Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

A human IAP-family gene, apollon, expressed in human brain cancer cells.

IAP is a family of protein that has baculovirus IAP repeat (BIR) domains and inhibits apoptosis. We found a human IAP family gene, which we named Apollon, encoding a huge protein (530 kDa) that contains a single BIR domain and a ubiquitin-conjugating enzyme domain, that is a human homolog of BRUCE. Apollon protein was expressed in four of six brain cancers (gliomas), and one of five ovarian cancers in 38 human cancer cell lines that we examined. Among the brain cancer cell lines, SNB-78 expressed a high level of Apollon, and this cell line shows resistance against various anticancer drugs. Treating SNB-78 cells with antisense oligonucleotide against Apollon reduced the expression of Apollon protein, and significantly sensitized the cells to apoptosis induced by cisplatin and camptothecin. These results suggest that Apollon protects SNB-78 cells from undergoing apoptosis and, at least in part, plays a role in tumorigenesis and drug resistance of this cell line.     

Daidzin inhibits growth and induces apoptosis through the JAK2/STAT3 in human cervical cancer HeLa cells

Daidzin, 4′, 7-dihydroxyisoflavone is an isoflavonic phytoestrogen present in leguminous plants. Traditional Chinese medicine utilizes daidzin to treat various diseases such diarrhea, fever, hepatitis, cardiac problems etc. In current study we examined the anticancer activity of daidzin against human cervical cancer in vitro. HeLa, human cervical cancer cell line was purchased from ATCC and the cells were cultured with DMEM medium. The cytotoxic effect of daidzin against HeLa cell line was analyzed with MTT assay. The IC-50 value was obtained at 20 µM hence the cells were treated with 20 µM of daidzin for further analysis. ROS generation was assessed with DCFH-DA staining and the induction of apoptosis was examined with Rhoadmine-123 staining. Acridine orange and ethidium bromide staining was done to examine the apoptotic and viable cells. Further the matrigel cell adhesion assay was done to analyze the inhibitory property of daidzin against cancer cell adhesion. Apoptotic induction of daidzin was examined by estimating the levels of Caspase 8 & 9 using ELISA technique. Inflammatory and cell proliferation signaling proteins were analyzed with qPCR analysis to confirm the anticancer activity of daidzin against human cervical cancer HeLa cell line. Daidzin significantly generated ROS and altered the mitochondrial membrane permeability in HeLa cell line. The results of AO/EtBr staining prove daidzin induced apoptosis in HeLa cell line and it also inhibited the cell adhesion property of HeLa which is reported in our matrigel cell adhesion assay. It also increased the caspases 8 & 9 which are key regulators of apoptosis. Daidzin significantly decreased the expression of inflammatory gene and cell proliferating signaling molecule. To, conclude our results confirm daidzin effectively decreased inflammation and induced apoptosis in human cervical cancer HeLa cell line.     

Synthesis and biological evaluation of cinnamyl compounds as potent antitumor agents

A series of cinnamyl compounds related to 2'-hydroxycinnamaldehyde were synthesized and their antitumor effects against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon tumor xenograft in nude mice. Its antitumor effects belong to the induction of apoptosis and arresting cell cycle at G(2)/M phase, which is confirmed by detection of apoptosis markers and cell cycle analysis.     

Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

Previous studies have suggested that 1,25(OH)2D3, the active form of vitamin D3, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D3 has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH)2D3 induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in this model. Moreover, 1,25(OH)2D3 failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D3.     

