Mechanistic Study of Macranthoside B Effects on Apoptotic Cell Death in Human Cervical Adenocarcinoma Cells.

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides, a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcysteine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ringfinger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis- inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

Excessive Oxidative Stress in the Synergistic Effects of Shikonin on the Hyperthermia-Induced Apoptosis.

BACKGROUND: Hyperthermia (HT) has been used widely for cancer therapy, and the development of modern devices has made it more efficient. Shikonin (SHK) is a natural naphthoquinone derivative from a Chinese herb. Although the anticancer properties of SHK are evident, the underlying molecular mechanisms are not fully understood. OBJECTIVE: In this study, the effects of combining low doses of SHK with mild HT were investigated in the U937 cell line. METHODS: The cells were subjected to HT at 44°C for 10 min with or without SHK pretreatment, and parameters reflecting apoptosis, ROS generation and intracellular calcium elevation were evaluated by using DNA fragmentation, flow cytometry, and western blot analyses. RESULTS: SHK 0.5 µM significantly enhanced HT-induced apoptosis as indicated by DNA fragmentation and caspase-3 activation with increased generation of ROS and elevation of intracellular calcium. The combined treatment also synergistically activated proapoptotic proteins and inactivated anti-apoptotic proteins. Furthermore, the phosphorylation of JNK and PKC- δ and the dephosphorylation of ERK and AKT were the upstream effects that may have compounded the induction of apoptosis. The modulatory effects of HT and SHK were abrogated with the employment of NAC and JNK-IN-8 by inactivating the MAPK pathway and cleavage of caspase-3. Intracellular calcium was also elevated and was found to be responsible for the induction of cell death evident by the DNA fragmentation with or without the employment of BAPTA-AM. CONCLUSION: Conclusively, this study provides persuasive evidence that SHK in combination with HT is a propitious therapeutic way for augmentation of apoptosis and hence suggest a novel strategy for treating cancers.

Multiple phase inversion of emulsions stabilized by in situ surface activation of CaCO3 nanoparticles via adsorption of fatty acids.

The in situ surface activation of raw CaCO(3) nanoparticles by interaction with a series of sodium carboxylates of chain length between 6 and 12 as well as sodium 2-ethylhexylsulfosuccinate (AOT) was studied, and the impact of this on the stabilization and phase inversion of toluene-water emulsions was assessed. By using complementary experiments including measurement of particle zeta potentials, adsorption isotherms of amphiphile, and relevant contact angles, the mechanism of this activation was revealed. The results show that hydrophilic CaCO(3) nanoparticles can be surface activated by interaction with sodium carboxylates and AOT even if they are not surface-active themselves. Both the electrostatic interaction between the positive charges on particle surfaces and the negative charges of anionic amphiphile headgroups and the chain-chain interactions of the amphiphile result in monolayer adsorption of the amphiphile at the particle-water interface. This transforms the particles from hydrophilic to partially hydrophobic such that they become surface-active and stabilize oil-in-water O/W(1) emulsions and induce O/W(1) → water-in-oil W/O phase inversion, depending on the chain length of the carboxylate molecules. At high amphiphile concentration, bilayer or hemimicelle adsorption may occur at the particle-water surface, rendering particles hydrophilic again and causing their desorption from the oil-water interface. A second phase inversion, W/O → O/W(2), may occur depending on the surface activity of the amphiphile. CaCO(3) nanoparticles can therefore be made good stabilizers of both O/W and W/O emulsions once surface activated by mixing with traces of suitable anionic amphiphile.

Effects of Asparagus officinalis extracts on liver cell toxicity and ethanol metabolism.

Asparagus officinalis is a vegetable that is widely consumed worldwide and has also long been used as a herbal medicine for the treatment of several diseases. Although A. officinalis is generally regarded as a supplement for the alleviation of alcohol hangover, little is known about its effects on cell metabolism. Therefore, this study was conducted to analyze the constituents of the young shoots and the leaves of asparagus and to compare their biochemical properties. The amino acid and inorganic mineral contents were found to be much higher in the leaves than the shoots. In addition, treatment of HepG2 human hepatoma cells with the leaf extract suppressed more than 70% of the intensity of hydrogen peroxide (1 mM)-stimulated DCF fluorescence, a marker of reactive oxygen species (ROS). Cellular toxicities induced by treatment with hydrogen peroxide, ethanol, or tetrachloride carbon (CCl(4)) were also significantly alleviated in response to treatment with the extracts of A. officinalis leaves and shoots. Additionally, the activities of 2 key enzymes that metabolize ethanol, alcohol dehydrogenase and aldehyde dehydrogenase, were upregulated by more than 2-fold in response to treatment with the leaf- and shoot extracts. Taken together, these results provide biochemical evidence of the method by which A. officinalis exerts its biological functions, including the alleviation of alcohol hangover and the protection of liver cells against toxic insults. Moreover, the results of this study indicate that portions of asparagus that are typically discarded, such as the leaves, have therapeutic use.

A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis.

The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.

A lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) enhances caspase-dependent apoptosis induced by hyperthermia.

PURPOSE: A free radical initiator, 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), was previously found to enhance apoptosis by hyperthermia. Here, but more lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) was investigated for its effects as a possible heat sensitizer. MATERIALS AND METHODS: Human myelogenous monocytic leukaemia U937 cells were treated with hyperthermia combined with a various concentration of AMVN for investigating its ability to induce apoptosis and various parameters to identify the pathway. RESULTS: Combined treatment of hyperthermia and AMVN induced DNA fragmentation markedly, while hyperthermia or AMVN alone induced marginal DNA fragmentation. Fractions of cells showed low mitochondrial membrane potential and increased superoxide production after the combined treatment. Experiments using various caspase inhibitors and a fluorogenic monitor of caspase 3 activities indicated that caspase acts both up- and down-stream of mitochondria. CONCLUSIONS: AMVN is suggested to be a potential heat sensitizer effective at a lower concentration than AAPH. The possible mechanism is discussed.