Colo205 Cells Research Articles

Biomarker Discovery for Cancer Sensitivity to CDK8 Inhibitors

BackgroundA novel piperazinylpyrimidine compound, Q12, was synthesized and tested for its potential to selectively inhibit the growth of certain tumor cell lines within the NCI‐60 cell line panel. Q12 is an ATP competitive inhibitor of cyclin dependent kinase 8 (CDK 8). We studied Q12 in the context of sensitive cell lines MDA‐MB‐468 (GI50 0.06 μM) and HCT‐116 (GI50 0.27 μM) and a non‐sensitive cell line COLO 205 (GI50 30 μM). The MDA‐MD‐468 cell line represents triple negative breast cancer, a difficult type of cancer to treat because of lacking HER2, estrogen and progesterone receptors. HCT‐116 and COLO 205 are human colon cancer cell lines, which harbor β‐catenin and APC mutations respectively. We hypothesized that Q12 is a promising molecule that can be used to probe biomarker discovery for CDK8 inhibitor sensitive tumor types.Material and MethodsIn vitro studies were performed by using of MDA‐MB‐468 breast cancer cell line, HCT‐116 and COLO 205 colon cancer cell lines. GI50 was measured and apoptosis assay was performed. The growth inhibitory effect of Q12 was assessed by Muse Cell Viability Kit. To evaluate the effects of Q12 on β‐catenin and STATs, Western Blotting were used.ResultsThe cell viability essay shows Q12 potently inhibited the growth and viability of MDA‐MB‐468 cells and HCT‐116 cells, but COLO 205 cell line was resistant. Western blotting shows the reduction of STAT1 phosphorylation, a robust indicator of CDK8 target engagement, in all three cell lines upon Q12 treatment. Western blotting also showed a reduction in β‐catenin in both HCT‐116 and COLO 205 cells, but unchanged in MDA‐MB‐468 cell line, upon treatment with Q12.ConclusionOur data shows that active β‐catenin is not necessarily a driver of colon tumor cell proliferation and viability nor is it a marker for CDK8 inhibitor sensitivity. Both HCT‐116 and COLO205 cells harbor mutations that stabilize β‐catenin (β‐catenin and APC respectively), and both experience reductions in β‐catenin upon treatment with CDK8, but have vastly different proliferation and viability responses. The MDA‐MD‐468 cell line is very sensitive to Q12, but does not experience a reduction in β‐catenin. What is interesting to note is that both HCT‐116 and MDA‐MB‐468 harbor mutations related to RB1 (CDKN2A and RB1 respectively). Disparate E2F1 transcriptional activity may hold the key to discovery of a biomarker of CDK8 inhibitor sensitivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells.

Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells. Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis. Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation. These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.

Differences in the Effects of EGCG on Chromosomal Stability and Cell Growth between Normal and Colon Cancer Cells.

The tea catechin epigallocatechin-3-gallate (EGCG) proved to be the most potent physiologically active tea compound in vitro. It possesses antioxidant as well as pro-oxidant properties. EGCG has the effect of inducing apoptosis of tumor cells and inhibiting cell proliferation. Whether this effect is associated with the antioxidant or pro-oxidative effects of EGCG affecting the genome stability of normal and cancer cells has not been confirmed. Here, we selected Human normal colon epithelial cells NCM460 and colon adenocarcinoma cells COLO205 to investigate the effects of EGCG (0–40 μg/mL) on the genome stability and cell growth status. Chromosomal instability (CIN), nuclear division index (NDI), and apoptosis was measured by cytokinesis-block micronucleus assay (CBMN), and the expression of core genes in mismatch repair (hMLMLH1 and hMSH2) was examined by RT-qPCR. We found that EGCG significantly reduced CIN and apoptosis rate of NCM460 at all concentrations (5–40 μg/mL) and treatment time, EGCG at 5 μg/mL promoted cell division; EGCG could significantly induce chromosome instability in COLO205 cells and trigger apoptosis and inhibition of cell division. These results suggest that EGCG exhibits different genetic and cytological effects in normal and colon cancer cells.

MiR-137 suppresses proliferation, migration and invasion of colon cancer cell lines by targeting TCF4.

