A weighted prior tensor train decomposition method for community detection in multi-layer networks.

Community detection in multi-layer networks stands as a prominent subject within network analysis research. However, the majority of existing techniques for identifying communities encounter two primary constraints: they lack suitability for high-dimensional data within multi-layer networks and fail to fully leverage additional auxiliary information among communities to enhance detection accuracy. To address these limitations, a novel approach named weighted prior tensor training decomposition (WPTTD) is proposed for multi-layer network community detection. Specifically, the WPTTD method harnesses the tensor feature optimization techniques to effectively manage high-dimensional data in multi-layer networks. Additionally, it employs a weighted flattened network to construct prior information for each dimension of the multi-layer network, thereby continuously exploring inter-community connections. To preserve the cohesive structure of communities and to harness comprehensive information within the multi-layer network for more effective community detection, the common community manifold learning (CCML) is integrated into the WPTTD framework for enhancing the performance. Experimental evaluations conducted on both artificial and real-world networks have verified that this algorithm outperforms several mainstream multi-layer network community detection algorithms.

Prognostic and Potential Therapeutic Roles of PRKDC Expression in Lung Cancer.

PRKDC is a key factor involved in the ligation step of the non-homologous end joining pathway. Its dysfunction has proven to be a biomarker for radiosensitivity of cancer cells. However, the prognostic value of PRKDC and its underlying mechanisms have not been clarified yet. In this study, we found that PRKDC overexpressed in lung adenocarcinoma (LUAD) and is significantly related to unfavorable survival, while downregulation of PRKDC is link to inflamed tumor immune signature. Our further in vitro results also showed a potent antitumor efficacy of PRKDC inhibitors alone or combined with cisplatin in human lung cancer cells. This study demonstrated that PRKDC is a potential prognostic biomarker, immunotherapy target, and promising combination candidate for chemotherapy for lung cancer, and highlighted the potential of PRKDC-targeted inhibitors for the treatment of lung cancer.

Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo

Objectives Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). Methods E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. Results CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. Conclusions C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC (AU)

Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo.

OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.

Novel EGFR­bispecific recombinant immunotoxin based on cucurmosin shows potent anti­tumor efficiency in vitro.

Epidermal growth factor receptor (EGFR) is overexpressed in various tumors and is associated with cancer initiation, progression, and poor prognosis. Despite the achievements made by tyrosine kinase inhibitors and monoclonal antibodies in certain cases, many patients have not benefited from such treatment due to resistance. Immunotoxins (ITs) are antibody­cytotoxin chimeric molecules with specific cell killing ability, which have achieved different degrees of success in the treatment of a wide range of cancers in clinical trials. The aim of the current study was to examine a novel targeting EGFR recombinant immunotoxin Bs/cucurmosin (CUS) generated by fusing CUS to the EGFR­specific nanobody 7D12­9G8. Bs/CUS was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. Furthermore, it retained binding capacity and specificity with EGFR and was superior to rE/CUS, a monospecific IT we reported previously. In vitro results showed that Bs/CUS could be internalized into the cytoplasm and selectively kill cells in the picomolar range. Flow cytometry showed that Bs/CUS killed the cells mediated by the apoptosis pathway. Taken together, results of the current study indicated that Bs/CUS is a promising candidate that should be further evaluated as a cancer therapeutic for the treatment of EGFR­positive tumors.

Cigarette smoking promotes keratinocyte malignancy via generation of cancer stem-like cells.

Objectives: Cigarette smoking is involved in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). However, the underlying molecular mechanisms of cigarette smoking-induced HNSCC carcinogenesis are unclear and may involve cancer stem-like cell generation. We examined the effects of cigarette smoke condensate (CSC) on the formation of cancer stem-like cells, which are rich in octamer-binding transcription factor (OCT)-4, inhibitor of differentiation 1 (ID1), nuclear factor (NF)-κB, and B lymphoma Mo-MLV insertion region 1 homolog (BMI-1). Materials and Methods: We used in vitro, in vivo, and archival human HNSCC tissue analysis to evaluate the effects of CSC on cancer stem-like cell formation. Results: We found that CSC regulated OCT-4 expression, which subsequently regulated ID1 and NF-κB, at the promoter, mRNA, and protein levels in vitro. Furthermore, OCT-4 knockdown with siRNA reduced ID1 expression. ID1 and NF-κB synergistically increased the expression of BMI-1 and stimulated keratinocyte sphere generation. In vivo, ID1 and NF-κB acted together to generate malignant xenograft tumors, which were aggressive locally and systemically metastatic. Clinical data confirmed that ID1- and NF-κB-positive patients had poor clinical outcomes and 5-year disease-free survival. Conclusion: Our data suggest that smoking cigarettes promoted cancer stem-like cell generation in the head and neck area via the OCT-4/ID1/NF-κB/BMI-1 signaling pathway.

