Tertiary lymphoid structures predict survival and response to neoadjuvant therapy in locally advanced rectal cancer.

Tertiary lymphoid structure (TLS) contributes to the anti-tumor immune response, and predicts the prognosis of colorectal cancer patients. However, the potential impact of TLS in shaping the immune status of rectal adenocarcinoma, and the intrinsic relationship between TLS and neoadjuvant therapies (neoTx) remain unclear. We performed hematoxylin-eosin staining, immunohistochemical and biomolecular analyses to investigate TLS and tumor-infiltrating lymphocytes (TILs) in 221 neoTx-treated and 242 treatment-naïve locally advanced rectal cancer (LARC) patients. High TLS density was significantly associated with the absence of vascular invasion, a lower neutrophil-to-lymphocyte ratio, increased TLS maturity, a longer recurrence-free survival (RFS) (hazard ratio [HR] 0.2985 95% confidence interval [CI] 0.1894-0.4706, p < 0.0001) and enhanced infiltration of adaptive immune cells. Biomolecular analysis showed that high TLS-score was strongly associated with more infiltration of immune cells and increased activation of immune-related pathways. TLS+ tumors in pre-treatment specimens were associated with a higher proportion of good respond (62.5% vs. 29.8%, p < 0.0002) and pathological complete remission (pCR) (40.0% vs. 11.1%, p < 0.0001), and significantly increased RFS (HR 0.3574 95%CI 0.1489-0.8578 p = 0.0213) compared with TLS- tumors in the neoTx cohort, which was confirmed in GSE119409 and GSE150082. Further studies showed that neoTx significantly reduced TLS density and maturity, and abolished the prognostic value of TLS. Our study illustrates that TLS may have a key role in mediating the T-cell-inflamed tumor microenvironment, which also provides a new direction for neoTx, especially neoadjuvant immunotherapy, in LRAC patients.

Gastric adenosquamous carcinoma with an elevated serum level of alpha-fetoprotein: A case report.

BACKGROUND: Gastric adenosquamous carcinoma (ASC) is rare and characterized by coexisting of adenocarcinoma andsquamous carcinoma within the same tumor. We present a female patient with gastric ASC who had an elevated serum level of alpha-fetoprotein (AFP), which decreased to normal levels after a laparoscopic distant radical gastrectomy in a short period. The clinicopathological features in AFP-producing gastric cancer (GC) are discussed, as well as potentially available prognostic predictors. CASE SUMMARY: A 50-year-old woman presented to our department with a chief complain of a 6-mo history of bloating. She had no basic diseases including heart diseases and respiratory diseases, and she also denied any prior history of dysphagia, hematemesis, melena, rectal bleeding, hematochezia, or unintentional weight loss. Based on her symptoms, an esophagogastroduodenoscopy was performed, showing an annular cavity lesion 3 cm from the pylorus with a diameter of 6 cm. A biopsy of the lesion showed gastric ASC, whereas the pylorus biopsy showed normal mucosa. The patient further received an enhanced computed tomography scan which demonstrated an invasive lesion close to the pylorus with a still clear margin of the tumor to peripheral organs such as the pancreas and liver. Scattered lymph nodes were visible around, whereas no sign of liver metastasis was discovered. Serum tumor markers including carcinoembryonic antigen (CEA), cancer antigen 199 (CA199), CA724, CA125, and CA242 were all normal, while the level of serum AFP increased to 172 ng/mL. A laparoscopic distant radical gastrectomy was performed after exclusion of surgical contraindications. Postoperative pathology results showed that the tumor displayed an ulcerated ASC phenotype (90% of medium to highly-differentiated squamous cell carcinoma, 10% of poorly differentiated adenocarcinoma. Surprisingly, the serum level of AFP decreased to normal level on post operation day 5. The tumor cells were positive for CK5/6, p63, and CEA, and negative for AFP and Epstein-Barr encoding region. CONCLUSION: We presented a rare case of gastric ASC with elevated serum AFP level, which may be new subtype of AFP-producing GC. Follow-up detection of serum AFP might be a useful tool to predict patient prognosis.

