Colorectal Cancer In Nude Mice Research Articles

MTHFD1 Regulates Autophagy to Promote Growth and Metastasis in Colorectal Cancer via the PI3K-AKT-mTOR Signaling Pathway.

Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is the enzyme with the activities of methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase. Our aim was to elucidate the function of MTHFD1 in colorectal cancer (CRC). In vitro assessments of the proliferation, invasion, and migration abilities of CRC cells were conducted using Immunohistochemistry, Transwell invasion assays, Western blot (WB), and Cell counting Kit-8 assays. WB was also utilized to measure autophagy protein levels and PI3K-AKT-mTOR signaling pathway expression. Furthermore, the role of MTHFD1 was evaluated invivo by using subcutaneous xenograft tumor models and lateral tail vein metastasis models of human CRC in nude mice. Overexpression of MTHFD1 promoted the abilities of tumorigenesis and metastasis in CRC invitro and invivo and reduced autophagy, attributing to the PI3K-AKT-mTOR signaling pathway in CRC cells. In contrast, the down-regulation of MTHFD1 increased autophagy and suppressed their proliferation, migration, and invasion. MTHFD1 can modulate the PI3K-AKT-mTOR signaling pathway to suppress autophagy and stimulate tumorigenesis and metastasis.

Silencing GDI2 inhibits proliferation, migration and invasion of colorectal cancer through activation of p53 signaling pathway

ObjectiveTo investigate the effect of silencing GDP dissociation inhibitor 2 (GDI2) on colorectal cancer development and possible mechanisms based on transcriptomic analysis. MethodsThe differences in the expression levels of GDI2 in normal colorectal tissues and tumor tissues of colorectal cancer (CRC) patients were detected. The correlation of GDI2 expression levels with survival and clinical characteristics of CRC patients was analyzed. The effects of GDI2 expression levels on the biological functions of CRC cells were examined by CCK-8 assay, plate clone formation assay, wound healing assay, and Transwell assay. The effect of GDI2 on the proliferation and growth of xenograft tumors was investigated by a xenograft tumor model of CRC in nude mice. Based on transcriptomics, we explored the possible mechanisms and associated pathways of the effect of silencing GDI2 on CRC cells. Cellular experiments and Western blot assays were performed to verify the potential mechanisms and related pathway of GDI2 action on CRC. ResultsThe expression levels of GDI2 in CRC tissues and cells were higher than those in normal tissues and cells. The expression level of GDI2 correlated with clinical characteristics such as lymphatic metastasis, tumor stage, tumor volume, and lymphocyte count. Silencing of GDI2 reduced the proliferative activity and migration and invasion ability of CRC cells, as well as inhibited the proliferation of CRC xenograft tumors. The differentially expressed genes were significantly enriched in biological processes such as cell cycle arrest and the p53 signaling pathway after GDI2 silencing. The percentage of G0/G1 phase cells in CRC cells was increased after silencing GDI2 as verified by flow cytometry. RAB5A was highly associated with the p53 pathway and could interact with TP53 via the ZFYVE20 protein. The mutual binding between GDI2 protein and RAB5A protein was verified by immunoprecipitation assay. Silencing GDI2 while overexpressing RAB5A reversed the reduced proliferation, migration, and invasion ability as well as cell cycle arrest of CRC cells. Meanwhile, the addition of p53 signaling pathway inhibitor Pifithrin-α (PFT-α) also reversed the biological effects of silencing GDI2 on CRC cells. The p-p21 and p-p53 protein expression levels were significantly greater in the sh-GDI2 group than in the sh-NC group. However, the p-p21 and p-p53 protein expression levels were reduced after silencing GDI2 while overexpressing RAB5A. ConclusionSilencing GDI2 activates the p53 signaling pathway by regulating RAB5A expression levels, which in turn induces cell cycle arrest and ultimately affects the proliferative activity, migration, and invasive ability of CRC cells.