Colorectal cancer is cancer of the colon or rectum and is the third most prevalent form of cancer. Currently, there are several shortcomings in the prognosis and early detection of colon cancer. The present study aims to address questions pertaining to the role of microRNA (miR)-137 in colon cancer progression and the mode of regulation. The endogenous and over-expressed levels of miR-137 in three colon cancer cell lines were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The MTT assay was used to assess cell proliferation. Cell migration and invasion assays were assessed using Transwell apparatus and Matrigel invasion chambers. The potential targets of miR-150 were predicted using TargetScan software, and one of the best scoring targets, transcription factor 4 (TCF4), was experimentally validated using western blot analysis and RT-qPCR. It was found that that miR-137 is expressed at extremely low levels in COLO205, HCT116 and SW480 cell lines. Cell proliferation, migration and invasion were inhibited subsequent to transfection of the colon cancer cell lines with miR-137. Using bioinformatics analysis, the best scoring putative targets were identified. One such target, TCF4, was experimentally validated, and it was shown that overexpression of miR-137 suppresses TCF4 in all three colon cancer cell lines. In conclusion, it was shown that miR-137 inhibits cell proliferation, migration and invasion in colon cancer cell lines by negatively regulating the expression of TCF4.

Design, synthesis and biological evaluation of tetrahydronaphthyridine derivatives as bioavailable CDK4/6 inhibitors for cancer therapy

CDK4/6 pathway is an attractive chemotherapeutic target for antitumor drug discovery and development. Herein, we reported the structure-based design and synthesis of a series of novel tetrahydronaphthyridine analogues as selective CDK4/6 inhibitors. Compound 5 was identified as a hit and then systematically structure optimization study was conducted. These efforts led to compound 28, which exhibited excellent in vitro potencies against CDK4/6 enzymatic activity with high selectivity over CDK1, and against Colo-205 cell growth. The compound demonstrated favorable in vitro metabolic and robust mice pharmacokinetic properties. In Colo-205 xenograft models, compound 28 showed potent tumor growth inhibition with acceptable toxic effects, which could serve as a novel anticancer agent for further preclinical study.

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells.

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.

ETV4 Facilitates Cell-Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1.

The ETS family transcription factor ETV4 is aberrantly expressed in a variety of human tumors and plays an important role in carcinogenesis through upregulation of relevant target gene expression. Here, it is demonstrated that ETV4 is overexpressed in pancreatic cancer tissues as compared with the normal pancreas, and is associated with enhanced growth and rapid cell-cycle progression of pancreatic cancer cells. ETV4 expression was silenced through stable expression of a specific short hairpin RNA (shRNA) in two pancreatic cancer cell lines (ASPC1 and Colo357), while it was ectopically expressed in BXPC3 cells. Silencing of ETV4 in ASPC1 and Colo357 cells reduced the growth by 55.3% and 38.9%, respectively, while forced expression of ETV4 in BXPC3 cells increased the growth by 46.8% in comparison with respective control cells. Furthermore, ETV4-induced cell growth was facilitated by rapid transition of cells from G1- to S-phase of the cell cycle. Mechanistic studies revealed that ETV4 directly regulates the expression of Cyclin D1 CCND1, a protein crucial for cell-cycle progression from G1- to S-phase. These effects on the growth and cell cycle were reversed by the forced expression of Cyclin D1 in ETV4-silenced pancreatic cancer cells. Altogether, these data provide the first experimental evidence for a functional role of ETV4 in pancreatic cancer growth and cell-cycle progression.Implications: The functional and mechanistic data presented here regarding ETV4 in pancreatic cancer growth and cell-cycle progression suggest that ETV4 could serve as a potential biomarker and novel target for pancreatic cancer therapy. Mol Cancer Res; 16(2); 187-96. ©2017 AACR.

Targeting scFv-Fc-scTRAIL fusion proteins to tumor cells.