Neutrophil Extracellular Trap is Surrogate Biomarker for Prognosis and Response to Neoadjuvant Therapy in Locally Advanced Rectal Cancer.

Purpose: To demonstrate the intrinsic association of Neutrophil extracellular traps (NETs) with outcome and neoadjuvant therapy response of locally advanced rectal cancer (LARC), and the mechanisms. Patients and Methods: We enrolled 240 patients with LARC who underwent surgery without neoadjuvant therapy in two independent sets (training and validation), and 153 patients who received neoadjuvant therapy with biopsy followed by surgery. Immunohistochemistry, immunofluorescence staining and bioinformatics analysis were performed in formalin-fixed paraffin-embedded sections. NETs were identified by costaining for myeloperoxidase and citrullinated histone H3. Results: NETs were associated with recurrence-free survival in the surgical training and validation sets. High-NET density predicted poor postoperative survival of patients with LARC. Multivariate analysis identified NETs, TNM stage, and neutrophil-to-lymphocyte ratio as independent prognostic factors for recurrence-free survival. Low-NETs LARC demonstrated increased CD8+ T cell and lower T regulatory cell infiltration, which indicated a tumor immune microenvironment with strong antitumor capacity. High-NET density was associated with epithelial-mesenchymal transition, which is considered to contribute to tumor progression. In the neoadjuvant therapy cohort, high-NET density on biopsy was significantly associated with reduced likelihood of complete/near complete response. Conclusion: NET was an independent prognostic factor in LARC that were associated with patients' survival, and NET density in pretreatment biopsies was an independent predictive biomarker of response to neoadjuvant therapy. This biomarker may be helpful in predicting survival in LARC with improved accuracy and selecting patients who will respond to neoadjuvant therapy.

Peritumoral tertiary lymphoid structure and tumor stroma percentage predict the prognosis of patients with non-metastatic colorectal cancer.

Background: Tertiary lymphoid structures (TLSs) are crucial in promoting and maintaining positive anti-tumor immune responses. The tumor stroma has a powerful immunosuppressive function that could exclude tumor-infiltrating lymphocytes from the tumor beds and lead to a "cold" phenotype. TLSs and tumor stroma percentage (TSP) are significantly associated with the prognosis of patients with certain cancers. However, the exact roles of TLSs and TSP and their intrinsic relationship are still largely unknown in colorectal cancer (CRC). Methods: TLSs and TSP were assessed using hematoxylin-eosin (H&E) and/or immunohistochemistry (IHC) staining from 114 CRC patients in the training set and 60 CRC patients in the external validation set. The correlation between TILs, TLS and clinicopathological characteristics and their prognostic values were assessed. Finally, we plotted a Nomogram including the TLS, TSP and tumor-node-metastasis (TNM) stage to predict the probability of recurrence-free survival (RFS) at 2- and 5-years in non-metastatic colorectal cancer (nmCRC) patients. Results: Peritumoral TLS (P-TLS), intratumoral TLS (In-TLS) and high TSP (H-TSP, >50%) were present in 99.1%, 26.3% and 41.2% patients, respectively. H-TSP tumor tends to be associated with lower P-TLS density (P =0.0205). The low P-TLS density (< 0.098/mm2) was significantly associated with reduced RFS (HR=6.597 95% CI: 2.882-15.103, P <0.001) and reduced overall survival (OS) (HR=6.628 95% CI: 2.893-15.183, P < 0.001) of nmCRC patients. In-TLS was not of significance in evaluating the clinical outcomes of nmCRC patients. H-TSP was significantly associated with reduced RFS (HR=0.126 95% CI: 0.048-0.333, P <0.001) and reduced OS (HR=0.125 95% CI: 0.047-0.332, P <0.001) of nmCRC patients. The 5-year RFS of the high P-TLS, low-TLS, H-TSP, and L-TSP groups were 89.7%, 47.2%, 53.2%, and 92.5%, respectively. The P-TLS density, TSP and TNM stage were independent prognosis factors of nmCRC patients. The Nomogram, including the P-TLS density, TSP and TNM stage, outperformed the TNM stage. Conclusions: High P-TLS density and low TSP (L-TSP) were independent and favorable prognostic factors of nmCRC patients, which might provide new directions for targeted therapy in the CRC tumor microenvironment, especially the tumor immune microenvironment.