Ring Finger Protein 38 Induces Colorectal Cancer Proliferation by Activating the Phosphatidyl- Inositol 3-Kinase/Serine-Threonine Kinase Signaling

Previous studies have identified that ring finger protein 38 (RNF38) induces proliferative potential in many types of tumors. However, its functions in colorectal cancer (CRC) are unclear. This study aims to elucidate the regulatory effects of RNF38 on CRC proliferation in vitro and in vivo. Expression pattern and prognostic potential of RNF38 in clinically collected CRC cases were analyzed. After intervening RNF38 levels in SW480 and HT29 cells, viability, cell cycle progression and apoptosis were examined. Subsequently, we generated RNF38 overexpressed transgenic mice and xenograft model in nude mice. in vivo regulation of RNF38 on CRC proliferation was assessed. RNF38 was upregulated in CRC tissues. Overexpression of RNF38 stimulated proliferative ability and inhibited apoptosis in CRC cells. In addition, in vivo overexpression of RNF38 triggered tumor growth of CRC in nude mice. Silence of RNF38 markedly suppressed in vivo proliferation of CRC and induced apoptosis by activating the PI3K/AKT signaling.

Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway.

Background Oxaliplatin (L-OHP) is a common chemotherapy drug used in the treatment of colorectal cancer (CRC). Our previous work showed that Zuo Jin Wan (ZJW), a traditional Chinese medicine prescription, could improve sensitivity to L-OHP in the treatment of CRC, but the detailed mechanism is not clear. In previous mechanistic studies, we found that the miR-200s expression in CRC is associated with L-OHP sensitivity through regulation of MDR1/p-gp and the downstream c-JunN-terminal kinase (JNK) signaling pathway. Moreover, lncRNA-MALAT1 offers great potential in the regulation of drug resistance by interacting with miR-200s. Therefore, in this work, we explored whether ZJW could reverse L-OHP resistance in CRC by regulating MALAT1, miR-200s, and the downstream signaling pathway. Methods Cell Counting Kit-8 and flow cytometry were used to detect the effects of ZJW combined with L-OHP on chemotherapy tolerance and cell apoptosis of HCT116/L-OHP cells. Western blotting and quantitative real-time PCR (qRT-PCR) were used to detect the activation of the JNK signaling pathway and the protein and mRNA expression levels of the drug resistance-related MDR1/ABCB1 gene in HCT116/L-OHP cells treated with ZJW. The binding sites of MALAT1 and miR-200s were predicted by bioinformatics tools and confirmed by qRT-PCR. qRT-PCR was used to detect the expression of miR-200s and MALAT1 in HCT116/L-OHP cells treated with ZJW. A xenograft model of CRC in nude mice was established to observe the effect of ZJW combined with L-OHP on the growth of subcutaneously transplanted tumors. Apoptosis in tumor cells was detected by TUNEL staining. The activation of the JNK signaling pathway and the expression of drug resistance-related proteins were detected by immunohistochemistry and immunofluorescence. qRT-PCR was used to detect the expression of miR-200s and the MALAT1 gene in the tumors. Results Our study showed that ZJW could significantly decrease the proliferation and promote apoptosis of HCT116/L-OHP cells treated with L-OHP. We further proved that ZJW could reverse the drug resistance of HCT116/L-OHP cells by reducing MALAT1, indirectly upregulating miR-200s, alleviating the activation of the JNK signaling axis, and downregulating the expression of resistance proteins such as MDR1/ABCB1 and ABCG2. ZJW combined with L-OHP inhibited the growth of subcutaneously transplanted tumors and induced apoptosis in nude mice. ZJW reduced the expression of MALAT1 and upregulated the expression of miR-200s in transplanted tumors. In addition, ZJW also alleviated the activation of the JNK signaling pathway while reducing the expression of MDR1/ABCB1 and ABCG2. Conclusions Our study identified that MALAT1 promotes colorectal cancer resistance to oxaliplatin by reducing the miR-200s expression. ZJW may reverse chemoresistance by inhibiting the expression of MALAT1 and regulating the miR-200s/JNK pathway, providing an experimental basis for the clinical application of ZJW in relieving chemotherapy resistance.

