Colo205 Cells Research Articles

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.

Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design, synthesis, biological investigation and docking studies

In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC50 value of 1.06µM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.

Antitumor activity of iNGR-GRIM-19 in colorectal cancer.

Gene associated with retinoid-interferon induced mortality-19 (GRIM-19) plays crucial roles in carcinogenesis. To explore the antitumor activity of internalizing NGR (iNGR) gene associated with GRIM-19 in colorectal cancer. Cells were incubated with fluorescein isothiocyanate-labeled fusion proteins followed by fluorescence microscopic analysis. Cell proliferation was determined by MTT assay. Cell cycle was analyzed by flow cytometric analysis. Cell migration and invasion capacity were evaluated by wound scratch and Transwell assays, respectively. Apoptosis was measured by Annexin V/PI staining and TUNEL assay. Gene expressions were determined by RT-PCR and Western blotting. Nude mice bearing colorectal cancer received vehicle, GRIM-19, or iNGR-GRIM-19 fusion protein injection, and the in vivo antitumor capacity of the fusion proteins was examined. iNGR-GRIM-19 was specifically taken up by human colorectal cancer Colo205 cells, but not corneal epithelial (HCEpic) cells, whereas GRIM-19 was not internalized by either cell type. Unlike GRIM-19, incubation with iNGR-GRIM-19 dose-dependently inhibited proliferation, induced G1 phase arrest, suppressed cell migration and invasion, and caused apoptosis in Colo205 cells. Additionally, injection of iNGR-GRIM-19 extended the lifespan of colorectal cancer-bearing nude mice and reduced in vivo tumor growth as compared with vehicle or GRIM-19 treatment. iNGR-GRIM-19 was localized only in the tumor mass, without affecting other tissues, such as liver or kidney. iNGR-GRIM-19 injection led to G1 phase arrest and induced cell apoptosis in xenografted colorectal cancer tissues. iNGR-GRIM-19 has an efficient antitumor activity in vitro and in vivo, and might be a promising agent for the treatment of colorectal cancer.

Abstract 1204: Enhanced efficacy of selumetinib by pretreatment of 5-FU in preclinical Ras/Raf-mutant colorectal cancer models

Abstract Optimal strategy for the application of MEK inhibitor as combination therapy has not been established in Ras/Raf-mutant colorectal cancer (CRC). As we preliminary observed that 5-FU-induced pharmacodynamic changes were restored by ERK inhibition, we hypothesized that post-treatment of MEK inhibitor would enhance antitumor activity of 5-FU. Treatment of 5-FU for 2 days followed by selumetinib for another 2 days (FS schedule) exhibited synergism for cell viability, whereas reverse or concomitant combination showed antagonism or inconsistent response, respectively, in 8 kinds of CRC cell lines including BRAFV600E colo205, BRAFV600E RKO, or KRASG13D HCT8 cells. Combination as FS schedule persistently down-regulated the phosphorylation of ERK and pS6 throughout treatment period, whereas selumininib alone or reverse combination transiently decreased within 24 hr. The cells treated as FS schedule exhibited higher apoptosis and lower capacity of anchorage-independent cell growth than the cells under single or reverse combination. Microarray analysis revealed the distinct groups of genes underlying different efficacy by schedule-dependent treatment. Tumor growth in mice administered 5-FU at 10 mg/kg/day for colo205 and at 30 mg/kg/day for HCT8 for 7 days followed by selumetinib at 10 mg/kg/day for colo205 and 25 mg/kg/day for HCT8 for another 7 days was significantly retarded than that in mice treated with single agents. But the combination in reverse sequence induced comparable tumor growth to single agents. Decreased expression of Ki67 was observed in tumors from mice treated as FS scheduling. Our results suggest that the schedule-dependent combination of 5-FU and selumetinib may be benefit in patients having CRC with hyper-activation of Ras/RAF pathway. Citation Format: Hye Yeon Jang, Haeng Jung Lee, Yeo Jin Nam, Won Dong Kim, Yong Ju Bae, Jin Hwang Jung, Seung Jin Lee, Dae Hyuk Moon. Enhanced efficacy of selumetinib by pretreatment of 5-FU in preclinical Ras/Raf-mutant colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1204. doi:10.1158/1538-7445.AM2017-1204

