Colon Cancer Cell Line SW480 Research Articles

Cytotoxicity of green-synthesized silver nanoparticles by Adansonia digitata fruit extract against HTC116 and SW480 human colon cancer cell lines

Abstract Nanoparticles synthesized from plants are being explored for cancer treatment therapies all over the world. This study reported the eco-friendly and low-cost method for the green synthesis of silver nanoparticles (AgNPs) from Adansonia digitata fruit as a reducing and capping agent. The anti-cancer potential of synthesized particles was explored against HTC116 and SW480 colon cancer cell lines. Prepared AgNPs were characterized by ultraviolet-visible spectroscopy, zeta potential, transmission electronic microscopy, scanning electronic microscopy, Fourier transform infrared, and energy-dispersive spectrum. The cytotoxicity was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and expression levels of four genes (CTNNB1, APC, LRP5, and LRP6) were checked by reverse transcription polymerase chain reaction. The sharp peak of surface plasmon resonance at 400 nm confirms the formation of AgNPs. Dynamic light scattering showed average sizes of 16.34 nm with a polydispersity index of 0.193. A. digitata AgNPs were spherical with slight aggregated. AgNPs were more cytotoxic than A. digitata extract and decrease the expression of CTNNB1 and LRP6 genes while LRP5 gene expression was increased in both cell lines. APC gene expression was decreased in SW480 but increased in HTC116 with treatment. Overall, this study suggested that AgNPs synthesized by A. digitata fruit extract can be an attractive candidate for anticancer applications.

Determination of anticancer potential of a novel pharmacologically active thiosemicarbazone derivative against colorectal cancer cell lines

Thiosemicarbazones have received noteworthy attention due to their numerous pharmacological activities. Various thiosemicarbazone derivatives have been reported to play a key role as potential chemotherapeutic agents for the management of cancer. Herein, we aimed to establish the anticancer efficacy of novel thiosemicarbazone derivative C4 against colon cancer in vitro. The MTT viability assay identified C4 as a promising anticancer compound in a panel of cancer cell lines with the most potent activity against colon cancer cells. Further, anticancer potential of C4 was evaluated against HT-29 and SW620 colon cancer cell lines considering the factors like cell adhesion and migration, oxidative stress, cell cycle arrest, and apoptosis. Our results showed that C4 significantly inhibited the migration and adhesion of colon cancer cells. C4 significantly increased the intracellular reactive oxygen species (ROS) and induced apoptotic cell death. Cell cycle analysis revealed that C4 interfered in the cell cycle distribution and arrested the cells at the G2/M phase of the cell cycle. Consistent with these results C4 also down-regulated the Bcl-XL and Bcl-2 and up-regulated the caspase-3 expression. These findings introduced C4 as the potential anticancer agent against colon cancer.

Protection against ulcerative colitis and colorectal cancer by evodiamine via anti‑inflammatory effects.

Evodiamine (Evo) is an alkaloid that can be extracted from the berry fruit Evodia rutaecarpa and has been reported to exert various pharmacological effects, such as antidiarrheal, antiemetic and antiulcer effects. In vivo, the potential effects of Evo were investigated in a mouse model of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) and in adenomatous polyposis coli (Apc)MinC/Gpt C57BL/6 mice with colorectal cancer (CRC), where the latter harbours a point-mutation in the Apc gene. Evo suppressed the degree of weight loss and colon shortening induced by DSS, decreased the disease activity index value and ameliorated the pathological alterations in the colon of mice with UC as examined via H&E staining of colon tissues. In addition, Evo decreased the number and size of colonic tumors in ApcMinC/Gpt mice. Proteomics (colon tissues), ELISA (colon tissues and serum) and western blotting (colon tissues) results revealed that Evo inhibited NF-κB to mediate the levels of various cytokines, including, in the DSS-induced UC model, IL-1β, IL-2, IL-6, IL-8, TNF-α, IFN-γ (ELISA of colon tissues and serum), NF-κB, IKKα+β, IκBα, S100a9, TLR4 and MyD88 (western blotting of colon tissues), and, in the colorectal cancer model, IL-1β, IL-2, IL-6, IL-15, IL-17, IL-22, TNF-α (ELISA of colon tissues and serum), NF-κB, IKKα+β, IκBα and S100a9 (western blotting of colon tissues), to achieve its anti-inflammatory and antitumor effects. In vitro, Evo also reduced the viability of the colon cancer cell line SW480, inhibited mitochondrial membrane potential (MMP detection), caused G2/M-phase arrest (cell cycle detection) and suppressed the translocation of phosphorylated-NF-κB from the cytoplasm into the nucleus (immunofluorescence of p-NF-κB). Theoretical evidence (MD simulations) suggest that Evo may bind to the ordered domain (α-helix) of NF-κB to influence this protein. The protein secondary structure changes were analyzed by the cpptraj module in Amber. In addition, these data provide experimental evidence that Evo may be an effective agent for treating UC and CRC.