The mechanism and tumor inhibitory study of Lagopsis supine ethanol extract on colorectal cancer in nude mice

BackgroundThis study was aimed to determination the tumor inhibitory effect and explore the potential mechanisms of Lagopsis supine ethanol extract (Ls) on colorectal cancer.MethodsThe cell growth inhibition experiment of Ls in colorectal cancer cell lines was determined by MTT method in the time course of 24, 48 and 72 h in four gradient drug concentrations. The protein expression levels of pSTAT3, pJAK2, STAT3, JAK2, Bcl-2 and caspase 3 were measured by Western blot method. The mRNA levels of the downstream genes of STAT3 were detected through semi-quantitative RT PCR. Sixty Balb/c-nude mice were xenograft with HCT116 colorectal cancer cells through subcutaneously. The xenografts were divided into five groups: model group, positive group (capecitabine 300 mg/kg) and three dosages of Ls treated groups (75, 150 and 300 mg/kg). Tumor size and tumor weight were calculated for evaluation the anti-tumor effects. H & E staining and immunohistochemical analysis were used to determine the histopathological changes and the levels of pSTAT3 and pJAK2 in the tumor tissues.ResultsLs exhibited a significant anti-proliferation effect in HCT116 and SW480 cells in vitro. The protein levels of pSTAT3, pJAK2 and Bcl-2, and the mRNA levels of Bcl-2 and Bak notably reduced with a dose-dependent manner. While the protein levels of caspase 3, and mRNA levels of Bax and caspase-3 remarkably increased in the gradient dosage of Ls in HCT116 cells. HCT116 in vivo xenografts experiment showed that the growth of the tumors significantly inhibited by Ls administration, which with no any significant body weight changes in each experiment group. The histopathology analysis displayed that Ls significantly reduced the inflammatory cells in tumor tissue. Furthermore, Ls also significantly down-regulate the protein levels of pSTAT3 and pJAK2 in the tumor tissues, compared with the model group.ConclusionsThis work shows that Ls inhibited the cell proliferation of colorectal cancer in vitro and significantly reduced the tumor growth in HCT116 xenografts in vivo, which is probably related with the JAK/STAT signal pathway.

Inhibitory effects of Chanling Gao on the proliferation and liver metastasis of transplanted colorectal cancer in nude mice.

This study aimed to explore the efficacy and mechanism of Chanling Gao (CLG), a compound Chinese medicine, on colorectal cancer (CRC). A model of transplanted CRC was established in nude mice. The mice were treated 7 days after CRC transplantation with either Capecitabine or CLG for 3 weeks. On the 28th day after the operation, CRC growth and liver metastasis were assessed by morphology, the changes in the expression of HIF-1α (hypoxia inducible factor-1α), stromal cell–derived factor-1 alpha (SDF-1α), CXCR4 (C-X-C chemokine receptor type 4), PI3K, and Akt in the transplanted tumor and SDF-1α and CXCR4 in the liver were detected by Western blot and immunohistochemistry. The protein contents of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and collagen IV in the serum and transplanted tumor and SDF-1α and CXCR4 in liver tissues were detected by enzyme-linked immunosorbent assay. In the Capecitabine and high dose CLG groups, the growth and liver metastasis of CRC were significantly inhibited, the protein levels of HIF-1α, SDF-1α, CXCR4, MMP-2, VEGF, PI3K, Akt, P-PI3K and P-Akt in the transplanted tumor were lower, while the content of collagen IV in the transplanted tumor was higher, than in Model group. A high dose of CLG inhibited the growth of transplanted tumor and liver metastasis of CRC in nude mice, probably by inhibiting the HIF-1α/SDF-1α-CXCR4/PI3K-Akt signaling pathway reducing the synthesis and release of VEGF and degradation of collagen IV.

