The induction of apoptosis in cancer cells is a therapeutic strategy for the treatment of cancer. In the present study, we investigated the regulatory mechanisms responsible for quercetin-induced apoptosis, mamely the increased expression of sestrin 2 and the activation of the 5′ AMP-activated protein kinase (AMPK)/p38 MAPK signaling pathway. Our results revealed that quercetin induced apoptosis by generating the production of intracellular reactive oxygen species (ROS) and increasing the expression of sestrin 2. The induction of apoptosis by quercetin occurred through the activation of the AMPK/p38 signaling pathway and was dependent on sestrin 2. However, the silencing of sestrin 2 using small interfering RNA (siRNA) targeting sestrin 2 revealed that quercetin did not regulate AMPK or p38 phosphorylation in the cells in which sestrin 2 was silenced. On the other hand, it has been previously reported that sestrin 2 expression is not dependent on p53 expression under hypoxic conditions, whereas DNA damage is dependent on p53. We demonstrate that the increase in the expression of sestrin 2 by quercetin-generated intracellular ROS is p53-independent. The increased expression of sestrin 2 induced apoptosis through the AMPK/p38 signaling pathway in the HT-29 colon cancer cells, which are p53 mutant, treated with quercetin. Thus, our data suggest that quercetin induces apoptosis by reducing mitochondrial membrane potential, generating intracellular ROS production and increasing sestrin 2 expression through the AMPK/p38 pathway. In addition, p53 is not a necessary element for an apoptotic event induced by sestrin 2.

Background:The suppression of abnormal cell proliferation is therapeutic strategies for the treatment of cancer. In this study, we investigated the regulatory mechanism of quercetin-induced apoptosis through regulation of Sestrin 2 and AMPK signaling pathway.Methods:After treatment of quercetin to colon cancer cells, intracellular ROS was detected using by DCFH-DA. To examine how quercetin and H2O2 induced apoptosis, we analyzed the change of Sestrin 2, p53 expression and p-AMPKα1, p-mTOR levels by Western blotting. To evaluate the effect of intracellular ROS generated by quercetin on colon cancer cells, NAC, anti-oxidative agent, was co-treated.Results:Quercetin increased apoptotic cell death though generating intracellular reactive oxygen species (ROS), and it was responsible for Sestrin 2 expression. Increased Sestrin 2 expression was accompanied by AMPK activation. Interestingly, mTOR activity by Sestirn 2 expression was dependent on AMPK phosphorylation. On the other hand, the expression of Sestrin 2 by quercetin-generated intracellular ROS was independent of p53.Conclusions:We suggested that quercetin-induced apoptosis involved Sestrin 2/AMPK/mTOR pathway, which was regulated by increased intracellular ROS by quercetin.

BackgroundThe extracts from Artemisia annua Linné (AAE) has been known to possess various functions including anti-bacterial, anti-virus and anti-oxidant effects. However, the mechanism of those effects of AAE is not well known. Pursuantly, we determined the apoptotic effects of extract of AAE in HCT116 cell. In this study, we suggested that AAE may exert cancer cell apoptosis through PTEN/PDK1/Akt/p53signal pathway and mitochondria-mediated apoptotic proteins.MethodsWe measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, Annexin V-PI staining, Mitopotential assay, immunofluorescence (IF) and Western blotting. Accordingly, our study showed that AAE treatment to HCT116 cells resulted in inhibition of PDK1, Akt, MDM2, Bcl-2, and pro-caspase 3 as well as activation of PTEN, p53-upregulated modulator of apoptosis (PUMA), Bax and Bak expression. Also we measured in vivo assay that xenograft model, H&E assay, TUNEL assay and IHC.ResultsAAE induced apoptosis via PTEN/p53/PDK1/Akt signal pathways through PTEN/p53-independent manner. AAE inhibit cell viability and increase LDH release in HCT116 colon cancer cell. Also, AAE increase apoptotic bodies, caspase −3,7 activation and reduces mitochondria membrane potential. AAE regulates cytochrome c translocation to the cytoplasm and Bax translocation to the mitochondrial membrane in an Immunofluorescence staining and increase PTEN and p53 expression in an in vivo tumor xenograft model. To elucidate the role of the PTEN/p53/PDK1/Akt signal pathways in cancer control, we conditionally inactivated PTEN/p53/PDK1/Akt signal pathways. We used inhibitors of PTEN, p53, PDK1, Akt. In consequence, these results indicate that AAE induced apoptosis by means of a mitochondrial event through the regulation of proteins such as Bax, Bak and cytochrome c in PDK1/Akt signaling pathways via PTEM/p53-independent manner.ConclusionsWe confirmed the apoptotic effect of extracts of AAE by Modulating PTEN/p53/PDK1/Akt/Signal Pathways through PTEN/p53-independent pathwaysin HCT116 colon cancer cell.

Abstract. Torilis japonica extract (TJE) has been reported to possess diverse medicinal properties including anti-inflammatory and antibacterial activities. However, the precise mechanism of its anticancer effect is not understood. Thus, we evaluated the apoptotic effects of TJE and examined its underlying molecular mechanisms in HCT116 colorectal cancer cells. Our results show that TJE induces apoptosis through the generation of intracellular reactive oxygen species (ROS), and that it regulates the mitochondrial outer membrane potential via the AMPK/p38 MAPK signaling pathway. Importantly, ~50% of cancer cells have p53 mutations. Thus, the ability to induce apoptosis in a p53-independent manner would be of great value in cancer treatment. Our results show that not only does TJE regulate the AMPK/p38 signaling pathway, but it induces apoptosis in cells in which p53 has been knocked down using siRNA. Moreover, as in in vitro studies, TJE induced apoptosis and regulated apoptosis related-proteins in an HCT 116 xenograft model. Taken together, our results demonstrate that TJE, a natural compound that may provide a substitute for chemotherapeutic drugs, has potential as an anticancer agent.

