The plant diterpene epoxysiderol targets Hsp70 in cancer cells, affecting its ATPase activity and reducing its translocation to plasma membrane.

The ATP-dependent molecular chaperone Hsp70 is over-expressed in cancer cells where it plays pivotal roles in stabilization of onco-proteins, promoting cell proliferation and protecting cells from apoptosis and necrosis. Moreover, a relationship between the ability of cancer cells to migrate and the abundance of membrane-associated Hsp70 was shown. However, although Hsp70 is a promising target for cancer therapy, there is a still unsatisfied requirement of inhibitors possibly blocking its cancer-associated activities. Moving from the evidence that the plant diterpene oridonin efficiently targets Hsp70 1A in cancer cells, we set up a small kaurane diterpenoids collection and subjected it to a Surface Plasmon Resonance-screening, to identify new putative inhibitors of this chaperone. The results obtained suggested epoxysiderol as an effective Hsp70 1A interactor; therefore, using a combination of bioanalytical, biochemical and bioinformatics approaches, this compound was shown to bind the nucleotide-binding-domain of the chaperone, thus affecting its ATPase activity. The interaction between epoxysiderol and Hsp70 1A was also demonstrated to actually occur inside cancer cells, significantly reduced the translocation of the chaperone to the cell membrane, thus suggesting a possible role of epoxysiderol as an anti-metastasis agent.

Identification of candidate biomarkers of the exposure to PCBs in contaminated cattle: A gene expression- and proteomic-based approach.

Dioxins and polychlorinated biphenyls (PCBs) are widespread and persistent contaminants. Through a combined gene expression/proteomic-based approach, candidate biomarkers of the exposure to such environmental pollutants in cattle subjected to a real eco-contamination event were identified. Animals were removed from the polluted area and fed a standard ration for 6 months. The decontamination was monitored by evaluating dioxin and PCB levels in pericaudal fat two weeks after the removal from the contaminated area (day 0) and then bimonthly for six months (days 59, 125 and 188). Gene expression measurements demonstrated that CYP1B1 expression was significantly higher in blood lymphocytes collected in contaminated animals (day 0), and decreased over time during decontamination. mRNA levels of interleukin 2 showed an opposite quantitative trend. MALDI-TOF-MS polypeptide profiling of serum samples ascertained a progressive decrease (from day 0 to 188) of serum levels of fibrinogen ß-chain and serpin A3-7-like fragments, apolipoprotein (APO) C-II and serum amyloid A-4 protein, along with an augmented representation of transthyretin isoforms, as well as APOC-III and APOA-II proteins during decontamination. When differentially represented species were combined with serum antioxidant, acute phase and proinflammatory protein levels already ascertained in the same animals (Cigliano et al., 2016), bioinformatics unveiled an interaction network linking together almost all components. This suggests the occurrence of a complex PCB-responsive mechanism associated with animal contamination/decontamination, including a cohort of protein/polypeptide species involved in blood redox homeostasis, inflammation and lipid transport. All together, these results suggest the use in combination of such biomarkers for identifying PCB-contaminated animals, and for monitoring the restoring of their healthy condition following a decontamination process.

Inhibition of bone resorption by Tanshinone VI isolated from Salvia miltiorrhiza Bunge.

During the last decade, a more detailed knowledge of molecular mechanisms involved in osteoclastogenesis has driven research efforts in the development and screening of compound libraries of several small molecules that specifically inhibit the pathway involved in the commitment of the osteoclast precursor cells. Natural compounds that suppress osteoclast differentiation may have therapeutic value in treating osteoporosis and other bone erosive diseases such as rheumatoid arthritis or metastasis associated with bone loss. In ongoing investigation into anti-osteoporotic compounds from natural products we have analyzed the effect of Tanshinone VI on osteoclasts differentiation, using a physiologic three-dimensional osteoblast/bone marrow model of cell co-culture. Tanshinone VI is an abietane diterpene extracted from the root of Salvia miltiorrhiza Bunge (Labiatae), a Chinese traditional crude drug, "Tan-Shen". Tashinone has been widely used in clinical practice for the prevention of cardiac diseases, arthritis and other inflammation-related disorders based on its pharmacological actions in multiple tissues. Although Tanshinone VI A has been used as a medicinal agent in the treatment of many diseases, its role in osteoclast-related bone diseases remains unknown. We showed previously that Tanshinone VI greatly inhibits osteoclast differentiation and suppresses bone resorption through disruption of the actin ring; subsequently, we intended to examine the precise inhibitory mechanism of Tanshinone VI on osteoclast differentiating factor. This study shows, for the first time, that Tanshinone VI prevents osteoclast differentiation by inhibiting RANKL expression and NFkB induction.

Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.

9-hydroxystearic acid (9-HSA) belongs to the class of endogenous lipid peroxidation by-products that greatly diminish in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity. In this paper our attention has not only been focused on HDAC1 inhibition but also on the hyperacetylation of other substrates such as p53, that is involved in inducing cell cycle arrest and/or apoptosis, and whose activity and stability are known to be regulated by posttranslational modifications, particularly by acetylation at the C-terminus region. 9-HSA administration to U2OS, an osteosarcoma cell line p53 wt, induces a growth arrest of the cells in G2/M and apoptosis via a mitochondrial pathway. In particular hyperacetylation of p53 induced by the HDAC1 inhibitory activity of 9-HSA has been demonstrated to increase Bax synthesis both at the transcriptional and the translational level. The subsequent translocation of Bax to the mitochondria is associated to a significant increase in caspase 9 activity. Our data demonstrate that the effects of 9-HSA on U2OS correlate with posttranslational modifications of p53.

Onconase: an unusually stable protein.

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.

Stem cell factor is localized in, released from, and cleaved by human mast cells.

Stem cell factor (SCF) is the most important cytokine regulating human mast cell growth and functions. The immunogold technique showed SCF in the secretory granules of skin mast cells and in lung parenchymal mast cells (HLMC). Immunoreactive SCF (iSCF) was detected in cell lysates of HLMC, but not in basophils; iSCF and histamine were detected in supernatants of HLMC 3 min after challenge with anti-FcepsilonRI or anti-IgE, and iSCF in supernatants rapidly declined after 30 min, whereas histamine remained unchanged for 120 min. HPLC and electrospray mass spectrometry (ES/MS) analysis of recombinant human SCF1-166 (18,656. 9 +/- 0.9 Da) treated with chymase showed a polypeptide of 17,977.1 +/- 0.6 Da and a minor component of 697.4 +/- 0.1 Da generated by specific cleavage at Phe159. SCF1-166 and SCF1-159 similarly activated HLMC, potentiated anti-IgE-induced activation of these cells, and stimulated HLMC chemotaxis. SCF159-166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE-activated HLMC incubated with recombinant human SCF1-166 showed that SCF1-166 was rapidly cleaved to SCF1-159 and SCF1-144. Experiments with supernatants of anti-IgE-activated HLMC incubated with SCF1-166 yielded similar results. In conclusion, SCF is stored in mast cell secretory granules and is immunologically released by human mast cells. SCF1-166 is rapidly and specifically cleaved to SCF1-159 by chymase, which retains its biological effect on mast cells. SCF is also cleaved by other proteases to several SCF species whose possible biological activities remain to be established.

Novel autocrine and paracrine loops of the stem cell factor/chymase network.

BACKGROUND: The aim of this study was to investigate whether the secretory granules of human mast cells store stem cell factor (SCF). We also addressed the question whether mast cell chymase, a chymotrypsin-like protease, also present in the secretory granules of human mast cells cleaves SCF at the peptide bound between Phe 158 and Met159. METHODS: The skin samples were obtained from patients with mastocytosis, undergoing skin biopsy for diagnostic purposes. Mast cells were isolated and purified from human lung parenchyma (human lung mast cells, HLMC) by countercurrent elutriation followed by discontinuous Percoll density gradient. SCF contents of human mast cells were assessed for immunoreactive SCF by ELISA. Western blot analysis of SCF and its cleavage products were performed with the MoAb anti-SCF 7H6. SCF and its proteolytic fragment were characterized by electrospray mass spectrometry (ES/MS). RESULTS: SCF is present in the secretory granules of human skin and lung mast cells. Immunoreactive SCF (iSCF) was detected in the cell lysates of HLMC, but not in basophils. iSCF was rapidly (3 min) released after challenge with anti-IgE, and iSCF in supernatants rapidly declined after 30 min. ES/MS analysis of rhSCF1-166 treated with recombinant human chymase showed a polypeptide of 17,977.1+/-0.6 Da and a minor component of 697.4+/-0.1 Da generated by specific cleavage at Phe159. SCF1-166 and SCF1-159 similarly activated HLMC and potentiated anti-IgE-induced activation of these cells. The cleavage product SCF160-166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE activated HLMC incubated for various intervals with rhSCF1-166 showed that rhSCF1-166 was converted to a faster-migrating form with a molecular weight compatible with SCF1-159 and to several SCF species. CONCLUSION: SCF is stored in human mast cell secretory granules and is immunologically released by mast cells. SCF1-166 is rapidly cleaved by chymase and other proteases to several SCF species.