Safety and efficacy of cabozantinib for patients with advanced hepatocellular carcinoma who advanced to Child-Pugh B liver function at study week 8: a retrospective analysis of the CELESTIAL randomised controlled trial

Background: Patients with hepatocellular carcinoma (HCC) and Child–Pugh B liver cirrhosis have poor prognosis and are underrepresented in clinical trials. The CELESTIAL trial, in which cabozantinib improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with HCC and Child–Pugh A liver cirrhosis at baseline, was evaluated for outcomes in patients who had Child–Pugh B cirrhosis at Week 8. Methods: This was a retrospective analysis of adult patients with previously treated advanced HCC. Child–Pugh B status was assessed by the investigator. Patients were randomised 2:1 to cabozantinib (60 mg once daily) or placebo. Results: Fifty-one patients receiving cabozantinib and 22 receiving placebo had Child–Pugh B cirrhosis at Week 8. Safety and tolerability of cabozantinib for the Child–Pugh B subgroup were consistent with the overall population. For cabozantinib- versus placebo-treated patients, median OS from randomisation was 8.5 versus 3.8 months (HR 0.32, 95% CI 0.18–0.58), median PFS was 3.7 versus 1.9 months (HR 0.44, 95% CI 0.25–0.76), and best response was stable disease in 57% versus 23% of patients. Conclusions: These encouraging results with cabozantinib support the initiation of prospective studies in patients with advanced HCC and Child–Pugh B liver function. Clinical Trial Registration: NCT01908426

Assessment of tumor response, AFP response, and time to progression in the phase 3 CELESTIAL trial of cabozantinib versus placebo in advanced hepatocellular carcinoma (HCC)

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Structural Basis of Allosteric and Intrasteric Regulation in Human Cystathionine β-Synthase and its Regulation by a CXXC Motif

The human cystathionine β-synthase (CBS) is a 5’ pyridoxal phosphate (PLP) dependent protein that catalyzes the condensation reaction between serine and homocysteine to yield cystathionine. This is the first step in the transsulfuration pathway which connects the methionine cycle to cysteine production. Mutations in CBS are the single most common cause of severe hereditary hyperhomocystenemia and over one hundred pathogenic mutations have been described so far. These mutations represent residues which are important for the structural and functional integrity of the enzyme. The biochemical characterization of these mutants thus leads to further insights into structure-function correlations in CBS. CBS has a subunit mass of 63 kDa and is a modular protein with 551 amino acids. The N-terminal half of the protein houses the two cofactors, PLP and heme and a CXXC motif comprised of residues C272 P273, G274 and C275. This motif is also found in the thioredoxin family of proteins where it is involved in thiol disulfide exchange reactions. The heme, which is coordinated by the axial ligands H65 and C52, is about 20 Å away from the CXXC motif as well as the PLP catalytic center. As such, heme does not contribute directly to catalysis, but is believed to be a center for redox regulation in CBS. The C-terminal half of the protein comprising residues 413 to 551 bear a tandem repeat of “CBS domains,” which are secondary structural motifs found in diverse families of proteins with no sequence similarities. In CBS, these domains are believed to be involved in the binding of the allosteric activator S-adenosyl methionine (AdoMet). Along with being involved in allosteric regulation, the C-terminal domain of CBS imposes intrasteric regulation on the catalytic core. Thus deletion of the C-terminal regulatory domain leads to the formation of a super-active enzyme, CBS-ΔC143 which has a 4-fold higher kcat than the full-length protein. It is also known that a truncated form of CBS is generated in cells exposed to proinflamatory agents such as TNFα. Although this observation suggests that the C-terminal domain interacts with the N-terminal catalytic core to effect this regulation, so far no experimental results were available which would characterize the nature of this interaction. In other words no information was available regarding the conformational changes associated with the allosteric and intrasteric regulation in CBS. In this study, we have mapped the regions of intrasteric and allosteric regulation in CBS. We have employed hydrogen/deuterium (H/D) exchange, mass spectrometric technique to locate conformational changes in the enzyme upon the binding of its allosteric activator, AdoMet. We have also used this approach to detect the surface involved in the interaction of the C-terminal domain with the catalytic core relevant to intrasteric regulation. A change in the kinetics of H/D exchange was located in a single peptide, extending from residues 511-531 upon AdoMet binding. CBS-D444N a patient mutant exhibits a similar change in the above peptide in its native state and thus samples a conformation which is acquired by the wild type upon AdoMet binding. Accordingly this mutant is not responsive to any further activation by the allosteric activator. Peptides 356-370 and 371-385 in the N-terminal half of the protein exhibited a change in H/D exchange kinetics when the full-length and its counter part in CBS-ΔC143, were compared by H/D exchange studies, associating this region with intrasteric regulation. We have also demonstrated that in addition to heme, the CXXC motif in CBS is a center for redox regulation. Thiol alkylation followed by mass spectrometric analysis demonstrated formation of an intramolecular disulfide bridge between C272 and C275, and identified the presence of a sulfenic acid intermediate under air oxidized conditions in CBS-ΔC143. The full length and the CBS-ΔC143 enzyme exhibited a 1.6 and 4.5 fold enhancement in specific activity respectively upon reduction of their disulfides at the CXXC center. A redox potential of -240 ± 4 mV was determined for the CXXC center using MAL-PEG for titrating thiol content at various ratios of oxidized and reduced dithiotreitol. Advisor: Ruma Banerje