Abstract 2866: Volumetric optoacoustic imaging of tumor cell death using a targeted imaging agent

Abstract Multispectral optoacoustic tomography (MSOT) generates high-resolution cross-sectional images in less than a second. MEDI3039, is a newly described agonist of the TNF-related apoptosis-inducing ligand receptor2 (TRAILR2), which has been shown in preclinical studies to preferentially induce cell death in cancer versus normal cells1. We show here that MSOT, when used with a near infra-red (NIR) fluorophore-labelled protein domain of Synaptotagmin-I (C2Am-750, ~15kDa) that binds to phosphatidylserine (PS) exposed by apoptotic and necrotic cells2, can be used to image MEDI3039-induced cell death within the entire tumor region. Non-specific probe retention was assessed using a site-directed mutant (iC2Am-680 or iC2Am-750). PS-specific binding of C2Am-750 to MEDI3039-treated TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 human colon adenocarcinoma cells in vitro was evaluated by flow cytometry and confocal microscopy. The capability of C2Am-750 to detect cell death in vivo was assessed in mice bearing Colo205 or HT-29 xenografts, following a single dose (0.4 mg/kg, i.v.) of MEDI3039. All mice then received an i.v. injection of a 1:1 mixture of 0.1 µmole/kg C2Am-750 and iC2Am-680 16 h post treatment, followed by fluorescence imaging (FLI) measurements using an IVIS 200 camera at 0 and 3 h post probe injection. FLI measurements in treated Colo205 xenografts showed significantly increased retention of C2Am-750 uptake versus size-matched untreated tumors (6.0-fold, P value <0.0001), but there was no significant difference between treated and untreated HT-29 xenografts. iC2Am-680 retention was negligible in all groups. Next, volumetric MSOT measurements of C2Am-750 and iC2Am-750 (0.2 µmole/kg) retention in MEDI3039-treated Colo205 tumors were made using an iTheraMedical inVision 256 system at different wavelengths (660-900nm) and time points (3-24 h). C2Am-750 signal was markedly increased in tumors at 3 h post probe injection. Maximal C2Am-750 signal appeared in the center of treated tumors, whereas there was negligible iC2Am-750 retention. Subsequent histological analyses showed that C2Am-750 signal was strongly correlated with both cleaved caspase-3 and TUNEL staining of dead cells. We have shown that real-time volumetric MSOT measurements with a NIR fluorophore-labelled C2Am, can be used to detect early tumor cell death within the entire tumor region following TRAILR2 agonist treatment. This study shows that this imaging technique can be used to characterize tumor heterogeneity and to determine the most appropriate therapy at an early stage post drug administration. 1. Swers, J.S. et al. Multivalent scaffold proteins as superagonists of TRAIL receptor 2-induced apoptosis. Molecular cancer therapeutics 12, 1235-1244 (2013). 2. Alam, I.S. et al. Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death. Bioconjugate chemistry 21, 884-891 (2010). Citation Format: Bangwen Xie, Michal Tomaszewski, André A. Neves, Stefanie R. Mullins, David Tice, Richard Sainson, Sarah Bohndiek, Robert W. Wilkinson, Kevin M. Brindle. Volumetric optoacoustic imaging of tumor cell death using a targeted imaging agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2866. doi:10.1158/1538-7445.AM2017-2866

Abstract 1183: Structure-activity studies and biological evaluations of ERGi-USU, a highly selective inhibitor for ERG-positive prostate cancer cells