Fusion proteins combining hexavalent TRAIL with antibody fragments allow for a targeted delivery and efficient apoptosis induction in tumor cells. Here, we analyzed scFv-Fc-scTRAIL molecules directed against EGFR, HER2, HER3, and EpCAM as well as an untargeted Fc-scTRAIL fusion protein for their potentials to induce cell death both in vitro and in a xenograft tumor model in vivo. The scFv-Fc-scTRAIL fusion protein directed against EGFR as well as the fusion protein directed against EpCAM showed targeting effects on the two tested colorectal carcinoma cell lines Colo205 and HCT116, while a fusion protein targeting HER3 was more effective than untargeted Fc-scTRAIL only on Colo205 cells. Interestingly, another anti-HER3 scFv-Fc-scTRAIL fusion protein exhibiting approximately 10-fold weaker antigen binding as well as the HER2-directed molecule were unable to increase cytotoxicity compared to Fc-scTRAIL. A comparison of EC50 values of cell death induction and antigen binding supports the assumption that high affinity antigen binding is one of the requirements for in vitro targeting effects. Furthermore, a minimal number of expressed target antigens might be required for increased cytotoxicity of targeted compared to non-targeted molecules. In a Colo205 s.c. xenograft tumor model, strongest antitumor activity was observed for the anti-HER3 scFv-Fc-scTRAIL fusion protein based on antibody 3-43, with complete tumor remissions after six twice-weekly injections. Surprisingly, a similar in vivo activity was also observed for untargeted Fc-scTRAIL in this tumor model, indicating that additional factors contribute to the potent efficacy of targeted as well as untargeted hexavalent Fc-scTRAIL fusion proteins in vivo.

Abstract A187: Exploring isoform-dependent functions of the mediator kinases in colorectal cancer

Abstract Introduction: CDK8 and its paralogue CDK19 are highly similar cyclin-dependent kinases that function as components of the kinase module of the Mediator complex. Despite their structural similarity, emerging data suggest that these proteins may have distinct functions in the regulation of transcription. Recently, we identified two chemical series of CDK8/19 ligands (1, 2); however, as these ligands have similar binding affinities for CDK8 and CDK19 it has not been possible, until now, to determine if the biologic effects observed were driven by inhibition of one or both kinases. Here, we describe a functional genomics approach that utilizes these ligands, and others described in the literature, to identify substrates of CDK8/19 and isoform-dependent functions of these kinases. Materials and Methods: Experiments were conducted on COLO205 and SW620 human colorectal cancer cell lines and SW620 cells harbouring CRISPR knockouts of CDK8 and/or CDK19. Transcription factor reporter assays were carried out to define signalling pathways and biologic processes modulated by CDK8/19 ligands. Cells were reverse-transfected with both an inducible transcription-factor-responsive construct encoding firefly luciferase under the control of a basal promoter element, and a construct constitutively expressing the Renilla luciferase. At 42 h post-transfection, cells were treated with one of our CDK8/19 ligands, CCT251545 or MSC2530818, or the CDK8/19 inhibitors Senexin B (3) or Selvita 120-34 (4), at a 10 x EC50 concentration. Reporter activity was quantified following 6 h continuous exposure. In addition, gene set enrichment analysis (GSEA) was employed to examine changes in gene expression resulting from ligand treatment or CDK8/19 knockout, including changes in the expression of those genes regulated by transcription factors identified in the pathway reporter assay. Results: The pathway reporter assay identified transcription factor activity that was modulated by compound treatment and/or CDK8/19 knockout. Enriched transcription factors included key players in Notch signalling, Wnt signalling, the interferon gamma response, the cellular stress response, and MAPK/Jnk signalling, all of which are known to be modulated directly or indirectly by CDK8. Some of the transcription factors were differentially regulated by the test compounds, as well as by CDK8, CDK19 and CDK8/CDK19 knockout. Data from the CRISPR-mediated CDK8/19 knockout line demonstrated that the majority of altered gene expression, and corresponding decreased phosphorylation of STAT1SER727, was driven by loss of CDK8 with some limited compensatory activity from CDK19. These observations suggest that there may be some functional differences between the two kinases and that the main biologic effects of ligand treatment result from CDK8 inhibition. Conclusions: These experiments have identified potential substrates of CDK8/19 and determined that the majority of the effects on gene expression or biomarker phosphorylation observed in colorectal cancer cell lines after treatment with CDK8/19 ligands are driven by the inhibition of CDK8.

