Preclinical study of cinobufagin as a promising anti-colorectal cancer agent.

Here, we assessed the anti-colorectal cancer (CRC) cell activity of cinobufagin (CBG). We found that CBG exerted potent cytotoxic and anti-proliferative activity against CRC lines (HCT-116 and HT-29) and primary human CRC cells. Meanwhile, it activated apoptosis, and disrupted cell-cycle progression in the cells. At the signaling level, CBG treatment in CRC cells provoked endoplasmic reticulum stress (ER stress), the latter was evidenced by caspase-12 activation, CHOP expression, as well as PERK and IRE1 phosphorylations. Contrarily, the ER stress inhibitor salubrinal, the caspase-12 inhibitor and CHOP shRNA remarkably attenuated CBG-induced CRC cell death and apoptosis. Further, CBG in-activated mammalian target or rapamycin complex 1 (mTORC1), which appeared responsible for proliferation inhibition in CRC cells. Introduction of a constitutively-active S6K1 ("ca-S6K1") restored proliferation of CBG-treated CRC cells. Finally, CBG intraperitoneal injection suppressed HCT-116 xenograft tumor growth in the nude mice. CHOP upregulation and mTORC1 in-activation were also noticed in CBG-treated HCT-116 tumors. The results of this preclinical study suggest that CBG could be tested as promising anti-CRC agent.

Modulatory effects of vitamin D on peripheral cellular immunity in patients with recurrent miscarriage.

PROBLEM: We aimed to investigate the modulatory effects of vitamin D on peripheral blood cellular immune response in patients with recurrent miscarriage (RM). METHOD OF STUDY: The effect of vitamin D on the number of peripheral blood cells, T helper 1 (Th1) cytokines, and NK cytotoxicity was measured in 99 women with RM. RESULTS: The percentage of CD19+ B cells and NK cytotoxicity at an effector-to-target cell (E:T) ratio of 50:1, 25:1, and 12.5:1 were significantly higher in the vitamin D insufficiency group (VDI) than in the vitamin D normal group (VDN) (P<.05 each). The proportion of TNF-α-expressing Th cells was significantly higher in the vitamin D deficiency group (VDD) than in VDN (P<.05). However, there were no significant differences between VDI and VDD. This dysregulation was significantly reduced with 1,25(OH)2 D supplementation. CONCLUSION: The data suggest that the abnormalities of cellular immune response were observed in RM patients with a low vitamin D level, which could be regulated to some extent with 1,25(OH)2 D supplementation.

Tryptophan mutants of cardiac troponin C: 3D structure, troponin I affinity, and in situ activity.

In situ fluorescence/NMR spectroscopic approaches have been used to elucidate the structure, mobility, and domain orientations of troponin C in striated muscle. This led us to consider complementary approaches such as solid-state NMR spectroscopy. The biophysical properties of tryptophan and Trp-analogues, such as fluorotryptophan or hydroxytryptophan, are often exploited to probe protein structure and dynamics using solid-state NMR or fluorescence spectroscopy. We have characterized Phe-to-Trp mutants in the 'structural' C-domain of cardiac troponin C, designed to immobilize the indole ring in the hydrophobic core of the domain. The mutations and their fluorinated analogues (F104W, F104(5fW), F153W, and F153(5fW)) were shown not to perturb the structural properties of the protein. In this paper, we characterize the mutations F77W and F77W-V82A in the 'regulatory' N-domain of cardiac troponin C. We used NMR to determine the structure and dynamics of the mutant F77W-V82A-cNTnC, which shows a unique orientation of the indole ring. We observed a decrease in calcium binding affinity and a weaker affinity for the switch region of TnI for both mutants. We present force recovery measurements for all of the N- and C-domain mutants reconstituted into skeletal muscle fibers. The F77W mutation leads to a reduction of the in situ force recovery, whereas the C-domain mutants have the same activity as the wild type. These results suggest that the perturbations of the N-domain caused by the Trp mutation disturb the interaction between TnC and TnI, which in turn diminishes the activity in fibers, providing a clear example of the correlation between in vitro protein structures, their interactions, and the resulting in situ physiological activity.