Fusobacterium nucleatum Promotes Colorectal Cancer Cell to Acquire Stem Cell-Like Features by Manipulating Lipid Droplet-Mediated Numb Degradation.

Fusobacterium nucleatum is a critical microbe that contributes to colorectal cancer progression and chemoresistance. However, whether and how F. nucleatum regulates colorectal cancer stem-like cells (CCSCs) remains unknown. Here, the authors show that F. nucleatum promotes CCSC self-renewal, and non-CCSCs to acquire CCSC features by manipulating cellular lipid accumulation. F. nucleatum infection decreases lipid accumulation in CCSCs by enhancing fatty acid oxidation, thus promoting CCSC self-renewal. In contrast, F. nucleatum increases lipid accumulation in non-CCSCs by promoting fatty acid formation. Lipids are deposited as lipid droplets, which recruits Numb, a key cell fate regulator, through the AP2A/ACSL3 complex, and MDM2, an E3 ubiquitin ligase, though VCP and UBXD8. On lipid droplets, Numb is degraded by MDM2, activating Notch signaling, thus promoting gain of stem-like cell features. Their findings demonstrate that F. nucleatum directly manipulates colorectal cancer cell fate and reveal the mechanism of lipid droplet-mediated Numb degradation for activating Notch signaling.

Creatine promotes cancer metastasis through activation of Smad2/3.

As one of the most popular nutrient supplements, creatine has been highly used to increase muscle mass and improve exercise performance. Here, we report an adverse effect of creatine using orthotopic mouse models, showing that creatine promotes colorectal and breast cancer metastasis and shortens mouse survival. We show that glycine amidinotransferase (GATM), the rate-limiting enzyme for creatine synthesis, is upregulated in liver metastases. Dietary uptake, or GATM-mediated de novo synthesis of creatine, enhances cancer metastasis and shortens mouse survival by upregulation of Snail and Slug expression via monopolar spindle 1 (MPS1)-activated Smad2 and Smad3 phosphorylation. GATM knockdown or MPS1 inhibition suppresses cancer metastasis and benefits mouse survival by downregulating Snail and Slug. Our findings call for using caution when considering dietary creatine to improve muscle mass or treat diseases and suggest that targeting GATM or MPS1 prevents cancer metastasis, especially metastasis of transforming growth factor beta receptor mutant colorectal cancers.

CD146 is a Novel ANGPTL2 Receptor that Promotes Obesity by Manipulating Lipid Metabolism and Energy Expenditure.

Obesity and its related complications pose an increasing threat to human health; however, targetable obesity-related membrane receptors are not yet elucidated. Here, the membrane receptor CD146 is demonstrated to play an essential role in obesity. In particular, CD146 acts as a new adipose receptor for angiopoietin-like protein 2 (ANGPTL2), which is thought to act on endothelial cells to activate adipose inflammation. ANGPTL2 binds to CD146 to activate cAMP response element-binding protein (CREB), which then upregulates CD146 during adipogenesis and adipose inflammation. CD146 is present in preadipocytes and mature adipocytes, where it is mediated by its ligands ANGPTL2 and galectin-1. In preadipocytes, CD146 ablation suppresses adipogenesis, whereas the loss of CD146 in mature adipocytes suppresses lipid accumulation and enhances energy expenditure. Moreover, anti-CD146 antibodies inhibit obesity by disrupting the interactions between CD146 and its ligands. Together, these findings demonstrate that ANGPTL2 directly affects adipocytes via CD146 to promote obesity, suggesting that CD146 can be a potential target for treating obesity.