Coprecipitation Method of Synthesis, Characterization, and Cytotoxicity of Pr3+:LaF3 (CPr = 3, 7, 12, 20, 30%) Nanoparticles

The Pr3+:LaF3 (CPr = 3, 7, 12, 20, 30%) nanoparticles were characterized by means of high-resolution transmission electron microscopy, X-ray diffraction, optical spectroscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, and MTT assay. It was revealed that the average diameter of all the NPs is around 14–18 nm. The hydrodynamic radius of the Pr3+:LaF3 (CPr = 7%) nanoparticles strongly depends on the medium. It was revealed that hydrodynamic radii of the Pr3+:LaF3 (CPr = 7%) nanoparticles in water, DMEM, and RPMI-1640 biological mediums were 18 ± 5, 41 ± 6, and 186 ± 8 nm, respectively. The Pr3+:LaF3 (CPr = 7%) nanoparticles were nontoxic at micromolar concentrations toward COLO-320 cell line. The lifetime curves were fitted biexponentially, and for the Pr3+:LaF3 (CPr = 7%) NPs, the luminescence lifetimes of Pr3+ ions were 480 ± 2 and 53 ± 5 nanosec.

Combination of novel DR5 targeting agonistic scFv antibody TR2-3 with cisplatin shows enhanced synergistic antitumor activity in vitro and in vivo

ObjectivesTo investigate the antitumor activity of a novel agonistic single chain fragment variable (scFv) antibody TR2-3 targeting death receptor 5 (DR5) combined with cisplatin in vitro and in vivo. MethodsThe in vitro cytotoxic effects of TR2-3 and cisplatin, alone or in combination on human cancer cell lines COLO205 and MDA-MB-231 were evaluated using the MTT assay. The apoptosis in cancer cells was evaluated by an Annexin V-PE apoptosis detection kit and flow cytometry. The mRNA and protein levels of DR5 were analyzed by real-time PCR and Western blot, respectively. Additionally, the in vivo antitumor activity of TR2-3 combined with cisplatin was evaluated in a xenograft model. ResultsThe combination treatment with TR2-3 and cisplatin for 24 h on COLO205 and MDA-MB-231 cells showed significant cytotoxicity effects by MTT assay, compared with the alone treatment. Consistent with cell viability results, the cisplatin enhanced the apoptosis-inducing effects of TR2-3 in the COLO205 cells and MDA-MB-231 cells by flow cytometry. In addition, treatment with cisplatin alone for 24 h resulted in significantly up-regulating the mRNA and protein levels of DR5 in both COLO205 and MDA-MB-231 cell lines by q-PCR and Western blot assay. Moreover, the cytotoxic effects of TR2-3 can be blocked by adding the soluble DR5, and the blocking rate can be greatly reduced by co-treatment with cisplatin. These results indicated that cisplatin sensitized COLO205 and MDA-MB-231 cancer cells to TR2-3-mediated apoptosis by up-regulation of DR5 expression. Furthermore, combination therapy with TR2-3 and cisplatin enhanced tumor growth inhibition compared to treatment with TR2-3 or cisplatin alone in mice bearing COLO205 xenograft tumors. ConclusionsOur findings suggest that cisplatin enhanced the antitumor activity of TR2-3 in COLO205 and MDA-MB-231 cancer cells through up-regulation of DR5 expression. The TR2-3 combined with cisplatin may be a promising treatment for cancer therapy.

Highly sensitive fluorogenic sensing of L-Cysteine in live cells using gelatin-stabilized gold nanoparticles decorated graphene nanosheets

A highly sensitive and selective fluorogenic sensing of L-Cysteine (L-Cys) was implemented based on gelatin stabilized gold nanoparticles decorated reduced graphene oxide (rGO/Au) nanohybrid. The rGO/Au nanohybrid was prepared by the one-pot hydrothermal method and well characterized by different physiochemical techniques. The nanohybrid exhibits a weak fluorescence of rGO due to the energy transfer from the rGO to Au NPs. The rGO/Au nanohybrid shows enhanced fluorescence activity due to the restoration of quenched fluorescence of rGO/Au nanohybrid in presence of L-Cys. The rGO/Au nanohybrid exhibits much lower detection limit of 0.51 nM for L-Cys with higher selectivity. The fluorescence sensing mechanism arose from the fluorescence recovery due to the stronger interaction between Au NPs and L-Cys, and consequently, the energy transfer was prevented between rGO and Au NPs. The practicability of rGO/Au sensor was implemented by invitro bioimaging measurements in Colo-205 (colorectal adenocarcinoma) and MKN-45 (gastric carcinoma) cancer live cells with excellent biocompatibility.