Eukaryotic Initiation Factor 5A2 Regulates Expression of Antiviral Genes

Translation factors are essential for regulation of protein synthesis. The eukaryotic translation initiation factor 5A (eIF5A) family is made up of two paralogues – eIF5A1 and eIF5A2 – which display high sequence homology but distinct tissue tropism. While eIF5A1 directly binds to the ribosome and regulates translation initiation, elongation, and termination, the molecular function of eIF5A2 remains poorly understood. Here, we engineer an eIF5A2 knockout allele in the SW480 colon cancer cell line. Using ribosome profiling and RNA-Sequencing, we reveal that eIF5A2 is functionally distinct from eIF5A1 and does not regulate transcript-specific or global protein synthesis. Instead, eIF5A2 knockout leads to decreased intrinsic antiviral gene expression, including members of the IFITM and APOBEC3 family. Furthermore, cells lacking eIF5A2 display increased permissiveness to virus infection. Our results uncover eIF5A2 as a factor involved regulating the antiviral transcriptome, and reveal an example of how gene duplications of translation factors can result in proteins with distinct functions.

Anti-tumor activity of a recombinant soluble Fzd7 decoy receptor in human gastric and colon cancer cells.

Objective(s):Frizzled-7, the most common receptor of the Wnt signaling pathway, was significantly over-expressed in gastric (GC) and colorectal (CRC) cancers and stimulated tumorigenesis. The extracellular domain of Fzd7 (sFzd7) as a decoy receptor, could competitively bound with ligands and antagonize the interaction between Fzd7 receptors and Wnt ligands. Materials and Methods:We expressed and purified the extracellular region of Fzd7 including cysteine-rich domain (33 aa–185 aa) from Escherichia coli by chromatography. The effect of sFzd7 was evaluated on AGS gastric and SW480 colon cancer cell lines expressing high levels of Fzd7 receptor. Accordingly, cell viability and apoptosis were measured using MTT and flow cytometry assays, respectively. Real-Time PCR determined the relative expression of the β-catenin and cyclin-D1 genes. Results:After three days of treatment with sFzd7, the viability of AGS and SW480 cell lines was decreased in a dose-dependent manner. In addition, sFzd7 at concentrations of 10 and 20 ug/ml increased the rate of apoptosis. Especially at the concentration of 20 ug/ml, the apoptosis rate was remarkably high in AGS (P-value= 0.003) and SW480 cells (P-value= 0.0007). Finally, the expressions of β-catenin (P-value= 0.01) and cyclin-D1 (P-value= 0.02) were obviously decreased in SW480 cells. The same results were obtained in AGS cells, although not statistically significant. Conclusion:sFzd7 decoy receptor inhibits tumor cell progression by attenuating the Wnt pathway through inhibiting Fzd7 receptors and Wnt ligand interaction. Hence, sFzd7 can be proposed as a candidate therapy for GC and CRC cells with high levels of Fzd7 expression.