Expression of HAX-1 in human colorectal cancer and its clinical significance.

Colorectal cancer (CRC) remains one of the most common cancers worldwide. HS1-associated protein X-1 (HAX-1) has been highlighted as an important marker in many types of cancers. However, little is known about the role of HAX-1 in CRC. The aim of this study was to analyze the correlation of HAX-1 expression with the clinicopathological features of CRC. The protein and mRNA levels of HAX-1 were examined by immunohistochemistry (IHC) and real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) in CRC tissues and adjacent noncancerous tissues. Survival curves were made with follow-up data. The relations of the prognosis with clinical and pathological characteristics were analyzed. Using IHC and RT-qPCR, we showed that HAX-1 expression was significantly higher in CRC tissues than in adjacent noncancerous tissues (P < 0.05). High HAX-1 expression was significantly associated with lymph node metastasis (P = 0.034) and tumor (T) node (N) metastasis (M) stage (P = 0.028) of patients with CRC. The Kaplan-Meier survival curves indicated that overall survival was significantly worse in CRC patients with HAX-1 overexpression. Multivariate analysis showed that high HAX-1 expression was an independent predictor of overall survival. In conclusion, our data for the first time provide a basis for the concept that overexpression of HAX-1 may contribute to the malignant progression of CRC and predict poor prognosis for patients with this disease. HAX-1 might be an important marker for tumor progression and prognosis, as well as a potential therapeutic target of CRC.

[Expression and clinical significance of Claudin-1 and Claudin-4 in colorectal cancer tissues].

OBJECTIVE: To investigate the expression of tight junction protein Claudin-1 and Claudin-4 in colorectal cancer tissues and its clinical significance. METHODS: Immunohistochemical staining detected the expression of tight junction protein Claudin-1 and Claudin-4 in 60 cases of colorectal cancer and 20 normal colorectal mucosa tissue. The clinical significance was analyzed. RESULTS: The positive rates of Claudin-1 and Claudin-4 in colorectal cancer tissues were 76.6%(46/60) and 85.0%(51/60), significantly higher than 20.0% (4/20) and 30.0%(6/20) in the normal colorectal mucosa(both P<0.01). The positive rates of Claudin-1 and Claudin-4 were associated with tumor differentiation degree, lymph node metastasis and TNM staging(all P<0.05). CONCLUSIONS: The high expression of the Claudin-1 and Claudin-4 may play a promoting role in colorectal cancer development and progression. Claudin-1 and Claudin-4 may become prognostic markers of colorectal cancer.

Proteomic analysis of differentially expressed proteins involving in liver metastasis of human colorectal carcinoma.

BACKGROUND: Early diagnosis of liver metastasis of colorectal carcinoma is very important for the appropriate treatment of such patients. However, there has been no effective approach available for clinical application. The present study aimed to investigate the differential expression of proteins in patients with liver metastasis of colorectal carcinomas using proteomic analysis and evaluate its potentiality in clinical diagnosis. METHODS: Fluorescence two-dimensional differential in-gel electrophoresis (2-D DIGE) was used to analyze and compare the protein expression between normal mucosa, the primary focus, and liver metastases. Proteomic analysis was made to identify the differentially expressed proteins. Immunohistological staining was used to confirm the expression of differentially expressed proteins in colorectal carcinomas and areas of liver metastasis. RESULTS: A 1.5-fold difference was found with 46 differentially expressed proteins. In 20 differentially expressed proteins, 3 were down-regulated and 17 up-regulated in liver metastases. Proteomic analysis showed that the S-adenosylmethionine transgelin variant was down-regulated in liver metastasis tissues. Zinc finger protein 64 homolog (Zfp64), guanine nucleotide exchange factor 4 (GEF4), human arginase, glutathione S-transferases (GSTs) A3, and tumor necrosis factor alpha (TNF-alpha)-induced protein 9 were up-regulated in liver metastasis tissues. Immunohistochemical staining confirmed that human arginase expression was higher in liver metastases than in the primary focus. CONCLUSIONS: There was a significant difference in protein expression between the primary focus of colorectal carcinoma and liver metastases. The differentially regulated proteins were closely related to liver metastasis of colorectal carcinoma. Elevated human arginase may be an important molecular marker for liver metastasis from colorectal carcinoma.

