Yang-Dan-Tang, Identified from 15 Chinese Herbal Formulae, Inhibits Human Lung Cancer Cell Proliferation via Cell Cycle Arrest

Lung cancer has long been one of the most deadly forms of cancer. The majority of lung cancers are of the non-small-cell lung cancer (NSCLC) type. Here we used the non-small-cell lung carcinoma cell line A549 to screen 15 different traditional Chinese herbal medicine (CHM) formulae to explore the possible mechanisms of alternative medicine in lung cancer therapy. We identified three formulae (Formulae 3, 5, and 14) that substantially decreased the survival of A549 cells but did not affect MRC5 normal lung tissue cells. Formula 14, Yang-Dan-Tang, a modified decoction of Ramulus Cinnamomi Cassiae, was chosen for further characterization. Flow cytometry analysis showed that treatment of Formula 14 induced cell cycle arrest in G1 and G2 phase without causing significant cell death. These results were also confirmed by Western blot analysis, with decreased expression of G1/S and G2/M promoting cell cycle machinery including cyclin D3, cyclin B1, CDK4, and CDK6. This study provides further insight into the possible working mechanism of Yang-Dan-Tang in patients

Functional Domains of a Pore-Forming Cardiotoxic Protein, Volvatoxin A2

Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N- terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site- directed mutagenesis, the amphipathic a- helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128-199 residues) was found to be a functional domain that mediates the membrane binding of VVA2 . We found a fragment localized at the C- terminal half of VVA2 containing beta6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166-194 residues ), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion

Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1-165 (wild type) and human stem cell factor1-141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor1-165 was significantly greater than that of recombinant human stem cell factor1-141 after heating the proteins (100 ng/mL) at 25-110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that β-sheet structures were altered in recombinant human stem cell factor1-141 but not recombinant human stem cell factor1-165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor1-165 and human stem cell factor1-141. Together, these data indicate that stem cell factor1-165 is more thermostable than stem cell factor1-141

Temperature-dependent changes in circular dichroism spectrum from 190 to 250 nm of (A) rhSCF^1–141 and (B) rhSCF^1–165.

<p>Conditions: 300 µg/mL in 100 mM phosphate buffer (pH 7.0); 1, 25°C as control; 2, 70°C for 10 min; 3, 90°C for 15 min; 4, 90°C for 30 min; and 5, 90°C for 60 min.</p

The expression and purification of rhSCF^1–141 and rhSCF^1–165.

<p>(A) PCR analysis of pPICZαC-rhSCF^1–141 (1011 bp) and pPICZαC/SCF^1–165 (1083 bp) using <i>5′AOX I</i> and <i>3′AOX I</i> primers (arrow). (B) Coomassie blue-stained SDS-PAGE of rhSCF^1–141 and rhSCF^1–165overexpressed from <i>Pichia</i> culture supernatants. Lane M: molecular weight markers. Numbers above the lanes represent hours after methanol induction. Arrows indicate the overexpressed rhSCF^1–141 or rhSCF^1–165 (C) Silver-stained 15% SDS–PAGE of purified rhSCF^1–141 and rhSCF^1–165 produced in <i>P. pastoris</i>. Lane M, molecular weight markers. Lanes 1 and 2, purified rhSCF^1–141 (200 and 100 ng, respectively); Lanes 3 and 4, purified rhSCF^1–165 (20 and 50 ng, respectively). (D) Western blot analysis of rhSCF^1–141-6His and rhSCF^1–165-6His from culture supernatants. The recombinant proteins were 6xHis tagged and detected with anti-His antibodies.</p

Thermostability study of SCF^1–141 and SCF^1–165 by molecular modeling simulation.

<p>(A) Potential energy of SCF^1–141 and SCF^1–165. The root mean square deviation (RMSD) of SCF^1–141 and SCF^1–165 at (B) 25°C; (C) 70°C; and (D) 90°C. (E) Snapshots of the evolving SCF^1–141 structure at different time points at the indicated temperatures.</p

The effect of temperature and time on the viability of TF-1 cells treated with rhSCF¹⁴¹ or rhSCF¹⁶⁵.

<p>(A) The effect of temperature on the viability of TF-1 cells treated with rhSCF^1–141 or rhSCF^1–165. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) for 10 minutes and the cell viability assayed. (B) Thermostability of rhSCF^1–141 and rhSCF^1–165 over time as indicated by cell viability. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) at 90°C for the times indicated. Results are expressed as mean ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001</p

Effects of rhSCF^1–141 and rhSCF^1–165 on downstream signaling targets MAPK and Akt.

<p>Cells were incubated with rhSCF^1–141 or rhSCF^1–165 in PBS (pH 7.4) at the concentrations indicated. A and B are Western blots using the indicated antibodies. Similar results were obtained in 3 independent experiments. C is the quantitation results of the Western blots shown in A and B.</p

SDS-PAGE analysis of limited proteolytic digestion of rhSCF^1–141 and rhSCF^1–165.

<p>Purified (A) rhSCF^1–141 or (B) rhSCF^1–165 (15 µg each in 0.1 M-Tris/HCl buffer, pH 8.2) was incubated with trypsin at 25°C or 90°C for 10 minutes (lanes 2 and 3), or preheated at 90°C 10 minutes (lanes 4 and 5), 90 minutes (lanes 6 and 7), or 120 minutes (lanes 8 and 9) and then treated with trypsin for 10 minutes at 25°C or 90°C as indicated above. Lanes 1 and 10 are rhSCF and trypsin only.</p