Expression and Clinical Significance of Micro Ribonucleic Acid-132 and Sex-Determining Region Y-Box 4 in Colon Cancer

This study aimed to investigate the expression and clinical significance of micro ribonucleic acid-132 and sex-determining region Y-box 4 gene in colon cancer. Luciferase reporter system was used to validate the target gene directly regulated by micro ribonucleic acid-132. Transwell invasion assay was used to test the effect of micro ribonucleic acid-132 on invasiveness of SW620 cells. Micro ribonucleic acid-132 and sex-determining region Y-box 4 expression levels in cancerous and peri-cancerous tissues were measured using in situ hybridization and EleVisionTM immunohistochemistry staining. Micro ribonucleic acid-132 downregulated sex-determining region Y-box 4 protein expression and inhibited the invasiveness of colon cancer cells. In the 72 cancerous tissue samples of colon cancer, micro ribonucleic acid-132 positive rate was 18.1 % (13/72) and sex-determining region Y-box 4 protein positive rate was 73.6 % (53/72), both expressions showed significant differences as compared to peri-cancerous tissues (p<0.01). Micro ribonucleic acid-132 expression was significantly lower in colon cancer tissues with lymph node metastasis than without lymph node metastasis (p<0.01). Sex-determining region Y-box 4 expression was elevated along with Dukes stage increase and higher expression was accompanied by more tumor invasion depth (both p<0.05). Sex-determining region Y-box 4 expression was significantly higher in colon cancer tissues with lymph node metastasis than without lymph node metastasis (p<0.01). Correlation analysis displayed significant negative correlation of micro ribonucleic acid-132 expression with sex-determining region Y-box 4 expression (r=-0.594, p=0.013). Micro ribonucleic acid-132 inhibits invasion and metastasis of colon cancer cell line SW620 via targeting sex-determining region Y-box 4. Low expression of micro ribonucleic acid-132 and high expression of sex-determining region Y-box 4 protein could be important biomarkers for malignant transformation of colonic mucosa and for invasion and metastasis of colon cancer. Measuring expression levels of both molecules is valuable for predicting invasion and metastasis of colon cancer.

Inhibitory Effect of Polyphyllin D on Proliferation of Human Colon Cancer Cell Line SW480 via the MicroRNA-21/Phosphatase and Tensin Homolog Deleted on Chromosome 10

To evaluate the effects of polyphyllin D on the proliferation and apoptosis of human colon cancer SW480 cell lines and to reveal whether this process is mediated by the micro ribonucleic acid-21/phosphatase and tensin homolog deleted on chromosome 10 pathway. Human colon cancer SW480 cells were transfected with micro ribonucleic acid-21-mimic or mimic-negative control and treated with polyphyllin D at different concentrations (control, 0.25, 0.5, 1 and 2 μM) for 24 h or 48 h. Compared with control polyphyllin D group, the viability of human colon cancer SW480 cells significantly declined but the apoptosis rate significantly rose in 0.5 μM polyphyllin D group and the colony number of SW480 cells significantly decreased in 0.25 μM polyphyllin D group (p<0.05). Compared with control group, 2 μM polyphyllin D group had significantly increased protein expressions of Bcl-2 associated X protein and cleaved caspase-3, but decreased protein expression of B-cell lymphoma-2 in SW480 cells (p<0.001). The protein levels of micro ribonucleic acid-21 and phosphorylated-protein kinase B in SW480 cells were significantly lower, while the phosphatase and tensin homolog deleted on chromosome 10 messenger ribonucleic acid and protein levels were higher in 2 μM polyphyllin D group than those in control group (p<0.001). However, the protein expression of protein kinase B was not changed in 2 μM polyphyllin D group (p>0.05). After transfection with micro ribonucleic acid-21-mimic, the level of micro ribonucleic acid-21 in SW480 cells was significantly higher, while the phosphatase and tensin homolog deleted on chromosome 10 messenger ribonucleic acid and protein levels were significantly lower than those in control group (p<0.001). Compared with control group, the antiproliferative and pro-apoptotic effects of polyphyllin D on SW480 cells were reversed by transfection with micro ribonucleic acid-2-mimic (p<0.001). Polyphyllin D inhibits proliferation and induces apoptosis of human colon cancer SW480 cells via the micro ribonucleic acid-21/phosphatase and tensin homolog deleted on chromosome 10 pathway.