Abnormal metastasis of carcinoma is associated with the loss of epithelial features and the acquisition of a mesenchymal phenotype. The stimulation of cells with epidermal growth factor (EGF) resulted in morphological changes and induced epithelial-mesenchymal transition (EMT). EGF stimulation resulted in increased mobility along with upregulated actin polarization related proteins, E-cadherin regulators and the mesenchymal markers. Treatment with Torilis japonica extract (TJE) along with stimulation by EGF prevented changes in cell morphology, mobility, expression of actin polarization proteins and EMT markers. Using specific inhibitors and siEGFR, it was demonstrated that TJE suppressed EMT through EGFR inactivation and regulation of its downstream signaling pathways. We suggest that TJE is a new potential reagent for EGFR-targeted therapy and anti-abnormal metastasis in MCF-7 breast cancer.

Neutrophil activity, including trapping of damage-associated molecular patterns by neutrophil extracellular traps (NETs), is an important response to microbial infection. Most activated neutrophils commit to apoptosis and are removed by activated macrophages in the process of efferocytosis. Improper clearance of apoptotic neutrophils often causes an unnecessary and exaggerated immune response and subsequent chronic inflammation. Effective macrophage mobility toward activated neutrophils, which is triggered by binding of 'find-me' signals to receptors such as P2Y2, is a crucial step for the timely clearance of apoptotic neutrophils. In this paper, we investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on efferocytosis and the underlying molecular mechanisms. In a coculture of apoptotic neutrophils with macrophages, PLAG treatment increased levels of efferocytosis of apoptotic neutrophils. PLAG induced faster translocation of P2Y2 from lipid rafts to nonlipid raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as a-tubulin and actin to improve the mobility of macrophages. The formation of vesicular, chylomicron-like structures by PLAG was a prerequisite for the induction of this macrophage activity, as none of these effects was seen when the vesicle receptor GPIHBP1 was absent. Taken together, these data showed that PLAG is a powerful immune resolvent that triggers the prompt clearance of apoptotic neutrophils by enhanced efferocytosis activity. PLAG could therefore be an effective lipid-based efferocytosis enhancer for use as a therapeutic drug to prevent inflammatory disease caused by uncontrolled immune responses.

Cnidium monnieri (L.) Cusson is a frequently used traditional Chinese medicine that treats gynecological diseases and carbuncles. However, the mechanism of action of C. monnieri remains to be fully elucidated. The present study examined the cell cycle arrest and apoptotic effects resulting from ethanol extract of C. monnieri (CME) in HepG2 (wild‑type p53) and Hep3B (p53‑null) hepatocellular carcinoma cells. An MTT assay was used to confirm the anti‑proliferative effect of CME. The cells were stained with Hoechst 33342 or propidium iodide. It was demonstrated that proliferation of HepG2 cells was suppressed by CME. Cell cycle arrest occurred in the G1 phase following treatment with CME and the number of apoptotic bodies was increased. The expression levels of cell cycle‑associated proteins, including protein kinase B (Akt), glycogen synthase kinase‑3β (GSK‑3β), p53, cyclin E and cyclin‑dependent kinase 2 (CDK2) were determined by western blot analysis. The protein levels of phosphorylated (p)‑Akt, p‑GSK‑3β, p‑MDM2 and cyclin E were decreased, whereas the protein levels of p53, p21 and p‑CDK2 (Thr14/Tyr15) were increased following treatment with CME. Furthermore, treatment or co‑treatment with LY294002 (phosphoinositide‑3‑kinase/Akt inhibitor) or Pifithrin‑α (p53 inhibitor) with CME resulted in CME‑induced G1 arrest which occurred through the p53‑independent signaling pathway in hepatocellular carcinoma cells. In conclusion, CME induces G1 arrest and apoptosis via the Akt/GSK‑3β signaling pathway which is regulated by MDM2‑induced degradation of p21, rather than p53.

The effectiveness of chemotherapy and radiotherapy to treat lung cancer is limited because of highly metastatic nature. Novel strategies and drugs to attenuate metastatic activity are urgently required. In this study, red fluorescence proteins (RFP)-labeled A549 human lung cancer cells were orthotopically implantation, where they developed primary tumors. Metastasis in brain and intestines were reduced by up to 80% by treatment with 100 mpk 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) compared with that in control mice. PLAG treatment also reduced the migration of the primary tumors. Interestingly, substantial neutrophil infiltration was observed in the tumors in control mice. The neutrophil contribution to A549 cell metastatic activity was examined in in vitro co-culture system. Metastatic activity could be achieved in the A549 cells through epidermal growth factor receptor (EGFR) transactivation mediated by protease activating receptor 2 (PAR2) receptor. Neutrophil elastase secreted from tumor-infiltrating neutrophils stimulated PAR2 and induced EGFR transactivation. However, this transactivation was inhibited by inducing PAR2 degradation following PLAG treatment and metastatic activity was effectively inhibited. PLAG attenuated cancer metastatic activity via modulated PAR2/EGFR transactivation by accelerating PAR2 degradation. These results suggest PLAG as potential therapeutic agent to combat tumor metastasis via regulating the activation signal pathway of PAR2 by tumor infiltrate-neutrophils.