Trichosanthin Research Articles

Trichosanthin inhibits integration of human immunodeficiency virus type 1 through depurinating the long-terminal repeats

Trichosanthin (TCS) is a type I ribosome-inactivating protein with potent inhibitory activity against human immunodeficiency virus type 1. However, the anti-viral mechanism remains elusive. By a well-accepted HIV-1 integration assay, we demonstrated that TCS prevents HIV-1 DNA integration in a dose dependent manner in cell culture. At the same condition, TCS fails to induce obvious cytotoxicity and is also unable to interference viral early events such as viral entry, uncoating or reverse transcription. The HIV-1 integrase can integrate HIV-1 long-terminal repeats into cellular chromosome. The interaction of TCS with these viral integration components was also examined, indicating that TCS does not interact with HIV-1 integrase by the GST-pull down assay, but binds to the long terminal repeats in a transient manner. We further revealed that TCS can efficiently depurinate HIV-1 long-terminal repeats, which may be responsible for the inhibitory activity on HIV-1 integration. In conclusion, we elucidated that TCS specifically inhibits HIV-1 integration by depurinating the long-terminal repeats.

CREB, a possible upstream regulator of Bcl-2 in trichosanthin-induced HeLa cell apoptosis

Our previous reports indicated that cyclic AMP response element-binding (CREB) protein was involved in the regulation of Bcl-2 expression in apoptotic HeLa cells induced by trichosanthin (TCS). Here we presented that blockade the binding site of CREB to Bcl-2 by a CRE decoy oligonucleotide abrogated the TCS-decreased Bcl-2 expression. Furthermore, overexpression of phosphorylated CREB (p-CREB) in cells transfected with p-CREB/GFP fusion construct resulted in an increase of Bcl-2 protein content, however, this increase was attenuated by TCS treatment. Therefore, this data supports the hypothesis that CREB is a possible upstream regulator of Bcl-2 in apoptotic HeLa cells induced by TCS. The study provides new insights into understanding the mechanism of TCS in the treatment of cervical cancer.

Trichosanthin functions as Th2-type adjuvant in induction of allergic airway inflammation

It is important to understand the pathogenesis of asthma induced by natural allergens, which could exclude the interference of artificial adjuvant and provide insights of natural immune response in the disease. In the present study, we show that Trichosanthin (TCS) could induce airway inflammation even without the help of alum. Furthermore, TCS appeared capable of replacing alum to promote OVA-specific airway inflammation. TCS induced accumulation of IL-4-producing eosinophils in peritoneum at an early stage and the adjuvant function of TCS was eliminated by blockage of IL-4 at this stage. Finally, the eosinophils triggered by TCS from WT mice, but not from IL-4-deficient mice were shown to function as adjuvant for the induction of OVA-specific Th2 responses. Our data indicate that TCS is not only an allergen, but also a Th2-type adjuvant modulating the switching of immune responses to a Th2 pathway. This chain of events results from IL-4 production by eosinophils at an early stage of TCS-priming. In conclusion, TCS may be useful as a Th2 adjuvant, and innate immune cells, such as eosinophils, may be a good target to study the initiation of Th2 response.

A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.

A Novel Strategy for the Invasive Toxin: Hijacking Exosome‐Mediated Intercellular Trafficking

Toxins penetrate mammalian cells through various means. In this study, we report a unique strategy used by trichosanthin (TCS), a plant toxin with ribosome-inactivating activity, to penetrate host cells. We found that in both JAR and K562 cells, endocytosed TCS is incorporated into intraluminal vesicles of the multivesicular body (MVB) and is then secreted in association with these vesicles upon fusion of the MVB with the plasma membrane. The secreted TCS-loaded vesicles secreted by K562 cells move throughout the intercellular space and target syngeneic and specific allogeneic cells. Subsequent internalization permits delivery of the toxin into the cytosol, resulting in ribosomal inactivation and cell death. Thus, our findings provide a novel mechanism by which foreign proteins pass between and penetrate into mammalian cells.

The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 Å crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs.

Induced apoptotic action of recombinant trichosanthin in human stomach adenocarcinoma MCG803 cells

Trichosanthin (TCS), is purified from the Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. The cDNA of trichosanthin was cloned and TCS was purified. The results showed that the proliferation of MCG803 cells were significantly suppressed by TCS in a dose-dependent manner at the concentration ranging from 20 to 100 microg/ml. The result of sequencing analysis indicates we obtained the TCS whole length gene. MTT assay was adopted to measure the growth inhibition ratio of MCG803 cells treated with TCS and apoptosis was assayed by agarose gel electrophoresis. DNA agarose gel electrophoresis showed a gradient, which confirmed that TCS could induce MCG803 cells apoptosis. The proportion of the periodic tumor cells were altered by TCS. Sub-G(1) curves were displayed by flow cytometry analysis. Results of Northern and Western blots showed that the transcription and expression of P21, was gradually up-regulated as treatment time increased. On the contrary, the transcription and expression of p53, was down-regulated. These data provided powerful evidences for the first time that recombinant TCS can induce the apoptosis of the MCG803 cells.

