Achyranthoside H methyl ester, a novel oleanolic acid saponin derivative from Achyranthes fauriei roots, induces apoptosis in human breast cancer MCF-7 and MDA-MB-453 cells via a caspase activation pathway.

Achyranthoside H methyl ester (AH-Me) is an oleanolic acid saponin derivative isolated from the roots of Achyranthes fauriei through diazomethane treatment. AH-Me exhibited significant cytotoxicity against human breast cancer MCF-7 and MDA-MB-453 cells, with respective ID(50) values of 4.0 and 6.5 muM: in the MTT assay. AH-Me is a unique saponin containing three methoxycarbonyl groups in the sugar moiety linked to the C-3 position of oleanolic acid. The demethylation of these methoxycarbonyl groups by alkaline hydrolysis caused a marked reduction of the cytotoxicity of AH-Me, suggesting that the methoxycarbonyl groups of AH-Me are key groups for the acquisition of cytotoxicity against human cancer cells. The staining of cancer cells with 4',6'-diamidino-2-phenylindole (DAPI) showed that the population of cells with altered nuclear morphology, for example chromatin condensation and fragmentation, increased markedly after AH-Me treatment. Exposure of MCF-7 and MDA-MB-453 cells to AH-Me resulted in a dose-dependent and time-dependent increase in the sub-G1 population, and in the cleavage of poly-ADP-ribose polymerase (PARP) followed by the formation of an 89 kD peptide. Pretreatment of the cells with the pan-caspase inhibitor z-VAD-fmk abolished the cleavage of PARP by AH-Me treatment and suppressed the antiproliferative effect of AH-Me on tumor cell growth. These results together led to the suggestion that AH-Me induces apoptosis via the caspase activation pathway in human breast cancer cells, and apoptosis is the major mode of the cytotoxic effect triggered by AH-Me.

Two new glucuronide saponins, Achyranthosides G and H, from Achyranthes fauriei root.

Two oleanolic acid saponins named achyranthosides G (1) and H (2) were newly isolated from Achyranthes fauriei root as methyl esters in addition to methyl esters of achyranthosides A - F and five oleanolic acid glucuronides (chikusetsusaponins IVa, V, 28-deglucosyl chikusetsusaponin V, pseudoginsenoside RT(1), and oleanolic acid 3-O-beta-D-glucuronopyranoside) as well as oleanolic acid 28-O-beta-D-glucopyranoside, beta-ecdysterone, and polypodine B. Their structures were characterized as follows on the basis of the chemical and spectroscopic evidences.

Critical role for mitochondrial oxidative phosphorylation in the activation of tumor suppressors Bax and Bak.

BACKGROUND: Activation of Bax and Bak, which act to permeabilize the mitochondrial membrane, is an essential step in the cell death response and therefore in the suppression of tumorigenesis. However, the mechanisms that regulate activation are poorly understood. METHODS: Bax and Bak activation (conformational change and dimerization) was monitored in Rat-1 fibroblasts and human cancer cells subjected to endoplasmic reticulum (ER) stress, DNA damage, or tumor necrosis factor-alpha (TNF-alpha) treatment. Pharmacologic inhibitors of reactive oxygen species production, electron transport in the respiratory chain, oxidative phosphorylation, and appropriate controls were used to identify potential modes by which Bax and Bak activation and the cell death response are controlled. The oligomerization state of Bax and Bak was determined by cross-linking and subsequent immunoblot analysis; Bax conformational change was analyzed by immunoprecipitation and immunoblotting with an antibody specific for the active conformation. Cell death was evaluated by dye exclusion. RESULTS: In both fibroblasts and human cancer cells subjected to cell death stimuli, inhibition of oxidative phosphorylation by use of antimycin A or oligomycin prevented ER stress-, DNA damage-, and TNF-alpha-induced Bax and Bak activation and cell death (UV-induced Rat-1 cell death at 15 hours: control, mean = 33.6%, 95% confidence interval [CI] = 18.8% to 48.4%; antimycin A, mean = 10.0%, 95% CI = 0% to 21.7%; oligomycin, mean = 13.1%, 95% CI = 5.7% to 20.5%; tunicamycin-induced MCF-7 cell death at 9 hours: control, mean = 29.2%, 95% CI = 21.6% to 36.8%; antimycin A, mean = 15.3%, 95% CI = 0.8% to 29.8%; oligomycin, mean = 11.5%, 95% CI = 3.9% to 19.1%; TNF-alpha-induced MCF-7 cell death at 6 hours: control, mean = 24.0%, 95% CI = 12.6% to 35.4%; antimycin A, mean = 8.9%, 95% CI = 3.9% to 13.9%; oligomycin, mean = 13.3%, 95% CI = 10.4% to 16.2%). Increasing and decreasing glycolytic adenosine triphosphate production, by adding glucose and 2-deoxy-D-glucose to the cell growth medium, respectively, neither reversed nor recapitulated, respectively, the effect of compromised oxidative phosphorylation on Bax and Bak activation. CONCLUSION: Oxidative phosphorylation is required for the activation of Bax and Bak and cell death triggered by disparate death stimuli. The reliance of tumor cells on glycolysis in preference to oxidative phosphorylation even under normoxic conditions (Warburg effect) may therefore be a potential means by which these cells evade programmed cell death.

