Driver gene KRAS aggravates cancer-associated stroke outcomes.

The incidence of cancer-associated stroke has increased with the prolonged survival times of cancer patients. Recent genetic studies have led to progress in cancer therapeutics, but relationships between oncogenic mutations and stroke remain elusive. Here, we focused on the driver gene KRAS, which is the predominant RAS isoform mutated in multiple cancer types, in cancer associated stroke study. KRASG13D/- and parental human colorectal carcinoma HCT116 cells were inoculated into mice that were then subjected to a photochemically-induced thrombosis model to establish ischemic stroke. We found that cancer inoculation exacerbated neurological deficits after stroke. Moreover, mice inoculated with KRASG13D/- cells showed worse neurological deficits after stroke compared with mice inoculated with parental cells. Stroke promoted tumor growth, and the KRASG13D/- allele enhanced this growth. Brain RNA sequencing analysis and serum ELISA showed that chemokines and cytokines mediating pro-inflammatory responses were upregulated in mice inoculated with KRASG13D/- cells compared with those inoculated with parental cells. STAT3 phosphorylation was promoted following ischemic stroke in the KRASG13D/- group compared with in the parental group, and STAT3 inhibition significantly ameliorated stroke outcomes by mitigating microglia/macrophage polarization. Finally, we compared the prognosis and mortality of colorectal cancer patients with or without stroke onset between 1 January 2007 and 31 December 2020 using a hospital-based cancer registry and found that colorectal cancer patients with stroke onset within 3 months after cancer diagnosis had a worse prognosis. Our work suggests an interplay between KRAS and ischemic stroke that may offer insight into future treatments for cancer-associated stroke.

Uev1A promotes breast cancer cell migration by up-regulating CT45A expression via the AKT pathway.

BACKGROUND: UEV1A encodes a ubiquitin-E2 variant closely associated with tumorigenesis and metastasis, but its underlying mechanism in promoting metastasis remains to be investigated. METHODS: In this study, we experimentally manipulated UEV1A and CT45A gene expression and monitored their effects on cancer-related gene expression, cell migration and the signal transduction cascade. RESULTS: It was found that UEV1A overexpression induces CT45A family gene expression in breast cancer cells. Indeed, ectopic expression of UEV1A was sufficient to induce CT45A and its downstream genes involved in tumorigenesis, epithelial-mesenchymal transition (EMT), stemness and metastasis, and to promote cell migration and EMT signaling. Consistently, depletion of CT45A abolished the above effects, indicating that CT45A is a critical downstream effector of Uev1A. The Uev1A-induced cell migration and EMT signaling was dependent on AKT but independent of NF-κB, indicating that CT45A acts downstream of the AKT pathway. CONCLUSIONS: Based on previous reports and observations in this study, we propose that the Ubc13-Uev1A complex activates AKT through K63-linked polyubiquitination, which leads to enhanced CT45A expression, stimulated cell migration and EMT signaling in breast cells. Since similar effects were also observed in a colorectal cancer cell line, the Ubc13/Uev1A-AKT-CT45A axis may also promote tumorigenesis and metastasis in other tissues.

Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

Pharmacological induction of mesenchymal-epithelial transition via inhibition of H2S biosynthesis and consequent suppression of ACLY activity in colon cancer cells.

