Normal CCD-18Co Cells Research Articles

Regulation of apoptosis and autophagy by albendazole in human colon adenocarcinoma cells

Albendazole (ABZ) was initially introduced as an anthelmintic, however, many studies have reported with its anticancer effects. We investigated the anti-tumor effects of ABZ in vitro in human colon adenocarcinoma HCT-15, HCT-116, HT-29, and SW480 cell lines in this study. The cytotoxicity of ABZ was analyzed in colon adenocarcinoma cell lines and normal CCD18Co cells. We found that ABZ induced the subG1 arrest during cell cycle progression, increased the late apoptotic cells, shifted of peak TUNEL-labeled cells peak, and induced apoptosis. Then effects on autophagy activation was confirmed by acridine orange (AO), MDC staining, and immunocytochemistry of LC3. It was observed that ABZ can induce the autophagy activation through modulating the levels of LC3, Atg7, and beclin-1. For mechanistic studies, apoptosis blocker (Z-DEVD-FMK) and autophagy inhibitor (3-MA) were used to confirm that whether ABZ has apoptosis and autophagy specific effects, and reversal in both these cell death processes were noted. The effects of ABZ on AMPK, MAPKs, and ULK induction was also evaluated. We noticed that N-acetyl cysteine (NAC), a broad spectrum antioxidant, can effectively inhibit both apoptosis and autophagy. However, ABZ could even recover suppression of apoptosis and autophagy caused by NAC in colon cancer cells. Therefore, ABZ can potentially up-regulate both the apoptosis and autophagy to significantly suppress tumorigenesis in colorectal cancer cell lines.

Synthesis of New Biscoumarin Derivatives, In Vitro Cholinesterase Inhibition, Molecular Modelling and Antiproliferative Effect in A549 Human Lung Carcinoma Cells.

A series of novel C4-C7-tethered biscoumarin derivatives (12a–e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC50 = 6.30 µM) and butyrylcholinesterase (BChE, IC50 = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood–brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer’s disease.

Pancratistatin Inhibits the Growth of Colorectal Cancer Cells by Inducing Apoptosis, Autophagy, and G2/M Cell Cycle Arrest.

BackgroundWorldwide, colorectal cancer is ranked as the third most prevalent cancer. The natural compound, pancratistatin, extracted from the spider lily, has previously been shown to target apoptosis in cancer cells lines. This study aimed to investigate the effects of pancratistatin in human colorectal cancer cells in vitro.Material/MethodsHuman colorectal cancer cell lines, including HTC-15 cells, were compared with a normal human colonic fibroblast cell line, CDD-18Co. Cells were treated with increasing doses of pancratistatin. The MTT assay was used to assess cell viability. Fluorescence microscopy using DAPI and Annexin-V/propidium iodide (PI) was used to detect cell apoptosis. Cell autophagy was detected by electron microscopy. Cell migration was evaluated using a wound healing assay, and Western blot determined the expression levels of cell cycle proteins.ResultsPancratistatin inhibited the growth of the colorectal cancer cells with an IC50 ranging from 15–25 μM, but had a limited effect in normal CCD-18Co cells, with an IC50 of >100 μM. Pancratistatin reduced HCT-15 cell migration. Growth inhibition due to pancratistatin was associated with morphological changes of HCT-15 cells and included autophagy and apoptosis, and increased expression the autophagic proteins, LC3II, beclin-1, and Bax. Pancratistatin induced arrest of HCT-15 cells at G2/M of the cell cycle and inhibited phosphorylation of cdc2/cyclin-dependent kinase 1 (CDK1) and Cdc25c and the expression of cyclin B1.ConclusionsPancratistatin inhibited the growth of colorectal cancer cells in vitro by inducing apoptosis, autophagy, and G2/M cell cycle arrest.

Downregulation of transcription factor EB inhibits the growth and metastasis of colorectal carcinomas

