Yuhong Hu

Curcumin Inhibits Proliferation and Induces Apoptosis of Human Colorectal Cancer Cells by Activating the Mitochondria Apoptotic Pathway

Curcumin, a natural plant extract from Curcuma longa, is known for its anti‐carcinogenic and chemopreventive effects on a variety of experimental cancer models. In this study, we evaluated the effects of curcumin and elucidated its mechanism in human colorectal carcinoma cells. Cell viability assay showed that curcumin significantly inhibited the growth of LoVo cells. Curcumin treatment induced the apoptosis accompanied by ultra‐structural changes and release of lactate dehydrogenase in a dose‐dependent manner. Moreover, treatment with 0–30 µg/mL curcumin decreased the mitochondrial membrane potential and activated the caspase‐3 and caspase‐9 in a dose‐ and time‐dependent manner. Nuclear and annexin V/PI staining showed that curcumin induced the apoptosis of LoVo cells. FACS analysis revealed that curcumin could induce the cell cycle arrest of LoVo cells at the S phase. Furthermore, western blotting analysis indicated that curcumin induced the release of cytochrome c, a significant increase of Bax and p53 and a marked reduction of Bcl‐2 and survivin in LoVo cells. Taken together, our results suggested that curcumin inhibited the growth of LoVo cells by inducing apoptosis through a mitochondria‐mediated pathway. Copyright

The novel antimicrobial peptides from skin of Chinese broad-folded frog, Hylarana latouchii (Anura:Ranidae).

Broad-folded frogs (Hylarana latouchii), one member of 12 species of the genus Hylarana in the Chinese frog fauna, are widely distributed in the South of China. In this study, we purified and characterized three antimicrobial peptides from the skin secretion of H. latouchii. Five different cDNA fragments encoding the precursors of these antimicrobial peptides were cloned, and five mature antimicrobial peptides belonging to two different families were deduced from the five cDNAs. Structural characterization of the mature peptides had identified them as members of the brevinin-1 and temporin families. They were named brevinin-1LTa (FFGTALKIAANVLPTAICKILKKC), brevinin-1LTb (FFGTALKIAANILPTAICKILKKC), temporin-LTa (FFPLVLGALGSILPKIF-NH(2)), temporin-LTb (FIITGLVRGLTKLF-NH(2)) and temorin-LTc (SLSRFLSFLKIVYPPAF-NH(2)). Brevinin-1LTa, temporin-LTa, temporin-LTb and temporin-LTc with different antimicrobial activities induced significant morphological alterations of the tested microbial surfaces as shown by scanning electron microscopy, which indicated strong membrane disruption.

Characterization of diverse antimicrobial peptides in skin secretions of Chungan torrent frog Amolops chunganensis

We have cloned, synthesized, and characterized 11 novel antimicrobial peptides from a skin derived cDNA library of the Chungan torrent frog, Amolops chunganensis. Seven of the 11 antimicrobial peptides were present in authentic A. chunganensis skin secretions. Sequence analysis indicated that the 11 peptides belonged to the temporin, esculentin-2, palustrin-2, brevinin-1, and brevinin-2 families. The peptides displayed potent antimicrobial activities against several strains of microorganisms. One peptide, brevinin-1CG5, demonstrated antimicrobial activity against all tested Gram-positive and Gram-negative bacteria and fungi, and showed high antimicrobial potency (MIC=0.6 μM) against Gram-positive bacterium Rhodococcus rhodochrous. Some peptides also demonstrated weak hemolytic activity against human erythrocytes in vitro. Phylogenetic analysis based on the amino acid sequences of brevinin-1, brevinin-2, and esculentin-2 peptides from family Ranidae confirmed that the current taxonomic status of A. chunganensis is correct.

Identification of multiple peptides with antioxidant and antimicrobial activities from skin and its secretions of Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus.

Amphibian skin and its secretions contain many kinds of peptides with different bioactivities. In this study, a large number of peptides including antioxidant and antimicrobial peptides were identified from three East Asian frog species Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus. The majority of these peptides were antimicrobial peptides, while eight antioxidant peptides were identified, which included two novel peptides taipehensin-1TP1 (TLIWEFYHQILDEYNKENKG) and taipehensin-2TP1 (CLMARPNYRCKIFKQC). These antioxidant peptides exhibited the ability to scavenge ABTS and/or DPPH free radicals. Moreover, six out of eight antioxidant peptides temporin-TP1, brevinin-1TP1, brevinin-1TP2, brevinin-1TP3, brevinin-1LF1, and palustrin-2GN1 also showed antimicrobial activity.

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae).

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.

Novel antimicrobial peptides isolated from the skin secretions of Hainan odorous frog, Odorrana hainanensis.