Abstract Introduction and objectives: While new prostate cancer (CaP) treatments (Abiraterone and Enzalutamide) have improved survival in castration-resistant prostate cancer (CRPC), their benefits are short-lived and drug resistance develops likely due to numerous adaptive mutations. Therefore it is a critical to develop effective novel inhibitors to target other signaling pathways that promote or contribute to prostate tumorigenesis. Accumulating evidence has established the androgen regulated TMPRSS2-ERG fusion as a common oncogenic driver that contributes to the early development and progression of over half of CaP. Therefore, ERG oncoprotein and ERG dependent pathways are promising targets for CaP therapy in early stages when cancer is most responsive to treatment. We previously identified a small molecule inhibitor, ERGi-USU, which selectively inhibits ERG protein and cell growth in ERG positive tumor cell lines and mouse xenograft models. In an effort to further develop ERGi-USU with enhanced efficacy we performed detailed structure-activity relationship (SAR) evaluation of ERGi-USU core structure and developed new derivatives. Methods: Based on SAR of the core structure of ERGi-USU, 48 new derivatives were designed and synthesized by substitutions with alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or hydroxyl groups. The new ERGi-USU derivatives were evaluated for inhibition of cell growth and ERG protein levels in the TMPRSS2-ERG fusion harboring CaP cell line, VCaP. Four of these compounds have been selected for evaluation of ERG selectivity by defining IC50 in ERG positive malignant cells (VCaP, KG1, MOLT-4 and COLO320), ERG negative CaP cell line (LNCaP) or ERG positive normal primary endothelium-derived cells (HUVEC). Result: Like parental compound, four new ERGi-USU derivatives exhibited inhibition of cell growth and ERG protein levels in ERG positive VCaP, KG1, MOLT-4 and COLO320 cell lines, with no or minimal effects on LNCaP and HUVEC cells. One of the new derivatives (ERGi-USU#6) showed increased efficacy for cell growth inhibition (IC50=0.074µM) compared to the parental ERGi-USU (IC50=0.200µM). Other three new compounds showed similar IC50 as the ERGi-USU. Conclusion: Comprehensive evaluation of ERGi-USU derivatives along with parental compound has continued to underscore selective inhibition of ERG positive tumor cells by these small molecules. Citation Format: Ahmed A. Mohamed, Charles P. Xavier, Gauthaman Sukumar, Samuel D. Banister, Vineet Kumar, Shyh-Han Tan, Shilpa Katta, Lakshmi Ravindranath, Muhammad Jamal, Taduru Sreenath, David G. McLeod, Gyorgy Petrovics, Albert Dobi, Meera Srivastava, Sanjay Malhotra, Clifton Dalgard, Shiv Srivastava. Structure-activity studies and biological evaluations of ERGi-USU, a highly selective inhibitor for ERG-positive prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2017-1183

Abstract 1702: Multimeric anti-DR5 IgM antibody displays potent cytotoxicity in vitro and promotes tumor regression in vivo

Abstract Death receptor 5 (DR5) is a member of the TNF receptor superfamily that induces apoptosis upon receptor trimerization. It is expressed on many tumor types and has therefore been an important target for developing antibody based treatments of epithelial, solid tumors. However several agonistic Anti-DR5 IgG antibodies that have demonstrated efficacy in preclinical models have been unsuccessful in clinical trials, likely due to insufficient receptor crosslinking by bivalent IgGs. We have developed a multimeric anti-DR5 IgM antibody which has strong avidity for the receptor. The IgM binds Colo205 cells and triggers apoptosis, and is 1000-fold more potent than the respective IgG and 100-fold more potent than crosslinked IgG in vitro. Anti-DR5 IgM displays strong in vitro potency across a panel of tumor cell lines, including ones that are IgG-resistant. Anti-DR5 IgM causes tumor regression and delays tumor growth in various in vivo xenograft models including Colo205, HCT15 and MDA-MB231. These results support the development of an human anti-DR5 IgM therapeutic with the potential to treat solid tumors. Citation Format: Beatrice Wang, Tasnim Kothambalwala, Paul Hinton, Dean Ng, Avneesh Saini, Ramesh Baliga, Bruce Keyt. Multimeric anti-DR5 IgM antibody displays potent cytotoxicity in vitro and promotes tumor regression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1702. doi:10.1158/1538-7445.AM2017-1702

Growth Inhibition of Colon Cancer and Melanoma Cells by Versiol Derivatives from a Paraconiothyrium Species.