To determine the roles of transcription factor EB (TFEB) in colorectal cancer (CRC), we collected samples of tumor tissues and normal tissues from 40 patients with CRC. The expression of TFEB in these samples was analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Furthermore, we explored the expression of TFEB mRNA in CCD-18Co normal cells and HT-29, HCT-8, C2BBe1 cancer cells. HT-29, HCT-8, and C2BBe1 cancer cells were transfected with a TFEB-specific small interference RNA (siRNA) and scrambled siRNA, then the TFEB expression was confirmed by Western blot. The migration and invasion abilities of cells transfected with TFEB-siRNA were examined by transwell method and wound-healing assay. The subsequent effect of TFEB silencing on the tumor growth was also detected in mice xenograft model in vivo. Our study found that TFEB expression was significantly increased ( P < 0.05) in colorectal tumor tissues compared with normal tissues. Consistent with TFEB expression in tissues, compared with the normal CCD-18Co cells, TFEB mRNA expression was also significantly augmented in CRC cells. TFEB protein expression was markedly reduced in HT-29, HCT-8, and C2BBe1 cells after TFEB-siRNA transfection. In addition, inhibition of TFEB expression resulted in decrease of cells migration and invasion abilities. In vivo study, compared with the negative control group, the tumor weight, and volume were also reduced after inhibiting the TFEB expression. Our research suggested that TFEB expression is related to the occurrence and development of colorectal adenocarcinoma. The migration and invasion abilities of cancer cells, the weight and volume of tumor were all decreased when inhibiting TFEB expression. Thus, TFEB serves as an important factor in the development of CRC by modulating cancer cell migration and invasion, showing the potential therapeutic target of CRC in clinical.

Macrophage and colon tumor cells as targets for a binuclear silver(I) N-heterocyclic carbene complex, an anti-inflammatory and apoptosis mediator

Chronic inflammation intensifies the risk for malignant neoplasm, indicating that curbing inflammation could be a valid strategy to prevent or cure cancer. Cancer and inflammation are inter-related diseases and many anti-inflammatory agents are also used in chemotherapy. Earlier, we have reported a series of novel ligands and respective binuclear Ag(I)–NHC complexes (NHC=N-heterocyclic carbene) with potential anticancer activity. In the present study, a newly synthesized salt (II) and respective Ag(I)–NHC complex (III) of comparable molecular framework were prepared for a further detailed study. Preliminarily, II and III were screened against HCT-116 and PC-3 cells, wherein III showed better results than II. Both the compounds showed negligible toxicity against normal CCD-18Co cells. In FAM-FLICA caspase assay, III remarkably induced caspase-3/7 in HCT-116 cells most probably by tumor necrosis factor-alpha (TNF-α) independent intrinsic pathway and significantly inhibited in vitro synthesis of cytokines, interleukin-1 (IL-1) and TNF-α in human macrophages (U937 cells). In a cell-free system, both the compounds inhibited cyclooxygenase (COX) activities, with III being more selective towards COX-2. The results revealed that III has strong antiproliferative property selectively against colorectal tumor cells which could be attributed to its pro-apoptotic and anti-inflammatory abilities.

The expression of frizzled-3 receptor in colorectal cancer and colorectal adenoma.

444 Background: Wnt signaling is well known for its role in colorectal cancer (CRC) formation through transcriptional activities of nuclear β-catenin. Although activation of Wnt signaling depends on specific Wnt/Frizzled receptors (FZD) combinations, the specificity of the interaction and the role of FZD in that particular interaction are still unknown. Among the 10 Wnt receptors of the FZD protein family, FZD-3 is involved in neurodevelopmental abnormalities and gastric cancer carcinogenesis. However, the expression of FZD-3 in CRC is not clear. Therefore in this study, we examined the expression of FZD-3 in CRC cell lines and CRC patient tissues with various pathological stages. The information obtained will be important for us to understand the role of FZD-3 in the development of CRC. Methods: FZD-3 mRNA expression was studied in CRC metastatic SW620, primary SW480 and normal CCD18co cell lines using quantitative real-time polymerase chain reaction with primers and a Taqman minor grove binder probe (Applied Biosystems, Foster City, USA). Moreover, paraffin-embedded specimens of 40 CRC patient tissues, 25 colorectal adenoma (CA) tissues were retrieved from the Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region for FZD-3 immunostaining using an anti-FZD-3 antibody (Catalog no: MAB1001, R&D systems Inc., Minneapolis, USA) in an automatic Ventana Benchmark XT immunostainer (Ventana Medical Systems Inc., Tucson, USA). Results: FZD-3 mRNA was up-regulated in metastatic SW620 cell line (fold-change: 622) and in primary SW480 cell line (fold-change: 820) when compared to that in normal CCD18co cell line. Furthermore, immunostaining showed that FZD-3 protein was expressed in 100% (40/40) of CRC specimens and 84% (21/25) of CA specimens. Detailed analysis showed that FZD-3 protein was significantly up-regulated in CRC, CA when compared to their adjacent normal colorectal epithelial tissues (p < 0.0005, Wilcoxon matched pairs test). Conclusions: This study provided evidence that FZD-3 is involved in CRC carcinogenesis and it is a potential therapeutic target in CRC. No significant financial relationships to disclose.