Hydrogen sulfide (H2S) is an important endogenous gaseous transmitter mediator, which regulates a variety of cellular functions in autocrine and paracrine manner. The enzymes responsible for the biological generation of H2S include cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). Increased expression of these enzymes and overproduction of H2S has been implicated in essential processes of various cancer cells, including the stimulation of metabolism, maintenance of cell proliferation and cytoprotection. Cancer cell identity is characterized by so-called "transition states". The progression from normal (epithelial) to transformed (mesenchymal) state is termed epithelial-to-mesenchymal transition (EMT) whereby epithelial cells lose their cell-to-cell adhesion capacity and gain mesenchymal characteristics. The transition process can also proceed in the opposite direction, and this process is termed mesenchymal-to-epithelial transition (MET). The current project was designed to determine whether inhibition of endogenous H2S production in colon cancer cells affects the EMT/MET balance in vitro. Inhibition of H2S biosynthesis in HCT116 human colon cancer cells was achieved either with aminooxyacetic acid (AOAA) or 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE). These inhibitors induced an upregulation of E-cadherin and Zonula occludens-1 (ZO-1) expression and downregulation of fibronectin expression, demonstrating that H2S biosynthesis inhibitors can produce a pharmacological induction of MET in colon cancer cells. These actions were functionally reflected in an inhibition of cell migration, as demonstrated in an in vitro "scratch wound" assay. The mechanisms involved in the action of endogenously produced H2S in cancer cells in promoting (or maintaining) EMT (or tonically inhibiting MET) relate, at least in part, in the induction of ATP citrate lyase (ACLY) protein expression, which occurs via upregulation of ACLY mRNA (via activation of the ACLY promoter). ACLY in turn, regulates the Wnt-ß-catenin pathway, an essential regulator of the EMT/MET balance. Taken together, pharmacological inhibition of endogenous H2S biosynthesis in cancer cells induces MET. We hypothesize that this may contribute to anti-cancer / anti-metastatic effects of H2S biosynthesis inhibitors.

Low-dose DNA demethylating therapy induces reprogramming of diverse cancer-related pathways at the single-cell level.

BACKGROUND: Epigenetic reprogramming using DNA demethylating drugs is a promising approach for cancer therapy, but its efficacy is highly dependent on the dosing regimen. Low-dose treatment for a prolonged period shows a remarkable therapeutic efficacy, despite its small demethylating effect. Here, we aimed to explore the mechanisms of how such low-dose treatment shows this remarkable efficacy by focusing on epigenetic reprograming at the single-cell level. METHODS: Expression profiles in HCT116 cells treated with decitabine (DAC) were analyzed by single-cell RNA-sequencing (scRNA-seq). Functional consequences and DNA demethylation at the single-cell level were analyzed using cloned HCT116 cells after DAC treatment. RESULTS: scRNA-seq revealed that DAC-treated cells had highly diverse expression profiles at the single-cell level, and tumor-suppressor genes, endogenous retroviruses, and interferon-stimulated genes were upregulated in random fractions of cells. DNA methylation analysis of cloned HCT116 cells revealed that, while only partial reduction of DNA methylation levels was observed in bulk cells, complete demethylation of specific cancer-related genes, such as cell cycle regulation, WNT pathway, p53 pathway, and TGF-ß pathway, was observed, depending upon clones. Functionally, a clone with complete demethylation of CDKN2A (p16) had a larger fraction of cells with tetraploid than parental cells, indicating induction of cellular senescence due to normalization of cell cycle regulation. CONCLUSIONS: Epigenetic reprogramming of specific cancer-related pathways at the single-cell level is likely to underlie the remarkable efficacy of low-dose DNA demethylating therapy.

Association between variation of circulating 25-OH vitamin D and methylation of secreted frizzled-related protein 2 in colorectal cancer.

