HCT116 Human Colon Cancer Cells Research Articles

Sasa quelpaertensis Nakai extract induces p53-independent apoptosis via the elevation of nitric oxide production in human HCT116 colon cancer cells.

Induction of apoptosis in human cancer cells by Sasa quelpaertensis Nakai has been considered to be a potential therapeutic target for cancer treatment; however, the underlying mechanisms of action are not well understood. The present study investigated the role of nitric oxide (NO•) and inhibitors of apoptosis (IAPs) during apoptosis induced by Sasa quelpaertensis Nakai extracts (SQE) in p53-wild type (WT) and p53-null HCT116 colon carcinoma cells. Trypan blue exclusion and Annexin V/propidium iodide assays were used to test for antiproliferation, and apoptosis and cell cycle. Griess and reverse transcription-polymerase chain reaction and western blotting assays were carried out to assay NO• production, and to detect the mRNA and protein levels of Bcl-2, PARP and IAPs. A colorimetric assay was utilized to measure the time-dependent increase in caspase-3 activity. SQE inhibited cell growth and promoted apoptosis by the elevation of NO• in a dose- and time-dependent manner. In addition, both cell types underwent a reduction in mRNA and protein levels of IAPs (survivin, CIAP-1 and -2, and X-linked inhibitor of apoptosis) as well as anti-apoptotic Bcl-2, whereas an increase in protein expression of poly (ADP-ribose) polymerase 1 and caspase 3 activity was observed; however, an equivalent cytotoxic and apoptotic effect by SQE was observed in p53-WT and p53-null cells. Collectively, the results indicated that SQE-induced apoptosis was independent of p53 status and associated with modulation of endogenous NO• and IAP family gene expression.

Synergistic effects of bevacizumab in combination with β-elemene on subcutaneous xenografts derived from HCT-116 human colon cancer cells.

BackgroundTo observe the effect of bevacizumab in combination with β-elemene on subcutaneous xenografts established from HCT-116 human colon cancer cells, explore the underlying mechanisms involved, and provide more options for the treatment of advanced colorectal cancer.MethodsMice were randomly divided into four groups: control, β-elemene, bevacizumab and combination treatment. Tumor volume was measured and the tumor inhibition rate and coefficient of drug interaction (CDI) were calculated. The pathological and morphological features of the tumor tissues were observed. Protein expression levels of ki-67, CD31, vascular endothelial growth factor (VEGF) and bcl-2 were measured by immunohistochemistry.ResultsThe tumor volumes in the four groups were 0.991±0.131, 0.833±0.145, 0.563±0.131 and 0.324±0.066 cm3, respectively. The tumor inhibition rates in the β-elemene, bevacizumab and combination groups were 15.9%, 43.2% and 67.3%, respectively. The degree of tumor reduction in the combination group was the most significant (P<0.05), with a CDI of 0.697. Immunohistochemistry showed that the ki-67 proliferation indexes in the four groups were 30.06%±3.37%, 19.79%±3.75%, 27.51%±2.16% and 16.9%±1.09%, the microvessel densities (MVDs) were 45.86±9.28, 31.14±5.58, 27.57±6.11 and 12.14±3.53, the rates of positive VEGF expression were 39.48%±6.72%, 28.86%±3.54%, 25.04%±2.64% and 19.5%±2.68%, and the rates of positive bcl-2 expression were 33.95%±6.38%, 20.11%±9.49%, 24.28%±6.57% and 11.18%±2.72%, respectively. Compared with the control group, the three experimental groups displayed different degrees of reduction in the above indicators (P<0.05), with the combination group exhibiting the lowest values (P<0.05).ConclusionsBevacizumab exerts a synergistic effect with β-elemene in suppressing the growth of tumors derived from HCT-116 cells, and the related mechanisms may include the inhibition of tumor cell proliferation and tumor angiogenesis and the promotion of tumor cell apoptosis.