EVALUATION OF CYTOTOXIC AND GENOTOXIC EFFECTS OF ZERUMBONE ON COLON ADENOCARCINOMA COLO205 CELLS AND HUMAN LYMPHOCYTES

Objective: The objective of the present study was to investigate the growth inhibitory effect, apoptosis initiation and genotoxic effect of zerumbone (ZER), a phytochemical and cisplatin, a chemotherapeutic drug on human colorectal cancer cell line COLO205 and normal human lymphocytes.Methods: The antiproliferative activity of ZER and cisplatin (positive control) on COLO205 cells and lymphocytes was analysed by 3( 4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. Morphological analysis of the cells was studied by using inverted phase contrast microscope. Propidium iodide staining method was used to observe the apoptotic morphological changes in the treated cells. Finally comet assay was conducted to observe the extent of DNA damage induced by ZER and cisplatin on COLO205 and lymphocytes.Results: ZER and cisplatin exhibited growth inhibition in a dose and time dependent manner against COLO205 with no considerable effect on lymphocytes. The IC50 values of ZER on COLO205 for 24h, 48h and 72h were 19 µg/ml, 10 µg/ml and 5 µg/ml. Comparatively the IC50 values of cisplatin on COLO205 for 24h, 48h and 72h were 38 µg/ml, 24 µg/ml and 15 µg/ml. Morphological changes such as cell shrinkage, membrane blebbing and nuclear condensation were observed in COLO205 while no significant change was observed in lymphocytes. Fluorescence imaging studies confirmed apoptotic cell death in treated COLO205 cells while no significant cell death was observed in treated lymphocytes. Comet assay revealed significant DNA damage in treated COLO205 cells.Conclusion: The present study demonstrated the cytotoxic and genotoxic effect of ZER and cisplatin on COLO205 cells. These drugs showed no significant effect on lymphocytes.

Lymphangiogenic Gene Expression Is Associated With Lymph Node Recurrence and Poor Prognosis After Partial Hepatectomy for Colorectal Liver Metastasis.

To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion. Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC. We designed a lymphangiogenic gene set (VEGFC, Nrp-2, PDPN, LYVE-1, MRC1, CCL-21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt-QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp-2 expression) and human dermal microvascular lymphatic endothelial cells (LECs). Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype-4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp-2. Blocking Nrp-2 significantly reduced invasion of Colo320 cells through an LEC monolayer. Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype-4 in both primary and liver metastatic CRC. VEGFC and Nrp-2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp-2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38MAPK.

Transforming growth factor (TGF)-β promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-β1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5,6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-β1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-β1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38MAPK signaling by TGF-β1 and that inhibition of p38MAPK reduced TGF-β1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-β1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38MAPK cascade.

Knockdown of CEMIP suppresses proliferation and induces apoptosis in colorectal cancer cells: downregulation of GRP78 and attenuation of unfolded protein response.

It has been suggested that cell migration inducing hyaluronan binding protein (CEMIP) contributes to the carcinogenesis of colorectal cancer (CRC). Cancer cells can adapt to endoplasmic reticulum (ER) stress by initiating an unfolded protein response (UPR). This study aimed to investigate whether CEMIP affects the UPR of CRC cells, with a focus on 78 kDa glucose-regulated protein (GRP78, a major ER chaperone). We found that knockdown of CEMIP inhibited cell proliferation and induced a G1 arrest in SW480 CRC cells. The levels of cyclin D1 and cyclin E1 and phospho-retinoblastoma, which are known to promote the cell cycle progression from G0 or G1 into S phase, were decreased in CEMIP-silenced cells. CEMIP shRNA induced apoptosis and inhibited GRP78 expression in SW480 and Colo205 cells. The basal UPR of cancer cells was attenuated by CEMIP shRNA, as evidenced by the decreased expression of UPR sensors, protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). Furthermore, CEMIP silencing sensitized CRC cells to thapsigargin-induced apoptosis. Our study demonstrates that the in-vitro anti-proliferative and pro-apoptotic effects in CRC cells that were induced by silencing CEMIP may be associated with GRP78 repression and UPR attenuation.

