Cordyceps species including Cordyceps bassiana are a notable anti-cancer dietary supplement. Previously, we identified several compounds with anti-cancer activity from the butanol fraction (Cb-BF) of Cordyceps bassiana. To expand the structural value of Cb-BF-derived anti-cancer drugs, we employed various chemical moieties to produce a novel Cb-BF-derived chemical derivative, KTH-13-amine-monophenyl [4-isopropyl-2-(1-phenylethyl) aniline (KTH-13-AMP)], which we tested for anti-cancer activity. KTH-13-AMP suppressed the proliferation of MDA-MB-231, HeLa, and C6 glioma cells. KTH-13-AMP also dose-dependently induced morphological changes in C6 glioma cells and time-dependently increased the level of early apoptotic cells stained with annexin V-FITC. Furthermore, the levels of the active full-length forms of caspase-3 and caspase-9 were increased. In contrast, the levels of total forms of caspases-3, caspase-8, caspase-9, and Bcl-2 were decreased in KTH-13-AMP treated-cells. We also confirmed that the phosphorylation of STAT3, Src, and PI3K/p85, which is linked to cell survival, was diminished by treatment with KTH-13-AMP. Therefore, these results strongly suggest that this compound can be used to guide the development of an anti-cancer drug or serve as a lead compound in forming another strong anti-proliferative agent.
Apoptosis ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Survival ; Cordyceps* ; Dietary Supplements ; Glioma ; Phosphorylation

BACKGROUND: Apoptosis protease activating factor-1 (Apaf-1), caspase-9, Bcl-2, p53, and survivin are important factors in the pathway of apoptosis, but their clinicopathologic significance remains unclear in human cutaneous melanoma. We investigated the expression of these proteins and their clinical value in human cutaneous melanocytic lesions. METHODS: We performed an immunohistochemical analysis to examine the expression and distribution of Apaf-1, caspase-9, Bcl-2, p53, and survivin in 36 cases of malignant melanoma (22 cases of primary melanoma and 14 cases of metastatic melanoma) and 41 cases of melanocytic nevus. RESULTS: The expression of p53 was significantly higher in malignant melanoma than in melanocytic nevus (p<0.01), however the expressions of Apaf-1 and caspase-9 were significantly lower in malignant melanoma compared with melanocytic nevus (p<0.01 and p=0.027, respectively). Also, there was a significant difference for Bcl-2 staining between primary melanomas and metastatic lesions (p=0.004). Nuclear staining for survivin were absent in nevus, but were positive in 14 of 36 melanomas (p<0.01). CONCLUSIONS: The altered expression of Apaf-1, caspase-9, p53, and survivin are considered to be related to malignant progression in human cutaneous melanocytic lesions. Loss of Bcl-2 can be considered as a prognostic marker of malignant melanomas.
Apoptosis ; Caspase 9 ; Humans ; Melanoma ; Nevus ; Nevus, Pigmented ; Proteins

PURPOSE: To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. METHODS: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 micro M of ceramide type II, VI and phytoceramide type II, VI. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin V for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor(IETD-CHO(R)) and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide. RESULTS: Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.
Annexin A5 ; Apoptosis* ; Caspase 8 ; Caspase 9 ; Ceramides ; Cornea ; Cytochromes c ; Cytoplasm ; Endothelial Cells* ; Protease Inhibitors

PURPOSE: The purpose of this study was to determine if ceramide, which is known as secondary messenger of programmed cell death (apoptosis), can cause apoptosis in lens epithelial cell (LEC) and if so, to identify the pathway by which apoptosis occurs. METHODS: After LECs were exposed to various concentrations of ceramide and phytoceramide, we evaluated the resulting apoptosis response using the Hoechst-EthD stain and Annexin stain. To search for the apoptosis pathway, LECs were preincubated in various concentrations of CPP32-like protease inhibitor, specific caspase-8 inhibitor, and specific caspase-9 inhibitor, then treated with ceramide and phytoceramide. We performed LDH assay 12 hours later. Cytochrome c immunostaining was done after exposure to the ceramide and phytoceramide. RESULTS: All kinds of ceramide induced time and concentration dependent apoptosis in LEC. Caspase 8 inhibitor and caspase 9 inhibitor reduced the apoptosis in ceramide VI, phytoceramide II, and phytoceramide VI. In all ceramides, cytochrome c staining was positive. CONCLUSIONS: Ceramide and phytoceramide can cause apoptosis in LEC. Ceramide and phytoceramide may be used to prevent the posterior capsular opacity after cataract surgery.
Apoptosis* ; Caspase 8 ; Caspase 9 ; Cataract ; Cell Death ; Ceramides ; Cytochromes c ; Epithelial Cells* ; Protease Inhibitors