[Effect of FasL gene expression on proliferation and apoptosis of rectal carcinoma cells in hypoxia state].

OBJECTIVE: To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. METHODS: The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348(B), HR-8348(L), HR-8348(F) and HR-8348(As)) with different invasive power were verified by Western blot. Hypoxia models for HR-8348(B), HR-8348(L), HR-8348(F) and HR-8348(As) were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptosis was assessed with TUNEL. RESULTS: FasL protein was pigmentized at the position of 40,000 by Western blot, and the expression level of FasL was significantly higher in HR-8348(F) cells than those in HR-8348(B), HR-8348(L) and HR-8348(As) cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348(F) cells than those in other groups(F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia(t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348(F)(60.43+/-3.72) than those in HR-8348(B)(40.01+/-3.30), HR-8348(L)(41.30+/-4.06) and HR-8348(As) cells(35.87+/-4.39), respectively (F=39.477,P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348(F)(17.30+/-1.98 and 13.10+/-1.04) than those in HR-8348(B)(33.70+/-4.33 and 21.60+/-1.31), HR-8348(L)(34.20+/-3.92 and 20.10+/-1.15), and HR-8348(As)(38.00+/-4.55 and 23.90+/-1.23), respectively(F=28.811 and 76.462, respectively, P<0.01). CONCLUSION: The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apoptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.

[The lethal effect of combined MDR1 antisense RNA with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells].

OBJECTIVE: To observe the lethal effect of multidrug resistance gene (MDR1) antisense RNA combined with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells. METHODS: PC-MDR1 plasmid including MDR1 was constructed with gene cloning techniques. The drug-resistant cancer cells (8348R) were transferred with the plasmids, and the positive neoplasm cells were selected with G418. Green fluorescent protein (GFP) gene was used as a reporting gene to monitor the gene transfer efficiency under the influence of oxaliplatin and 5-FU. The cytotoxicity and therapeutic effects of MDR1 anti-sense RNA combined with oxaliplatin and 5-FU were evaluated by colony-forming rate and MTT assay. RESULTS: A significant decrease of biological activity was observed in 8348R cells transferred with PC-MDR1, cell cycles were blocked in S phase, or in G2/M phase, and apoptosis rate of the cells increased. With treatment of oxaliplatin, the plasmid transfer efficiency in the drug-resistant cancer cells was improved about 18 times. Using an IC(50) dose of oxaliplatin and 5-FU combined with (MDR1) anti-sense RNA, 75 percent of 8348R cells were killed, which was significant higher than that of the control cells. CONCLUSIONS: Combined MDR1 antisense RNA with oxaliplatin and 5-FU has a synergistic effect of killing drug-resistant cancer cells and may be a promising method for treating drug-resistant rectal carcinoma.

[Proteome study of colorectal cancer genesis and hepatic metastasis].