The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts

The α2-adrenoceptor inducer dexmedetomidine protects against acute lung injury (ALI), but the mechanism of this effect is largely unknown. The present study investigated the effect of dexmedetomidine on apoptosis induced by lipopolysaccharide (LPS) and the relationship between this effect and gap junction intercellular communication in human lung fibroblast cell line. Flow cytometry was used to detect apoptosis induced by LPS. Parachute dye coupling assay was used to measure gap junction function, and western blot analysis was used to determine the expression levels of connexin43 (Cx43). The results revealed that exposure of human lung fibroblast cell line to LPS for 24 h increased the apoptosis, and pretreatment of dexmedetomidine and 18α-GA significantly reduced LPS-induced apoptosis. Dexmedetomidine exposure for 1 h inhibited gap junction function mainly via a decrease in Cx43 protein levels in human lung fibroblast cell line. These results demonstrated that the inhibition of gap junction intercellular communication by dexmedetomidine affected the LPS-induced apoptosis through inhibition of gap junction function by reducing Cx43 protein levels. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting ALI.     

A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.     

Hormonemate,a new cytotoxic and apoptosis-inducing compound from the endophytic fungus Hormonema dematioides. I. Identification of the producing strain,and isolation and biological properties of hormonemate

In a search for new fungal compounds inducing apoptosis of the colon cancer derived cell line COLO-320, hormonemate (1) was purified from fermentations of an endophytic fungus isolated from living needles of a Pinus species. The producing strain was identified as Hormonema dematioides by microscopy and ITS rDNA sequence analysis. The structure of hormonemate was determined by spectroscopic methods. The compound exhibited cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 as well as five other human cell lines (HL-60, JURKAT, HEP-G2, MCF-7, HeLa S3). It also induced apoptosis in COLO-320 cells as detected by a caspase-activity assay and morphological changes, and it triggered morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently died by apoptosis.     

Viral FLICE inhibitory protein of rhesus monkey rhadinovirus inhibits apoptosis by enhancing autophagosome formation

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to human herpesvirus 8 (HHV8). RRV encodes viral FLICE inhibitory protein (vFLIP), which has death effector domains. Little is known about RRV vFLIP. This study intended to examine its function in apoptosis. Here we found that RRV vFLIP inhibits apoptosis induced by tumor necrosis factor-α (TNF-α) and cycloheximide. In HeLa cells with vFLIP expression, the cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than those in controls. Cell viability of HeLa cells with vFLIP expression was significantly higher than control cells after apoptosis induction. However, RRV vFLIP appears unable to induce NF-κB signaling when tested in NF-κB reporter assay. RRV vFLIP was able to enhance cell survival under starved conditions or apoptosis induction. At early time points after apoptosis induction, autophagosome formation was enhanced and LC3-II level was elevated in cells with vFLIP and, when autophagy was blocked with chemical inhibitors, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis by enhancing autophagy to maintain cell survival. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These results indicate that vFLIP protects cells against apoptosis by enhancing autophagosome formation to extend cell survival. The finding of vFLIP's inhibition of apoptosis via the autophagy pathway provides insights of vFLIP in RRV pathogenesis.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

Dehydrocostus lactone inhibits cell proliferation and induces apoptosis by PI3K/Akt/Bad and ERS signalling pathway in human laryngeal carcinoma

The anti-cancer effect of dehydrocostus lactone (DHL) derived from Saussurea costus (Falc.) Lipech against laryngeal carcinoma was assessed. The cytotoxic activity of DHL against laryngeal carcinoma is still obscure. Therefore, our study investigated the role of DHL in the growth inhibition of laryngeal carcinoma in vitro and in vivo, and the molecular mechanism of DHL-induced apoptosis in cancer cells of the larynx. The results showed that DHL inhibits the viability, migration and proliferation of Hep-2 and TU212 cells with little toxic effects on human normal larynx epithelial HBE cell line. Flow cytometry analysis (FAC) analysis and staining assay (Hoechst 33258) indicated that DHL stimulated Hep-2 and TU212 cell apoptosis in a dose-dependent manner. Mechanistically, DHL is capable of inhibiting Hep-2 and TU212 cell viability via promoting p53 and P21 function, meanwhile DHL dose-dependently induces Hep-2 and TU212 cells apoptosis via activating mitochondrial apoptosis by inhibiting PI3K/Akt/Bad pathway and stimulating endoplasmic reticulum stress-mediated apoptosis pathway. In vivo, DHL inhibited the growth of the Hep-2 nude mouse xenograft model and observed no significant signs of toxicity in the organs of nude mice. In vivo experiments further confirmed the anti-cancer effect of DHL on laryngeal carcinoma cells in vitro, and DHL-treated nude mice can reduce the volume of tumours. Together, our study indicated that DHL has the potential to inhibit human laryngeal carcinoma via activating mitochondrial apoptosis pathway by inhibiting PI3K/Akt/Bad signalling pathway and stimulating endoplasmic reticulum stress-mediated apoptosis pathway, providing a strategy for the treatment of human laryngeal carcinoma.     

Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.     

Gas1 Knockdown Increases the Neuroprotective Effect of Glial Cell-Derived Neurotrophic Factor Against Glutamate-Induced Cell Injury in Human SH-SY5Y Neuroblastoma Cells

Growth arrest-specific 1 (Gas1) protein acts as an inhibitor of cell growth and a mediator of cell death in nervous system during development and is also re-expressed in adult neurons during excitotoxic insult. Due to its structural similarity to the glial cell-derived neurotrophic factor family receptors α (GFRα), Gas1 is likely to interfere with the neuroprotective effect of GDNF. In the present study, we investigated the expression profile of Gas1 during glutamate insults in human SH-SY5Y neuroblastoma cells as well as the influence of Gas1 inhibition on the protective effect of GDNF against glutamate-induced cell injury. Our data showed that Gas1 expression was significantly increased with the treatment of glutamate in SH-SY5Y cells. The silencing of Gas1 by small interfering RNA promoted the protective effect of GDNF against glutamate-induced cytotoxicity as well as cell apoptosis, which effect was likely mediated through activating Akt/PI3 K-dependent cell survival signaling pathway and inhibiting mitochondrial-dependent cell apoptosis signaling pathway via Bad dephosphorylation blockade. In summary, this study showed the synergistic effect of Gas1 inhibition and GDNF against glutamate-induced cell injury in human SH-SY5Y neuroblastoma cells, which information might significantly contribute to better understanding the function of Gas1 in neuronal cells and form the basis of the therapeutic development of GDNF in treating human neurodegenerative diseases in the future.     

Upregulation of cellular triacylglycerol - free fatty acid cycling by oleate is associated with long-term serum-free survival of human breast cancer cells.

We previously showed that exogenous oleate protects human breast cancer cells against palmitate-induced apoptosis in part by increasing esterification of this free fatty acid (FFA) into triacylglycerol (TG). Here, we studied the mechanism whereby oleate protects these cells against apoptosis induced by serum withdrawal. The metabolism of FFA, TG, and glucose, in parallel with long-term cell survival in the absence of serum, was investigated in a panel of human breast cancer cell lines and in nontransformed MCF-10A cells after treatment with exogenous oleate. Short-term (3-24 h) exposure of MDA-MB-231 human breast cancer cells to exogenous oleate resulted in a dose-dependent long-term (10 day) serum-free survival that correlated with the accumulation of TG in lipid droplets and with upregulation of lipolysis. Both effects persisted for several days after oleate removal. Rapid TG lipolysis and FFA re-esterification, supported by high rates of glycolysis that provide the glycerol backbone for TG synthesis, are consistent with the presence of very active TG-FFA cycling in human breast cancer cells. Only the cancer cell lines capable of accumulating TG showed long-term serum-free survival after oleate treatment. The results suggest that upregulation of TG-FFA cycling induced by oleate may be involved in maintenance of human breast cancer cell survival.     