N-1,2,3-Triazole-isatin derivatives: anti-proliferation effects and target identification in solid tumour cell lines.

Molecular hybridization approaches have become an important strategy in medicinal chemistry, and to this end, we have developed a series of novel N-1,2,3-triazole-isatin hybrids that are promising as tumour anti-proliferative agents. Our isatin hybrids presented high cytotoxic activity against colon cancer cell line SW480, lung adenocarcinoma cell line A549, as well as breast cancer cell lines MCF7 and MDA-MB-231. All tested compounds demonstrated better anti-proliferation (to 1-order of magnitude) than the cis-platin (CDDP) benchmark. In order to explore potential biological targets for these compounds, we used information from previous screenings and identified as putative targets the histone acetyltransferase P-300 (EP300) and the acyl-protein thioesterase 2 (LYPLA2), both known to be involved in epigenetic regulation. Advantageous pharmacological properties were predicted for these compounds such as good total surface area of binding to aromatic and hydrophobic units in the enzyme active site. In addition, we found down-regulation of LYPLA2 and EP300 in both the MCF7 and MDA-MB-231 breast cancer cells treated with our inhibitors, but no significant effect was detected in normal breast cells MCF10A. We also observed upregulation of EP300 mRNA expression in the MCF10A cell line for some of these compounds and the same effect for LYPLA2 mRNA in MCF7 for one of our compounds. These results suggest an effect at the transcriptional regulation level and associated with oncological contexts.

HRMS Characterization, Antioxidant and Cytotoxic Activities of Polyphenols in Malus domestica Cultivars from Costa Rica.

There is increasing interest in research into fruits as sources of secondary metabolites because of their potential bioactivities. In this study, the phenolic profiles of Malus domestica Anna and Jonagold cultivars from Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (HRMS) using a quadrupole-time-of-flight analyzer (UPLC-QTOF-ESI MS), on enriched-phenolic extracts from skins and flesh, obtained through Pressurized Liquid Extraction (PLE). In total, 48 different phenolic compounds were identified in the skin and flesh extracts, comprising 17 flavan-3-ols, 12 flavonoids, 4 chalcones, 1 glycosylated isoprenoid and 14 hydroxycinnamic acids and derivatives. Among extracts, the flesh of Jonagold exhibits a larger number of polyphenols and is especially rich in procyanidin trimers, tetramers and pentamers. Evaluating total phenolic content (TPC) and antioxidant activities using ORAC and DPPH procedures yields higher values for this extract (608.8 mg GAE/g extract; 14.80 mmol TE/g extract and IC50 = 3.96 µg/mL, respectively). In addition, cytotoxicity evaluated against SW620 colon cancer cell lines and AGS gastric cancer cell lines also delivered better effects for Jonagold flesh (IC50 = 62.4 and 60.0 µg/mL, respectively). In addition, a significant negative correlation (p < 0.05) was found between TPC and cytotoxicity values against SW620 and AGS adenocarcinoma (r = −0.908, and −0.902, respectively). Furthermore, a significant negative correlation (p < 0.05) was also found between the number of procyanidins and both antioxidant activities and cytotoxicity towards SW620 (r = −0.978) and AGS (r = −0.894) cell lines. These results align with Jonagold flesh exhibiting the highest abundance in procyanidin oligomers and yielding better cytotoxic and antioxidant results. In sum, our findings suggest the need for further studies on these Costa Rican apple extracts—and particularly on the extracts from Jonagold flesh—to increase the knowledge on their potential benefits for health.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin β-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The βA-subunit and activin-A increased, whilst βB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Diosmin in combination with naringenin enhances apoptosis in colon cancer cells.