Potential neurotrophic activity and cytotoxicity of selected C21 steroidal glycosides from Cynanchum otophyllum

Cynanchum otophyllum was traditionally used for treating muscle and skeletal pain, epilepsy, abdominal pain, and tightness. Four known C21 steroidal glycosides, namely caudatin 3-O-β-cymaropyranoside (1), caudatin 3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside (2), otophylloside B (3), and otophylloside A (4) were isolated from the roots of this plant and their neurotrophic activities were examined in rat neuronal PC12 cell model, while their cytotoxicities were evaluated in human cancer cells and normal fibroblasts. Differential promoting effects on neurite-bearing cells were found among testing compounds, with 2 the most potent. On the other hand, selective cytotoxicity against human colon cancer cells HCT-116 of 2 was firstly demonstrated here. Besides, 2 induced inhibitory activities on cancer cell proliferation, sphere formation and interfered the cell cycle. The new potential pharmacological activities of 2 on colon cancer cells were revealed in the present study.

The Influence of In Vitro Gastrointestinal Digestion on the Anticancer Activity of Manuka Honey.

Manuka honey (MH) is a natural food with many beneficial properties to human health, thanks to its high variety of bioactive compounds; however, little is known about its bioaccessibility. The aim of this study was to evaluate and compare the polyphenol compounds, the antioxidant capacity and the anticancer activity of MH subjected to an in vitro gastrointestinal digestion in human HCT-116 colon cancer cells. Raw MH and digested MH (DMH) were assessed for total polyphenols and flavonoids by spectrophotometric and HPLC-ESI-MS/MS analysis, and total antioxidant capacity (TAC) using different methods. Cell viability, intracellular ROS production, apoptosis, cell cycle and colony formation capacity were tested after treatment with MH or DMH. Results showed that total polyphenols, total flavonoids and TAC were significantly (p < 0.05) reduced after in vitro digestion. In addition, MH and DMH at 8, 16 and 24 mg/mL had similar effects in inducing intracellular ROS production and in inhibiting the colon formation ability; MH induced a more marked apoptosis compared to DMH, while cell cycle was blocked in S phase by MH and in Sub G1 phase by DMH. Our results increase knowledge of the effect of gastrointestinal digestion on the biological effect of honey against colorectal cancer.

Mechanism of a Novel Camptothecin-Deoxycholic Acid Derivate Induced Apoptosis against Human Liver Cancer HepG2 Cells and Human Colon Cancer HCT116 Cells.

Camptothecin (CPT) is known as an anticancer drug in traditional Chinese medicine. However, due to the lack of targeting, low solubility, and instability of CPT, its therapeutic applications are hampered. Therefore, we synthesized a series of CPT-bile acid analogues that obtained a national patent to improve their tumour-targeting chemotherapeutic effects on liver or colon cancers. Among these analogues, the compound G2 shows high antitumor activity with enhanced liver targeting and improved oral absorption. It is significant to further investigate the possible anticancer mechanism of G2 for its further clinical research and application. We aimed to unearth the anticancer mechanism of G2 in HepG2 and HCT116 cells. Cell viability was measured using MTT assay; cell cycle, Mitochondrial Membrane Potential (MMP), and cell apoptosis were detected by flow cytometer; ROS was measured by Fluorescent Microplate Reader; the mRNA and protein levels of cell cycle-related and apoptosis-associated proteins were examined by RT-PCR and western blot, respectively. We found that G2 inhibited cells proliferation of HepG2 and HCT116 remarkably in a dosedependent manner. Moreover, G2-treatment led to S and G2/M phase arrest in both cells, which could be elucidated by the change of mRNA levels of p21, p27 and Cyclin E and the increased protein level of p21. G2 also induced dramatically ROS accumulated and MMP decreased, which contributed to the apoptosis through activation of both the extrinsic and intrinsic pathways via changing the genes and proteins expression involved in apoptosis pathway in both of HepG2 and HCT116 cells. These findings suggested that the apoptosis in both cell lines induced by G2 was related to the extrinsic and intrinsic pathways.

Synthesis and Cytotoxicity Evaluation of Panaxadiol Derivatives.