Bioassay-guided fractionation of a colon cancer growth inhibitory extract of the fungus Paraconiothyrium sp. led to the isolation of eight new versiol derivatives (1, 3-8, 10) along with two known compounds. The structures were elucidated by interpretation of combined MS and 2D NMR spectroscopic data. Compounds 8, 9, and 10 showed cell growth inhibition against COLO205 and KM12 cells, and both 8 and 9 displayed selectivity in their inhibition of melanoma cell lines in the NCI 60 one-dose test. In addition, compound 8 and the crude Paraconiothyrium sp. extract showed potent dose-dependent inhibitory effects in the five-dose NCI 60 cell line assay.

Synthesis of Bifunctional Furano-Allocolchicinoids

An efficient seven-step semisynthetic approach towards non-racemic bifunctional furano-allocolchicinoids, starting from naturally occurring colchicine is presented. The Pd-catalyzed domino Sonogashira coupling/5-endo-dig cyclization was employed as the key step. The prepared compounds exhibited substantial cytotoxicity against T3M4, MiaPaCa-2, Colo-357, and PANC-1 cell lines. The presence of two functionalities with different reactivity (hydroxyl and amino groups) in the target molecules allows for an easy conjugation of furano-allocolchicinoids with drug delivery carriers, and opens promising opportunities for their further exploitation in the search of therapeutics.

Characterizing the detection threshold for optical imaging in surgical oncology.

Optical imaging to guide cancer resections is rapidly transitioning into the operating room. However, the sensitivity of this technique to detect subclinical disease is yet characterized. The purpose of this study was to determine the minimum range of cancer cells that can be detected by antibody-based fluorescence imaging. 2LMP (breast), COLO-205 (colon), MiaPaca-2 (pancreas), and SCC-1 (head and neck) cells incubated in vitro with cetuximab-IRDye800CW (dose range 8.6-86 nM) were implanted subcutaneously in mice (n = 3 mice, 5 tumors/mouse). Following incubation with 8.6 × 10-2 µM of cetuximab-IRDye800CW in vitro, serial dilutions of each cell type (1 × 103 -1 × 106 ) were implanted subcutaneously (n = 3, 5 tumors/mouse). Tumors were imaged with Pearl Impulse and Xenogen IVIS 100 imaging systems. Scatchard analysis was performed to determine receptor density and kinetics for each cell line. Under conditions of minimal cetuximab-IRDye800CW exposure to low cellular quantity, closed-field fluorescence imaging theoretically detected a minimum of 4.2 × 104 -9.5 × 104 2LMP cells, 1.9 × 105 -4.5 × 105 MiaPaca-2 cells, and 2.4 × 104 -6.7 × 104 SCC-1 cells; COLO-205 cells could not be identified. Higher EGFR-mediated uptake of cetuximab correlated with sensitivity of detection. This study supports the clinical utility of cetuximab-IRDye800CW to sensitively localize subclinical disease in the surgical setting.

TGF-β Signal Transduction in Pancreatic Carcinoma Cells is Sensitive to Inhibition by the Src Tyrosine Kinase Inhibitor AZM475271.