BACKGROUNDS: Colorectal cancer (CRC) results from the accumulation of epigenetic and genetic changes in colon cells during neoplasic transformation, which the activation of Wingless (Wnt) signaling pathway is a common mechanism for CRC initiation. The Wnt pathway is mainly regulated by Wnt antagonists, as secreted frizzled-related protein (SFRP) family. Indeed, SFRP2 is proposed as a noninvasive biomarker for CRC diagnosis. Vitamin D also antagonizes Wnt signaling in colon cancers cells. Several studies showed that vitamin D was able to alter DNA methylation, although this mechanism is not yet clear. Therefore, the aim of this study was to find an association between circulating 25-OH vitamin D (30th percentile of vitamin D) and the SFRP2 methylation. METHODS: A total of 67 CRC patients were included in the study. These patients were subdivided into two groups based on their 30th percentile vitamin D (20 patients were below, and 47 participants were above the 30th percentile of vitamin D). We investigated the SFRP2 methylation in peripheral blood mononuclear cells (PBMCs), visceral adipose tissue (VAT), CRC tumor tissue, and adjacent tumor-free area. We also determined the relationship between SFRP2 methylation and methylation of carcinogenic and adipogenic genes. Finally, we tested the effect of vitamin D on the SFRP2 methylation in human colorectal carcinoma cell lines 116 (HCT116) and studied the association of neoadjuvant therapy under the 30th percentile vitamin D with SFRP2 promoter methylation. RESULTS: SFRP2 methylation in tumor area was decreased in patients who had higher levels of vitamin D. SFRP2 promoter methylation was positively correlated in tumor area with insulin and homeostasis model assessment of insulin resistance (HOMA-IR) but negatively correlated with HDL-c. SFRP2 methylation was also correlated with T cell lymphoma invasion and metastasis 1 (TIAM1) methylation in tumor area and CCAAT/enhancer-binding protein alpha (C/EBPα) in VAT. Treatment with vitamin D did not affect SFRP2 methylation in HCT116 cell line. Finally, neoadjuvant treatment was correlated with higher circulating 25-OH vitamin D and SFRP2 methylation under linear regression model. CONCLUSION: Our results showed that higher circulating vitamin D is associated with low SFRP2 promoter methylation. Therefore, our results could suggest that vitamin D may have an epigenetic effect on DNA methylation. Finally, higher vitamin D could contribute to an improvement response to neoadjuvant treatment.

p53 dependent LGR5 inhibition and caspase 3 activation are critically involved in apoptotic effect of compound K and its combination therapy potential in HCT116 cells.

Though ginsenoside metabolite compound K was known to have antitumor effect in several cancers, its underlying apoptotic mechanism still remains unclear so far. Thus, in the present study, the apoptotic mechanism of compound K was explored in colorectal cancer cells (CRCs) in association with leucine rich repeat containing G protein-coupled receptor 5 (LGR5) that was overexpressed in colorectal cancers with poor survival rate. Here compound K significantly reduced viability of HCT116p53+/+ cells better than that of HCT116p53-/- cells. Consistently, compound K increased sub G1 population and attenuated the expression of LGR5, c-Myc, procaspase3, Pin1 in HCT116p53+/+ cells more than in HCT116p53-/- cells. Conversely, caspase 3 inhibitor Z-DEVD-FMK reversed inhibitory effect of compound K on LGR5, c-Myc and procaspase3 in HCT116 cells. Consistently, inhibition of LGR5 using transfection method enhanced suppression of pro-PARP, Bcl-xL c-Myc, Snail and Pin1 in compound K treated HCT116p53+/+ cells. Furthermore, compound K synergistically potentiated antitumor effect of 5-fluorouracil (5-FU) or Doxorubicin to reduce the survival genes and cytotoxicity in HCT116p53+/+ cells. Overall, our findings provide scientific insight that compound K induces apoptosis in colon cancer cells via caspase and p53 dependent LGR5 inhibition with combination therapy potential with 5-FU or doxorubicin.