In this study, 13 panaxadiol (PD) derivatives were synthesized via reactions with aromatic compounds and amino acids. Following this, the cytotoxicity of these compounds was evaluated against four cancer cell lines (human hepatoma cells HepG-2, human lung cancer cells A549, human breast cancer cells MCF-7, and human colon cancer cells HCT-116) and one normal cell lines (human gastric epithelial cells GES-1). The results showed that the panaxadiol derivatives 3, 12, and 13 showed significant inhibition of cellular proliferation against cancer cells compared with PD, and the panaxadiol derivative 12 had the lowest IC50 value for A549 (IC50 =18.91±1.03 μm). For MCF-7 cells, most compounds exhibited good inhibition of cellular proliferation, and the panaxadiol derivative 13 showed the strongest inhibitory effect (IC50 =8.62±0.23 μm), which significantly increased the cytotoxicity of PD and was stronger than the positive control (mitomycin). For normal cells, all compounds exhibited low or no toxic effects; thus, these derivatives can be used to develop novel antiproliferative agents.

Cembrane-type diterpenoids from the gum resin of Boswellia carterii and their biological activities.

Eight new cembrane-type diterpenoids, boscartins AH–AK (1–8), along with two known ones (9-10), were isolated from the gum resin of Boswellia carterii. Compounds 1–3 were characteristic of high oxidation assignable to three epoxy groups, while compounds 4–8 were characteristic of two epoxy groups. Spectroscopic examination was used to elucidate their structures. All isolates were evaluated for antiproliferative activity against HCT-116 human colon cancer cells, anti-inflammatory activity against nitric oxide (NO) production, and hepatoprotective activity in vitro. All of them showed weak antiproliferative activity (IC50 > 100 μM), 8 exhibited potent inhibitory effects on NO production (IC50 of 14.8 μM), with the others showing weak anti-inflammatory activity (IC50 > 30 μM), and 1 exhibited more potent hepatoprotective activity than the positive control, bicyclol, at 10 μM against the damage induced by paracetamol in HepG2 cells.

TRAF6-Mediated Inflammatory Cytokines Secretion in LPS-induced Colorectal Cancer Cells Is Regulated by miR-140.

Colorectal cancer (CRC) cells secrete inflammatory cytokines that affect CRC progression. The aim of the present study was to determine if micro-RNA-140(miR-140) regulates inflammatory cytokine secretion induced by lipopolysaccharide (LPS) in colorectal cancer cells by targeting tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Fifty fresh colon-cancer specimens and normal colorectal tissues were collected from patients with CRC and tested for the expression miR-140. Human CRC cell lines SW480 and HCT116 were treated with various concentrations and times with LPS. miR-140 and mRNA expression of potentially related genes were analyzed by qPCR. Protein expression was analyzed using western blot or ELISA. Overexpression plasmids with pcDNA3.1-TRAF6, pGL4.10-wtTRAF6 and pGL4.10-mutTRAF6 were constructed. miRNA target gene prediction and a dual luciferase assay were used to analyze miR-140-targeted TRAF6. miR-140 expression was up-regulated in CRC tissues. In CRC cells, LPS could increase miR-140 expression in a time- and concentration-dependent manner. LPS increased inflammatory cytokine mRNA expression levels in SW480 and HCT116 human colon-cancer cells. miRNA-140 suppressed TRAF6 expression via targeting the 3'UTR. TRAF6 affected miR-140-mediated inflammatory cytokine expression of SW480 and HCT116 cells under LPS treatment. miR-140 regulates inflammatory cytokine secretion of LPS-induced colorectal cancer cells by targeting TRAF6.

