer drugs within the tumor cells, and thereby exerts an improved synergistic anti-lung cancer effect.     

Selective proapoptotic activity of polyphenols from red wine on teratocarcinoma cell,a model of cancer stem-like cell

Cancer stem cells are expected to be responsible for tumor initiation and metastasis. These cells are therefore potential targets for innovative anticancer therapies. However, the absence of bona fide cancer stem cell lines is a real problem for the development of such approaches. Since teratocarcinoma cells are totipotent stem cells with a high degree of malignancy, we used them as a model of cancer stem cells in order to evaluate the anticancer chemopreventive activity of red wine polyphenols (RWPs) and to determine the underlying cellular and molecular mechanisms. We therefore investigated the effects of RWPs on the embryonal carcinoma (EC) cell line P19 which was grown in the same culture conditions as the most appropriate normal cell line counterpart, the pluripotent embryonic fibroblast cell line NIH/3T3. The present study indicates that RWPs selectively inhibited the proliferation of P19 EC cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, RWPs treatment specifically triggered apoptosis of P19 EC cells in association with a dramatic upregulation of the tumor suppressor gene p53 and caspase-3 activation. Our findings suggest that the chemopreventive activity of RWPs on tumor initiation and development is related to a growth inhibition and a p53-dependent induction of apoptosis in teratocarcinoma cells. In addition, this study also shows that the EC cell line is a convenient source for studying the responses of cancer stem cells to new potential anticancer agents.     

Fisetin‐induced apoptosis of human oral cancer SCC‐4 cells through reactive oxygen species production,endoplasmic reticulum stress,caspase‐, and mitochondria‐dependent signaling pathways

Oral cancer is one of the cancer‐related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin‐induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin‐induced cell death and associated signal pathways on human oral cancer SCC‐4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca²⁺, mitochondria membrane potential (ΔΨ_m), and caspase‐8, ‐9, and ‐3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca²⁺ production, and decreased the level of ΔΨ_m and increased caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin‐induced cell apoptosis in SCC‐4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl‐2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC‐4 cells. We also used ATF‐6α, ATF‐6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria‐, and caspase‐dependent pathways.     

A new semisynthetic 1-O-acetyl-6-O-lauroylbritannilactone induces apoptosis of human laryngocarcinoma cells through p53-dependent pathway

Initiation of apoptosis is an important event for chemoprevention and chemotherapy of cancer. Naturally derived products had drawn growing attention as lead compounds for anticancer drug discovery. ABL-L, a semisynthetic analogue of natural sesquiterpenoid 1-O-acetylbritannilactone (ABL) isolated from Inula britannica, showed stronger suppression against three solid tumor cell lines with 4–10 fold improvement than ABL. However, its molecular mechanism of cell death induction has still not been determined. The present study evaluated the anticancer efficacy of ABL-L and its biological activities mechanism on human laryngocarcinoma cells HEp-2 in vitro. We found that ABL-L-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest. Typical apoptotic morphological and biochemical features were also observed in treated cells. Furthermore, the levels of the anti-apoptotic Bcl-2, pro-caspase 3/8/9 and poly(ADP-ribose) polymerase PARP decreased, and the level of pro-apoptotic Bax increased. Involvement of the caspase-mediated apoptosis was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. In addition, ABL-L induced a tumor suppressor p53 and its target genes expression p21, fas, noxa and puma. The results of p53 knockdown suggest that caspase-mediated apoptosis induced by ABL-L was in p53-dependent pathway on HEp-2 cells. Our data indicate that the cytotoxicity of the novel semisynthetic analogue ABL-L involved G1 cell cycle arrest and apoptosis via a p53-dependent, caspase-mediated pathway on human laryngocarcinoma cells.     

Gambogenic acid inhibits proliferation of A549 cells through apoptosis inducing through up-regulation of the p38 MAPK cascade

Gamboge is a dry resin secreted from Garcinia hanburryi, and gambogenic acid (GNA) is one of the main active compounds of gamboge. We have previously demonstrated the anticancer activity of GNA in A549 cells and pointed out its potential effects in anticancer therapies. Previous studies reported that GNA induced apoptosis in many cancer cell lines and inhibited A549 tumor growth in xenograft of nude mice in vivo. However, the anticancer mechanism of GNA has still not been well studied. In this paper, we have investigated whether GNA-induced apoptosis is critically mediated by the p38 mitogen-activated protein kinase (MAPK) pathway. Our findings revealed that GNA could induce apoptosis, inhibit proliferation, down-regulate the expression of p38 and MAPK, increase the activations of caspase-9, caspase-3, and cytochrome c release. Furthermore, using SB203580, an adenosine triphosphate-competitive inhibitor of p38 MAPK, inhibit the expression of p-p38 and the experimental results show that it may promote the occurrence of apoptosis induced by GNA. Taken together, these results suggested that up-regulation of the p38 MAPK cascade may account for the activation of GNA-induced apoptosis.