Trichosanthin suppresses HeLa cell proliferation through inhibition of the PKC/MAPK signaling pathway

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-tumor activities. Our previous studies have shown that TCS inhibits HeLa cell proliferation by activating the apoptotic pathway. In particular, the transcriptional factor cAMP response element binding (CREB) protein plays a pivotal role in apoptotic HeLa cells. However, no information, to date, is available about the signaling pathways involved in the inhibition of cell proliferation induced by TCS. The present study showed that PKA and PKC activities were significantly inhibited by TCS treatment. However, specific inhibitor of PKA activity failed to affect the inhibition of HeLa cell proliferation induced by TCS, even in the presence of cAMP agonists. In contrast, PKC activator/inhibitor significantly attenuated/enhanced the inhibitory effect of TCS on cell proliferation. In particular, the reversed effect of cAMP agonist on cell proliferation was partly prevented by PKC, ERK1/2, and p38 MAPK blockade. Consistent with these results, the reversed effect of cAMP agonists on CREB phosphorylation was significantly decreased by inhibitors of these kinases, but not PKA inhibitor. Therefore, our results suggested that HeLa cell proliferation was inhibited by TCS via suppression of PKC/MAPK signaling pathway.

Trichosanthin, an initiator of the alternative complement activation pathway.

Trichosanthin (TCS), a protein purified from the plant Trichosanthes Kirilowii Maxim, activates normal human serum complement via the alternative pathway, as shown by TCS-induced C3 conversion in normal serum and its prevention by depletion of factor B, but not with the addition of EGTA. Injection of TCS to BALB/c mice consumed the complement alternative pathway (APC) activity in serum, implying in vivo activation of the alternative pathway by TCS. Elevation of peripheral blood leucocyte count as well as protein exudation and neutrophil accumulation in the peritoneal cavities could be induced by peritoneal injection of TCS. The main effect of complement activation by TCS was demonstrated to be induction of neutrophil accumulation.

TYchi, a novel chitinase with RNA N-glycosidase and anti-tumor activities

Chitinases which catalyze hydrolysis of chitin are believed to be antifungal proteins in plant. Nevertheless, a variety of functions and some new enzymatic activities of chitinases have been found in recent years. We cloned a novel protein from Trichosanthes kirilowii Maximowicz (Family Cucurbitaceae) named TYchi. Expression of TYchi gene in T. kirilowii plants was induced by F. oxysporum, an important cucurbitaceous fungal pathogen, which indicated that TYchi involved in the pathogen-induced plant defense reaction. In addition to its chitin-hydrolytic activity, the recombinant TYchi protein also had RNA N-glycosidase property. In cell-free rabbit reticulocyte lysate system, TYchi inhibited protein synthesis with an IC50 of approximate 5 nM. TYchi also exhibited efficient cytotoxicities to leukemia U937 and choriocarcinoma JAR cells with IC50 about 54 microg ml(-1) and 73 microg ml(-1), respectively. Structure analyses indicated that the putative domain of TYchi is highly similar to the well known active domain of the N-glycosidase trichosanthin (TCS). This bifuntional protein should be useful in diverse applications like RIP-based immunotoxin agent and genetic engineering of plant resistance.

Effect of site-directed PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with multiple biological and pharmacological activities. It has been approved effective in the clinical treatment of AIDS and tumor, but its strong immunogenicity and short plasma half-life have limited the clinical administration. To reduce the immunogenicity and prolong the plasma half-life of this compound, three TCS muteins (M 1, M 2 and M 3) and two PEGylated TCS muteins (PM 1 and PM 2) were constructed by site-directed mutagenesis and PEGylation, respectively. Compared with the unmodified TCS, both PEGylated TCS showed a 3- to 4-fold decrease in immunogenicity, a 0.5- to 0.8-fold decrease in non-specific toxicity, and a 4.5- to 6-fold increase in plasma half-life. But there is a problem of activity reduction. The increased circulating half-life in vivo may compensate for the reduced activity. Together with the other benefits of PEGylation such as reduced immunogenicity and toxicity, it is worthwhile to further explore the potential application of the PEGylated TCS as a better therapeutic agent for AIDS and tumor.

Triton, a novel family of miniature inverted-repeat transposable elements (MITEs) in Trichosanthes kirilowii Maximowicz and its effect on gene regulation

Miniature inverted-repeat transposable elements (MITEs) have a broad impact on genome structure and function. Although MITEs are found associated to genes, little is known about their effect on gene regulation. We have identified a novel MITE family, named Triton, whilst analyzing two independent trichosanthin (TCS) gene promoters (TP9 and TP12) cloned from Trichosanthes kirilowii Maximowicz. Triton1 and Triton2 are nested in TP9, and Triton3 (with 93% sequence similarity to Triton2) is in TP12. To assess the effect of MITE insertion on TCS promoters, we excised Triton1 from TP9 and inserted it into TP12. GUS activity analysis revealed that nested Triton1 is required for effective repression of promoter activity. Detailed analyses of a series of 5′-truncated promoters concerning Triton1 showed that a dark-specific repressor and some constitutive elements endow Triton1 with ability to response to light conditions. These results suggest that Triton1 MITE, which contains cis-regulatory elements, could mediate gene expression.