Screening and characterization of marine actinomycetes from the northern Oman Sea sediments for cytotoxic and antimicrobial activity

A total of 168 actinomycete colonies were isolated from 14 sediment samples of the northern parts of the Oman Sea and were screened for cytotoxic and antimicrobial activity. Among four media and two treatments, the glucose arginine agar medium (18%) and heat treatment (28.3%) showed maximum isolation rate of actinomycetes. Preliminary characterization revealed that the members of Streptomycetaceae were widely distributed (66%) in the most of the sampling stations followed by Micromonosporaceae (14%), Nocardiaceae (6%), and Pseudonocardiaceae (4%), respectively. Approximately, 23.8% of the isolates inhibited the growth of at least one of the microbial test strains, while the majority of them belonged to the Streptomycetaceae family. Minimum inhibitory concentrations of the ethyl acetate culture extracts of the five most putative isolates varied from 64 μg/mL against Micrococcus luteus and Candida albicans to 1 mg/mL against Aspergillus niger. These extracts showed significant cytotoxic activity at18.74-193.5 μg/mL on the human breast (MCF7), colon (HCT 116), and liver (HepG2) tumor cell lines while exhibited less or no cytotoxicity on the normal cell line (HUVEC). Interestingly, IFSRI 193 extract selectively inhibited the growth of HCT 116 cell line and gram-positive bacteria. 16S rRNA gene sequencing revealed that the potent isolates have 97 to 99% similarity with S. chartreusis, S. cacaoi, S. sampsonii, S. qinglanensis, and S. diastaticus. These results suggested that the five Streptomyces strains could be considered candidates for discovering the antitumor antibiotics

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Chelidonine inhibits TNF-α-induced inflammation by suppressing the NF-κB pathways in HCT116 cells.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a complex that regulates several hundreds of genes, including those involved in immunity and inflammation, survival, proliferation, and the negative feedback of NF-κB signaling. Chelidonine, a major bioactive, isoquinoline alkaloid ingredient in Chelidonium majus, exhibits antiinflammatory pharmacological properties. However, its antiinflammatory molecular mechanisms remain unclear. In this work, we explored the effect of chelidonine on TNF-induced NF-κB activation in HCT116 cells. We found chelidonine inhibited the phosphorylation and degradation of the inhibitor of NF-κB alpha and nuclear translocation of RELA. Furthermore, by inhibiting the activation of NF-κB, chelidonine downregulated target genes involved in inflammation, proliferation, and apoptosis. Chelidonine also inhibited mitogen-activated protein kinase pathway activation by blocking c-Jun N-terminal kinase and p38 phosphorylation. These results suggest that chelidonine may be a potential therapeutic agent against inflammatory diseases in which inhibition of NF-κB activity plays an important role.

Phenethyl isothiocyanate suppresses cancer stem cell properties in vitro and in a xenograft model.

BACKGROUND: Cancer stem cells (CSCs) are a subset of cells within the bulk of a tumor that have the ability to self-renew and differentiate, and are thus associated with cancer invasion, metastasis, and recurrence. Phenethyl isothiocyanate (PEITC) is a natural compound found in cruciferous vegetables such as broccoli and is used as a cancer chemopreventive agent; however, its effects on CSCs are little known. PURPOSE: To evaluate the effect of PEITC on CSCs in this study by examining CSC properties. METHODS: NCCIT human embryonic carcinoma cells were treated with PEITC, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by luciferase assay and western blot. Effect of PEITC on self-renewal capacity and clonogenicity were assessed with the sphere formation, soft agar assay, and clonogenic assay in an epithelial cell adhesion molecule (EpCAM)-expressing CSC model derived from HCT116 colon cancer cells using a cell sorting system. The effect of PEITC was also investigated in a mouse xenograft model obtained by injecting nude mice with EpCAM-expressing cells. RESULTS: We found that PEITC treatment suppressed expression of the all three pluripotency factors in the NCCIT cells, in which pluripotency factors are highly expressed. Moreover, PEITC suppressed the self-renewal capacity and clonogenicity in the EpCAM-expressing CSC model. EpCAM was used as a specific CSC marker in this study. Importantly, PEITC markedly suppressed both tumor growth and expression of three pluripotency factors in a mouse xenograft model. CONCLUSION: These results demonstrate that PEITC might be able to slow down or prevent cancer recurrence by suppressing CSC stemness.

Metastasis regulation by PPARD expression in cancer cells.