Novel hybrid molecules of 3,5-bis(benzylidene)-4-piperidones and dichloroacetic acid which demonstrate potent tumour-selective cytotoxicity

A novel class of hybrid molecules 2a-o was designed as candidate antineoplastic agents from dichloroacetic acid which is a known inhibitor of pyruvate dehydrogenase kinase and a number of cytotoxic 3,5-bis(benzylidene)-4-piperidones 1. In general these new hybrid molecules are potent cytotoxins towards human HCT116 colon cancer cells. A number of lead molecules emerged having the IC50 values in the double digit nanomolar range. Most of these compounds are less toxic to human CRL1790 non-malignant colon cells and hence the selectivity index (SI) figures for most of the compounds are huge; in the case of 2c-g, m, n, the SI values are in excess of 100. Compounds 2g, 2j, 2m and 2n displayed >100-fold higher potency than the reference drug 5-FU. Quantitative structure-activity relationships revealed that the potencies of the compounds in series 2 increase as the magnitude of the Hammett σ and Taft σ* values rise. X-ray crystallographic of a representative compound 2c revealed various structural features which may influence cytotoxic potencies. Several representative compounds lowered the mitochondrial membrane potential and increased the production of reactive oxygen species in HCT116 cells. A minimal effect was noted in altering the percentage of cells in different phases of the cell cycle. Some futuredirections have been outlined for analog development.

Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and PI3K/Akt signaling pathway.

Cocoa tea (Camellia ptilophylla), a natural gallocatechin gallate (GCG)-rich and low caffeine-containing tea species, has been recently reported to possess various bioactivities. However, the anti-colon cancer effects of Cocoa tea and its underlying mechanisms remain virtually unknown. This study aimed to assess the anti-proliferative and pro-apoptotic effects of water extract of Cocoa tea (CWE) on human colon cancer HCT116 cells compared with Yunnan Daye tea (YWE). Primarily, CWE showed stronger anti-proliferation and apoptosis induction than YWE. Moreover, reduction of mitochondrial membrane potential (MMP), up-regulation of Bax/Bcl-2 ratio, release of cytochrome c, activation of caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP) were observed, suggesting that mitochondrial apoptotic pathway was activated by CWE. Furthermore, CWE-induced apoptosis in HCT116 cells was dependent on the generation of intracellular reactive oxygen species (ROS) and down-regulation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Pretreatment with ROS scavenger N-acetyl cysteine (NAC) attenuated the impact of CWE on mitochondria-related apoptosis proteins, and partially recovered the inhibition of Akt phosphorylation. These results indicated that ROS generation mediated mitochondrial dysfunction and inactivation of PI3K/Akt pathway in CWE-induced HCT116 cell apoptosis. Additionally, CWE significantly inhibited tumor growth in HCT116 tumor-bearing mice, suggesting that Cocoa tea could act as a potential functional beverage to prevent or treat colorectal cancer.

Abstract B079: Inhibitory role of NFE2L2/NRF2-silencing in hypoxia-induced metabolic adaptation of cancer cells

Abstract The nuclear factor erythroid 2-like-2 (NFE2L2/NRF2) pathway contributes to the environmental resistance of cancers by enhancing the antioxidant capacity. Hypoxia, a common element in the tumor environment, leads to hypoxia-inducible factor-1α (HIF-1α) stabilization to induce the adaptive response to hypoxic condition, including metabolic reprogramming and blood vessel formation. Our global miRNA expression analysis of HT29 and HCT116 human colon cancer cells identified that NRF2 silencing upregulated miR-181c, and this increase was associated with the reduction in mitochondria-encoded cytochrome c oxidase subunit-1 (MT-CO1), a catalytic core subunit of the complex IV of the electron transport chain. Therefore, NRF2-silenced cancer cells exhibited the decreases in the mitochondrial membrane potential, oxygen consumption rate, and cellular ATP contents. Notably, as a result of low oxygen consumption rate, NRF2-inhibited cancer cells failed to accumulate HIF-1a protein under hypoxic conditions. This phenomenon led to differential responses of NRF2-inhibted cancer cells to hypoxic condition when compared to control cells. First, stable knockdown of NRF2 in human colon cancer cells suppressed tumor growth in mouse xenograft settings with a concomitant reduction in blood vessel formation and vascular endothelial growth factor expression. Second, NRF2-silencing in breast cancer cells inhibited metabolic adaptation upon hypoxia, and hypoxic-inducible levels of metabolites involved in glycolysis and pentose phosphate pathway were reduced. Collectively, targeting NRF2/miR-181c could be an effective strategy to counteract HIF-1α-orchestrated adaptation of cancer cells to hypoxic microenvironment. Citation Format: Mi-Kyoung Kwak, Sujin Lee, Steffanus Pranoto Hallis. Inhibitory role of NFE2L2/NRF2-silencing in hypoxia-induced metabolic adaptation of cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B079. doi:10.1158/1535-7163.TARG-19-B079

