TKP, a serine protease extracted from Trichosanthes kirilowii, inhibits the migration and invasion of colorectal adenocarcinoma cells by targeting Wnt/ß-catenin and Hedgehog/Gli1 signalings.

Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/ß-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/ß-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9.

A serine protease extracted from Trichosanthes kirilowii induces apoptosis via the PI3K/AKT-mediated mitochondrial pathway in human colorectal adenocarcinoma cells.

Trichosanthes kirilowii exhibits various biological functions including anti-inflammatory, antidiabetic and anticancer activities. In this study, a novel protein with anti-cancer activity, named as TKP, was purified from Trichosanthes kirilowii fruit by cell-base screening. Mass spectrometry and protease assays revealed that TKP is a serine protease. TKP decreased cell viability in a concentration-and time-dependent manner in human colorectal adenocarcinoma cells. Notably, TKP presented very little effect on human colon epithelial cell line NCM460. Furthermore, TKP inhibited colorectal adenocarcinoma cells to aggregate into viable colony clusters and induced cell apoptosis. The loss of mitochondrial membrane potential, upregulation of cytochrome c and Bax, downregulation of Bcl-2, and activation of caspase-9 and -3 were observed, indicating mitochondria-dependent apoptosis induced by TKP. We found that TKP activated MAPK/ERK and suppressed PI3K/AKT signaling. Inhibition of MAPK/ERK signaling by employing inhibitor PD98059 did not antagonize the effects of TKP. Treatment with PI3K inhibitor LY294003 increased the impact of TKP on mitochondria-related apoptosis proteins (cytochrome c, Bcl-2, Bax, caspase-9 and caspase-3) and cell apoptosis. On the contrary, PI3K/AKT activator insulin-like growth factor-1 reversed the effects of TKP. Taken together, our results demonstrated that TKP has potential anti-colorectal cancer activity by inducing apoptosis, which was regulated by the PI3K/AKT-mediated mitochondria-dependent pathway.

[Study on isozymes of farm cultivars of Fructus trichosanthis from Shandong].

OBJECTIVE: To determine different cultivars of Fructus trichosanthis of Shandong, and explore their genetic relationships. METHODS: The peroxidase isozyme and esterase isozyme of cultivars and wild plant were studied by means of polyarcylamide gel electrophresis, and the finger-prints were established. RESULTS: The zymogram of the peroxidase isozyme and the esterase isozyme of different cultivars and wild plant of Frustus trichosanthis consisted of common bands, but they had more or less difference in the number of isozymic bands, their positions and activity intensity. CONCLUSION: There are closer genetic relationships between Niuxin Gualou with Ren Gualou, and Xiao Guang-dan with wild sample; Mingpi and Xiao Guang-dan are two different cultivars; The isozyme analysis can provide reliable basis for determining different cultivars of Fructus trichosanthis of Shandong.

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica.

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.

Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds.

Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds. Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12-oleate desaturase activity. Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds.

Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp. Bac Kan 8-98.

We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin. The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS. It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM. The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline.