HCT116 Colon Cancer Research Articles

Abstract 261: Released exosomes from colon cancer cells regulate formation and progression of perineural invasion

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer and cause of cancer related mortality. CRC that metastasizes to distance sites results in poor prognosis, diminished survival, and reduced effectiveness of treatments. Perineural invasion (PNI) is an emerging field that aims to understand cancer-neuronal crosstalk that enables cancer to spread via nerves. Several studies have indicated that tumor derived exosomes (TDEs) play significant roles in the initiation and spread of cancer, along with driving chemoresistance. We hypothesize that CRC derived exosomes alter nerves within the surrounding environment and result in recruitment of nerves towards tumors for invasion along the nerve. Exosomes from both HCT116 and SW837 colon cancer cells were isolated and purified using a modified iodixanol ultracentrifugation gradient technique. A Proteomic analysis conducted on cancer derived exosomes revealed several potential proteins involved in neuronal growth and migration, including Vinculin (VCL), Periostin (POSTN or OSF-2), DCLK1, and USP9X. In another set of experiments, conditioned media (CM) from HCT116 and SW837 cells were added to both PC12 neuronal-like cells and dorsal root ganglion (DRG) from C57BL/6J mice to understand the effects of excreted cancer factors on neuronal outgrowth and differentiation. CM studies showed how signaling factors excreted from colon cancer cells induce increased nerite outgrowth in both PC12 cells and DRG. These results were not seen in CM from cancer cells that were grown in the presence of an exosome inhibitor. Altogether, these results indicate that CRC utilizes exosomes to alter the adjacent neuronal networks as a means to employ surrounding nerves for invasion. Citation Format: Colewyn Knoblich, Khosrow Rezvani. Released exosomes from colon cancer cells regulate formation and progression of perineural invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 261.

Abstract 3300: Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors

Abstract Oncolytic virotherapy represents a promising therapeutic strategy for cancers that do not respond well to immune checkpoint inhibitors. Oncolytic viruses (OV) preferentially replicate in and lyse tumor cells, leading to systemic immune stimulation. Studies have shown that hypersialylation promotes tumor growth and metastasis, as well as the suppression of immune cells. Cancer-associated fibroblasts (CAFs) are known to facilitate tumor invasion and angiogenesis and maintain immunosuppressive microenvironment in solid tumors. We are developing vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase, to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager, to eliminate glycoimmune checkpoint and tumor stroma, respectively. vvDD-Sial-FAP/CD3 is an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) genes for greater dependence on the cell cycling status of the cancer cells. Our results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the Fc fused to the sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay. In vitro efficacy studies were conducted using HCT-116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. In both tumor models, vvDD-Sial-FAP/CD3 induced activation of both CD4+ and CD8+ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroids. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing. Taken together, our results have demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated. Citation Format: Winnie M. Chan, Mariya Viskovska, Micaela McArthur, Xue Mary Yu, Wei Liang, Zane Norman, Xiaotong Song, Nancy T. Chang, Haiyan Jiang. Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3300.

Discovery of dearomatized isoprenylated acylphloroglucinols with colon tumor suppressive activities in mice via inhibiting NFκB-FAT1-PDCD4 signaling activation

Dearomatized isoprenylated acylphloroglucinols (DIAPs) are specific natural products mainly distributed in the plants of genus Hypericum. In this study, guided by HPLC-UV screening, 46 DIAPs (approximately 70% of all DIAPs) including 20 new ones and an unprecedented architecture, were discovered from the roots of Hypericum henryi, which were elucidated by comprehensive spectroscopic, X-ray crystallography, and ECD methods. Compounds 1–7, 39, and 41–42 exhibited remarkable cytotoxicities (IC50 = 0.84–5.63 μM) in human colon cancer HCT116 cells, in which 2 and 6 possessed selective cytotoxicities towards colon cancer cells. The preliminary structure-activity relationships of these tested compounds were discussed. In addition, mechanistic investigations demonstrated that 2 and 6 could significantly suppress the expressions of NFκB, FAT1, and promoted novel tumor suppressor gene PDCD4 in HCT116 cells. Furthermore, in HCT116 colon xenograft-bearing mouse model, treatments with 2 and 6 reduced the growth of xenograft tumors in dose-dependent manner. Expressions of FAT1 in tumors were also decreased in mice treated with 2 and 6, suggesting their anti-tumor effects were via FAT1 signaling pathway. In conclusion, this is the first report on the mechanistic and in vivo studies of DIAP, indicating that these metabolites can be considered as a new type of anti-colon cancer lead agents for further drug development.