RASAL1 induces to downregulate the SCD1, leading to suppression of cell proliferation in colon cancer via LXR\u03b1/SREBP1c pathway

BackgroundRecent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated.ResultsWe collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3′-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo.ConclusionTaken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

Composition and activity changes of the soluble water and ethanol extracts from white mulberry (Morus alba L.) fruits in response to thermal treatment

This study investigated the impact of a thermal treatment at 100 °C on the compositions and activities of the two soluble extracts from white mulberry fruits. Lyophilized white mulberry fruits were extracted using water and ethanol–water (75%, v/v) solvents, followed by various extract treatments like ethanol removal, activated carbon-mediated dephenolization, or 100 °C thermal treatment of 15–45 min. The thermal treatment caused increased values for total phenols and flavonoids contents; subsequently, the values for total phenol contents (expressed as gallic acid equivalent) of the water- and ethanol-extracts were enhanced from 110.0 and 146.5 to 120.9 and 161.3 mg/L, while the values for total flavonoid contents (expressed as quercetin equivalent) were increased from 19.0 and 23.4 to 28.2 and 30.4 mg/L, respectively. However, the thermal treatment time-dependently decreased the anti-oxidation of these heated extracts that consistently showed lower activities to scavenge the DPPH, ABTS, and hydroxyl radicals, or lower efficiencies to reduce Fe3+. The water- and ethanol-extracts also showed three activities in human colon cancer HCT-116 cells, resulting in growth inhibition, morphology change, and the generation of intracellular reactive oxygen species. Moreover, the heated extracts received activity increases in the cells, compared with the unheated extracts. In brief, the Maillard reaction during the thermal treatment might partly make contribution for the activity increases. It is thus concluded that the thermal treatment of these extracts at 100 °C resulted in composition and activity changes due to the Maillard reaction and other unidentified reactions, which might endow the heated extracts with weakened anti-oxidation but increased activity against HCT-116 cells.

Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells

Advanced colon cancer is extremely difficult to cure, underscoring the need to develop novel therapeutic agents. Prenylated curcumins that are semisynthetic curcumin derivatives with significant anti-cancer potential have been studied herein to assess their therapeutic potential for colon cancer and tested to this aim in vitro for their growth inhibitory properties against 5-fluorouracil + oxaliplatin resistant human colon cancer CR-HT29 and HCT-116 cells. The resulting most active product, gercumin (mono-O-geranylcurcumin), has been further tested for its synergistic effects with FOLFOX (a combination of 5-fluorouracil and oxaliplatin) on the same cell lines. Activity of this combination on colonosphere formation was also investigated. Gercumin was able to suppress the growth of cancer cells with a potency similar to that of curcumin. A synergistic effect of this compound and FOLFOX was also observed. doses tested for synergy in the colonosphere assays did not show greater suppression of colonosphere formation than independent treatment with either reagent alone. Only one of the combinations was shown to be more effective at suppressing colonosphere formation [gercumin 5 μM + FOLFOX (2x)]. Thus, the growth inhibitory effects of curcumin against human cancer cells can be modulated and enhanced by the introduction of hydrophobic chains, normally found in several natural compounds, like the geranyl one. Such compounds are also able to synergize with known chemotherapeutics.

D-Ring-Modified Analogues of Luotonin A with Reduced Planarity: Design, Synthesis, and Evaluation of Their Topoisomerase Inhibition-Associated Cytotoxicity.