OBJECTIVE: To study differential proteins and their biological functions associated with colorectal cancer genesis and hepatic metastasis by proteomics and molecular biology techniques. METHODS: Isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis was used to analyse the expression of differential proteins from normal colorectal mucosa, primary cancer lesion and hepatic metastasis region. Liquid chromatography/mass spectrometry (LC-MS/MS) was used to identify the differential proteins. Transfection of colorectal cancer lovo cells was performed with the differential protein cDNA, and the changes of cell biological behavior was observed. RESULTS: Significant difference in protein expression was found on two-dimensional electrophoresis. Thirteen differential protein spots were analysed and identified. Human carbonic anhydrase II was detected in normal colorectal mucosa but not in primary cancer lesion and hepatic metastasisnegion. Phosphoglycerate kinase 1, fumarate hydratase and aldolase A were expressed in primary cancer. Expression of homo sapiens arginase and homo sapiens glutathione S-transferase A3 was found in hepatic metastasisnegion, but not in primary cancer lesion. After transfection with human carbonic anhydrase II cDNA, the lovo cells changed obviously with reduction in invasiveness, chemotaxy motor ability and tolerance. CONCLUSION: Differential expression of proteins was found between colorectal cancer genesis and hepatic metastasisnegion. No carbonic anhydrase II expression and enhanced expression of sapiens arginase and sapiens glutathione S-transferase A3 are related with biological behavior of colorectal cancer cell and facilitate hepatic metastasis.

[Expression of cathepsin B and its clinical importance in colorectal cancer].

OBJECTIVE: To investigate the expression of cathepsin B (CatB) in colorectal cancer tissues and serum levels of CatB in patients with colorectal carcinoma and to study the association of CatB expression with lymph node and li ver metastasis. METHODS: Immunohistochemistry was used to detect CatB expression in tissues, and enzyme linked immunosorbent assay was applied to test CatB levels in peripheral vein blood in 83 patients with colorectal cancer. RESULTS: The expression rates of CatB in primary lesions, normal colon mucosa, lymph node metastases and hepatic metastases were 56.6%, 31.3%, 88.4%, 85.0% respectively. The positive rates of CatB in primary lesions, hepatic and lymph node metastases were higher than that in normal mucosa (chi (2)=45.6124, P< 0.01). The CatB expression rates in lymph node and hepatic metastases were higher than that in primary lesions chi (2)=11.5982, 4.3747, P< 0.05). The positive rate of CatB was higher in Dukes C and D tumors than that in Dukes A and B tumors (chi (2)=18.8871, 25.1650, P< 0.01), higher in poorly differentiated and mucous adenocarcinomas than that in well-moderately differentiated adenocarcinomas chi (2)=14.2338, P< 0.05). The mean serum level of CatB in 83 patients with colorectal cancer was (5.9+/- 2.9) ng/ml, higher than (2.3+/- 1.1) ng/ml in the controls of 30 healthy volunteers (t=6.6975, P< 0.01). The serum level of CatB in the patients with Dukes C, D stages were higher than that with Dukes A, B stages. CONCLUSIONS: Enhanced expression of CatB in colorectal cancer tissues is associated with tumor infiltration and metastasis. Monitoring serum CatB level in patients with colorectal cancer is important in the prediction and diagnosis of lymph node and hepatic metastasis,and valuable for evaluation of the therapeutic effect.

[Expression of metallothionein and FasL in colorectal cancer and its clinical significance].