Long time geographical isolation of Hainan Island from the China continent has resulted in appearance of many novel frog species. As one of them, Hainan odorous frog, Odorrana hainanensis possesses some special antimicrobial peptides distinct from those found in other Odorrana. In this study, three antimicrobial peptides have been purified and characterized from the skin secretion of O. hainanensis. With the similarity to the temporin family, two peptides are characterized by amidated C-terminals, so they are named as temporin-HN1 (AILTTLANWARKFL-NH(2)) and temporin-HN2 (NILNTIINLAKKIL-NH(2)). The third antimicrobial peptide belongs to the brevinin-1 family which is widely distributed in Eurasian ranids, and thus, it is named as brevinin-1HN1 (FLPLIASLAANFVPKIFCKITKKC). Furthermore, after sequencing 68 clones, eight cDNAs encoding antimicrobial peptide precursors were cloned from the skin-derived cDNA library of O. hainanensis. These eight cDNAs can encode seven mature antimicrobial peptides including the above three, as well as brevinin-1V, brevinin-2HS2, odorranain-A6, and odorranain-B1. Twelve different species of microorganisms were chosen, including Gram-positive, Gram-negative and fungi, to test the antimicrobial activities of temporin-HN1, temporin-HN2, brevinin-1HN1, brevinin-1V, and brevinin-2HS2. The result shows that, in addition to their activities against Gram-positive bacteria, temporin-HN1 and temporin-HN2 also possess activities against some Gram-negative bacteria and fungi. However, the two antimicrobial peptides, brevinin-1HN1 and brevinin-1V of the brevinin-1 family have stronger antimicrobial activities than temporin-HN1 and temporin-HN2 of the temporin family. Brevinin-1HN1 possesses activity against Staphylococcus aureus (ATCC25923), Rhodococcus rhodochrous X15, and Slime mould 090223 at the concentration of 1.2 μM.

Peptidomic Analysis of Antimicrobial Peptides in Skin Secretions of Amolops mantzorum

Amphibian skin secretions contain abundant bioactive peptides that are valuable natural resources for human beings. However, many amphibians are disappearing from the world, making relevant scientific studies even more important. In this study, 24 cDNA sequences encoding antimicrobial peptide (AMP) precursors were initially cloned by screening a cDNA library derived from the skin of the Sichuan torrent frog, Amolops mantzorum. Eighteen mature AMPs belonging to 11 different families were deduced from these cDNA clones. Biological function was confirmed in each family of these AMPs. Some of them were purified from the skin secretions, and their molecular structures were determined by Edman degradation. Liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS)-based peptidomics was used to further confirm the actual presence and characteristics of mature AMPs in the skin secretions of A. mantzorum. Incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase the peptidome coverage and reproducibility of peptide ion selection.

Diverse families of antimicrobial peptides isolated from skin secretions of three species of East Asian frogs, Babina daunchina, Babina adenopleura, and Rana omeimontis (Ranidae).

Twenty-two novel cDNAs encoding 22 peptide precursors for 19 mature peptides including antimicrobial peptides (AMPs) were identified from East Asian frog species Babina daunchina, Babina adenopleura, and Rana omeimontis skin-derived cDNA libraries. Two atypical members of the brevinin-1 family AMPs, named brevinin-1AN1 (FLTGVLKLASKIPSVLCAVLKTC) and brevinin-1DN1(FLKGVINLASKIPSMLCAVLKTC), were purified from the skin secretions of B. adenopleura and B. daunchina, respectively. A member of the ranatuerin-2 family AMP named ranatuerin-2DN1 (GLFDSITQGLKDTAVKLLDKIKCKLSACPPA) was also purified from the skin secretion of B. daunchina. One AMP named japonicin-2OM1 (FIVPSIFLLKKAFCIALKKNC) was purified from the skin secretion of R. omeimontis. The antimicrobial tests showed that brevinin-1DN1, brevinin-1DN2, brevinin-1AN1, and japonicin-2OM1 possess higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria.

Identification and Functional Analysis of Novel Bradykinin-Related Peptides (BRPs) from Skin Secretions of Five Asian Frogs

In recent decades, various types of bioactive substances have been identified from amphibian skin and its secretions. Bradykinin-related peptides (BRPs) are among these compounds that make up the host defence system of amphibians. In the present study, we identified six novel BRPs, amolopkinin-GN1, amolopkinin-RK1, amolopkinin-TR1, amolopkinin-LF1, ranakinin-MS1, and ranakinin-MS2, from five East Asian amphibians, Amolops granulosus, Amolops ricketti, Amolops torrentis, Amolops lifanensis, and Hylarana maosonensis. This is the first report on BRPs in the skin of these species. Physiological assays reveal that these peptides have a contractive effect on the smooth muscle of rat ileum.