A- and D-ring-modified luotonin-inspired heterocycles have been synthesized and were evaluated for their activity against the viability of four cancer cell lines in vitro, namely, MCF7, HCT116, JURKAT, and NCI-H460. The analysis of results indicated that two of the synthesized derivatives displayed good inhibition against the growth of the human colon cancer HCT116 cell line, with potencies lower than but in the same order of magnitude as camptothecin (CPT). These two luotonin analogues also showed an activity similar to that of the highly potent alkaloid CPT as inhibitors of topoisomerase I and also inhibited topoisomerase II. These results show that complete planarity is not a strict requirement for topoisomerase inhibition by luotonin-related compounds, paving the way to the design of analogues with improved solubility.

Cells Deficient in the Shwachman-Diamond Syndrome Protein SBDS or the Diamond-Blackfan Anemia Protein RPS19 Have Impaired Homologous Recombination

The inherited bone marrow failure syndromes (IBMFS) are rare genetic disorders caused by mutations in critical components of fundamental cellular processes such as ribosome biogenesis, DNA repair, and telomere maintenance. The IBMFS Shwachman-Diamond syndrome(SDS) and Diamond-Blackfan anemia (DBA) are classified as ribosomopathies due to etiologic mutations in genes encoding factors involved in ribosome biogenesis (SBDSin the majority of patients with SDS) or ribosomal proteins (RPS19most commonly in patients with DBA). Although these disorders can be distinguished clinically and from the other IBMFS, they share with each other and with other IBMFS increased predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas genomic instability due to defective DNA repair or telomere maintenance is thought to underlie cancer predisposition in the IBMFS Fanconi anemia and dyskeratosis congenita, respectively, the molecular mechanisms driving cancer in SDS and DBA are not fully understood. Our research has focused on DNA repair in SDS and DBA. A prior report suggested lymphoblastoid cell lines (LCLs) derived from patients with SDS arehypersensitive to ionizing radiation (IR). Consistent with this, we found SDS-LCLs had decreased survival following IR compared to control-LCLsin colony survival assays. To determine if this cellular phenotype was unique to SDS or present in the other IBMFS ribosomopathy, DBA, we examined LCLs derived from patients with DBA, including those with mutations in RPS19, RPS26, RPL5and RPL11. We found that the DBA-LCLs were similarly hypersensitive to IR as compared to control-LCLs. Further examination of γ-H2AX, a DNA damage response (DDR) factor and marker of DNA double strand breaks (DSBs), revealed that SDS- and DBA-LCLs had delayed resolution of γ-H2AX foci and increased protein levels at 24 hrs after IR as compared to control LCLs. p53, phospho-ATM, and DNA-PKcs protein levels were also higher in SDS-LCL compared to controls. The decreased survival and increased and sustained DDR following IR led us to hypothesize that SDS and DBA cells have a defect in DSB repair. There are two major pathways of DSB repair in mammals, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), and loss of either results in hypersensitivity to IR. To examine each pathway, we employed U2OS (human osteosarcoma) and HCT116 (human colon cancer) cells containing an integrated green fluorescent protein HDR or NHEJ reporter transgene. Interestingly, we found that knockdown of either SBDS or RPS19 proteins resulted in an approximately 50% reduction in HDR efficiency but no change in NHEJefficiency compared to the scrambled control in both cell lines. We next sought to determine the mechanism underlying the effect of SBDS and RPS19 deficiency on HDR. A survey of proteins required for HDR revealed a reduction in the recombinase RAD51 in SDS-LCLs and in SBDS-depleted HCT116 and U2OS cells, whereas, an initial survey in SDS-LCLs[e1] of factors involved in NHEJ did not reveal a specific NHEJ factor deficiency. Knockdown of eiF6 is known to rescue the defect in 40S and 60S ribosome subunit joining that manifests in SDS patient cells. However, we found eIF6 depletion failed to rescue the level of RAD51 protein and had no impact on HDR in SBDS-deficient cells. We conclude that decreased RAD51 levels in SBDS-deficient cells might contribute to impaired HDR, however, this decrease is independent of the ribosome subunit joining defect. Similarly, RPS19 knock down resulted in a reduction in RAD51 protein level, suggesting a potentially common pathway. We also asked whether SBDS or RPS19 might be more directly involved in the DDR or repair of DSBs. Consistent with this, we found SBDS and RPS19 recruited to chromatin surrounding an I-Sce1 site following DSB induction in chromatin immunoprecipitation assays. Collectively, these findings provide evidence that SBDS and RPS19 may be directly involved in the DDR or DSB repair and raise the possibility that loss of this function may contribute to MDS/AML predisposition in SDS and DBA patients. Disclosures No relevant conflicts of interest to declare.