Chemotherapeutic efficacy of cucurmosin for pancreatic cancer as an alternative of gemcitabine: a comparative metabolomic study.

BACKGROUND: As the preferred drug for single chemotherapeutic application in pancreatic cancer, gemcitabine often demonstrated low sensitivity and strong chemotherapy resistance in patients. Therefore, the search for other drugs with high efficiency and low side effects has become of high importance. The aim of this study was to assess the therapeutic effects of cucurmosin on pancreatic cancer as an alternative of gemcitabine and explore its underlying biochemical mechanism. METHODS: The subcutaneous xenograft mice with pancreatic cancer were treated by high- and low-dose cucurmosin and gemcitabine, respectively. A comparative metabolomic analysis was performed on the serum samples from the different groups by 1H nuclear magnetic resonance (NMR) techniques and then subjected to univariate and multivariate statistical analysis. RESULTS: Cucurmosin demonstrated a dose-dependent inhibition to the pancreatic tumors. High-dose cucurmosin provided similar chemotherapeutic efficacy with gemcitabine by positively regulating pyruvate metabolism, glycolysis or gluconeogenesis, and cysteine and methionine metabolism. Inactivating GFR signaling pathway and further inducing apoptosis of tumor cells are the important mechanism of anti-tumor function of cucurmosin. CONCLUSIONS: Cucurmosin is a promising chemotherapeutic drug for pancreatic cancer. However, the dose selection and surface modification should be optimized according to the stage of pancreatic cancer, and an expanded study in both laboratory and clinical regimes needs to be performed.

Novel cucurmosin-based immunotoxin targeting programmed cell death 1-ligand 1 with high potency against human tumor in vitro and in vivo.

Immunotoxins are Ab-cytotoxin chimeric molecules with mighty cytotoxicity. Programmed cell death 1-ligand 1 (PD-L1), is a transmembrane protein expressed mainly in inflammatory tumor tissues and plays a pivotal role in immune escape and tumor progression. Although PD-L1 immune checkpoint therapy has been successful in some cases, many patients have not benefited enough due to primary/secondary resistance. In order to optimize the therapeutic efficacy of anti-PD-L1 mAb, we used durvalumab as the payload and CUS245C , a type I ribosome-inactivating protein isolated from Cucurbita moschata, as the toxin moiety, to construct PD-L1-specific immunotoxin (named D-CUS245C ) through the engineered cysteine residue. In vitro, D-CUS245C selectively killed PD-L1+ tumor cells. In vivo studies also showed that D-CUS245C had obvious antitumor effect on PD-L1+ human xenograft tumors in nude mice. In conclusion, in the combination of the toxin with mAb, this study developed a new immunotoxin targeting PD-L1, emphasizing a novel and promising treatment strategy and providing a valuable way to optimize cancer immunotherapy.

Recombinant cucurmosin-based immunotoxin targeting HER-2 with potent in vitro anti-cancer cytotoxicity.

Trastuzumab is a humanized monoclonal antibody against HER2 approved by FDA for breast and gastric cancer therapy. However, only a quarter of patients have the potential to benefit from it, and most of them develop resistance within therapy. The main purpose of this study is to broaden trastuzumab's therapeutic window by conjugating trastuzumab with recombinant cucurmosin to form an immunotoxin called T-CUS245C. T-CUS245C was chemically conjugated and the purification of T-CUS245C was evaluated by SDS-PAGE. SRB tests showed a remarkable cytotoxicity of T-CUS245C with IC50 values in picomolar range on HER2 positive cancer cells without significantly proliferation inhibition on HER2 negative cells (P < 0.01). Confocal microscopy verified the time-dependent internalization effects of T-CUS245C and revealed that the lethal efficacy can be increased by provoking the internalization. These results indicate the therapeutic potential of T-CUS245C for the HER-2 targeted therapy.