Peroxisome proliferator-activated receptor-δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity for metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer.

Apigenin induces both intrinsic and extrinsic pathways of apoptosis in human colon carcinoma HCT-116 cells.

Apigenin is one of the plant-originated flavones with anticancer activities. In this study, apigenin was assessed for its in vitro effects on a human colon carcinoma line (HCT­116 cells) in terms of anti-proliferation, cell cycle progression arrest, apoptosis and intracellular reactive oxygen species (ROS) generation, and then outlined its possible apoptotic mechanism for the cells. Apigenin exerted cytotoxic effect on the cells via inhibiting cell growth in a dose-time-dependent manner and causing morphological changes, arrested cell cycle progression at G0/G1 phase, and decreased mitochondrial membrane potential of the treated cells. Apigenin increased respective ROS generation and Ca2+ release and thereby, caused ER stress in the treated cells. Apigenin shows apoptosis induction towards the cells, resulting in enhanced portion of apoptotic cells. A mechanism involved ROS generation and endoplasmic reticulum stress was outlined for the apigenin-mediated apoptosis via both intrinsic mitochondrial and extrinsic pathways, based on the assayed mRNA and protein expression levels in the cells. With this mechanism, apigenin resulted in the HCT-116 cells with enhanced intracellular ROS generation and Ca2+ release together with damaged mitochondrial membrane, and upregulated protein expression of CHOP, DR5, cleaved BID, Bax, cytochrome c, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9, which triggered apoptosis of the cells.

Resveratrol analogue, HS-1793, induces apoptotic cell death and cell cycle arrest through downregulation of AKT in human colon cancer cells.

Resveratrol, a polyphenolic compound, is a naturally occurring phytochemical and is found in a variety of plants, including grapes, berries and peanuts. It has gained much attention for its potential anticancer activity against various types of human cancer. However, the usefulness of resveratrol as a chemotherapeutic agent is limited by its photosensitivity and metabolic instability. In this study the effects of a synthetic analogue of resveratrol, HS-1793, on the proliferation and apoptotic cell death were investigated using HCT116 human colon cancer cells. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of HS-1793 on human colon cancer and its mechanisms of action have not been extensively studied. HS-1793 inhibited cell growth and induced apoptotic cell death in a concentration-dependent fashion. Induction of apoptosis was determined by morphological changes, cleavage of poly(ADP-ribose) polymerase, alteration of Bax/Bcl-2 expression ratio, and caspase activations. Flow cytometric analysis revealed that HS-1793 induced G2/M arrest in the cell cycle progression in HCT116 cells. Furthermore, HS-1793 showed more potent anticancer effects in several aspects than resveratrol in HCT116 cells. In addition, HS-1793 suppressed Akt and the phosphatidylinositol-3 kinase/Akt inhibitor LY294002 was found to enhance its induction of apoptosis. Thus, these findings suggest that HS-1793 have potential as a candidate chemotherapeutic agent against human colon cancer.

[ID1 suppress the apoptosis of HCT116 cells induced by chemotherapeutic drugs and ultraviolet radiation].