Ascorbate induced cross-linking of oxyhemoglobin subunits

280-282Ascorbic acid during oxidation in vitro can generate H2O2 which induces non-disulphide covalent cross-linking of coincubated oxyhemoglobin. The cross-linking phenomenon mediated by H2O2 takes place possibly without the involvement, of hydroxyl radicals as evident from the failure of radical scavengers like mannitol and dimethyl sulphoxide as well as metal-chelator, to inhibit the process. This pro-oxidant effect of ascorbic acid may have physiological significance in red blood cells in vivo.</i

Induction of a feed forward pro-apoptotic mechanistic loop by nitric oxide in a human breast cancer model.

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells

Induction of a feed forward pro-apoptotic mechanistic loop by nitric oxide in a human breast cancer model.

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells

Plasma membrane vesicles (IOV) and cytosols from cancer cells together can form ATP.

<p>(A) Schematic showing the procedure for IOV assay with reverse acid gradient. Vesicles were sealed at pH 6.34. To this was added ³²P_i (300 μCi/ml, 0.1 mM) and ADP (0.1 mM) and the pH was shifted to the indicated pH by adding small aliquot of alkali. The reaction was quenched after 90 seconds and then analyzed by TLC. (B) Formation of PP_i by inside-out vesicles (IOV) with reverse acid gradient. Intensity of PP_i bands were corrected for load by intensity of ³²P_i and then represented as relative increase with the maximum as 100. The pH profile was repeated three times. Two point test (single addition of alkali to pH 8) yielding PP_i was performed more than ten times with similar results. (C) Schematic showing IOV and cytosol combinations used in panel D and the abbreviations used. Vesicles were sealed at pH 6.34. To this was added ³²P_i (200 μCi/ml, 0.2 mM) and ADP (100 μM). Cytosolic extract (5 μg protein) at pH 7.5 was added to 25 μl reaction and the pH was immediately shifted to the indicated pH by adding small aliquot of alkali. The reaction was quenched after 10 seconds and analyzed by TLC. (D) IOV and cytosol combination experiment. The TLCs of the experiment are shown. The amount of ATP (%) formed relative to ³²P_i were estimated and are represented as bars. The data represents mean of 3 similar experiments (error bar = 2.s.d. The p values were calculated using student’s two-tailed test: *p<0.05).</p