Colon cancer is one of the most commonly diagnosed malignancies, which begins as a polyp and grows to become cancer. Diosmin (DS) and naringenin (NR) are naturally occurring flavonoids that exhibit various pharmacological activities. Although several studies have illustrated the effectiveness of these flavonoids as anti‑cancerous agents individually, the combinatorial impact of these compounds has not been explored. In the present study, the combined effect of DS and NR (DiNar) in colon cancer cell lines HCT116 and SW480 were assessed by targeting apoptosis and inflammatory pathways. The MTT assay was used to evaluate the effect of DiNar on cell proliferation, while Chou‑Talalay analysis was employed to determine the combination index of DS and NR. Moreover, flow cytometry was used to monitor cell cycle arrest and population study. The onset of apoptosis was assessed by DAPI staining, DNA fragmentation, and AnnexinV‑fluorescein isothiocyanate/propidium iodide (AnnexinV‑FITC/PI). The expression levels of apoptotic pathway markers, Bcl‑2, Bax, caspase3, caspase8, caspase9 and p53, and inflammatory markers, NF‑κβ, IKK‑α and IKK‑β, were assessed using western blotting and reverse transcription‑quantitative PCR. These results suggested that DiNar treatment acts synergistically and induces cytotoxicity with a concomitant increase in chromatin condensation, DNA fragmentation and cell cycle arrest in the G0/G1 phase. AnnexinV‑FITC/PI apoptosis assay also showed increased number of cells undergoing apoptosis in the DiNar treatment group. Furthermore, the expression of apoptosis and inflammatory markers was also more effectively regulated under the DiNar treatment. Thereby, these findings demonstrated that DiNar treatment could be a potential novel chemotherapeutic alternative in colon cancer.

G-749 Promotes Receptor Tyrosine Kinase TYRO3 Degradation and Induces Apoptosis in Both Colon Cancer Cell Lines and Xenograft Mouse Models.

G-749 is an FLT3 kinase inhibitor that was originally developed as a treatment for acute myeloid leukemia. Some FLT3 kinase inhibitors are dual kinase inhibitors that inhibit the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and are used to treat solid cancers such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). AXL promotes metastasis, suppression of immune response, and drug resistance in NSCLC and TNBC. G-749, a potential TAM receptor tyrosine kinase inhibitor, and its derivative SKI-G-801, effectively inhibits the phosphorylation of AXL at nanomolar concentration (IC50 = 20 nM). This study aimed to investigate the anticancer effects of G-749 targeting the TAM receptor tyrosine kinase in colon cancer. Here, we demonstrate the potential of G-749 to effectively inhibit tumorigenesis by degrading TYRO3 via regulated intramembrane proteolysis both in vitro and in vivo. In addition, we demonstrated that G-749 inhibits the signaling pathway associated with cell proliferation in colon cancer cell lines HCT15 and SW620, as well as tumor xenograft mouse models. We propose G-749 as a new therapeutic agent for the treatment of colon cancer caused by abnormal TYRO3 expression or activity.

Dibenzocyclooctadiene lignans from the root bark of Schisandra sphenanthera

• Two new dibenzocyclooctadiene lignans were obtained from the root bark of Schisandra sphenanthera . • The structures were elucidated by NMR, MS, IR and CD data analysis. • Nine compounds showed cytotoxic activities with IC 50 values ranging from 13.5 to 48.8 μM. Two new dibenzocyclooctadiene lignans, schisphenlignan M and N ( 1 and 2 ), together with nine known analogues, schisantherin B ( 3 ), gomisin G ( 4 ), schisantherin D ( 5 ), schisantherin A ( 6 ), epigomisin O ( 7 ), gomisin O ( 8 ), (+)-gomisin K3 ( 9 ), schisanhenol B ( 10 ) and gomisin A ( 11 ) were isolated from the 80 % ethanol extract of root bark of Schisandra sphenanthera . Their structures were determined on the basis of spectroscopic analysis including IR, HR-ESI-MS, 1D and 2D NMR and CD spectra data. Compounds 1 - 11 were evaluated for their cytotoxic activity against human lung cancer cell line A549, and human colon cancer cell lines HTC116 and SW620, and the results showed that all of the lignans, except 3 and 8 (>50 μM), exhibited cytotoxic activities with IC 50 values ranging from 13.5 to 48.8 μM.

