Characterization of colonic dendritic cells in normal and colitic mice

AIM： Recent studies demonstrating the direct involvement of dendritic cells （DC） in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS： Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient （IL2-/-） mice that develop colitis. RESULTS： In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type i myeloid （CD11c^＋, CD11b^＋, B220, CD8） DC made up the largest （40-45%） population and all DC expressed low levels of CD80, CD86, and CD40, and had high endooltic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes （MLN）. The majority （〉85%） of DC in the colon and MLN of IL2./ mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION： These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.

CD74 is a survival receptor on colon epithelial cells

AIM: To investigate the expression and function of CD74 in normal murine colon epithelial cells (CEC) and colon carcinoma cells. METHODS: Expression of CD74 mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and fluorescence-activated cell sorter (FACS). The effect of migration inhibitory factor (MIF) on the survival of normal CEC from C57BL/6, NOD/SCID, and CD74 def icient mice both in vitro and in vivo, and on the CT26 carcinoma cell line was analyzed by (quantitative) qRT-PCR, RTPCR, Western blotting and FACS. RESULTS: CD74 was found to be expressed on normalCEC. Stimulation of CD74 by MIF induced a signaling cascade leading to up-regulation of Bcl-2 expression, resulting in a signif icant increased survival of CEC. CD74 was also expressed on the CT26 colon carcinoma cell line and its stimulation by MIF resulted in enhanced cell survival, up-regulation of Akt phosphorylation and Bcl-2 expression.

Therapeutic implications of colon cancer stem cells

Colorectal cancer is the second most common cause of cancer-related death in many industrialized countries and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support with regard to several solid tumors, including colorectal cancer. According to the cancer stem cell hypothesis, cancer can be considered a disease in which mutations either convert normal stem cells into aberrant counterparts or cause a more differentiated cell to revert toward a stem cell-like behaviour; either way these cells are thought to be responsible for tumor generation and propagation. The statement that only a subset of cells drives tumor formation has major implications for the development of new targeted therapeutic strategies aimed at eradicating the tumor stem cell population. This review will focus on the biology of normal and malignant colonic stem cells, which might contribute to our understanding of the mechanisms responsible for tumor development and resistance to therapy.

Anti-cancer effect of ethylacetate fraction from Orostachys japonicus on HT-29 human colon cancer cells by induction of apoptosis through caspase-dependent signaling pathway

Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus(0. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry.Morphological changes in the nucleus were observed, using a fluorescence microscope with4',6-diamidino-2-phenylindole(DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner,while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G_1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from0. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.

Tetrandrine:A Potent Abrogator of G_2 Checkpoint Function in Tumor Cells and Its Mechanism

Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio （SER） of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis （M phase）. Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

Intestinal stem cells and the colorectal cancer microenvironment

Colorectal cancer (CRC) remains a highly fatal condition in part due to its resilience to treatment and its propensity to spread beyond the site of primary occurrence. One possible avenue for cancer to escape eradication is via stem-like cancer cells that, through phenotypic heterogeneity, are more resilient than other tumor constituents and are key contributors to cancer growth and metastasis. These proliferative tumor cells are theorized to possess many properties akin to normal intestinal stem cells. Not only do these CRC &#x0201c;stem&#x0201d; cells demonstrate similar restorative ability, they also share many cell pathways and surface markers in common, as well as respond to the same key niche stimuli. With the improvement of techniques for epithelial stem cell identification, our understanding of CRC behavior is also evolving. Emerging evidence about cellular plasticity and epithelial mesenchymal transition are shedding light onto metastatic CRC processes and are also challenging fundamental concepts about unidirectional epithelial proliferation. This review aims to reappraise evidence supporting the existence and behavior of CRC stem cells, their relationship to normal stem cells, and their possible dependence on the stem cell niche.

Testosterone induced apoptosis in colon cancer cells is regulated by PI3K/Racl signaling

Recently, it has been reported that testosterone membrane signaling regulates actin reorganization and induces pro-apoptotic responses in colon tumor cells. In the present study the membrane androgen receptors （mARs）-induced activation of Rac I GTPase and the involvement of PI3K/Racl signaling in controlling the apoptotic responses in testosterone treated Caco2 colon cancer cells has been analyzed. In line with previous findings, activation of mAR by testosterone conjugates triggered early and transient actin reorganization as indicated by the significant decrease of the G/Total actin ratio after 15- and 30-min treatment of the cells. Interestingly, stimulation of mAR rapidly activated the Racl GTPase. This effect was evident after 15 min and persisted for at least 24 h. Testosterone induced Rac I activation was fully blocked in Caco2 cells pre-treated with the PI3K inhibitor wortmannin, indicating that Racl signaling is acting downstream of the PI3K pathway. Remarkably, when cells were pre-treated with wortmannin that blocks the PI3K/Racl signaling, apoptotic response was almost fully inhibited. These finding suggest that Racl activation, triggering actin redistribution, is involved in testosterone induced pro-apoptotic responses governed by mAR activation and emphasize the regulatory role of PI3K/Racl signaling in colon tumors.