OBJECTIVE: To study the expression of metallothionein (MT) and FasL in colorectal cancer and their relation to lymph node and liver metastasis. METHODS: Immunohistochemistry and quantitative RT-PCR were used to detect expression of MT and FasL in protein and mRNA levels in 93 cases of colorectal cancer. RESULTS: The rates of MT expression in primary foci, non-cancerous colon mucosa, lymph node metastasis and liver metastasis were 58.1%, 32.3%, 81.1%, 64.3% respectively. And the rates of FasL expression were 41.9%, 19.4%, 62.3%, and 92.9% respectively. The positive rates of MT and FasL in primary foci, liver and lymph node metastasis were higher than that in non-cancerous mucosa (chi(2) = 35.2421, 57.5152, P < 0.01). MT expression rate in lymph node metastasis was higher than that in primary foci (chi(2) = 8.0565, P < 0.01). In liver metastasis, FasL expression rate was higher than in lymph node metastasis and primary foci (chi(2) = 8.6674, 22.4455, P < 0.01). The positive rates of MT and FasL in Dukes stage C and D were higher than that in Dukes stage A and B (chi(2) = 18.8871, 25.1650, P < 0.01). And higher rates of MT and FasL expression were observed in low differentiation adenocarcinoma and mucus adenocarcinoma than in middle-high differentiation adenocarcinoma (chi(2) = 11.1546, 9.2239, P < 0.05). High MT mRNA level was found in lymph node metastasis and high FasL mRNA level in liver metastasis. CONCLUSIONS: Enhanced expression of MT and FasL was associated significantly with lymph node and liver metastasis of colorectal cancer. Assay of MT and FasL expression has prognostic values for colorectal cancer patients.

Combined liposome-mediated cytosine deaminase gene therapy with radiation in killing rectal cancer cells and xenografts in athymic mice.

PURPOSE: The aim of this study was to assess the antitumor efficacy of combination of cytosine deaminase (CD) suicide gene therapy with radiation and to grope for new therapeutic strategy for local recurrent rectal cancer. EXPERIMENTAL DESIGN: HR-8348 cell line of human rectal cancer was used to assess efficiency of transfection with plasmid pEGFP-N1 and PXJ41-CD. The cells were exposed to radiation followed by liposome-mediated transfection. Cell inhibition assay was done with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antitumor efficacy of combined liposome-mediated CD suicide gene therapy with radiation was determined by treatment of nude mice bearing HR-8348 cancer cell xenograft. RESULTS: The efficiency of liposome-mediated CD gene transfection can be improved by radiation. With radiation at 2, 4, 6, and 8 Gy, the efficiency of liposome-mediated transfection increased from 21.3% to 62.2%, 78.0%, 83.2%, and 87.8%, respectively. CD expression was enhanced as well. Cancer cell inhibition experiment showed that combined liposome-mediated CD gene therapy with radiation had much stronger antitumor effect. With HR-8348 tumor xenograft model, suppression of tumor xenograft was observed. Compared with control group, tumor volume was inhibited by 81.5%, 48.5%, and 37.4%, respectively, in the combined CD/5-fluorocytosine with radiation group, CD/5-fluorocytosine group, and radiation group and the wet weight of tumor was decreased by 80%, 41.7%, and 37.7%, respectively. CONCLUSION: These findings suggested that combination of liposome-mediated CD gene therapy with radiation is a safer and efficient anticancer method. Its therapeutic efficacy may meet clinical treatment on local recurrent rectal cancer.

Comparative study of proteome between primary cancer and hepatic metastatic tumor in colorectal cancer.

AIM: To identify the differential proteins associated with colorectal cancer genesis and hepatic metastasis. METHODS: Hydrophobic protein samples were extracted from normal colorectal mucosa, primary cancer lesion and hepatic metastatic foci of colorectal cancer. With two-dimensional electrophoresis and image analysis, differentially expressed protein spots were detected, and the proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and peptide mass fingerprint analysis. RESULTS: Significant alterations of the proteins in number and expression levels were discovered in primary cancer and hepatic metastatic foci, the expression of a number of proteins was lost in 25-40 ku, but protein spots was increased in 14-21 ku, compared with normal mucosa. Nine differentially expressed protein spots were identified. Three proteins expressed in normal mucosa, but lost in primary cancer and hepatic metastasis, were recognized as calmodulin, ribonuclease 6 precursor and mannosidase-alpha. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. The differentially expressed protein of beta-globin was found in normal mucosa and hepatic metastatic tumor, but lost in primary cancer lesion. Cdc 42, a GTP-binding protein, was identified in hepatic metastasis. The protein spots of C4 from primary cancer, M7 and M9 from hepatic metastasis had less homology with the proteins in database. CONCLUSION: Variations of hydrophobic protein expression in colorectal cancer initiation and hepatic metastasis are significant and can be observed with two-dimensional electrophoresis. The expression of calmodulin, ribonuclease 6 precursor and mannosidase-alpha is lost but the expression of proapolipoprotein is enhanced which is associated with colorectal cancer genesis and hepatic metastasis. Cdc 42 and beta-globin are expressed abnormally in hepatic metastasis. Protein C4, M7 and M9 may be associated with colorectal cancer genesis and hepatic metastasis.