Anti-colon Cancer Activity of Parkia javanica (Lamk.) Merr. Bark Extract: An In-vitro Study

The Methanolic extract of Parkia javanica bark (MEPJB) was examined in vitro to see whether it has any cytotoxic or chemotherapeutic potential against the colon cancer cell lines HCT116 and SW480. Cell viability or proliferation assay and migration assay were performed, which showed significant results. The bark extract exhibited significant cytotoxic potential against both the colon cancer cell lines, and it also inhibited cell migration in a dose-dependent and time-dependent experiment. The IC₅₀ values of MEPJB on the HCT116 cell line for 24 hours, 48 hours, and 72 hours were 83.33 μg/mL, 37.5 μg/mL, and 34.37 μg/mL, respectively, whereas on the SW480 cell line, the IC₅₀ values were 87.5 μg/mL, 40 μg/mL and 31.25 μg/mL for 24 hours, 48 hours, and 72 hours respectively. However, IC₅₀ doses of MEPJB were inactive against the normal, healthy human fibroblast cell line. Cytotoxicity screening of 5-fluorouracil (5-FU), a commonly used drug in colon cancer treatment, was also done on both the colon cancer cell lines HCT116 and SW480, which showed much higher IC₅₀ than MEPJB after 24 hours, 48 hours, and 72 hours of incubation. MEPJB changed the cell morphology and induced apoptosis in colon cancer cell lines, confirmed by a morphological study of treated and untreated colon cancer cell lines and the acridine orange/ethidium bromide (AO/EB) dual staining procedure, respectively. After 48 hours of incubation with respective IC₅₀ concentrations of MEPJB, the treated colon cancer cells showed early and late apoptotic features. So this current research work indicates that the methanolic extract of P. javanica bark has chemotherapeutic potential against colon cancer cells and promotes apoptosis in cancer cells, which results in cell death.

HnRNPA2B1 Promotes Colon Cancer Progression via the MAPK Pathway.

HNRNPA2B1, an RNA-binding protein, plays a key role in primary microRNA processing, alternative splicing, mRNA metabolism and transport. Interestingly, hnRNPA2B1 also works as an N6-methyladenosine (m6A) reader and is critical during tumorigenesis of various tissue types. However, its role in colon cancer is still unclear. In this study, we aimed to elucidate the biological functions of hnRNPA2B1 and to explore its underlying mechanisms in colon cancer. We examined the expression of hnRNPA2B1 in Oncomine and TCGA databases. Then verified the findings in colon cancer cells and clinical samples with western blotting and immunohistochemistry (IHC). We used CRISPR/Cas9 directed gene editing to knockout hnRNPA2B1 expression in human colon cancer cell line SW480 and HCT-116 and carried out both in vivo and in vitro experiments. The results were further confirmed by RNA-seq analyses. We found that hnRNPA2B1 significantly promoted colon cancer cell proliferation both in vitro and in vivo, while knockout of hnRNPA2B1 induced apoptosis and cell cycle arrest in SW480. RNA-seq analyses revealed that the ERK/MAPK pathway was activated by hnRNPA2B1 upregulation. In addition, both hnRNPA2B1 and MAPK pathway were activated in clinical colon cancer specimens and positively correlated. Mechanistically, hnRNPA2B1 appeared to be an upstream regulator of the ERK/MAPK pathway and inhibition of MAPK signaling blocked the effects of hnRNPA2B1. Taken together, our data demonstrated that the RNA-binding protein hnRNPA2B1 promotes cell proliferation and regulates cell cycle and apoptosis of human colon cancer by activating the ERK/MAPK signaling, which may provide a new insight into the development of hnRNPA2B1 as a potential therapeutic target for treatment of colon cancer.

487P Targeting PDL-1 in colon cancer using nuclear receptor 4A1 (NR41) antagonist

The nuclear orphan receptor 4A1 (NR4A1, Nur77, TR3) is overexpressed in multiple solid tumors including colorectal tumors and is a negative prognostic factor for patient survival. NR4A1 is expressed in colon cancer cells and exhibit pro-oncogenic activity and results of examination of several colon cancer cell lines show that PD-L1 expression is limited and NR4A1 and PD-L1 are co-expressed in SW480 and RKO colon cancer cell lines. Previous studies showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter.