Growth inhibition of colon cancer cells by compounds affecting AMPK activity

AIM:To determine if other molecules reported to modulate AMP-dependent protein kinase(AMPK)activ-ity would have effects resembling those of metformin and phenformin on colon cancer cell proliferation and metabolism.METHODS:Studies were performed with four hu-man colon cancer cell lines,Caco-2,HCT116,HT29 and SW1116.The compounds that were studied included A-769662,5-aminoimidazole-4-carboxamide-1-ribofu-ranoside,butyrate,(-)-epigallocatechin gallate(EGCG),KU-55933,quercetin,resveratrol and salicylates.The parameters that were measured were cell proliferation and viability,glucose uptake,lactate production and acidification of the incubation medium.RESULTS:Investigations with several molecules that have been reported to be associated with AMPK activa-tion(A-769662,5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside,EGCG,KU-55933,quercetin,resve-ratrol and salicylates)or AMPK inhibition(compound C)failed to reveal increased medium acidification and increased glucose uptake in colon cancer cells as previ-ously established with metformin and phenformin.The only exception was 5-aminosalicylic acid with which there were apparently lower glucose levels in the me-dium after incubation for 72 h.Further study in the absence of cells revealed that the effect was an artifact due to inhibition of the enzyme-linked glucose assay.The compounds were studied at concentrations that inhibited cell proliferation.CONCLUSION:It was concluded that treatment with several agents that can affect AMPK activity resulted in the inhibition of the proliferation of colon cancer cells under conditions in which glucose metabolism is not enhanced,in contrast to the effect of biguanides.

Effects of silkworm pupa protein on apoptosis and energy metabolism in human colon cancer DLD-1 cells

Objective:To investigate the effects of silkworm pupa(Bombyx mori) protein(SPP) on cell proliferation,apoptosis and energy metabolism in human colon cancer cells DLD-1.Methods:CCK-8 was used to detect cell proliferation rate after 72 h of cell culture for the control group(normal cultured DLD-1 cells) and SPP dose groups;Annexin-V/PI was applied to observe cell apoptosis;XFe24 Extracellular Flux Analyzer was used to detect cell mitochondrial respiratory function and glycolytic function.Results:Comparing with the control,SPP significantly inhibited the proliferation of DLD-1 cells with all the dosage tested(P <0.01);flow cytometry showed that SPP significantly promoted apoptosis(P<0.05).Additionally,SPP could significantly inhibited mitochondrial metabolism and glycolysis of DLD-1 cells and decreased cell energy metabolism in all groups treated with different doses.Conclusion:SPP can cause oxidative damage,promote apoptosis,and reduce mitochondrial respiratory and glycolysis rate in colon cancer DLD-1 cells,which reveals that SPP has the potential to serve as the anti-cancer drugs in the future,but further experimental evidence is needed.

Targeting BCRP/ABCG2 by RNA Interference Enhances the Chemotherapy Sensitivity of Human Colon Cancer Side Population Cells

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.

Identification of a Native Novel Oncolytic Immunoglobulin on Exfoliated Colon Epithelial Cells: A Bispecific Heterodimeric Chimera of IgA/IgG*

Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5 μM - 5.0 μM and 5.0 μM - 8.0 μM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

Identification of integrin β6 gene promoter and analysis of its transcription regulation in colon cancer cells

BACKGROUND The integrinβ6 gene,which is expressed in epithelial cancer,plays a pivotal role in various aspects of cancer progression.The present research for integrinβ6 regulation mainly focuses on the post-transcription and translation related regulation mechanism and its role in tumorigenesis.The mechanisms of how the integrinβ6 gene is regulated transcriptionally,and the promoter and transcription factors responsible for basic transcription of integrinβ6 gene remain unknown.AIM To clone and characterize the integrinβ6 promoter.METHODS Software analysis was used to predict the region of integrinβ6 promoter.Luciferase reporter plasmids,which contained the integrinβ6 promoter,were constructed.Element deletion analysis was performed to identify the location of core promoter and binding sites for transcription factors.RESULTS The regulatory elements for the transcription of the integrinβ6 gene were located between-286 and-85 and contained binding sites for transcription factors such as STAT3 and Ets-1.CONCLUSION For the first time,we found the region ofβ6 core promoter and demonstrated the binding sites for transcription factors such as Ets-1 and STAT3,which are important for integrinβ6 promoter transcription activity.These findings are important for investigating the mechanism of integrinβ6 activation in cancer progression.

Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its possible mechanism of action. Methods： Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca2＋ was assessed using fluorescent intensity （FI） of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results： In the in vitro experiment, RS （0.02, 0.2, 2, 20 g/L） inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations （P 〈 0.01） for both Peak （the maximal contraction amplitude） and Area （the area under curves）. Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant （P 〈 0.01）, as was the inhibition of the Area values in all RS groups （P 〈 0.05）. Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect （P 〈 0.01）. In experiments using primary smooth muscle cell cultures in Ca2＋ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values （P 〈 0.05）, and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant （P 〈 0.01）. However, in Ca2＋-free buffer, FS had no significant effect on cell fluorescence. Conclusion： RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α -adrenoceptors, but not β -adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2＋ from extracellular sources, but may had no effect on the release of Ca2＋ from sarcoplasmic reticulum and endoplasmic reticulum.

Colon signet-ring cell carcinoma with chylous ascites caused by immunosuppressants following liver transplantation:A case report

BACKGROUND Chylous ascites is caused by disruption of the lymphatic system,which is characterized by the accumulation of a turbid fluid containing high levels of triglycerides within the abdominal cavity.The two most common causes are cirrhosis and tuberculosis,and colon signer ring cell carcinoma(SRCC)due to the use of immunosuppressants is extremely rare in cirrhotic patients after liver transplantation,making it prone to misdiagnosis and missed diagnosis.CASE SUMMARY A 52-year-old man who underwent liver transplantation and was administered with immunosuppressants for 8 months was admitted with a 3-month history of progressive abdominal distention.Initially,based on lymphoscintigraphy and lymphangiography,lymphatic obstruction was considered,and cystellar chyli decompression with band lysis and external membrane stripping of the lymphatic duct was performed.However,his abdominal distention was persistent without resolution.Abdominal paracentesis revealed allogenic cells in the ascites,and immunohistochemistry analysis revealed adenocarcinoma cells with phenotypic features suggestive of a gastrointestinal origin.Gastrointestinal endoscopy was performed,and biopsy showed atypical signet ring cells in the ileocecal valve.The patient eventually died after a three-month follow-up due to progression of the tumor.CONCLUSION Colon SRCC,caused by immunosuppressants,is an unusual but un-neglected cause of chylous ascites.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel

AIM:To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoliposomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G2/M cells and induced apoptosis, but significantly decreased the percentage of cells in G2/G1 and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

Growth hormone releasing peptide 2 reverses anorexia associated with chemotherapy with 5-fluoruracil in colon cancer cell-bearing mice

The cancer-associated anorexia-cachexia syndrome is observed in 80% of patients with advanced-stage cancer, and is one of the major obstacles in chemo- therapy. Ghrelin is a orexigenic hormone that has been proposed to prevent anorexia. Aim of the study was to determine whether the addition of the ghrelin agonist growth hormone releasing peptide 2 (GHRP-2) to cytotoxic therapy with 5-fluoruracil (5-FU) prevents the anorexia associated with chemotherapy in cancer cachectic mice. Thirty-three BALB/c female tumourbearing mice were randomized to receive a solution containing: (a) placebo; (b) GHRP-2; (c) 5-FU; or (d) 5-FU + GHRP-2. Ten BALB/c no tumour-bearing mice received placebo solution. Food intake and survival were checked. Six hours after the drug injection the cumulative food intake was signifi cantly increased in mice treated with the combination of 5-FU + GHRP-2 versus the 5-FU alone (P = 0.0096). On day 3, the cumulative food intake of mice treated with GHRP-2,5-FU and 5-FU + GHRP-2 signifi cantly increased com- pared with naive and vehicle groups (P = 0.0007, P = 0.0038 and P = 0.0166, respectively). The median survival time was longer in 5-FU + GHRP-2 treated mice than in those with 5-FU, although it was not signifi cant (18 d versus 15.5 d, P = 0.7). For the fi rst time, we demonstrated that the addition of GHRP-2 to cytotoxic therapy with 5-FU improved appetite in tumour-bearing mice with anorexia/cachexia syndrome in early stage. These data suggest that GHRP-2 may improve the effi cacy of therapy and the quality of life of cancer patients thank to the amelioration of their nutritional state.