OBJECTIVE: To investigate the changes of ID1 expression in tumor cells treated with etoposide, cisplatin and ultraviolet (UV) irradiation, and explore the effect of ID1 on chemotherapeutic drug- and UV-induced apoptosis. METHODS: In the present study, upon onset of apoptosis induced by various kinds of inducers such as etoposide, cisplatin and UV irradiation, the expression level of ID1 was detected by Western blot and real-time PCR. We also analyzed the half-life of ID1 protein and stability of ID1 mRNA respectively by cycloheximide inhibition test and RT-PCR. Annexin-V assay was carried out to evaluate the contribution of ID1 protein to chemotherapeutic drug- and UV-induced apoptosis. RESULTS: ID1 expression presented a profound down-regulation in the HCT116 cells treated with etoposide, cisplatin and UV irradiation(P<0.05 for all). The apoptosis in the UV irradiation group, cisplatin group, etoposide group was (58.70±1.55)%, (35.80±0.92)% and (21.00±0.72)%, respectively, significantly higher than that of the control group(1.10±0.07)%, (1.20±0.13)% and (3.50±0.23)% (P<0.05 for all). Upon etoposide treatment, ID1 expression level was decreased via induction of mRNA instability, but not the protein degradation changes. Additionally, ectopic expression of ID1 in the HCT116 cells alleviated etoposide-, cisplatin- and UV-induced apoptosis. The results of flow eytometry revealed that the percentage of apoptotic cells in the ID1 group under the treatment of etoposide, cisplatin and UV irradiation was (23.80±0.82)%, (17.80±1.34)% and (13.40±0.53)%, respectively, significantly lower than that in the empty vector group (41.10±1.61)%, (30.40±2.67)% and (22.50±3.47)% (P<0.05 for all). CONCLUSIONS: These observations indicate that the treatment with etoposide reduces the amount of ID1 by induction of mRNA instability, and exogenously introduced ID1 protects cells against etoposide-, cisplatin- and UV irradiation-induced apoptosis. Inhibition of the ID1 bioactivity may become a new strategy in cancer treatment.

Dose-dependent effect of 2-deoxy-D-glucose on glycoprotein mannosylation in cancer cells.

High glucose consumption due to Warburg effect is one of the metabolic hallmarks of cancer. Consequently, glucose antimetabolites, such as 2-deoxy-glucose (2DG), can induce substantial growth inhibition of cancer cells. However, the inhibition of metabolic pathways is not the sole effect of 2DG on cancer cells. As mannose-mimetic molecule, 2DG is believed to interfere with normal glycosylation of proteins in cells. Here, we address how 2DG influences protein glycosylation in cancer cells and discuss possible implications of the consequences of this influence. In detail, six colorectal cancer cell lines were examined for alterations of protein glycosylation by measuring monosaccharide incorporation into cellular glycoproteins and cell surface glycosylation by lectin FACS. A significant increase in mannose incorporation was observed on treatment with 2DG (1 mM for 48 h), which was also reflected by an increased binding of the mannose-binding lectin Concanavalin A in FACS analysis. This phenomenon, which could be reversed by external addition of mannose, was not caused by 2DG-mediated mannosidase inhibition, as shown by pulse-chase experiments, arguing in favor of the hypothesis that 2DG directly influenced the incorporation of mannose. Increased mannose content was generally observed in cellular glycoproteins, including glycoproteins isolated from the plasma membrane fraction. Our results indicate that 2DG at low doses, which have only a limited metabolism-related effect on glycosylation, induces a strong increase in mannose incorporation into cellular glycoproteins. On the other hand, higher 2DG concentrations (10 and 20 mM) led to a significant decrease of absolute mannose incorporation accompanied by a dramatically reduced protein synthesis rate. 2DG-induced alterations of glycosylation may represent a novel mechanism potentially explaining the varied effects of 2DG on cancer cells. Moreover, 2DG treatment may open a path toward novel diagnostic and cancer therapeutic approaches, which specifically target altered glycoantigen structures induced by 2DG.

Regenerating gene 1B silencing inhibits colon cancer cell HCT116 proliferation and invasion.