Examination of the differences between sulforaphane and sulforaphene in colon cancer: A study based on next-generation sequencing.

Sulforaphane and sulforaphene are isothiocyanate compounds derived from cruciferous vegetables that have demonstrated antiproliferative properties against colon cancer. However, the underlying mechanism of action of these two compounds has yet to be elucidated. The aim of the present study was to examine the effects of sulforaphane and sulforaphene on colon cancer using next-generation sequencing (NGS). The SW480 colon cancer cell line was cultured with 25 µmol/l sulforaphane or sulforaphene. Total RNA was extracted from the cells following 48 h of incubation with these compounds, and NGS was performed. Pearson's correlation and principal component analyses were performed on the NGS data in order to determine sample homogeneity followed by hierarchical clustering, chromosomal location, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A total of 873 probes in the sulforaphene group were differentially expressed compared with the control group. Similarly, 959 probes in the sulforaphane group were differentially expressed compared with the control group. The differentially expressed genes were dispersed on the chromosomes, across 22 pairs of autosomes, as well as the X and Y chromosomes. GO and KEGG analyses demonstrated that both drugs affected the ‘p53 signaling pathway’, ‘MAPK signaling pathway’, ‘FOXO signaling pathway’ and ‘estrogen signaling pathway’, while ‘Wnt signaling pathway’ was enriched in the sulforaphane group, and ‘ubiquitin mediated proteolysis’ and ‘estrogen signaling pathway’ in the sulforaphene group. Thus, sulforaphane and sulforaphene exhibited similar biological activities on colon cancer cells. Sulforaphane and sulforaphene may be associated with Wnt and estrogen signaling, respectively.

Abstract 1149: Nuclear receptor 4A1 (NR4A1) antagonists target PD-L1 in colon cancer

Abstract The nuclear orphan receptor 4A1 (NR4A1, Nur77, TR3) is overexpressed in multiple solid tumors including colorectal tumors and is a negative prognostic factor for patient survival. NR4A1 is expressed in colon cancer cells and exhibit pro-oncogenic activity and results of examination of several colon cancer cell lines show that PD-L1 expression is limited and NR4A1 and PD-L1 are co-expressed in SW480 and RKO colon cancer cell lines. Previous studies in breast cancer cells showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter. Knockdown of NR4A1 or Sp1 by RNA interference or treatment with mithramycin an inhibitor of Sp-mediated transcription decreased expression of PD-L1 in RKO and SW480 colon cancer cell lines. These data coupled with ongoing gene expression and PD-L1 promoter studies demonstrate that PD-L1 expression is regulated by NR4A1/Sp1 in colon cancer cells. Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways. Treatment of RKO and SW480 colon cancer cell lines with a series of potent 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl) analogs decreased expression of PD-L1. These results show that bis-indole derived NR4A1 antagonists act as small molecule mimics of immunotherapeutics that target PD-L1. In vivo applications of NR4A1 ligands that target PD-L1 and their effects on tumor growth and immune surveillance are currently being investigated. Citation Format: Kumaravel Mohankumar, Keshav Karki, Stephen Safe, Maen Abdelrahim. Nuclear receptor 4A1 (NR4A1) antagonists target PD-L1 in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1149.

Abstract LB108: Validation of computationally predicted synthetic lethal interactions for KRAS