Earlier results from our group have shown that in pancreatic ductal adenocarcinoma (PDAC)-derived cells transforming growth factor (TGF)-β1-dependent epithelial-mesenchymal transition (EMT) and cell motility was inhibited by the Src inhibitors PP2 and PP1 both of which targeted the TGF-β receptors for inhibition. In this study we evaluated the impact of another Src inhibitor, AZM475271, on various TGF-β responses in PDAC cells. The effect of AZM475271 on TGF-β1-induced random cell migration (chemokinesis), the expression of EMT and migration/invasion-associated genes, TGF-β-induced luciferase activity, and C-terminal phosphorylation of Smad2 and Smad3 was measured in the PDAC-derived Panc-1 and Colo357 cell lines using real-time cell migration assays, quantitative real-time PCR, luciferase reporter gene assays and phosphoimmunoblotting, respectively. AZM475271 effectively blocked TGF-β1-induced chemokinesis of Panc-1 cells in a dose-dependent fashion and inhibited the high chemokinetic activity of Panc-1 cells with ectopic expression of a constitutively active ALK5T204D mutant. AZM475271 but not another Src inhibitor, SU6656, partially relieved the suppressive effect of TGF-β1 on E-cadherin and inhibited TGF-β1-induced upregulation of the MMP2, MMP9, N-cadherin and vimentin genes, activity of a TGF-β1-dependent reporter gene, and activation of Smad2 and Smad3. Our data suggest that AZM475271 cross-inhibits tumor-promoting TGF-β signaling and may thus function as an inhibitor of both TGF-β and Src in both experimental and clinical therapies against metastatic dissemination in late-stage PDAC.

P62/sequestosome 1 in human colorectal carcinoma as a potent prognostic predictor associated with cell proliferation.

p62/sequestosome 1 (p62) is a multi‐domain protein that functions as a receptor for ubiquitinated targets in the selective autophagy and serves as a scaffold in various signaling cascades. p62 have been reported to be up‐regulated in several human malignancies, but the biological roles and significance of p62 are still poorly understood in colorectal carcinoma. We immunohistochemically evaluated p62 in 118 colorectal adenocarcinoma and 28 colorectal adenoma cases. We used four colon carcinoma cells (HCT8, HT29, COLO320, and SW480) in the in vitro studies. p62 immunoreactivity was detected in 11% of colorectal adenoma cases and 31% of adenocarcinoma cases, while it was negligible in the normal epithelium. The immunohistochemical p62 status was significantly associated with synchronous liver metastasis, and it turned out to be an independent adverse prognostic factor in colorectal cancer patients. Following in vitro studies revealed that HCT8 and HT29 cells transfected with p62‐specific siRNA showed significantly decreased cell proliferation activity, whereas COLO320 and SW480 cells transfected with p62 expression plasmid showed significantly increased cell proliferation activity. The p62‐mediated cell proliferation was not associated with the autophagy activity. These findings suggest that p62 promotes the cell proliferation mainly as a scaffold protein, and that the p62 status is a potent prognostic factor in colorectal carcinoma patients.

AKT is critically involved in the antagonism of BRAF inhibitor sorafenib against dabrafenib in colorectal cancer cells harboring both wild-type and mutant (V600E) BRAF genes

BRAF, one of the key factors in mitogen-activated protein kinase (MAPK) signaling pathway, plays an important role in cell functions including growth and proliferation. Inhibition of BRAF represents a promising antitumor strategy. Dabrafenib, a type I inhibitor of BRAF interrupting RAF/MEK interaction, has been approved by FDA as a single agent or combined with MEK inhibitor trametinib for the treatment of patients with BRAF V600E mutation-positive advanced melanoma. In the present study, we investigated the feasibility of combined treatment with dabrafenib and sorafenib, type I and type II BRAF inhibitor respectively, on colorectal cancer cells with BRAF V600E mutation. Unexpectedly, sorafenib significantly antagonized the inhibition effect of dabrafenib on the proliferation of colorectal cancer HT-29 and Colo205 cells. The antagonism relied on co-existence of wild-type and mutant (V600E) BRAF, for no antagonism was observed in tumor cells expressing homozygous wild-type or mutant (V600E) BRAF. BRAF, but not CRAF, was required for this antagonism. Moreover, we found that sorafenib reversed dabrafenib inhibition of AKT in HT-29 cells, and phosphatidylinositol-3-kinase (PI3K) inhibitor GDC0941 significantly restored this antagonistic effect when combined with dabrafenib and sorafenib, indicating that AKT is critically involved in this antagonism. Collectively, we found that significant antagonism was observed when dabrafenib was combined with sorafenib in colorectal cancer cells harboring heterozygous genotype of BRAF and AKT is critically involved in this antagonism. We suggest that BRAF inhibitor dabrafenib and sorafenib should not be combined in clinic.