Selenium-functionalized cyclic ethers derived from natural terpenic alcohols – biological in vitro profile

Terpenic alcohols linalool, nerolidol and α-terpineol were subjected to the PhSe-induced cyclization, where corresponding cyclic ethers were obtained. The heterocyclic product derived from α-terpineol is a derivate of the natural product eucalyptol, while linalool cyclization produces precursors of the natural product karahanaenone. All three cyclic ether products have an organoselenium moiety in the side chain, which can represent a significant source of bioactivities. Biological evaluation of obtained products, in vitro cytotoxicity and redox status parameters, was performed on two model systems: HCT-116 – immortalized colon cancer cell line, and MRC-5 – healthy fibroblasts isolated from lung pleura. The results indicate a strong prooxidative character of all compounds on colon cancer HCT-116 and healthy MRC-5 cells in the highest applied concentrations. Selectivity towards cancer cells was not observed, except when nerolidol-derived product was used for longer exposure time. A biological evaluation was conducted to establish the quantitative relationship between enhanced radical species formation and cell viability.

Efficient Strategy for the Preparation of Chemical Probes of Biologically Active Glycosides Using a Boron-Mediated Aglycon Delivery (BMAD) Method

Abstract Development of an efficient method for the analysis and identification of the target proteins with which biologically active glycosides directly interact is highly desirable in many research fields. In this article, we report an efficient strategy for the preparation of chemical probes of biologically active glycosides using a reaction sequence of i) a boron-mediated aglycon delivery (BMAD) with an N3-functionalized 1,2-anhydroglucose donor, ii) deprotection, and iii) strain-promoted azide-alkyne cycloaddition. Using the synthesized chemical probes, we successfully demonstrated that the target proteins of a cardiac glycoside, lanatoside C (1), can be visualized and identified in human colon cancer HCT116 cells.

C-MYC and HIF1α promoter G-quadruplexes dependent metabolic regulation mechanism of berberine in colon cancer.

G-quadruplexes are molecular switches regulating gene transcription. c-MYC and hypoxia-inducible factor 1-alpha (HIF1α) play important roles in cell proliferation, apoptosis, and metabolic regulation in colon cancer. Whether berberine can regulate metabolism by interacting with c-MYC and HIF1α G-quadruplexes in colon cancer needs to be explored. The binding mode of berberine with c-MYC and HIF1α G-quadruplexes were explored by ultraviolet and visible absorption spectroscopy and fluorescence spectroscopy. Circular dichroism (CD) spectroscopy was performed to evaluate the effects of berberine on the stability of c-MYC and HIF1α G-quadruplexes. After different concentrations of berberine acting on HCT116 cells for 24 h, cell proliferation and apoptosis were detected by MTT assay and flow cytometry; quantitative real-time polymerase chain reaction and western blot were performed to detect mRNA and protein expression of c-MYC and HIF1α; transcriptome sequencing was used to analyze the metabolic pathways. For the effects of berberine on colon cancer mouse model with dose of 50 mg·kg-1 for 14 days, tumor growth were monitored, hematoxylin and eosin staining and immunofluorescence staining were performed to analyze histopathology and protein expression of c-MYC and HIF1α, central carbon metabolism was detected in tumor tissues. The binding ability of berberine with c-MYC G-quadruplex was different to that of berberine with HIF1α G-quadruplex. Both binding modes involved π-π stacking. The stoichiometric ratios were 1:1, 1:3, and 3:1 for berberine with c-MYC G-quadruplex and only 1:1 for berberine with HIF1α G-quadruplex. Temperature had a greater effect on the binding of berberine to c-MYC G-quadruplex. Berberine could improve the thermal stability of both c-MYC and HIF1α G-quadruplexes. Berberine inhibited the gene transcription and protein expression of c-MYC and HIF1α in colon cancer HCT116 cells. In vivo, berberine delayed tumor progression and inhibited the protein expression of c-MYC and HIF1α. Twelve differential metabolites such as decreased adenosine triphosphate were obtained, indicating that berberine could regulate the metabolic pathways of the tricarboxylic acid (TCA) cycle and glycolysis/gluconeogenesis, among others. Berberine may inhibit colon cancer by regulating the TCA cycle and glycolysis/gluconeogenesis based on the interaction with c-MYC and HIF1α G-quadruplexes.