Inhibition of ultraviolet B (UVB) induced apoptosis in A431 cells by mimosine is not dependent on cell cycle arrest

Ultraviolet (UV) radiation is a strong apoptotic trigger in many cell types. We have previously reported that a plant amino acid, mimosine (beta [N-(3-hydroxy-4-pyridone)]-alpha-aminopropionic acid), with a well-known reversible G1 cell cycle arrest activity can inhibit apoptosis induced by UV irradiation and RNA polymerase II blockage in human A431 cells. Here, apoptosis was measured with a fluorimetric caspase activation assay. Interestingly, the protective state was effective up to 24 h following removal of mimosine from the culture medium while cells were progressing in the cell cycle. Our results demonstrate that the protective effect of mimosine against UV-induced apoptosis can be dissociated from its G1 cell-cycle arrest activity.     

Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.     

Anti-Ki-67 peptide nucleic acid affects the proliferation and apoptosis of human renal carcinoma cells in vitro

We treated in vitro human renal carcinoma cells (cell line 786-0) with the lipid-delivered peptide nucleic acids (PNAs) against Ki-67 gene. Corresponding control groups were treated with the antisense oligonucleotides (ASOs) of the same nucleobase sequence, and with mismatched PNAs. In cells treated by anti-Ki-67 PNAs, the Ki-67 expression rate, Ki-67 protein level, cell growth and the DNA synthesis-indicative 3H-thymidine incorporation rate were lower than in the ASO-treated groups, and reduced significantly compared to untreated controls, whereas the rate of apoptosis was markedly increased by PNA treatment. We conclude that anti-Ki-67 PNA has more strong (than ASO) and dose-dependent effects on the proliferation and apoptosis of human renal carcinoma cells. Our results indicate that the strategy of using PNA against the Ki-67 gene might be a promising approach in renal carcinoma therapy.     

Insulin-like growth factor I (IGF-I) stimulates proliferation but also increases caspase-3 activity, Annexin-V binding, and DNA-fragmentation in human MG63 osteosarcoma cells: co-activation of pro- and anti-apoptotic pathways by IGF-I.

Insulin-like growth factor-I (IGF-I) was found to promote proliferation, cell survival, and inhibition of apoptosis. But in some instances, IGF-I was found to mildly induce apoptosis, i. e. Fas-mediated apoptosis in human MG63 osteosarcoma cells. In the present study, we intended to further investigate IGF-I dependent pathways leading either to proliferation and cell survival or to cell death. MG63 osteosarcoma cells were treated with serum free medium alone or in combination with IGF-I, a neutralizing antibody against the human IGF-I receptor (alphaIR-3) or non-immune control IgG (1) for two to six days. We investigated cell survival (cell count), proliferation (CD71-FACS), apoptosis (Annexin-V-FACS, Caspase-3 activity, PCD) and anti-apoptosis (112-Ser Bad phosphorylation), and regulation of IGF-I receptor surface expression (IGF-I receptor-FACS). We found that IGF-I treatment (48 h) stimulated cell growth and proliferation, but also mildly induced apoptosis. IGF-I activated specific apoptotic pathways (Caspase-3 activation, Annexin-V binding and DNA degradation), as well as anti-apoptotic signals (Bad phosphorylation at serine 112). alphaIR-3 blocked cell proliferation, strongly induced apoptosis, and inhibited Bad-phosphorylation. Thus, IGF-I treatment overall resulted in increased tumour cell mass, despite a detectable stimulation of apoptosis; in other words proliferation exceeded cell death. If IGF-I was first added on day 0, 2, or 4 of serum free culture, we found decreasing IGF-I specific effects on proliferation and apoptosis. In parallel, we found a down-regulation of IGF-I receptors (FACS) by serum withdrawal, which was partly reversed if IGF-I was added. Therefore receptor number might have an impact on IGF-I function in MG63 cells. In conclusion, co-activation of apoptosis and proliferation by IGF-I might result in higher cell turnover in MG63 osteosarcoma cells. Furthermore, in sarcomas or carcinomas showing clinical association to IGF-I levels and malignancy, IGF-I dependent apoptosis and proliferation could be a significant mechanism of malignant tumour growth.