Anti-tumor effects of an antagonistic mAb against the ASCT2 amino acid transporter on KRAS-mutated human colorectal cancer cells.

KRAS mutations are detected in numerous human cancers, but there are few effective drugs for KRAS‐mutated cancers. Transporters for amino acids and glucose are highly expressed on cancer cells, possibly to maintain rapid cell growth and metabolism. Alanine‐serine‐cysteine transporter 2 (ASCT2) is a primary transporter for glutamine in cancer cells. In this study, we developed a novel monoclonal antibody (mAb) recognizing the extracellular domain of human ASCT2, and investigated whether ASCT2 can be a therapeutic target for KRAS‐mutated cancers. Rats were immunized with RH7777 rat hepatoma cells expressing human ASCT2 fused to green fluorescent protein (GFP). Splenocytes from the immunized rats were fused with P3X63Ag8.653 mouse myeloma cells, and selected and cloned hybridoma cells secreting Ab3‐8 mAb were established. This mAb reacted with RH7777 transfectants expressing ASCT2‐GFP proteins in a GFP intensity‐dependent manner. Ab3‐8 reacted with various human cancer cells, but not with non‐cancer breast epithelial cells or ASCT2‐knocked out HEK293 and SW1116 cells. In SW1116 and HCT116 human colon cancer cells with KRAS mutations, treatment with Ab3‐8 reduced intracellular glutamine transport, phosphorylation of AKT and ERK, and inhibited in vivo tumor growth of these cells in athymic mice. Inhibition of in vivo tumor growth by Ab3‐8 was not observed in HT29 colon and HeLa uterus cancer cells with wild‐type KRAS. These results suggest that ASCT2 is an excellent therapeutic target for KRAS‐mutated cancers.

Doxorubicin-Loaded Delta Inulin Conjugates for Controlled and Targeted Drug Delivery: Development, Characterization, and In Vitro Evaluation.

Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid–labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.

General toxicity assessment of the novel aldose reductase inhibitor cemtirestat.

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b] indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 “Inactive” outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic β-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 μM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies.

Numerical analysis of apparent decitabine uptake in HCT116 cells: Incorporation of a bidirectional first-order kinetic parameter for ENT1 transport and Michaelis-Menten parameters for subsequent phosphorylation

Decitabine (DAC), a DNA methylation inhibitor, is transported into cancer cells mainly via equilibrative nucleoside transporter 1 (ENT1) and subsequently phosphorylated by deoxycytidine kinase (dCK). We previously reported that apparent DAC uptake into cells may be described using a simple compartment model with clearance for facilitated diffusion (PS) and subsequent phosphorylation (CLmet). In the present study, time course of apparent intracellular [3H]-DAC uptake was analyzed numerically, and PS and CLmet values were calculated using the compartment model in human colon cancer HCT116 cells. PS at 0.1 μM [3H]-DAC was markedly decreased in the presence of 100 μM irinotecan or etoposide, while CLmet was markedly decreased in the presence of 100 μM cytarabine or gemcitabine. CLmet at 0.1–10 μM [3H]-DAC varied in a concentration-dependent manner and was described by Michaelis-Menten parameters Km,met and Vmax,met. In conclusion, DAC uptake mainly via ENT1 may be described by a bidirectional first-order kinetic parameter, while phosphorylation by dCK may be described by Michaelis-Menten parameters.