Endoplasmic reticulum stress contributed to inflammatory bowel disease by activating p38 MAPK pathway.

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn’s disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.

Cytotoxic benzylidene hydrazides of terephthalic acid and related compounds.

The present investigation involved the synthesis of a number of novel benzylidene hydrazides as candidate cytotoxic agents. The preparation of these compounds from terephthalic acid and isophthalic acid proceeded satisfactorily. However, the reaction of phthalic acid hydrazide with various aryl aldehydes was unsuccessful in general. Some of the unexpected products were identified. The shapes and also the distances between the centers of the aryl rings designated B and C of three representative compounds 1b, 2b and 3b were determined. The compounds designated 1a-e, 2a-e and 3b were screened against human HCT116 and HT29 colon cancer cells as well as human CRL1790 non-malignant colon cells which revealed the tumor-selective toxicity displayed by these compounds.

Effects of Artonin E on Cell Growth Inhibition and Apoptosis Induction in Colon Cancer LoVo and HCT116 Cells.

Today, colon cancer is the leading cause of cancer death. In Thailand, colon cancer is the third most common cancer in men and the second in women. Currently, the treatments for colon cancer include surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, targeted drug therapy, and stem cell therapy. However, some treatments have side effects for cancer patients, causing unwanted symptoms. In addition, targeted therapy comes with a high cost for patients. Therefore, bioactive compounds might be a good choice for colon cancer treatment. In this study, we investigated the effect of artonin E on apoptosis induction in colon cancer LoVo and HCT116 cells. The concentration ranges of artonin E at 3, 5, 10, and 30 µg/mL in LoVo cells and 1, 1.5, 2, and 3 µg/mL in HCT116 cells were examined. The results implied that artonin E decreased cell viability and increased apoptotic cells in a dose-dependent manner. In addition, artonin E stimulated mitochondrial membrane potential (ΔΨm) changes associated with apoptosis by increasing the sub-G1 population analyzed by flow cytometry. Western blotting showed that artonin E increased the proapoptotic protein, Bax, and decreased anti-apoptotic proteins’ (Bcl-2 and Bcl-x) expression. Moreover, artonin E also increased cleaved caspase-7 and cleaved-PARP expression in both LoVo and HCT116 cells. Interestingly, artonin E induced apoptosis through p-ERK1/2, p-p38/p38, and p-c-Jun expression in both cells. Our results suggested that artonin E induced apoptosis via caspase activation associated with the MAPKs signaling pathway. Therefore, artonin E might be used as a potential anticancer drug for colon cancer in the future.

In PD-1+ human colon cancer cells NIVOLUMAB promotes survival and could protect tumor cells from conventional therapies

BackgroundColorectal cancer (CRC) is one of the most prevalent and deadly tumors worldwide. The majority of CRC is resistant to anti-programmed cell death-1 (PD-1)-based cancer immunotherapy, with approximately 15% with...

Effect of Sodium Butyrate on p16INK4a, p14ARF, p15INK4b, Class I HDACs (HDACs 1, 2, 3) Class II HDACs (HDACs 4, 5, 6), Cell Growth Inhibition and Apoptosis Induction in Pancreatic Cancer AsPC-1 and Colon Cancer HCT-116 Cell Lines.

Background:In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition. Materials and Methods:The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively. Results:The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell. Conclusion: The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.

Selective radiosensitization by nitazoxanide of quiescent clonogenic colon cancer tumour cells.