BACKGROUND: The human regenerating gene 1B (REG1B) is found to be frequently up-regulated in many types of human tumors. It is unclear whether REG1B expression may have therapeutic value in colorectal carcinoma. Additionally, how REG1B is associated with the clinical features of colorectal carcinoma is not known. To investigate the relationship between REG1B and colorectal cancer, we analyzed REG1B expression in clinical specimens and cell lines and the effect of down-regulation of REG1B by short hairpin RNA (shRNA) in HCT116 cells. METHODS: Paraffin-embedded specimens from 30 pairs of colorectal cancer tissues and adjacent colon tissues were used to investigate the expression of REG1B by immunohistochemistry. We also examined whether REG1B itself may be related to cell proliferation, cell cycle arrest, apoptosis, migration and invasion in colon cancer HCT116 cells. RESULTS: Our results showed that REG1B was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status. The results also illustrated that REG1B silencing with shRNA inhibited cell proliferation, migration and invasion but did not induce apoptosis. Furthermore, down-regulation of REG1B induces G1-phase cell cycle arrest in colon cancer cells. CONCLUSIONS: Knockdown of REG1B can inhibit cell proliferation, migration and invasion. It may act by a mechanism regulating cell cycle progression. Thus, REG1B may be a novel candidate therapeutic target for colorectal cancer.

Antheraea pernyi silk fibroin-coated PEI/DNA complexes for targeted gene delivery in HEK 293 and HCT 116 cells.

Polyethylenimine (PEI) has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF), a natural protein rich in arginyl-glycyl-aspartic acid (RGD) peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney) 293 and HCT (human colorectal carcinoma) 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH) release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors.

Establishment and characterization of a mutagenized cell line exhibiting the 'cell-in-cell' phenotype at a high frequency.

Cell-in-cell structures represent live cell events in which one cell internalizes another. Because formation of cell-in-cell structures is a rare event in most cell types and the event is associated with cell death, there has been limited clarification of this phenomenon, and its physiological role and molecular mechanism are yet to be precisely elucidated. In this study, we established a mutagenized cell line that exhibited cell-in-cell structures at a more than 10-fold higher frequency as compared to the parent cells. Interestingly, both engulfment and invasion were increased in the mutagenized cell line as compared with that in the parent cell line in the suspension culture condition. This finding indicates that this mutagenized cell line showed an interchangeable status in terms of its ability to form cell-in-cell structures, and the system described here could be useful for elucidation of the mechanisms regulating the formation of cell-in-cell structures, including engulfment and invasion, in a given cellular environment. Further studies using this cell line are warranted to understand the mechanism of formation and biological significance of the cell-in-cell formation.

Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human urine and modulation of the glutathione levels in cancer cells by [6]-shogaol.

SCOPE: Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anticancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans. METHODS AND RESULTS: We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using LC/ESI-MS/MS. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. CONCLUSION: Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer-preventive activity of ginger.

Estrogen receptor ß potentiates the antiproliferative effect of raloxifene and affects the cell migration and invasion in HCT-116 colon cancer cells.

BACKGROUND: Estrogen receptor ß (ERß) is the predominant ER in the colorectal epithelium, whose expression is greatly reduced in colorectal cancer compared with normal colon tissue. Recent in vitro studies suggested that ERß may suppress tumor growth. No research was reported whether ERß can be used as therapeutic agent for colon cancer. METHODS: In this study, ERß gene constructed into adenoviral (Ad) vectors was used to treat colon cancer HCT-116 cells alone or in combination with raloxifene. In vitro and in vivo studies were conducted to investigate the therapeutic effects of ERß and raloxifene in HCT-116 cells. RESULTS: Our results indicated that, although Ad-ERß alone had no effect on the proliferation of HCT-116 cells, the combination of Ad-ERß with raloxifene significantly inhibited the proliferation of HCT-116 cells. The apparently apoptotic induction effects may partly explain the cytotoxicity of the two agents. The results of the study of ERß on migration and invasion of HCT-116 cells demonstrated that overexpression of ERß significantly decreased cell migration and increased invasion of cells. The antitumor efficacies of ERß as well as raloxifene were further investigated on HCT-116 tumor bearing mice. Results demonstrated that both Ad-ERß and raloxifene individually inhibited tumor growth. The combination group showed the highest inhibitory efficiency compared with other three groups. CONCLUSION: These findings demonstrated that combined administration of Ad-ERß with raloxifene represents a promising colon cancer therapeutic strategy.