Novel recombinant immunotoxin of EGFR specific nanobody fused with cucurmosin, construction and antitumor efficiency in vitro.

Epidermal growth factor receptor (EGFR) overexpression is related to the increased aggressiveness, metastases, and poor prognosis in various cancers. In this study, we successfully constructed a new EGFR nanobody-based immunotoxin rE/CUS containing cucurmosin (CUS), The immunotoxin was expressed by prokaryotic system and we obtained a yield of 5 mg protein per liter expression medium. The percentage of it's binding ability totumor cell lines A549, HepG2, SW116, which highly expressed EGFR was 55.6%, 79.6% and 97.1%, respectively, but SW620 was only 4.45%. rE/CUS has the ability to bind A549, HepG2, SW116 cells specifically, and the antigen binding capability was not affected because of extra part of CUS component. The rE/CUS significantly inhibited the cell viability against EGFR over expression tumor cell lines in a dose-and time-dependent manner. Moreover, rE/CUS also induced apoptosis of HepG2 and A549 mightily. Our results demonstrate that rE/CUS is a potential therapeutic strategy for treating EGFR-positive solid tumors.

Establishment of human pancreatic cancer gemcitabine­resistant cell line with ribonucleotide reductase overexpression.

A gemcitabine (GEM)-resistant human pancreatic cancer cell line (PANC-1RG7) was established in vitro by gradually increasing GEM concentrations and cloning cell cultures to develop a cellular model of acquired drug resistance studies. We found that PANC-1RG7 cells exhibited significantly different morphological characteristics from parental cells. PANC-1RG7 cells grew slowly (p<0.05), yet the cell cycle remained unchanged (p>0.05). PANC-1RG7, with a resistance index to GEM of 39.9, showed cross-resistance characteristics to methotrexate, gefitinib, cisplatin and 5-fluorouracil. The proliferation inhibition of GEM was significantly reduced in vivo (p<0.05). The known resistance-associated genes and proteins we detected remained unchanged, with the exception of cytidine deaminase, multidrug resistance-related protein and breast cancer resistance protein genes, which decreased; by contrast, 5'-nucleotidase, ribonucleotide reductase (RRM) 1 and RRM2 proteins increased (p<0.05). Therefore, a cell line with acquired GEM resistance was established successfully. Resistance was acquired by overexpressing RRM1 and RRM2 proteins.

[Inhibitory effect of pumpkin protein on expression of Notch signal in RPMI8226 myeloma cells].

This study was aimed to explore the inhibitory effect of pumpkin protein (cucurmosin, CUS) on proliferation of RPMI8226 myeloma cells in vitro and its mechanism. Western blot was used to detect the expression level of Notch-1, Jagged-2, P-Akt and NF-KB in the myeloma cells treated by different concentrations of CUS. The results demonstrated that CUS could down-regulate the protein expression levels of Notch1, Jagged-2, P-Akt and NF-KB in the myeloma cells and with time-and concentration-dependent way, at the same time CUS could also decrease the expressions of BCL-2 and P-Akt. It is concluded that CUS can obviously inhibit the RPMI8226 cell proliferation in vitro, down-regulate the expression levels of Notch signal and its down-stream target genes. Therefore, Notch signaling pathway can be used as a new treatment target for multiple myeloma, and CUS may be become a potential new drug for regulating Notch signaling pathway.

PANC-1 pancreatic cancer cell growth inhibited by cucurmosin alone and in combination with an epidermal growth factor receptor-targeted drug.