The p300/YY1/miR-500a-5p/HDAC2 signalling axis regulates cell proliferation in human colorectal cancer

The biological role of miR-500a-5p has not yet been reported in the context of colorectal cancer (CRC). Here, we show that miR-500a-5p expression is decreased in CRC tissues compared with adjacent normal tissues. Low miR-500a-5p expression is associated with malignant progression. Moreover, transfection of CRC cells with miR-500a-5p induces G0/G1 cell cycle arrest and inhibits their growth and migration. Mechanistically, miR-500a-5p directly targets HDAC2 and inhibits HDAC2-mediated proliferation in CRC in nude mice. Furthermore, YY1 binds to the promoter of miR-500a-5p and negatively regulates its transcription. Restoration of miR-500a-5p expression is up-regulated via the p300/YY1/HDAC2 complex. Besides, therapeutic delivery of miR-500a-5p significantly suppresses tumour development in a xenograft tumour model and a HDAC2 inhibitor FK228-treated CRC model. Our studies demonstrate that miR-500a-5p functions as a tumour suppressor in CRC by targeting the p300/YY1/HDAC2 axis, which contributes to the development of and provides new potential candidates for CRC therapy.

Development of EGFR-targeted evodiamine nanoparticles for the treatment of colorectal cancer.

Invasion and metastasis of colorectal cancer (CRC) are leading causes of death of CRC patients. Previous findings demonstrate that evodiamine (Evo), an indolequinone alkaloid, is effective in combating CRC; however, its poor aqueous solubility and low oral bioavailability limit its application in the prevention of invasion and metastasis of CRC. It is known that selectively targeting cancer-specific receptors highly expressed on the surface of cancer cells by nanocarriers loaded with cytotoxic drugs is a viable strategy in nanobiotechnology to enhance cancer cell killing and minimize side effects. In this study, we report the development of a new class of nanotherapeutics: EGFR-targeting Evo-encapsulated poly(amino acid) nanoparticles (GE11-Evo-NPs). These nanoparticles exhibited good aqueous solubility, slow release, and active targeting capability. Their inhibitory effect on human colon cancer cells and therapeutic efficacy against invasion and metastasis of CRC in nude mice were systematically investigated. Mechanisms of the GE11-Evo-NPs against EGFR mediated invasion and metastasis of CRC were also explored. Compared with free Evo, the GE11-Evo-NPs showed significantly increased cytotoxicity to colon cancer cells and potently inhibited CRC LoVo cell adhesion, invasion, and migration. The expression of EGFR, VEGF, and MMP proteins was dramatically down-regulated, which may partially account for their inhibition of invasion and metastasis of CRC. Moreover, in vivo studies show that the GE11-Evo-NPs exhibited much greater potency than other control groups in inhibiting CRC invasion and metastasis, tumor volume, and growth in nude mice, leading to a significantly prolonged tumor-bearing survival duration (P < 0.01).

Development of patient-derived orthotopic xenografts from metastatic colorectal cancer in nude mice

Liver metastasis is the major cause of death for patients with colorectal cancer. Despite treatment with surgery and chemotherapy, patient outcomes are quite unfavourable. Thus, there is an urgent need to develop new treatment strategies with the associated establishment of good animal models. Metastatic disease can be modelled using patient-derived orthotopic xenografts, which accurately replicate intra-tumoral heterogeneity so that various chemotherapeutic agents can be tested on individual tumours to aid in clinical decision-making. The objective of this study was to develop metastatic colorectal tumours in athymic nude mice by implanting fresh tumour fragments into mouse liver parenchyma. Metastatic tumours were successfully propagated in mice following transplantation from human patients, then serially implanted in second and third-generation mice. Morphologic and immunohistochemical characteristics indicate that xenografts recreate the tumour architecture and mismatch repair gene expression for MLH1, MSH2, MSH1, and PMS2. After tumour implantation during the first passage, the time of tumour growth decreased without loss of tumour identity. Post-transplantation lymphoproliferative disease was observed in one case. This pilot study was successful in establishing the institutional PDX preclinical platform to study new therapeutic strategies, disease progression biomarkers, and treatment responsiveness.

