Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells

Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis

Cordycepin induces apoptosis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells

86-91In this study, the effect of cordycepin (3’-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 mM and 80 mM, respectively. Cordycepin treatment at 120 mM and 80 mM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma

Ionizing Radiation Selectively Increases CXC Ligand 10 Level via the DNA-Damage-Induced p38 MAPK-STAT1 Pathway in Murine J774A.1 Macrophages

Ionizing radiation (IR) is an important means of tumor treatment in addition to surgery and drugs. Attempts have been made to improve the efficiency of radiotherapy by identifying the various biological effects of IR on cells. Components of the tumor microenvironment, such as macrophages, fibroblasts, and vascular endothelial cells, influence cancer treatment outcomes through communication with tumor cells. In this study, we found that IR selectively increased the production of CXC motif chemokine ligand 10 (CXCL10), which is emerging as an important biomarker for determining the prognosis of anticancer treatments, without changing the levels of CXCL9 and CXCL11 in murine J774A.1 macrophages. Pretreatment with KU55933, an ataxia telangiectasia mutated (ATM) kinase inhibitor, significantly inhibited IR-induced CXCL10 production. In contrast, pretreatment with N-acetyl-cysteine or glutathione, a reactive oxygen species scavenger, did not inhibit IR-induced CXCL10 production. Further, we attempted to identify the intracellular molecular target associated with the IR-induced increase in CXCL10 secretion by J774A.1 macrophages. IR phosphorylated p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1) in J774A.1 macrophages, and p38 MAPK and STAT1 were involved in CXCL10 via IR using pharmacological inhibitors (SB203580 and fludarabine, respectively) and the siRNA technique

Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

<div><p>Anticancer properties and mechanisms of mimulone (MML), <i>C</i>-geranylflavonoid isolated from the <i>Paulownia tomentosa</i> fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.</p></div

A rare case of multiple pituitary adenomas in an adolescent Cushing disease presenting as a vertebral compression fracture

Cushing disease in children and adolescents, especially with multiple pituitary adenomas (MPAs), is very rare. We report 17-year-old boy with MPAs. He presented with a vertebral compression fracture, weight gain, short stature, headache, and hypertension. On magnetic resonance imaging (MRI), only a left pituitary microadenoma was found. After surgery, transient clinical improvement was observed but headache and hypertension were observed again after 3 months later. Follow-up MRI showed a newly developed right pituitary microadenoma 6 months after the surgery. The need for careful clinical and radiographic follow-up should be emphasized in the search for potential MPAs in patients with persistent Cushing disease

MML induces autophagy through p53-mediated AMPK/mTOR pathway.

<p>(A) A549 cells were treated with or without MML (60 µM) for 24 h and Western blot analysis was performed with antibodies as indicated above (p53, p-AMPK-α, AMPK-α, p-ACC, ACC, p-mTOR, mTOR, LC3, PARP-1/2, caspase-3). (B) A549 cells were treated with MML (60 µM) for the different time period as indicated above. Immunoblot analysis was performed with antibodies as indicated before. Bar graphs show the densitometry analysis of p53, p-AMPK and mTOR (bottom). Each graph indicates the ratios of p53/GAPDH, p-AMPK/AMPK, p-mTOR/mTOR and LC3 II/GAPDH, respectively. (C) A549 cells were treated with or without compound C (10 µM) for 1 h and treated with MML (60 µM) for 24 h. Immunoblotting analysis was performed with antibodies as indicated above. Cells were treated with same conditions as indicated before and then cell viability was measured by MTT assay. Bar graph represents the percent of cell viability (bottom). (D) Control cells and shp53 knockdown cells were treated with MML (60 µM) for 24 h and then Western blot analysis was performed with antibodies as indicated before. Cell viability was measured by MTT assay under the same conditions as mentioned above. Bar graph indicates the percent of cell viability (bottom). All data were expressed as mean ± SEM of three independent experiments. **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.</p

MML-induced autophagy is associated with the decrease in p53 levels.

<p>(A) A549 cells were co-treated with or without pifithrin-α (PFT-α, 20 µM) and MML (60 µM) for 24 h. After that, immunoblot analysis was performed with antibodies against p53, LC3, PARP-1/2 and caspase-3, respectively. Bar graphs indicate the ratio of LC3-II/GAPDH and the cleaved caspase-3/GAPDH, respectively. (B) A549 cells were co-treated with or without PFT-α (20 µM) and MML (60 µM) for 24 h and then immunofluorescence staining was performed using LC3 (green) antibody. Nuclei were stained with DAPI (blue). Fluorescent images were obtained using confocal microscope (Bar; 10 µm). (C) Cells were co-treated with or without PFT-α (20 µM) and MML (60 µM) for 24 h and apoptotic cells were stained with FITC-conjugated Annexin V/PI and analyzed by flow cytometry. Bar graph indicates the percentage of apoptotic cells. Percentage of apoptotic cells was evaluated by Annexin V positive and PI negative (AV+/PI-, white) and Annexin V and PI double positive (AV+/PI+, black) apoptotic cells. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.</p

MML treatment induces autophagy in A549 cells.

<p>(A) A549 cells were treated with 60 µM of MML for 24 h and then morphological images were captured by phase contrast microscope (400×, P.C.). Arrows indicate the endogenous autophagosome-like vacuoles (left) and bar graph indicates the number of vacuoles per cell. Cells were treated with MML (60 µM, 24 h) and then cells were stained with MDC and LC3 antibody, respectively. MDC (bright color, middle) images were captured by fluorescence microscope and LC3 (green, right) fluorescent images were detected by confocal microscope (bar; 10 µm). Bar graph indicates the fluorescent intensity of LC3 FITC. (B) Cells were treated with MML (60 µM) for different time periods (0–24 h) and LC3 immunofluorescence staining was performed to detect autophagosomes. Nuclei were stained with DAPI. Images were captured using confocal microscope (bar; 10 µm). (C) A549 cells were treated with various concentrations of MML (0–60 µM) for 24 h. Western blot analysis was then performed with antibodies against ATG7, Beclin-1 and LC3, respectively. GAPDH was used as loading control. Bar graph indicates densitometry analysis of LC3-II/GAPDH ratio. A549 cells were treated with different concentrations of MML (0–60 µM) for 24 h (D) or 60 µM of MML for the different time course (E) and then immunoblot analysis was carried out using antibodies against p62 and LC3, respectively. GAPDH was used as loading control. (F) A549 cells were pre-incubated with or without chloroquine (CQ, 25 µM) for 1 h, then treated with MML (60 µM) for 24 h. Western blot analysis was performed using antibodies as indicated above. GAPDH was used as loading control of Western blotting. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control.</p

The molecular structure of MML and its cytotoxic effects on various cancer cell lines.

<p>(A) Molecular structure of the mimulone. Various human cancer cells, non-small cell lung cancer A549 (B), breast cancer MCF7 (C), colon cancer HCT116 (D) and osteosarcoma U2OS (E) cells were treated with the MML as indicated concentrations (0-80 µM) for 12 h or 24 h and then cell viability was measured by MTT assay. Bar graphs indicate the percentage of viability. (F) A549 cells were treated with the MML at various concentrations (0-80 µM) for 24 h and viable cells were counted by trypan blue staining. Live cells (non-stained cells) were calculated using hemocytometer and bar graph indicates the percentage of viable cells. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control.</p

Knockdown of <i>Beclin-1</i> gene suppresses MML-induced apoptotic cell death.

<p>(A) Control and Beclin-1 knockdown cells were treated with MML (60 µM) for 24 h and then LC3 immunofluorescence staining was carried out for detecting autophagosomes. Nuclei were stained with DAPI. Images were captured by confocal microscope (bar; 10 µm). (B) Knockdown cells (shControl, shBeclin-1) were treated with MML (60 µM) for 24 h. Western blot analysis was performed with antibodies against Beclin-1, LC3, PARP-1/2 and caspase-3, respectively. GAPDH was used as loading control. Bar graphs indicate the ratio of LC3-II/GAPDH and cleaved caspase-3/GAPDH, respectively. (C) Control and Beclin-1 knockdown cells were treated with MML (60 µM) for 24 h and then cell viability was measured by MTT assay. Bar graph represents the percentage of cell viability. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control; ^# p<0.05 compared with MML-treated group.</p