OBJECTIVES: To investigate the inhibition of PANC-1 pancreatic cancer cell growth by cucurmosin (CUS) and its possible mechanism. METHODS: We observed the inhibition of PANC-1 cell growth by sulforhodamine B and colony-forming experiments in vitro and established nonobese diabetic/severe combined immunodeficiency mouse subcutaneous tumor models in vivo. We used Western blot to analyze protein levels related to apoptosis and epidermal growth factor receptor (EGFR) signaling pathways after drug intervention, whereas the messenger RNA expression of EGFR was analyzed by quantitative real-time polymerase chain reaction. RESULTS: Sulforhodamine B and colony-forming experiments indicated that CUS inhibited PANC-1 cell proliferation in a dose- and time-dependent manner. A stronger inhibitory effect was observed when CUS was combined with gefitinib. The subcutaneous tumor growth was also inhibited. Western blot showed that all the examined proteins decreased, except for 4E-BP1 and the active fragments of caspase 3 and caspase 9 increased. Epidermal growth factor receptor expression did not change significantly in quantitative real-time polymerase chain reaction. CONCLUSIONS: Cucurmosin can strongly inhibit the growth of PANC-1 cells in vitro and in vivo. Cucurmosin can down-regulate EGFR protein expression, but not at the messenger RNA level. Cucurmosin can also inhibit the ras/raf and phosphatidylinositol 3-kinase/Akt downstream signaling pathways and enhance the sensitivity of the EGFR-targeted drug gefitinib.

[Effect of cucurmosin on chronic myeloid leukemia K562 cell line].

This study was aimed to investigate the antitumor effect of pumpkin protein (cucurmosin, CUS) on subcutaneous transplant tumor in chronic myeloid leukemia K562 cell-NOD/SCID mice and leukemia model. The subcutaneous transplant tumor in K562-NOD/SCID mice and leukemia model were established; using two models, the antitumor activity of CUS in mice was evaluated. The results indicated that the inhibitory rate of 0.5 mg/kg and 1 mg/kg CUS on subcutaneous transplant tumor were 53.45% and 59.43% respectively; survival time of mice received 0.25 mg/kg and 0.5 mg/kg CUS was 39.8 ± 5.5 d and 43.4 ± 6.6 d, antitumor rate was 24.9% and 36% respectively. It is concluded that CUS has significant inhibitory effect on mice with CML cell line K562.

Cucurmosin induces the apoptosis of human pancreatic cancer CFPAC-1 cells by inactivating the PDGFR-ß signalling pathway.

BACKGROUND: Pancreatic cancer treatment is limited and effective drugs are needed. We investigated cucurmosin (CUS)-induced apoptosis in cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and a possible mechanism of action to evaluate the clinical application potential of this new Type I ribosome-inactivating protein. METHODS: We analyzed the growth inhibition and apoptosis of CFPAC-1 cells via methylthiazol tetrazolium assay and fluorescence-activated cell sorting. Western blot was used to analyze the protein levels of caspase 3, bcl-2, caspase 9, platelet-derived growth factor receptor (PDGFR)-ß, PI3K, Akt, p-Akt, the mammalian target of rapamycin (mTOR), p-mTOR, P70S6K-α, p-P70S6K-α, 4E-BP1, p-4E-BP1 and p-Bad after CUS intervention. The mRNA expression of PDGFR-ß was analyzed using reverse transcription polymerase chain reaction. RESULTS: CUS inhibited the proliferation of pancreatic cancer cells. The induction of apoptosis depended on the CUS dose and incubation time. The drug inhibited all of the examined proteins in the PI3K/Akt/mTOR signalling pathway and induced the active fragments of caspase 3 and caspase 9. CUS downregulated PDGFR-ß expression but no significant change was observed at the mRNA level. CONCLUSION: CUS strongly inhibits the growth of CFPAC-1 by inducing cell apoptosis. CUS downregulated the expression of PDGFR-ß at the protein level and induced the apoptosis of CFPAC-1 through the PI3K/Akt/mTOR signalling pathway.

Cucurmosin kills human pancreatic cancer SW-1990 cells in vitro and in vivo.

OBJECTIVE: To investigate the effect of cucurmosin (CUS) on proliferation inhibition in the human pancreatic cancer cell line SW-1990 in vitro and in vivo. METHODS: 1. MTT assay was used to analyse the proliferation inhibition of CUS in SW-1990 cells compared with gemcitabine (GEM) in vitro. 2. We established an NOD-SCID mice orthotopic transplantation model and estimated the proliferation inhibition effect of CUS in SW-1990 cells in vivo. 3. Western blot was used to determine the protein expressions of Caspase 3, Bcl-2, Caspase 9, PI3K, Akt, mTOR, P70S6k, and 4E-BP1 after CUS intervention. RESULTS: 1. CUS inhibited the proliferation of pancreatic cancer cells and induced apoptosis in CUS dose- and time-dependent manners. 2. NOD-SCID mice models were established successfully, and the tumour proliferation inhibition rates of these models increased compared with the control group. 3. CUS inhibited all of the examined proteins in the PI3K/Akt/mTOR signalling pathway and induced active fragments of Caspase 3 and Caspase 9. CONCLUSION: 1. CUS can inhibit the growth of SW-1990 cells in vitro and in vivo. 2. CUS can induce apoptosis in SW-1990 cells to inhibit cell growth.