Prediction of tumor biological characteristics in different colorectal cancer liver metastasis animal models using 18F-FDG and 18F-FLT

BackgroundPositron emission tomography (PET) is a noninvasive method to characterize different metabolic activities of tumors, providing information for staging, prognosis, and therapeutic response of patients with cancer. The aim of this study was to evaluate the feasibility of 18F-fludeoxyglucose (18F-FDG) and 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET in predicting tumor biological characteristics of colorectal cancer liver metastasis. MethodsThe uptake rate of 18F-FDG and 18F-FLT in SW480 and SW620 cells was measured via an in vitro cell uptake assay. The region of interest was drawn over the tumor and liver to calculate the maximum standardized uptake value ratio (tumor/liver) from PET images in liver metastasis model. The correlation between tracer uptake in liver metastases and VEGF, Ki67 and CD44 expression was evaluated by linear regression. ResultsCompared to SW620 tumor-bearing mice, SW480 tumor-bearing mice presented a higher rate of liver metastases. The uptake rate of 18F-FDG in SW480 and SW620 cells was 6.07% ± 1.19% and 2.82% ± 0.15%, respectively (t = 4.69, P = 0.04); that of 18F-FLT was 24.81% ± 0.45% and 15.57% ± 0.66%, respectively (t = 19.99, P < 0.001). Micro-PET scan showed that all parameters of FLT were significantly higher in SW480 tumors than those in SW620 tumors. A moderate relationship was detected between metastases in the liver and 18F-FLT uptake in primary tumors (r = 0.73, P = 0.0019). 18F-FLT uptake was also positively correlated with the expression of CD44 in liver metastases (r = 0.81, P = 0.0049). ConclusionsThe uptake of 18F-FLT in metastatic tumor reflects different biological behaviors of colon cancer cells. 18F-FLT can be used to evaluate the metastatic potential of colorectal cancer in nude mice.

Evaluation of therapeutic effectiveness of (131)I-antiEGFR-BSA-PCL in a mouse model of colorectal cancer.

To investigate the biological effects of internal irradiation, and the therapeutic effectiveness was assessed of (131)I-labeled anti-epidermal growth factor receptor (EGFR) liposomes, derived from cetuximab, when used as a tumor-targeting carrier in a colorectal cancer mouse model. We described the liposomes and characterized their EGFR-targeted binding and cellular uptake in EGFR-overexpressing LS180 colorectal cancer cells. After intra-tumor injections of 74 MBq (740 MBq/mL) (131)I-antiEGFR-BSA-PCL, we investigated the biological effects of internal irradiation and the therapeutic efficacy of (131)I-antiEGFR-BSA-PCL on colorectal cancer in a male BALB/c mouse model. Tumor size, body weight, histopathology, and SPECT imaging were monitored for 33 d post-therapy. The rapid radioiodine uptake of (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL reached maximum levels at 4 h after incubation, and the (131)I uptake of (131)I-antiEGFR-BSA-PCL was higher than that of (131)I-BSA-PCL in vitro. The (131)I tissue distribution assay revealed that (131)I-antiEGFR-BSA-PCL was markedly taken up by the tumor. Furthermore, a tissue distribution assay revealed that (131)I-antiEGFR-BSA-PCL was markedly taken up by the tumor and reached its maximal uptake value of 21.0 ± 1.01 %ID/g (%ID/g is the percentage injected dose per gram of tissue) at 72 h following therapy; the drug concentration in the tumor was higher than that in the liver, heart, colon, or spleen. Tumor size measurements showed that tumor development was significantly inhibited by treatments with (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL. The volume of tumor increased, and treatment rate with (131)I-antiEGFR-BSA-PCL was 124% ± 7%, lower than that with (131)I-BSA-PCL (127% ± 9%), (131)I (143% ± 7%), and normal saline (146% ± 10%). The percentage losses in original body weights were 39% ± 3%, 41% ± 4%, 49% ± 5%, and 55% ± 13%, respectively. The best survival and cure rates were obtained in the group treated with (131)I-antiEGFR-BSA-PCL. The animals injected with (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL showed more uniform focused liposome distribution within the tumor area. This study demonstrated the potential beneficial application of (131)I-antiEGFR-BSA-PCL for treating colorectal cancer. (131)I-antiEGFR-BSA-PCL suppressed the development of xenografted colorectal cancer in nude mice, thereby providing a novel candidate for receptor-mediated targeted radiotherapy.