Forced expression of antisense 14-3-3beta RNA suppresses tumor cell growth in vitro and in vivo.

The 14-3-3 family proteins are key regulators of various signal transduction pathways including malignant transformation. Previously, we found that the expression of the 14-3-3beta gene is deregulated as well as c-myc gene in aflatoxin B1 (AFB1)-induced rat hepatoma K1 and K2 cells. To elucidate the implication of 14-3-3beta in tumor cell growth, in this paper we analyzed the effect of forced expression of antisense 14-3-3beta RNA on the growth and tumorigenicity of K2 cells. K2 cells transfected with antisense 14-3-3beta cDNA expression vector diminished their growth ability in monolayer culture and in semi-solid medium. Expression level of vascular endothelial growth factor mRNA was also reduced in these transfectants. Tumors that formed by the transfectants in nude mice were much smaller and histologically more benign tumors, because of their decreased level of mitosis compared with those of the parental cells. Frequency of apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was increased in the transfectant-derived tumors accompanying the inhibition of angiogenesis. In addition, over-expression of 14-3-3beta mRNA was observed in various murine tumor cell lines. These results suggest that 14-3-3beta gene plays a pivotal role in abnormal growth of tumor cells in vitro and in vivo.

Guanine nucleotide exchange factor, Tiam1, directly binds to c-Myc and interferes with c-Myc-mediated apoptosis in rat-1 fibroblasts.

The transcription factor c-Myc is important for the control of cell growth, cell cycle progression, neoplasia, and apoptotic cell death. Recently, c-Myc-binding proteins, which bind either to the N-terminal domain or the C-terminal domain of c-Myc, have been proposed as the key molecules to realize the mechanisms of these multiple c-Myc functions. We report in the present study on another protein, Tiam1, which is a specific guanine nucleotide exchange factor of Rac1 and which binds to c-Myc and modulates several of its biological functions. We were able to detect the direct binding and in vivo association between c-Myc and Tiam1. The necessary role in this interaction of the Myc box II of c-Myc was revealed in the cell extracts. The additional discovery of the intranuclear localization of Tiam1 in Rat1 cells and in neuronal cells of the mouse brain suggests this interaction may occur in the nucleus. Overexpression of Tiam1 repressed the luciferase activity of c-Myc and also inhibited the c-Myc apoptotic activity through this protein-protein interaction. Taken together, we concluded that Tiam1 is another c-Myc regulator, working in the nuclei to control c-Myc-related apoptosis.

Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression.

BACKGROUND: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. METHODS: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. RESULTS: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P =.006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. CONCLUSIONS: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.

A cellular mechanism that reversibly inactivates pancaspase inhibitor zAsp-CH(2)-DCB: a potential pitfall causing discrepancy between in vitro and in vivo caspase assays.

Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.

Akt protein kinase inhibits non-apoptotic programmed cell death induced by ceramide.

A growing body of evidence now suggests that programmed cell death (PCD) occurs via non-apoptotic mechanisms as well as by apoptosis. In contrast to apoptosis, however, the molecular mechanisms involved in the regulation of non-apoptotic PCD remain only poorly understood. Here we show that ceramide induces a non-apoptotic PCD with a necrotic-like morphology in human glioma cells. Characteristically, the cell death was not accompanied by loss of the mitochondrial transmembrane potential, cytosolic release of cytochrome c from mitochondria, or the activation of the caspase cascade. Consistent with these characteristics, this ceramide-induced cell death was inhibited neither by the overexpression of Bcl-xL nor by the pan-caspase inhibitor zVAD-fmk. However, strikingly, the ceramide-induced non-apoptotic cell death was inhibited by the activation of the Akt/protein kinase B pathway through the expression of a constitutively active version of Akt. The results for the first time indicate that the Akt kinase, known to play an essential role in survival factor-mediated inhibition of apoptotic cell death, is also involved in the regulation of non-apoptotic PCD.