Autophagy (A) inhibition to enhance combined immunotherapy (I) and anti-EGFR treatment (E) in colorectal cancer (CRC).

e14554 Background: Resistance to E impairs survival of metastatic CRC (mCRC) patients. The aim of this study is to assess the preclinical activity of triple inhibition of E, I and A in CRC. Methods: RKO and Colo-205 cell lines were treated with E [cetuximab (C) / panitamumab (P)] and/or I [pembrolizumab (PE) or nivolumab (NI) with/without ipilimumab (IPI)] and/or autophagy inhibitor 3-Methyladenine (3-MA). Phosphorylated EGFR (pEGFR), p62 (SQSTM1), MAP1LC3 (LC3B) and PARP protein levels were detected by Western blotting. Cell viability was determined by MTT Assay. Tumor volumes were determined from 3D cell culture on Corning Matrigel Matrix as well as the apoptotic cell death. Results: E+I combination compared to E alone decreased pEGFR and increased A activation through p62 downregulation and LC3II activation. Similar results were shown for cell viability/apoptosis. Cells were also treated with C or P combined with I plus 3-MA. Triple inhibition suppresses EGFR and blocks A. Compared to E+I, triple inhibition reduced more cell growth/viability. Caspase-3 activity was increased as well as proportion of apoptotic cells (Table). Conclusions: Τhese results indicate that the combinatorial inhibition of A, E and I represents a promising treatment strategy in mCRC that needs further testing. [Table: see text]

Potent Pan-Raf and Receptor Tyrosine Kinase Inhibitors Based on a Cyclopropyl Formamide Fragment Overcome Resistance

While selective BRafV600E inhibitors have been proven effective clinically, acquired resistance rapidly develops through reactivation of the mitogen-activated protein kinase (MAPK) pathway. Simultaneous targeting of multiple nodes in the pathway offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Replacement pyridine group of Y-1 by a cyclopropyl formamide group afforded I-01 as a novel multitargeted kinase inhibitor template. I-01 displayed enzyme potency against Pan-Raf and receptor tyrosine kinases (RTKs). Based on the binding mode of I-01, analogues I-02-I-18 were designed and synthesized. The most promising compound I-16 potently inhibits all subtypes of Rafs with IC50 values of 3.49 (BRafV600E), 8.86 (ARaf), 5.78 (BRafWT), and 1.65 nM (CRaf), respectively. I-16 not only exhibit comparable antiproliferative activities with positive control compounds against HepG2, SW579, MV4-11, and COLO205 cell lines, but also suppress the proliferation of melanoma SK-MEL-2 harboring overexpressed BRafWT with IC50 values of 0.93 μM. The Western blot results for the ERK inhibition in human melanoma SK-MEL-2 cell lines show that I-16 inhibits the proliferation of SK-MEL-2 cell lines without paradoxical activation of ERK, which support the hypothesis that the inhibition of Pan-Raf and RTKs might be a tractable strategy to overcome the resistance of melanoma induced by the therapy with the current selective BRafV600E inhibitors.

Antitumor Activity of a Mesenchymal Stem Cell Line Stably Secreting a Tumor-Targeted TNF-Related Apoptosis-Inducing Ligand Fusion Protein.

Mesenchymal stem cells (MSCs) are currently exploited as gene delivery systems for transient in situ expression of cancer therapeutics. As an alternative to the prevailing viral expression, we here describe a murine MSC line stably expressing a therapeutic protein for up to 42 passages, yet fully maintaining MSC features. Because of superior antitumoral activity of hexavalent TNF-related apoptosis-inducing ligand (TRAIL) formats and the advantage of a tumor-targeted action, we choose expression of a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) as a model. The bioactivity of Db-scTRAIL produced from an isolated clone (MSC.TRAIL) was revealed from cell death induction in Colo205 cells treated with either culture supernatants from or cocultured with MSC.TRAIL. In vivo, therapeutic activity of MSC.TRAIL was shown upon peritumoral injection in a Colo205 xenograft tumor model. Best antitumor activity in vitro and in vivo was observed upon combined treatment of MSC.TRAIL with bortezomib. Importantly, in vivo combination treatment did not cause apparent hepatotoxicity, weight loss, or behavioral changes. The development of well characterized stocks of stable drug-producing human MSC lines has the potential to establish standardized protocols of cell-based therapy broadly applicable in cancer treatment.