Curcumin Combined with Oxaliplatin Effectively Suppress Colorectal Carcinomain vivoThrough Inducing Apoptosis

Studies have shown chemopreventive and/or chemotherapeutic effects of several curcumin-based combinatorial treatments on colorectal cancer cells. However, their in vivo effects remain unclear. This study has demonstrated the therapeutic effect of curcumin and oxaliplatin, alone or in combination, on subcutaneously xenografted LoVo human colorectal cancer cells in immunodeficient (nu/nu) mice in vivo. Combinatorial administration of curcumin and oxaliplatin evidently inhibited the growth of colorectal cancer in nude mice, which was significantly more effective than either agent alone. Curcumin combined with oxaliplatin treatment induced apoptosis, accompanied by ultrastructural changes and cell cycle arrest in S and G2/M phases. Further mechanism analysis indicated that while the number of apoptotic tumor cells and the expression of Bax, caspase-3, and poly (ADP-ribose) polymerase (PARP) increased significantly, the expression of Bcl-2, survivin, HSP70, pro-caspase-3, and pro-PARP were dramatically suppressed in tumor cells after the treatment with combinatorial curcumin and oxaliplatin for 22 days. Taken together, the present study has demonstrated that administration of combined curcumin and oxaliplatin effectively suppressed colorectal carcinoma in vivo through inducing apoptosis and thus may provide an effective treatment for colorectal carcinoma.

Ursolic Acid Inhibits Growth and Metastasis of Human Colorectal Cancer in an Orthotopic Nude Mouse Model by Targeting Multiple Cell Signaling Pathways: Chemosensitization with Capecitabine

Development of chemoresistance, poor prognosis, and metastasis often renders the current treatments for colorectal cancer (CRC) ineffective. Whether ursolic acid, a component of numerous medicinal plants, either alone or in combination with capecitabine, can inhibit the growth and metastasis of human CRC was investigated. The effect of ursolic acid on proliferation of CRC cell lines was examined by mitochondrial dye uptake assay, apoptosis by esterase staining, NF-κB activation by DNA-binding assay, and protein expression by Western blot. The effect of ursolic acid on the growth and chemosensitization was also examined in orthotopically implanted CRC in nude mice. We found that ursolic acid inhibited the proliferation of different colon cancer cell lines. This is correlated with inhibition of constitutive NF-κB activation and downregulation of cell survival (Bcl-xL, Bcl-2, cFLIP, and survivin), proliferative (cyclin D1), and metastatic (MMP-9, VEGF, and ICAM-1) proteins. When examined in an orthotopic nude mouse model, ursolic acid significantly inhibited tumor volume, ascites formation, and distant organ metastasis, and this effect was enhanced with capecitabine. Immunohistochemistry of tumor tissue indicated that ursolic acid downregulated biomarkers of proliferation (Ki-67) and microvessel density (CD31). This effect was accompanied by suppression of NF-κB, STAT3, and β-catenin. In addition, ursolic acid suppressed EGF receptor (EGFR) and induced p53 and p21 expression. We also observed bioavailability of ursolic acid in the serum and tissue of animals. Overall, our results show that ursolic acid can inhibit the growth and metastasis of CRC and further enhance the therapeutic effects of capecitabine through the suppression of multiple biomarkers linked to inflammation, proliferation, invasion, angiogenesis, and metastasis.