Nitazoxanide is a Food and Drug Administration-approved antiprotozoal drug recently demonstrated to be selectively active against quiescent and glucose-deprived tumour cells. This drug also has several characteristics that suggest its potential as a radiosensitizer. The present study aimed to investigate the interaction between nitazoxanide and radiation on human colon cancer cells cultured as monolayers, and to mimic key features of solid tumours in patients, as spheroids, as well as in xenografts in mice. In the present study, colon cancer HCT116 green fluorescent protein (GFP) cells were exposed to nitazoxanide, radiation or their combination. Cell survival was analysed by using total cell kill and clonogenic assays. DNA double-strand breaks were evaluated in the spheroid experiments, and HCT116 GFP cell xenograft tumours in mice were used to investigate the effect of nitazoxanide and radiation in vivo. In the clonogenic assay, nitazoxanide synergistically and selectively sensitized cells grown as spheroids to radiation. However, this was not observed in cells cultured as monolayers, as demonstrated in the total cell kill assays, and much less with the clinically established sensitizer 5-fluorouracil. The sensitizing effect from nitazoxanide was confirmed via spheroid γ-H2A histone family member X staining. Nitazoxanide and radiation alone similarly inhibited the growth of HCT116 GFP cell xenograft tumours in mice with no evidence of synergistic interaction. In conclusion, nitazoxanide selectively targeted quiescent glucose-deprived tumour cells and sensitized these cells to radiation in vitro. Nitazoxanide also inhibited tumour growth in vivo. Thus, nitazoxanide is a candidate for repurposing into an anticancer drug, including its use as a radiosensitizer.

Synthesis and Antimicrobial, Anticancer and Anti-Oxidant Activities of Novel 2,3-Dihydropyrido[2,3-d]pyrimidine-4-one and Pyrrolo[2,1-b][1,3]benzothiazole Derivatives via Microwave-Assisted Synthesis.

In our attempt towards the synthesis and development of effective antimicrobial, anticancer and antioxidant agents, a novel series of 2,3-dihydropyrido[2,3-d]pyrimidin-4-one 7a–e and pyrrolo[2,1-b][1,3]benzothiazoles 9a–e were synthesized. The synthesis of 2-(1,3-benzo thiazol-2-yl)-3-(aryl)prop-2-enenitrile (5a–e) as the key intermediate was accomplished by a microwave efficient method. Via a new variety oriented synthetic microwave pathway, these highly functionalized building blocks allowed access to numerous fused heteroaromatic such as 7-amino-6-(1,3-benzo thiazol-2-yl)-5-(aryl)-2-thioxo-2,3dihydropyrido [2,3-d]pyrimidin-4(1H)-one 7a–e and 1-amino-2-(aryl)pyrrolo[2,1-b][1,3]benzothiazole-3-carbonitrile derivatives 9a–e in order to study their antimicrobial and anticancer activity. The present investigation offers effective and rapid new procedures for the synthesis of the newly polycondensed heterocyclic ring systems. All the newly synthesized compounds were evaluated for antimicrobial, anticancer and antioxidant activity. Compounds 7a,d, and 9a,d showed higher antimicrobial activity than cefotaxime and fluconazole while the remaining compounds exhibited good to moderate activity against bacteria and fungi. An anticancer evaluation of the newly synthesized compounds against the three tumor cell lines (lung cell NCI-H460, liver cancer HepG2 and colon cancer HCT-116) exhibited that compounds 7a, d, and 9a,d have higher cytotoxicity against the three human cell lines compared to doxorubicin as a reference drug. These compounds also exhibited higher antioxidant activity and a great ability to protect DNA from damage induced by bleomycin.

Synthesis, solubility and antitumor activity of maslinic acid derivatives

Maslinic acid (MA), a pentacyclic triterpenoid obtained from olives that is characterized by its antiproliferative activity in tumor cells, has become a promising molecule that could be modified to improve cancer treatment. In this work we have synthesized in good yields several new MA conjugates, including glycerin, oligo(ethylene glycol), and amino acid derivatives (compounds 3a-f). The synthesis offers the possibility of recovering unreacted MA, and thus the scaling up of the process. For the tyramine-MA conjugate, compound 3f or TMA, the preparation has been optimized to a one-pot reaction. Solubility of conjugates in polar solvents has been measured, showing a marked increase of solubility with respect to MA. Moreover, we selected the tyramidyl maslinic acid conjugate (3f or TMA) to determine antitumor capacity over a wide range of cancer cell lines, including glioblastoma, melanoma, breast, lung, colorectal and pancreatic cancer. Our results clearly demonstrated that TMA induced higher cytotoxicity in all cancer cell types compared to MA. TMA was more effective than MA, especially in breast cancer cells (MCF-7) and melanoma cells (B16–F10) where IC50 reductions of 4.12 and 4.72, respectively, was detected. Interestingly, TMA showed a remarkable antitumor ability against the resistant HCT-15 colon cancer cell line. Furthermore, we demonstrated for the first time a relevant effect of a MA derivative against glioblastoma cells (A172 and SF-268). These results suggest that TMA is able to improve the antitumor characteristics of MA in a wide range of cancers and that it may be a promising compound for various tumor types, including resistant cancer.