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

The small heat‐shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene‐addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET‐addicted EBC‐1 lung cancer cells, epidermal growth factor receptor (EGFR)‐addicted colorectal carcinoma (CRC) DiFi cells and BRAF‐addicted CRC COLO205 and OXCO‐1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET‐addicted gastric carcinoma MKN45 and EGFR‐addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro‐apoptotic proteins of the BCL2 family and of active caspase‐3 and lamin B. Together, these data suggest that oncogene‐addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents.

Selection and optimization of nano-formulation of P-glycoprotein inhibitor for reversal of doxorubicin resistance in COLO205 cells.

The prime objective of current work was to develop a strategy for preparation of combinational nano-formulation for reversal of drug resistance. As a model system, doxorubicin (DOX)-resistant COLO205 cells were developed and validated. From co-treatment studies with DOX, curcumin was selected as it reversed DOX-resistance at lowest concentration. In an attempt to increase its solubility, curcumin was encapsulated into hydroxypropyl-β-cyclodextrin (HP-β-CD). Here, we propose that presence of stabilizer overcomes its low encapsulation efficiency. Thus, we evaluated curcumin encapsulation in HP-β-CD in presence of different stabilizers and organic solvents. Finally, the effect of nanocurcumin with liposomal DOX was studied for reversal of resistance in COLO205 cells. In the process encapsulation, selective optimization of organic solvent by freeze-drying was found to be appropriate among other methods. From optimization studies with different organic solvent (acetone and dichloromethane) and stabilizer [polyvinyl alcohol (PVA) and Pluronics], HP-β-CD-encapsulated curcumin prepared using acetone in PVA-stabilized dispersion increased encapsulation (60%) with size of ~40nm. Prepared nano-curcumin reversed the DOX resistance effectively in combination with liposomal DOX. Curcumin reversed DOX resistance in COLO205 cells at low concentration and enhanced curcumin encapsulation in HP-β-CD was noted in presence of PVA. Further, it was observed that prepared HP-β-CD-encapsulated curcumin is equi-efficacious to nano-dispersed curcumin.

Antitumor activity, cell cycle arrest and apoptosis induction in human colon cancer cell line by Primula macrophylla extracts

The primary objective of the current work was to investigate the antitumor potential of Primula macrophylla extracts in human colon cancer cell line (Colo-205) along with evaluating the effects on apoptosis induction, cell cycle arrest and mitochondrial membrane potential. Cell viability was assessed by tetrazolium-based MTT assay. Flow cytometry measurement was carried out to assess the effect of the extract on cell cycle phase distribution and mitochondrial transmembrane potential. Results showed that P. macrophylla methanol extract was effective and exhibited highest cell growth inhibition (IC50 value, 26.17 ?g/mL). Methanol extract significantly increased the side-scattering profile of Colo-205 cells in concentration-dependent pattern. Exposure of Colo-205 cells with different concentrations of the methanol extract (0-80 ?g/mL) caused dose-dependent G0/G1 cell cycle arrest along with inducing apoptotic cascade by increasing the population of cells at G0/G1 phase. Furthermore, methanol extract treatment caused an increase in mitochondrial membrane depolarization in Colo-205 cells.
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Connexin-43 channels are a pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells

Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H+-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H+ ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 μm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore, lactate entry into the better-perfused recipient cells has a favourable alkalinizing effect and supplies substrate for oxidative phosphorylation. Cx43 is thus a novel target for influencing metabolite handling in junctionally-coupled tumours.