Abstract Despite dedicated research in precision medicine, identification of therapeutic strategies for oncogenic drivers remains a major challenge as many mutations are difficult to target directly. Synthetic lethality-based target prediction becomes a powerful approach for drug discovery. We previously developed a computational platform, Mining Synthetic Lethals (MiSL), that can output a list of possible synthetic lethal partners of a specific gene when fed with primary human tumor genomic and transcriptomic data. The principal logic of the MiSL algorithm is based on the Boolean implication that the SL partners of a mutation will never be deleted in the presence of the mutation in the primary tumors. High frequency of KRAS mutation in lung and colon cancers makes it an important therapeutic target. While drugs directly targeting KRAS are yet to be fully developed, we applied the principle of synthetic lethality using the MiSL platform to predict potential KRAS specific therapeutic targets. We identified 556 putative SL partners of KRAS mutations combining lung and colon cancers. To efficiently test all predictions, we used customized shRNA and CRISPR lentiviral libraries to validate all the predicted SL interactions for KRAS in colon cancer cell lines HT29 (KRAS WT) and SW620 (KRAS G12V mutant). Eight shRNAs and four sgRNAs were designed for each gene in the shRNA and CRISPR libraries. The shRNAs and sgRNAs expressed at baseline and at the end of each screen were analyzed by barcode sequencing. Synthetic lethal interactions with KRAS were determined by identifying individual sh/sgRNAs whose mean values at the end relative to the start of the screen were significantly reduced in KRAS Mut cell lines compared to WT. We also compared the results of the in vitro to an in vivo screen of the focused libraries in human tumors xenografted into mice using the same cell lines. We compared the top 50 SL candidates from each screen as ranked by MAGeCK (Model-based Genome-wide Analysis of CRISPR/Cas9 KO) and found that only 19 were shared between the in vitro and in vivo screens. Further validation of several top SL candidates coming from both in vitro and in vivo screens was performed by testing individual shRNAs/sgRNAs for growth inhibition in KRAS Mut (SW620) vs. KRAS WT (HT29) cell lines in vitro and in vivo. Individual in vitro CRISPR/Cas9 knock out of two of the candidate SL partners reduced cell viability by 50% and 57% respectively in KRAS mutant cells compared to KRAS WT cells. Furthermore, induced individual KD or KO of four SL partners resulted in inhibition of tumor growth by 84%, 68%, 95% and 60%, respectively. In summary, MiSL has shown to be a powerful platform for discovering novel targets for specified cancer types. This study has identified and validated several genes as promising targets of KRAS dependent colon cancer for further therapeutic investigation. Citation Format: He Gong, Claire Repellin, Puja Patel, Merrill Knapp, Mingze He, Jun Li, Lisa Wu, Xiaohe Liu, Subarna Sinha, Lidia Sambucetti. Validation of computationally predicted synthetic lethal interactions for KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB108.

Study on effects of miR-141-3p in proliferation, migration, invasion and apoptosis of colon cancer cells by inhibiting Bcl2.

This study aimed to investigate the relationship between miR-141-3p and B lymphocyte-2 gene (Bcl2) gene and its biological behavior on colon cancer cell line SW480. qRT-PCR was used to detect the expression level of miR-141-3p in colon cancer tissues and adjacent tissues, as well as in colon cancer cell line and normal human colonic epithelial cell line FHC. MTT assay, wound assay, and Transwell demonstrated the effects of miR-141-3p on colon cancer proliferation, migration and invasion. Targetscan7.1 predictive software and dual luciferase reporter assays were used to detect the targeted regulation of miR-141-3p on the apoptosis-related gene Bcl2. MTT assay, wound assay, Transwell and flow cytometry were used to detect the effect of Bcl2 on miR-141-3p on colon cancer proliferation, migration, invasion and apoptosis. Compared with adjacent tissues, the expression of miR-141-3p in colon cancer tissues was significantly down-regulated. Colon cancer patients with low expression of miR-141-3p had poorer prognosis. Compared with normal colonic epithelial cells, miR-141-3p expression was significantly down-regulated in colon cancer cell lines, and overexpression of miR-141-3p significantly attenuated the proliferation, migration and invasion of colon cancer cells. Knockdown of miR-141-3p significantly promoted the proliferation, migration and invasion of colon cancer cells. miR-141-3p targets the negative regulation of Bcl2. Knockdown of Bcl2 significantly attenuated the promotion of miR-141-3p inhibitor on proliferation, migration and invasion of colon cancer cells and inhibition of apoptosis. Knockdown of Bcl2 significantly enhanced the inhibition effect of miR-141-3p inhibitor on proliferation, migration and invasion of colon cancer cells. In conclusion, miR-141-3p can inhibit the cancer by regulating Bcl2, and miR-141-3p has the potential to become a potential therapeutic target for colon cancer.