Abstract 1778: Anticancer activities and regulatory effects on apoptotic gene expression of Bufalin in the orthotopic transplantation model of human colorectal cancer in nude mice

Abstract Background: Bufalin, a natural small-molecule compound from the Traditional Chinese Medicine “Chansu”, shows inhibitory effects on many human cancer cells. However, whether or not Bufalin has anticancer activity in human colorectal cancer (CRC), and its possible underlying mechanism, remains unclear. Objectives: 1) To investigate anticancer activities of Bufalin in human CRC using an orthotopic transplantation model in nude mice and 2) to study possible underlying mechanisms related to apoptosis. Materials and methods: An orthotopic transplantation tumor model was established by implanting human CRC HCT-116 cells into the colon of nude mice. Sixty mice were randomly divided into five treatment groups (12 mice in each group): normal saline group, 5-FU group, and 3 Bufalin groups with low (BL), medium (BM), and high (BH) doses. After cell transplantation, 0.2 ml normal saline, 25 mg/kg 5-Fu, or 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg Bufalin was injected intraperitoneally in mice in each corresponding group, daily for 7 days (days 15 to 21). Six mice in each group were sacrificed on day 24. Tumor size, growth inhibition rate, and morphological changes of tumor cell under microscopy were recorded. Apoptosis was determined by TUNEL staining, mRNA and protein expressions of Bcl-xl and Bax were evaluated with real-time RT-PCR and immunohistochemical staining, respectively. The overall survival (OS) was determined in the remaining 6 mice in each group. Results: The tumor size in mice treated with 5-Fu, BL, BM, or BH was significantly smaller than that in mice treated with normal saline (p&amp;lt;0.05), with the tumor inhibitor rates of 64.3%, 51.4%, 48.7%, and 36.3%, respectively. Under microscopy, increased tumor necrosis was observed in the 5-Fu, BM and BH groups. The tumor apoptotic rate in each of 3 Bufalin treated groups was significantly higher than that in the normal saline group (p&amp;lt;0.05). Real time RT-PCR and immunohistochemical staining showed both mRNA and protein expressions of Bcl-xl were decreased, whereas the expression of Bax increased, in tumor treated with Bufalin. The OS was prolonged in groups of BL and BM (p&amp;lt;0.05). Conclusion: Bufalin has significant anticancer activity in a human CRC orthotopic transplantation model in nude mice, and induces tumor cell apoptosis, which may be associated with the down-regulation of Bcl-xl and up-regulation of Bax in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2011-1778

Effect of APRIL on growth and apoptosis in transplanted tumor with human colorectal cancer cell line SW480 in nude mice

To study the effect of pGCsi-H1-APRIL on the growth of human colorectal cancer cells in transplated tumor in nude mice and to improve the effect of APRIL on proliferation and apoptosis of colorectal cancer (CRC). Human CRC model was established in nude mice, and the nude mice were treated with APRIL siRNA twice per week for 2 weeks. APRIL mRNA expression was surveyed by PCR and APRIL protein expression was detected by immunohistochemistry. The expression of PCNA protein was detected by ELISA. The expression of bcl-2 and bcl-xl was assessed by immunohistochemical staining, and TUNEL staining was used to detect apoptosis. The expression of APRIL mRNA in the APRIL siRNA group was (0.13 ± 0.05) × 10(-3), significantly lower than that in the vector group (0.95 ± 0.04) × 10(-3) and the PBS group (0.96 ± 0.05) × 10(-3). The expression of APRIL protein in the APRIL siRNA group was (87.5 ± 5.0)% lower than that in the vector and PBS groups (P < 0.05). APRIL siRNA significantly suppressed the growth of SW480 tumor: the IR (inhibitory rate) of APRIL siRNA group was (60.7 ± 1.5)% (P < 0.05). The expression of PCNA in APRIL siRNA group was (176.8 ± 18.1) ng/ml, was (56.5 ± 2.0)% lower than that of PBS group (328.4 ± 22.8) ng/ml. Furthermore, the expressions of anti-apoptosis proteins bcl-2 and bcl-xl of APRIL siRNA group were (82.6 ± 4.5)% and (79.2 ± 3.5)% lower than those of the PBS group. The apoptotic rate of the APRIL siRNA group was 40.1% ± 2.5%, significantly higher than that in the vector group (2.5 ± 0.1)% and PBS group (2.5 ± 0.2)% (P < 0.05). APRIL siRNA may significantly suppress the growth and promote apoptosis in transplanted tumor of human colorectal cancer in nude mice. APRIL may become a candidate gene of gene therapy of human colorectal cancer.

