A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF).

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

Cytotoxic effects of secosterols and their derivatives on several cultured cells.

The cytotoxic effects of various oxysterols on several culture cells were examined. Ozonolysis products of cholesterol, secosterols (3ß-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product (3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde) and their keto alcohol and acid derivatives, were found to have potent cytotoxic activities, as compared with major endogenous oxysterols such as 5ß,6ß-epoxycholesterol, 7ß-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol. Secosterols might play important roles in tissue damage and inflammation-associated diseases.

A new 9,11-secosterol from the soft coral Sinularia granosa.

Chemical investigations on the EtOAc-soluble fractions from the EtOH extract of Formosa soft coral afforded a new 9,11-secosteroid, 8αH-3ß,11-dihydroxy-5α,6α-expoxy-24-methylene-9,11-secocholestan-9-one (1), along with one known steroid 3ß,11-dihydroxy-5ß,6ß-expoxy-24-methylene-9,11-secocholestan-9-one (2) from Sinularia granosa. The structure of the new metabolite was elucidated on the basis of extensive spectroscopic analysis and by comparison of their NMR data with the known compounds, including 2. Both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase protein, and 1 also was found to effectively reduce the level of cyclooxygenase-2 protein, in lipopolysaccharide-stimulated RAW264.7 macrophage cells at 10 µM. Furthermore, cytotoxic activity of 1 and 2 toward a limited panel of cancer cell lines was also discovered.

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde--role of hydrogen peroxide.

3ß-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer's disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H(2)O(2) in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H(2)O(2) in ChSeco-induced cytotoxicity in cardiomyoblasts.

Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde.

Cholesterol secoaldehyde (ChSeco or 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Abeta aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 microM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-L-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Abeta42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Abeta42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species.

A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts.

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimer's disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC(50) of 8.9+/-1.29 microM (n=6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 microM CSeco for 24h, for instance, resulted in 30.8+/-3.28% apoptotic and 1.8+/-1.11% of necrotic cells as against DMSO controls that only showed 1.3+/-0.33% of apoptosis and 1.6+/-0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.

New secosteroids from an undescribed gorgonian of the genus Muricella.

Three new 9,10-secosteroids, calicoferols C-E [2-4] have been isolated from an undescribed gorgonian of the genus Muricella, and their structures determined by a combination of spectroscopic methods. Calicoferol D [3] exhibited potent antiviral activity and brine-shrimp lethality.

Doisynolic-type acids--uterotropically potent estrogens which compete poorly with estradiol for cytosolic estradiol receptors.

Doisynolic acids, a class of seco-steroid acids some of which exhibit greater uterotropic estrogenicity than estradiol-17 beta, are D-ring cleavage products of steroidal estrogens formed by fusion with KOH above 200 degrees C. We have found that electron-transfer reactions between estrone or estradiol and CCl4 or CBrCl3 in KOH-t-BuOH at 25 degrees C rapidly provide 16,16-dichloro- or -dibromodoisynolic acid, respectively, the former approaching estradiol in uterotropic potency. Simple esters from these highly hindered tertiary carboxylic acids, easily prepared via phase-transfer-catalyzed alkylations, also rival estradiol in uterotropic activity. Unlike natural steroidal estrogens or their commonly used artificial equivalents (DES, hexoestrol, ethynylestradiol, etc.) whose uterotropic activity is accompanied by substantial binding affinity for cytosolic estradiol receptors, these highly uterotropic doisynolic-type acids and esters exhibit binding affinities for this receptor of only about 1% that of estradiol-17 beta as determined by the usual competitive binding-inhibition studies with [3H]estradiol. Other highly uterotropic carboxylic acids may exhibit similar characteristics. These unusual results leave open the possibilities that uterotropic seco-steroid and related carboxylic acids undergo some unknown metabolic activation, are exceptionally persistent estrogens, bind to a cytosolic receptor site other than the conventional (type I) estradiol site, or bind directly to type I or type II nuclear receptor sites. At dosages of 1000 times those required for a uterotropic effect, the doisynolic-type acids (24 doses over an 8-week period) were neither toxic nor carcinogenic.