Trypanosoma cruzi: High ribosomal resistance to trichosanthin inactivation

Trypanosoma cruzi is the parasite causing Chagas Disease. Several results already published suggest that T. cruzi ribosomes have remarkable differences with their mammalian counterparts. In the present work, we showed that trypanosomatid (T. cruzi and Crithidia fasciculata) ribosomes are highly resistant to inactivation by trichosanthin (TCS), which is active against mammalian ribosomes. Differential resistance is an intrinsic feature of the ribosomal particles, as demonstrated by using assays where the only variable was the ribosomes source. Because we have recently described that TCS interacts with the acidic C-terminal end of mammalian ribosomal P proteins, we assayed the effect of a TCS variant, which is unable to interact with P proteins, on trypanosomatid ribosomes. This mutant showed similar shifting of IC50 values on rat, T. cruzi and C. fasciculata ribosomes, suggesting that the resistance mechanism might involve other ribosomal components rather than the C-terminal end of P proteins.

CREB-mediated Bcl-2 expression in trichosanthin-induced Hela cell apoptosis

Bcl-2 plays a pivotal role in the control of cell death and is down-regulated in trichosanthin (TCS)-induced cell apoptosis. Because Bcl-2 expression is regulated by the transcription factor cyclic AMP response element-binding protein (CREB), we investigated the role of CREB activation in TCS-induced Hela cells apoptosis. Our results showed that TCS-caused Hela cell apoptosis was accompanied by the decrease of Bcl-2 and phosphorylated CREB protein levels. Interesting, this inhibitive effect can be abolished by the combined treatment of TCS/cAMP agonists. Furthermore, TCS-mediated Bcl-2 protein was abrogated by the suppression of CREB expression with antisense treatment, and blocking the interaction between CREB-binding protein and the Bcl-2 cyclic AMP-responsive element (CRE) by a CRE decoy oligonucleotide. Therefore, these data support the hypothesis that CREB plays a critical role in the regulation of Bcl-2 expression in TCS-induced Hela cell death.

Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of γ-actin mRNA preceded that of β-actin; α- and β-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.

Different neuronal toxicity of single-chain ribosome-inactivating proteins on the rat retina

Objective To investigate the neurotoxicity of two structurally similar single chains of ribosome-inactivating proteins (RIPs): trichosanthin (TCS) and ricin A chain (RTA). Methods TCS, RTA and Ricinus communis agglutinin (RCA, a multi-chain RIP for comparison) were separately injected into rat eyes. Saline was used as control. The data on cell counts, retinal thickness and histopathological scores were collected, and the TUNEL method (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) was used to study the mode of cell death. Results TCS caused distinct retinal changes at 1 nmol. Its toxic effects were most pronounced on the cells of the outer nuclear layer, less so on those of the inner nuclear layer, and little on the ganglion cells. Apoptosis was the predominant type of cell death induced by TCS. In contrast, RTA and RCA, both at 0.01 nmol, brought about acute retinal inflammation and necrosis. Conclusion TCS can eliminate specific retinal cells by apoptosis, while RTA and RCA cause retinitis. The B chain of type II RIPs is not obligatory for their neurotoxicity. The RIPs may be useful for creating retinal models and TCS may be useful for the chemical treatment of retinoblastoma.

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.

Trichosanthin down-regulated p210Bcr-Abl and enhanced imatinib-induced growth arrest in chronic myelogenous leukemia cell line K562

Trichosanthin (TCS), an active component extracted from the root tubers of traditional Chinese medical herb Tian-Hua-Fen of the Cucurbitaceae family, has long been used for medical purpose in China; there is increasing interest in developing TCS as cancer therapeutic agents. The present study was to investigate the growth arrest of K562 cells and its molecular mechanisms, which the drugs induced by TCS and the possible functional interaction of TCS with imatinib (STI571) to K562 cells. Trypan blue exclusive staining was used to access the cell growth inhibition; western blot was used to evaluate the p210(Bcr-Abl), phosphorylated tyrosine kinase (PTK), and some signaling molecules involving in cell proliferation and apoptosis in K562 cells. TCS and imatinib inhibited K562 cells at a time- and dose-dependent manners, respectively; TCS down-regulated p210(Bcr-Abl) at a time- and dose-dependent manners; TCS synergistically enhanced imatinib-induced K562 cell growth arrest and down-regulation of p210(Bcr-Abl), PTK activities, procaspase-3, Hsp90,NF-kappaB and PKC. The results suggest that TCS not only by itself involves but also synergizes activities of imatinib to induce K562 cell growth arrest, down-regulation of p210(Bcr-Abl) and its downstream signals and to stimulate the effect of the tyrosine kinase inhibition.

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A4324 at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81-83 and KR173-174) were identified by computer modeling, and then three TCS mutants namely TCS(YFF81-83ACS), TCS(KR173-174CG), and TCS(YFF-KR) were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD(50) of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.