PURPOSE: We investigated the effect of ceramide on keratocyte apoptosis and pathway of ceramide-induced keratocyte apoptosis. METHODS: Cultured Newzealand White Rabbit keratocytes were exposed to various concentrations of ceramide type II, VI and phytoceramide type II, VI. LDH level was measured for the evaluation of time and concentration related apoptosis. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK, diffuse caspase inhibitor), specific caspase-8 inhibitor (IETD-CHO) and specific caspase-9 inhibitor (Z-LEHD-FMK), then were exposed to 20 micro M of 4 types of ceramide. Cytochome C immune stainining was done after exposure of keratocyte to 4 types of ceramide. RESULTS: The lower effective dose of 4 types of ceramide was 20 micro M. Apoptosis of keratocytes was dependent on ceramide exposure time. Ceramide induced keratocyte apoptosis was inhibited by CPP32-like protease inhibitor, specific caspase-8 inhibitor and specific caspase-9 inhibitor. Apoptotic keratocytes induced by ceramide were immune-stained with cytochrome C antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal keratocytes. This apoptosis developed according caspase cascade, especially via mitochondria.
Apoptosis* ; Caspase 8 ; Caspase 9 ; Corneal Keratocytes ; Cytochromes c ; Mitochondria ; Protease Inhibitors

OBJECTIVETo study the effect of ginsenoside on apoptosis of human leukemia-60 (HL-60) cells and its mechanism.

METHODSMTT cytotoxicity assay was used to determine the growth inhibition activity of ginsenoside (100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μmol/L) on HL-60 cells. The apoptosis of HL-60 cells after treatment with ginsenoside (0,5,10 and 20 μmol/L) was determined by Annexin V-FITC/PI staining and flow cytometry. The cleavage of total proteins by caspase-8, caspase-9 and caspase-3 was evaluated by Western blot. The cleavage of caspase-3 protein was detected by Western blot after treatment with 10 μmol/L ginsenoside and caspase-8 and 9 inhibitors.

RESULTSGinsenoside had potent cytotoxicity on HL-60 cells, with an IC50 value of 7.3±1.2 μmol/L. After treatment with ginsenoside (0, 5, 10 and 20 μmol/L) for 48 hours, the apoptotic rate displayed a dose dependency, as shown by flow cytometry, with significant differences between the groups (F=12.67, P<0.01). Western blot showed that there were caspase-9 and caspase-3 cleavage bands, but without caspase-8 cleavage band. The specific inhibitor of caspase-9 Z-LEHD-FMK could block the caspase-3 cleavage induced by 10 μmol/L ginsenoside, but the specific inhibitor of caspase-8 Z-IETD-FMK did not have this effect.

CONCLUSIONSGinsenoside can induce apoptosis of HL-60 cells, which may be related to a mitochondria-dependent pathway.

Apoptosis ; drug effects ; Caspase 9 ; physiology ; Caspase Inhibitors ; pharmacology ; Ginsenosides ; pharmacology ; HL-60 Cells ; Humans

OBJECTIVE: To investigate the effect of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis (KOA). METHODS: A total of 32 rats were divided into a normal group, a model group, a control treatment group and a treatment group by random number grouping method, 8 rats in each one. The rats in the normal group received no intervention. The rats in the remaining three groups adopted modified Videman method to develop KOA model, the ankle joint of left posterior leg was fully extended and fixed with a resin bandage for 6 weeks. After successful modeling, the rats in the model group received no intervention. The rats in the control treatment group were treated with medium-frequency pulse electrotherapy. The rats in the treatment group were treated with inner- heating acupuncture, 30 min each treatment, once a day, five days per week, and totally 3-week treatment was given. After 3 weeks, the damaged cartilage tissue was collected, and HE staining was used to observe the pathological changes of the cartilage tissue of the knee joint. ELISA was used to detect the content of cytochrome-C in the tissue homogenate supernatant. The chondrocytes in damaged cartilage tissue were isolated, flow cytometer was used to detect the changes of apoptosis and mitochondrial membrane potential. The mRNA and protein expression of Caspase-3 and Caspase-9 in chondrocytes were detected by real-time quantitative PCR (qRT-PCR) and Western blot (WB), respectively. RESULTS: Compared with the normal group, the damage of cartilage tissue in the model group was significant, and the expression level of Cyt-C in the homogenate supernatant of damaged cartilage tissue was increased (<0.01); the chondrocyte apoptosis was increased significantly (<0.01); the chondrocyte mitochondrial membrane potential was decreased significantly (<0.01); the mRNA and protein expression of Caspase-3 and Caspase-9 was increased significantly (all <0.01). Compared with the model group, the cartilage injury in the control treatment group and the treatment group was significantly relieved; the expression level of Cyt-C in the supernatant of damaged cartilage tissue homogenate was decreased (both <0.01); the chondrocyte apoptosis was significantly reduced (both <0.01); the chondrocyte mitochondrial membrane potential was increased significantly (both <0.01). Moreover, the mRNA and protein expression of Caspase-3 and Caspase-9 was significantly reduced (all <0.01). Compared with the control treatment group, the treatment group was more effective in the treatment of KOA. CONCLUSION The inner-heating acupuncture could significantly improve the pathological changes of KOA rats, inhibit the apoptosis of chondrocytes, which may be closely related to the suppression of Caspase-3 and Caspase-9 expression.
Animals ; Apoptosis ; Cartilage, Articular ; Caspase 3 ; Caspase 9 ; Chondrocytes ; Heating ; Osteoarthritis, Knee ; Rats

Chios Gum Mastic (CGM) is a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios. It has been used by traditional healers, and it has antibacterial, antifungal properties, and therapeutic benefits for the skin. The CGM reduces the formation of dental plaque and bacterial growth in oral saliva, and recent studies have demonstrated the role of antioxidant activity of CGM. Although CGM has been widely investigated, its protective effect against oxidative-damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against H2O2-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes. The pretreatment with CGM significantly reduced apoptosis in H2O2-exposed HaCaT cells. It promoted the degradation of caspase-3, caspase-8, and caspase-9; and it induced the formation of the processed PARP. The treatment with CGM caused an increase in vesicle formation compared to control group. The level of p62 was reduced and the conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. In summary, CGM helps the cells to survive under stressful conditions by preventing apoptosis and enhancing autophagy. Besides, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.
Apoptosis ; Autophagy* ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Dental Plaque ; Gingiva* ; Humans ; Keratinocytes ; Oxidative Stress* ; Pistacia ; Plants ; Saliva ; Skin

Chronic hypoxia has been associated with change in neurovascular behavior, mediated, in part, by erythropoietin (EPO). EPO, a hematopoietic growth factor, could act as a neurotrophic factor. In the present study, we investigated the characteristics of EPO and erythropoietin receptor (EPOR) expressions by cortical neuron in vivo and in vitro and tested the hypothesis that EPO serves protective functions under chronic hypoxia. E18, P5 and P7 mice for 3 days and primary cultured neurons for 6 days were incubated in hypoxic conditions consisted of a mixture of 10% O2, 5% CO2, 85% N2. To study expressions of EPO, EPOR, caspases, pAKT, pERK, and PARP, immunohistochemical stainning and western blotting were carried out. In addition to expressing EPO and EPOR under normoxic conditions, neurons increased their expression of EPO and EPOR under hypoxia. The effects of recombinant EPO appeared to be mediated via the phosphatidylinositol (PI) 3- kinase-AKT pathway, correlated directly with activation of caspase 3. Also recombinant EPO decreased expression of caspase 8, but not caspase 9. Finally, recombinant EPO decreased apoptosis of cultured neurons as evaluated by expression of PARP. These data support a role for EPO in maintenance of cortical neuron under chronic hypoxia.
Animals ; Anoxia* ; Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspases ; Erythropoietin ; Mice ; Neurons* ; Phosphatidylinositols ; Receptors, Erythropoietin

Columbianadin (CBN), a natural coumarin from Angelica decursiva (Umbelliferae), is known to have various biological activities including anti-inflammatory and anti-cancer effects. In this study, the anti-proliferative mechanism of actions mediated by CBN was investigated in HCT-116 human colon cancer cells. CBN effectively suppressed the growth of colon cancer cells. Low concentration (up to 25 μM) of CBN induced apoptosis, and high concentration (50 μM) of CBN induced necroptosis. The induction of apoptosis by CBN was correlated with the modulation of caspase-9, caspase-3, Bax, Bcl-2, Bim and Bid, and the induction of necroptosis was related with RIP-3, and caspase-8. In addition, CBN induced the accumulation of ROS and imbalance in the intracellular antioxidant enzymes such as SOD-1, SOD-2, catalase and GPx-1. These findings demonstrate that CBN has the potential to be a candidate in the development of anti-cancer agent derived from natural products.
Angelica ; Apoptosis* ; Biological Products ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Catalase ; Cell Proliferation* ; Colon* ; Colonic Neoplasms* ; Humans ; Oxidative Stress