Effects of APRIL-targeted RNA interference on colorectal cancer in nude mice in vivo

Objective To study the effects of a proliferation-inducing hgand (APRIL) siRNA on the growth of human colorectal tumors in nude mice. Methods SW480 cells were transplanted into nude mice,and when the tumors were about 100-200 mm~3 , all nude mice were divided into three groups: APRIL siRNA injection group, pGC-vector injection group and PBS injection group, 5 nude mice in each group. Then intratumoral injections were respectively performed. Tumor volumes were calculated every before in-jection. In the end of experiment, all nude mice were sacrificed. APRIL mRNA and protein expressions was detected by reverse transcription-pelymerase chain reaction (RT-PCR) and immunohistochemistry, re-spectively. HE strain of Tumor, the lung and liver tissues were stained with HE for pathological examina-tion. By using immunohistochemistry, the expression of APRIL, Ki-67 and matrix metalloproteinase (MMP) -2 was detected. Results The intratumoral injection of APRIL siRNA significantly suppressed the growth of tumors compared with controls [(0.78±0.04) vs (1.60±0.07), (1.66±0.06)cm~3] (P 0.05 ). Conclusion Knockdown of APRIL expression in colorectal cancer by RNAi significantly suppressed the tumor cell proliferation and metastasis activity. RNAi targeting APRIL would be a potential therapy for human colorectal cancer. Key words: RNA interference; Colorectal carcinoma; Proliferation; Metastasis

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice

To investigate the inhibitory and anti-metastatic effect of mutant p27 gene (p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism. Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intra-tumoral injection of Ad-p27mt in a pre-established transplantation model of human colorectal cancer in nude mice. Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor. Expression of MMP-9 in tumor tissue was detected by immunohistochemistry. The average sizes of transplantation tumors were 1.94 +/- 0.67 cm(3), 2.75 +/- 0.83 cm(3) and 3.01 +/- 0.76 cm(3) in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average proliferation rates were 37.34% +/- 1.45%, 53.16% +/- 3.27% and 54.48% +/- 2.43%, in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average apoptosis rates were 19.79% +/- 3.32%, 6.38% +/- 4.91% and 7.25% +/- 5.20% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). The average MMP-9 expression rates were 20%, 75% and 66.7% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells, as well as decrease transpl-anted tumor metastasis.

Anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability in nude mice

To study the anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability (MSI) in nude mice and to explore its mechanism. A colostomy was performed on the caecum of nude mice. Tumor fragments collected from the subcutaneous tumor of hMSH2-absence colon carcinoma Lovo cell line were surgically implanted onto the submucosa of the caecum during colostomy to establish the model. Then, the nude mice were divided into untreated group and 50, 75 and 100 mg/kg tea polyphenols groups. The mice in tea polyphenols-treated groups were given intra-abdominal injection of 50, 75 and 100 mg/kg tea polyphenols respectively. The inhibition rates of tumors were calculated, and microsatellite instability (MSI) and the alteration of transforming growth factor-beta1 (TGF-beta1), TGF-beta2 and insulin-like growth factor (IGF) were detected by Genescan method at different times after the injection. The tumor volumes of the three groups began to decrease at the 1st week and decreased most greatly from 2 to 3 weeks after treatment, and then the tumors tended to increase. The study found that tea polyphenols could inhibit the tumor growth. The tumor inhibition rates in the three treated groups were significantly higher than those in untreated group 1, 2, 3 and 4 weeks after treatment (P<0.01). Detection of MSI showed that the colorectal tumor in the untreated group presented with four MSI signs, including BAT-25, D2S123, D5S346 and D17S250, and TGF-beta1, TGF-beta2, IGF expressions. After using the tea polyphenols, the microsatellite tended to become stable. Tea polyphenols can inhibit the mismatch-repair-gene deficient colorectal cancer in nude mice by down-regulating the microsatellite instability.