Cordycepin inhibits colon cancer proliferation by suppressing MYC expression

BackgroundCordycepin is a purine nucleoside anti-metabolite and anti-biotic isolated from the fungus Cordyceps militaris, which has potential anti-neoplastic activities. This study aimed to investigate the effect of cordycepin in inhibiting colon cancer development.MethodsThe proliferation of cordycepin-treated HCT116 and Caco-2 colon cancer cell lines was assessed with 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the viability was measured with colony formation assay. At the same time, cordycepin responsive gene and microRNAs (miRNAs, miRs) were screened by qRT-PCR. MYC over-expressing HCT116 and Caco-2 cell lines were constructed, which were further transfected with miR-26a. Inhibitory effect of cordycepin on cell proliferation was evaluated with cell viability assay, cell number count, and colony formation assay. The relative expression of MYC and miR-26a was detected by qRT-PCR and Western blot.ResultsCordycepin inhibited colon cancer cell proliferation by down-regulating MYC mRNA/protein expression and up-regulating miR-26a in both HCT116 and Caco-2 cells. MYC over-expression could suppress the expression of miR-26a, which could be restored by cordycepin treatment. Additional miR-26a transfection in MYC over-expressing cells could reverse MYC over-expression-promoted proliferation, which could be further potentiated by cordycepin treatment.ConclusionCordycepin is able to suppress colon cancer cell proliferation, likely mediated by the MYC/miR-26a pathway, supporting its potential for the treatment of colon cancer.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Hinokiflavone, as a MDM2 inhibitor, activates p53 signaling pathway to induce apoptosis in human colon cancer HCT116 cells

Hinokiflavone (HF), a natural biflavonoid that possesses various biological activities, has reported that HF could be a pre-mRNA splicing modulator, whereas its underlying mechanisms remain elusive. In the present study, we identified HF as a potential MDM2 inhibitor. What's more, we found that HF suppressed mdm2 mRNA synthesis at the transcriptional level. Then, this MDM2 inhibition led in turn to increase p53 protein expression and activate p53 pathway, which could decrease the survival of HCT116 colon cells by G2/M phase arrest and apoptosis induction. Then, bioinformatics suggested that ESR1 was a predicted and potential target of HF. Finally, we used molecular docking and molecular dynamics simulation to demonstrate the binding patterns of HF and ESR1. To sum up, our study unearthed that HF was a feasible agent for MDM2 inhibitor through down-regulating mdm2 RNA level and activating p53 signaling pathway.

Effect of 5'-fluoro-2'-deoxycytidine, 5-azacytidine, and 5-aza-2'–deoxycytidine on DNA Methyltransferase 1, CIP/KIP Family, and INK4a/ARF in Colon Cancer HCT-116 Cell Line

Background: Cyclin-dependent kinase inhibitors (CKIs) are the negative regulator of cell cycle progression, which inhibits cyclin-cdk complexes, resulting in cell cycle arrest. Recently, we evaluated the effect of 5-Aza-CdR on DNMT1 gene expression in the WCH-17 hepatocellular carcinoma (HCC) cell line. Objectives: The current study was designed to analyze the effects of 5-aza-2'–deoxycytidine (5-Aza-CdR, decitabine), 5-azacytidine (5-AzaC, vidaza), and 5'-fluoro-2'-deoxycytidine (FdCyd) on INK4a/ARF, CIP/KIP, and DNA methyltransferase 1 gene expression, apoptosis induction, and cell growth inhibition in colon cancer HCT-116 cell line. Methods: The colon cancer HCT-116 cell line was treated with 5-azaC, 5-Aza-CdR, and FdCyd at 24 and 48h. To determine colon cancer HCT-116 cell viability, cell apoptosis, and the relative expression level of the INK4a/ARF, CIP/KIP, and DNA methyltransferase 1 genes, MTT assay, flow cytometry, and qRT-PCR were done, respectively. Results: 5-azaC, 5-Aza-CdR, and FdCyd significantly inhibited colon cancer HCT-116 cell growth and induced apoptosis. Besides, they significantly increased CIP/KIP (p21CIP1, p27KIP1, and p57KIP2) and INK4 (p14ARF, p15INK4b, and p16INK4a) and decreased DNMT1 gene expression. Besides, minimal and maximal apoptosis were seen in the groups treated with FdCyd and 5-Aza-CdR, respectively. The IC50 for CAF for FdCyd was 1.72 ± 0.23 and 1.63 ± 0.21μM at 24 and 48h, respectively. The IC50 for CAF for 5-AzaC was 2.18 ± 0.33 and 1.98 ± 0.29 μM at 24 and 48h, respectively. The IC50 for CAF for 5-Aza-CdR was 4.08 ± 0.61 and 3.18 ± 0.50 μM at 24 and 48h, respectively. Conclusions: The 5-azac, 5-Aza-CdR, and FdCyd can reactivate the INK4a/ARF and CIP/KIP families through inhibition of DNMT1 activity.

Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1H-Imidazol-5-One Variants.

Structural changes of small-molecule drugs may bring interesting biological properties, especially in the field of kinase inhibitors. We sought to study tirbanibulin, a first-in-class dual Src kinase (non-ATP competitive)/tubulin inhibitor because there was not enough reporting about its structure–activity relationships (SARs). In particular, the present research is based on the replacement of the outer ring of the biphenyl system of 2-[(1,1′-biphenyl)-4-yl]-N-benzylacetamide, the identified pharmacophore of KX chemotype, with a heterocyclic ring. The newly synthesized compounds showed a range of activities in cell-based anticancer assays, agreeing with a clear SAR profile. The most potent compound, (Z)-N-benzyl-4-[4-(4-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl]phenylacetamide (KIM-161), demonstrated cytotoxic IC50 values at 294 and 362 nM against HCT116 colon cancer and HL60 leukemia cell lines, respectively. Profiling of this compound (aqueous solubility, liver microsomal stability, cytochrome P450 inhibition, reactivity with reduced glutathione, and plasma protein binding) confirmed its adequate drug-like properties. Mechanistic studies revealed that this compound does not depend on tubulin or Src kinase inhibition as a factor in forcing HL60 to exit its cell cycle and undergo apoptosis. Instead, KIM-161 downregulated several other kinases such as members of BRK, FLT, and JAK families. It also strongly suppresses signals of ERK1/2, GSK-3α/β, HSP27, and STAT2, while it downregulated AMPKα1 phosphorylation within the HL60 cells. Collectively, these results suggest that phenylacetamide-1H-imidazol-5-one (KIM-161) could be a promising lead compound for further clinical anticancer drug development.

Osthole Exerts Inhibitory Effects on Hypoxic Colon Cancer Cells via EIF2[Formula: see text] Phosphorylation-mediated Apoptosis and Regulation of HIF-1[Formula: see text

Hypoxic microenvironment and dysregulated endoplasmic reticulum stress/unfolded protein response (UPR) system are considered important factors that promote cancer progression. Although osthole extracted from Cnidium monnieri(Fructus Cnidii) has been confirmed to exhibit an anticancer activity in various cancers, the effects of osthole in hypoxic colon cancer cells have not been explored. Therefore, the aim of this study was to examine whether osthole has an inhibitory effect on hypoxic colon cancer HCT116 cells and further investigate the underlying molecular mechanisms. Treatment with osthole significantly attenuated the cell viability, proliferation, and migration in hypoxic HCT116 cells. Osthole also activated UPR signaling such as phospho-eukaryotic initiation factor 2 alpha (EIF2[Formula: see text]/ATF4/CHOP/DR5 cascade accompanied by upregulation of pro-apoptotic proteins. Moreover, the tubule-like formation of human umbilical vein endothelial cells, the secretion of vascular endothelial growth factor A, and the expression and activity of hypoxia-inducible factor-1[Formula: see text] (HIF-1[Formula: see text] in hypoxic HCT116 cells were markedly suppressed by osthole. However, suppressing EIF2[Formula: see text] phosphorylation with salubrinal or ISRIB markedly reversed the effects of osthole on the expressions of pro-apoptotic proteins and HIF-1[Formula: see text]. Co-treatment of hypoxic HCT116 cells with osthole greatly increased the sensitivity to cisplatin and the expressions of phospho-EIF2[Formula: see text] and cleaved caspase 3. Collectively, the inhibitory effect of osthole in hypoxic HCT116 cells may be associated with EIF2[Formula: see text] phosphorylation-mediated apoptosis and translational repression of HIF-1[Formula: see text]. Taken together, osthole may be a potential agent in the treatment of colon cancer.