Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt.

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.

Reduction in lipopolysaccharide-induced apoptosis of fibroblasts obtained from a patient with gingival overgrowth during nifedipine-treatment.

OBJECTIVE: We have previously demonstrated that the mechanism of nifedipine (NIF)-induced gingival overgrowth is related to the observation that proliferation and cell cycle progression of gingival fibroblasts derived from NIF reactive patient (NIFr) are greater than those from NIF non-reactive patient (NIFn). Gingival overgrowth has also been reported to be a result of inhibited apoptosis of gingival fibroblasts. Apoptosis in fibroblasts is induced by lipopolysaccharide (LPS). Thus, we focused upon evaluating whether there is a difference in LPS-induced apoptosis between NIFn and NIFr. METHODS: Both NIFn and NIFr were arrested in DMEM containing 0.5% FBS, stimulated by LPS, and assayed for apoptosis, cell cycle analysis, Western blotting, and caspase activity. RESULTS: Compared to NIFn, the number of apoptotic cells was significantly decreased and the percentage of cells in S and G(2)/M phase was significantly increased in NIFr. The levels of Bax and cytochrome c proteins in NIFr were not up-regulated by LPS compared with NIFn. Both NIFn and NIFr displayed the following changes in protein expression: increased Bad, decreased Bcl-xL, and unchanged Bcl-2 and p53. Caspase-3 and -9 activities were significantly increased by LPS in NIFn but were unchanged in NIFr. Caspase-2 activity remained constant whilst caspase-8 activity significantly increased upon LPS treatment in both NIFn and NIFr. CONCLUSION: Bad, Bax, cytochrome c, p53, and caspases-2, -3, -8, and -9 are pro-apoptotic proteins. Bcl-2 and Bcl-xL are anti-apoptotic proteins. Thus, the mechanism of NIF-induced gingival overgrowth might be related to decreased apoptosis in NIFr through a reduction of Bax, cytochrome c, and caspase-3 and -9.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac).

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin alphavbeta3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Formosanin C-induced apoptosis requires activation of caspase-2 and change of mitochondrial membrane potential.

Formosanin C is a pure compound isolated from Paris formosana Hayata (Liliaceae). The antitumor efficacy of formosanin C has been observed in cultured cells and animal systems. However, the molecular mechanisms of formosanin C remain unknown. The results of the present study indicate that formosanin C induced apoptosis of HT-29 cells characterized by exposure of phosphatidylserine, accumulation of cells at the sub-G(1) phase, fragmentation of DNA, and change of nuclear morphology in a time- and dose-related manner. The apoptotic signaling cascades may proceed via proteolytic activation of caspase-2, change of mitochondrial membrane potential (Deltapsi(m)), release of cytochrome c and second mitochondria-derived activator of caspase/direct IAP binding protein with low pI (Smac/DIABLO), activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). Increase in apoptosis-inducing factor and endonuclease G expressions in nuclei, and increase in Bax and Bak expressions and decrease in Bcl-X(L) expression on mitochondria were also observed in formosanin C-treated HT-29 cells. Attenuation of formosanin C-induced change of Deltapsi(m) by caspase-2 inhibitor (Z-VDVAC) implies that caspase-2 acts upstream of the mitochondria. Blockage of formosanin C-induced apoptotic process by using either permeability transition pore inhibitor (cyclosporine A) or caspase-9 inhibitor (Z-LEHD) demonstrates the necessity of mitochondria and caspase-9 in formosanin C-induced apoptosis of HT-29 cells. Taken together, the apoptotic mechanism of formosanin C in human colorectal cancer HT-29 cells involves activation of caspase-2 and the dysfunction of mitochondria.

A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells.

A novel small molecule inhibitor, 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB), competes with the Bak BH3 peptide to bind Bcl-2 protein with a binding affinity of IC(50) = 0.70 microM, as assessed by a fluorescence polarization based binding assay. HL-60 cells express the highest levels of Bcl-2 among the cell lines examined. Treated with 5 microM of MNB only for 6 h, 85% of HL-60 cells were detected to undergo apoptosis. Pan-caspase inhibitor, Z-VAD-FMK, blocks MNB-induced apoptosis in HL-60 cells. Caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, and PARP activation were observed at as early as 4 to 6 h of MNB treatment. In addition, it has been confirmed that the caspase-3 specific inhibitor, Z-DEVD-FMK, blocks the activation of caspase-8 in MNB-treated HL-60 cells. MNB treatment does not change Bcl-2 or Bax expression level in HL-60 cells, but causes Bid cleavage. Further experiments have illustrated that MNB inhibits the heterodimerization of Bcl-2 with Bax or Bid, reduces the mitochondrial membrane potential (DeltaPsimt), and induces cytochrome c release from mitochondria in HL-60 cells. These results suggest that MNB induces apoptosis in HL-60 by inhibiting the heterodimerization of Bcl-2 with pro-apoptosis Bcl-2 members, resulting in a decrease in the mitochondrial membrane potential and cytochrome c release, activation of caspases and PARP; it is a caspase-dependent process in which the activation of caspase-8 is dependent on the mitochondrial apoptosis signal transduction pathway. MNB prolongs the life spans of HL-60 bearing mice, potently kills fresh AML and ALL cells, indicating that it has the potential to be developed to treat leukemia.

Phellinus linteus activates different pathways to induce apoptosis in prostate cancer cells.

It is known that polysaccharides extracted from the Phellinus linteus (PL) mushroom possess antitumour activity. We previously have demonstrated that high doses of PL render murine or human lung cancer cells susceptible to apoptosis. However, the molecular mechanisms of PL-mediated apoptosis have not been fully explored. In this study, we demonstrate that LNCaP cells expressing the androgen receptor (AR) are highly susceptible to apoptosis in response to treatment with high doses of PL. In this process, caspase 8 and its downstream effectors (such as BID), as well as ER stress-related, apoptotic signalling, are activated. In contrast, a moderate amount of apoptosis occurs in PC3 cells (that lack AR) after the same treatment, which does not activate ER-mediated apoptotic signalling. We also show that, in the process of PL-induced apoptosis, caspase 2 is induced in LNCaP cells, but not in PC3 cells. However, LNCaP cells that express a mutated AR or LNCaP cells treated with a caspase 2 inhibitor blocked ER stress-induced apoptotic signals. The magnitudes of the induction of apoptosis in these cells are comparable with what occurred in the PC3 cells. The data demonstrate that high doses of PL activate the AR-dependent and independent apoptotic pathways. Our study also suggests that caspase 2 is a key target in the determination of the susceptibility of prostate cancer cells to PL-induced apoptosis.