Effects of cell membrane phospholipid level and protein kinase C isoenzyme expression on hepatic metastasis of colorectal carcinoma.

BACKGROUND: The molecular mechanism of hepatic metastasis of colorectal cancer is not well understood. The aim of this study was to assess the relations between phospholipid contents of cellular membrane and isoenzyme expression of protein kinase C (PKC) and their effects on hepatic metastasis of colorectal cancer. METHODS: High performance liquid chromatography was used to detect contents of cell membrane phospholipids: phosphatidylinosital (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in primary foci, paratumor mucosa and hepatic metastatic foci in patients with colorectal carcinoma. The mRNA expression levels of PKC-alpha, -betaII, -delta, -epsilon, -lambda, -zeta isoenzymes were detected with the QRT-PCR technique. RESULTS: The levels of PI, PC and PE in primary foci and hepatic metastatic foci were higher than those in paratumor mucosa. The level of PE in hepatic metastatic foci was much higher than that in primary foci (t=98.88, P<0.01); but the levels of PI and PC were not significantly different between primary foci and hepatic metastatic foci (t=1.73, 1.36, P>0.05). The expression levels of PKC-betaII, -delta, -epsilon, -lambda, -zeta were enhanced in primary foci and hepatic metastatic foci, but the level of PKC-alpha in primary foci was decreased as compared with that in paratumor mucosa. The levels of PKC-delta, -epsilon, -lambda, -zeta in hepatic metastatic foci were higher than those in primary foci. A positive correlation was observed between the expression levels of PI, PC and PKC-betaII and also between those of PE and PKC-delta, -epsilon, -lambda, -zeta. However, there was a close negative correlation between PE and PKC-alpha. CONCLUSION: Increased levels of PI and PC and decreased ratio of PKC-alpha to PKC-betaII are related to colorectal cancer genesis. Increased levels of PE, increased expression of PKC-delta, -epsilon, -lambda, -zeta isoenzymes and decreased level of PKC-alpha are related to hepatic metastasis in colorectal carcinoma.

[Proteome study of colorectal cancer genesis and hepatic metastasis].

OBJECTIVE: To study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques. METHODS: Using isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins. RESULTS: Significant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP_040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis. CONCLUSION: Loss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.

[The construction of yeast two-hybrid method in the protein-interactions and its significance in hepatic metastasis of colorectal carcinoma].

OBJECTIVE: To construct the yeast two-hybrid system, and screen the proteins which interact with FasL, and investigate the relationship of FasL and hepatic metastasis of colorectal carcinoma. METHODS: We have cloned the FasL gene into the pGBKT7 vector as the bait, then screened the fetal liver cDNA library, and have got a series of specific proteins that interact with FasL protein. Using the bioinformatics, we analyzed the interacting proteins in the mechanism of hepatic metastasis of colorectal carcinoma. RESULTS: We have screened several proteins that interaction with FasL protein, including metallothionein 1K, 1G, 2A, cathepsin B, fatty acid synthase, interferon alpha-inducible protein 27, phospholipid scramblase, Ser/Thr-like kinase, anchor attachment protein, fibulin-5. CONCLUSIONS: We have successfully constructed the yeast two-hybrid system, and preliminary identified that the interaction between FasL, metallothionein, cathepsin and anchor attachment protein is radically related to the hepatic metastasis of colorectal carcinoma.