Cucurmosin induces apoptosis of BxPC-3 human pancreatic cancer cells via inactivation of the EGFR signaling pathway.

Pancreatic cancer remains the fourth most common cause of cancer-related death in the United States. Potent therapeutic strategies are urgently needed for pancreatic cancer. Cucurmosin is a novel type 1 ribosome-inactivating protein (RIP) isolated from the sarcocarp of Cucurbita moschata (pumpkin). Due to its cytotoxicity, cucurmosin can inhibit tumor cell proliferation through induction of apoptosis on tumor cells, but the specific mechanism is still unclear. We explored the function of cucurmosin in BxPC-3 pancreatic cancer cells using multiple cellular and molecular approaches such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), Western blotting and transmission electron microscopy for observing typical changes and formation of apoptotic bodies. We found that cucurmosin inhibited the proliferation of BxPC-3 cells in a time- and dose-dependent manner, and increased the cell population in the G0-G1 phase. With increasing concentration of cucurmosin, the expression of EGFR, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, P70S6K-α, p-P70S6K-α, 4E-BP1 and p-4E-BP1 at the protein level was decreased, whereas the expression of p-Bad and caspase-9 was elevated. However, the mRNA expression of EGFR did not change. These findings suggest that cucurmosin can down-regulate the expression of EGFR by targeting. Cucurmosin induces the apoptosis of BxPC-3 pancreatic cancer cells via the PI3K/Akt/mTOR signaling pathway.

Anti-proliferative effects of cucurmosin on human hepatoma HepG2 cells.

We extracted cucurmosin (CUS) from the sarcocarp of Cucrubita moschata (pumpkin). Recently, a number of studies have indicated that CUS has cytotoxic properties and induces apoptosis in a number of human tumor cells. However, the detailed mechanisms are largely unknown. The aim of this study was to confirm CUS's anticancer activity on human hepatoma HepG2 cells in vitro and in vivo, and to elucidate the mechanism of its activity. MTT was used to detect the cytotoxic effects of CUS. Flow cytometry was used to analyze cell apoptosis and the cell cycle. Transmission electron microscopy was used to observe the morphology of apoptotic cells. Western blot analysis was performed to measure the protein expression of bax, bcl-2 and procaspase-3. The established orthotopic transplantation models of human hepatoma in NOD/SCID mice were tested for anticancer activities in vivo. The results showed that CUS inhibited the proliferation of HepG2 cells in vitro and in vivo. CUS induced apoptosis and arrested the cell cycle. In addition, CUS increased the protein expression of bax, but decreased the bcl-2 and procaspase-3 expression in HepG2 cells. Our data indicate that CUS has potential anticancer activity for human hepatoma, which can be attributed in part to its inhibition of proliferation and apoptosis, induced by decreasing the bcl-2:bax ratio and caspase-3 activation.

[Studies on the triterpenoids of Vitex trifolia].

OBJECTIVE: To study the chemical constituents of Vitex trifolia. METHODS: Chromatography and spectroscopic analysis were employed to isolate and elucidate the chemical constituents in the plant. RESULTS: Five triterpenoids were obtained and identified as ursolic acid (I),2alpha,3alpha-dihydroxyurs-12-en-28-oic acid (II), betulinic acid (11), taraxerol (IV), 2alpha,3beta, 19-trihydroxyurs-12-en-28-oic acid (V). CONCLUSION: Compounds 1-5 are isolated from this plant for the first time.

Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata.

A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04A, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven alpha-helices and eight beta-strands, and a smaller C-terminal domain consisting of three alpha-helices and two beta-strands. The high resolution structure established a glycosylation pattern of GlcNAc(2)Man(3)Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.