Anti-proliferation activity of terpenoids isolated from Euphorbia kansui in human cancer cells and their structure-activity relationship

AbstractEuphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC50 values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells.

Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

ATP fuels the removal of metabolic end-products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low-yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid-neutralizing HCO3- ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43-positive pancreatic cancer Colo357 cells during light-controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3-, but this process requires a restorative HCO3- flux. The effect of CO2/HCO3- was ablated by connexin43 inhibition or knockdown. In connexin-decoupled spheroids, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an inhibitor of HCO3- uptake, had no effect on cytoplasmic [H+] in the H+-uncaging region, indicating that DIDS-sensitive transport is not an adequate pH-regulatory strategy therein. With intact connexin-coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3- ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors.-Dovmark, T. H., Hulikova, A., Niederer, S. A., Vaughan-Jones, R. D., Swietach, P. Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

Structural characterization and evaluation of antioxidant, anticancer and hypoglycemic activity of radiation degraded oat (Avena sativa) β- glucan

Oat β-d-glucan after extraction was degraded at doses of 3, 6, 9, 12 and 15kGy. The average molecular weight decreased to 45kDa at dose of 15kGy from an initial value of 200kDa in native sample. XRD analysis revealed no significant change in diffraction pattern of irradiated samples when compared with control, except a decrease in intensity of x-ray diffraction. The results of the antioxidant activity revealed decrease in EC50 values and corresponding increase in antioxidant activity of radiation degraded oat β-d-glucan. Results of the anticancer studies indicated that cytotoxicity of gamma irradiated oat β-d-glucan in cancer cell lines was highest against colo-205 and MCF7 cancer cells compared to T47D cell and no cytotoxicity was observed in normal cell lines at all concentrations used. Evaluation of hypoglycemic activity showed highest inhibition in α-glucosidase activity compared to α-amylase activity due to gamma irradiation of oat β-d-glucan. Comparison of the EC50 values of known standards and gamma irradiated oat beta-glucan samples indicates that radiation treatment significantly modified the biological activity of the beta-glucan samples. Therefore, it is suggested that gamma irradiation can be used for producing low molecular weight oat β-d-glucan; which can help in modifying the biological activities.

Novel histone deacetylase 8-selective inhibitor 1,3,4-oxadiazole-alanine hybrid induces apoptosis in breast cancer cells.

Identification of isoform-specific histone deacetylase inhibitors (HDACi) is a significant advantage to overcome the adverse side effects of pan-HDACi for the treatment of various diseases, including cancer. We have designed, and synthesized novel 1,3,4 oxadiazole with glycine/alanine hybrids as HDAC8-specific inhibitors and preliminary evaluation has indicated that 1,3,4 oxadiazole with alanine hybrid [(R)-2-amino-N-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)propanamide (10b)] to be a potent HDAC8 inhibitor. In the present study, the in vitro efficacy of the molecule in inhibiting the cancer cell proliferation and the underlying molecular mechanism was studied. 10b inhibited the growth of MDA-MB-231 and MCF7 breast cancer cells, with a lower IC50 of 230 and 1000nM, respectively, compared to K562, COLO-205 and HepG2 cells and was not cytotoxic to normal breast epithelial cells, MCF10A. 10b was specific to HDAC8 and did not affect the expression of other class I HDACs. Further, a dose-dependent increase in H3K9 acetylation levels demonstrated the HDAC-inhibitory activity of 10b in MDA-MB-231 cells. Flow cytometric analysis indicated a dose-dependent increase and decrease in the percent apoptotic cells and mitochondrial membrane potential, respectively, when treated with 10b. Immunoblot analysis showed a modulation of Bax/Bcl2 ratio with a decrease in Bcl2 expression and no change in Bax expression. 10b treatment resulted in induction of p21 and inhibition of CDK1 proteins along with cytochrome c release from mitochondria, activation of caspases-3 and -9 and cleavage of poly ADP-ribose polymerase leading to apoptotic death of MDA-MB-231 and MCF7 cells. In conclusion, our results clearly demonstrated the efficacy of 10b as an anticancer agent against breast cancer.