COLO 320DM Cells Research Articles

Development, characterization, and assessment of PLAROsomal vesicular system of curcumin for enhanced stability and therapeutic efficacy

BackgroundCurcumin (CUR) is a natural polyphenol and one of the key phytoconstituents found in the rhizomes of Curcuma Longa. It exhibits various pharmacological properties, encompassing antioxidant, anticancer effects, antiseptic, and anti-inflammatory, among several others.A significant drawback of using CUR is its limited bioavailability, which primarily depends on gut microorganisms responsible for converting it into its bioavailable form. Therefore, the contemporary study intended to formulate a novel PLAROsomal vesicular delivery of CUR, i.e., CUR-PLAROsomes employing a design of experiments approach to examine the influence of various process parameters, such as particle size and drug percentage release.ResultThe prepared CUR-PLAROsomes were characterized for their physicochemical properties using various hyphenated tools. The CUR-PLAROsomes exhibited sizes ranging from 40 to 300 nm, and the optimized batch demonstrated a drug entrapment of 86.38 ± 0.22%.In-vitro anticancer studies were conducted using human colorectal adenocarcinoma cells (COLO320DM) and human breast adenocarcinoma (MCF-7). CUR-PLAROsomes exhibited significant in-vivo anti-inflammatory potential against carrageenan-induced paw edema. CUR-PLAROsomes were more potent against COLO320DM and MCF-7 cell lines, even at lower concentrations, than pure CUR.ConclusionFurthermore, based on the observations, it exhibits potential as an anti-inflammatory agent, suggesting that PLAROsomes are an effective vesicular drug delivery system.HighlightsNewly introduced PLARosome is a next generation of Liposomes which have gain popularity owing to its better adaptability to overcome leakage problem of vesicular drug delivery system.This is the pioneer attempt to prepare Curcumin-loaded PLARosome as an anti-cancer and anti-inflammatory activity.Nano size of the PLAROsomes may contribute to enhance the efficacy of Curcumin as a target specific drug delivery system.Site specific delivery of phytoconstituents is possible by use of PLAROsomes as a novel drug delivery system.Graphical abstract

Abstract 1810: Development of new phthalic hydrazide scaffold for potent TNKS inhibitors

Abstract Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modifications of target proteins through poly-ADP-ribosylation. Inhibition of TNKS stabilizes AXIN and induces β-catenin degradation in adenomatous polyposis coli (APC)-mutated colorectal cancer (CRC) cells. Therefore, TNKS emerges as a therapeutic target for APC mutant CRC, and there is a critical need for drug development against it. Tankyrase 1 and 2 (TNKS1/2) catalyze the post-translational modification of target proteins through poly-ADP-ribosylation. Their inhibition stabilizes AXIN and induces β-catenin degradation. Therefore, TNKS is a therapeutic target for APC mutant colorectal cancer (CRC), and there is a need for drug development against it. However, reported TNKS inhibitors have serious limitations in that they cause intestinal toxicity. To address these issues and enhance efficacy, we synthesized 60 compounds based on the tetrahydrophthalazine scaffold using protein structure modeling-Al technology. Using an in vitro TNKS1 and TNKS2 enzyme assay and derivatizing Hit compounds, we confirmed that TI-61987 significantly inhibits TNKS enzyme activity at low nanomolar levels. TI-61987 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM, APC-mutated CRC cells. Additionally, TI-61987 exhibited excellent anticancer effects not only in COLO320DM cells but also in in vivo xenograft mouse models. Our findings demonstrate that TI-61987 is a therapeutic candidate targeting TNKS for the treatment of APC-mutated CRCs, and this anticancer drug may represent an effective therapy regimen for APC-mutated CRCs. Citation Format: Hwani Ryu, Hye-Ran Seo, Ju Han Lee, Kyu Myung Lee, Jiyeon Ahn. Development of new phthalic hydrazide scaffold for potent TNKS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1810.

Reduced chemokine C-C motif ligand 1 expression may negatively regulate colorectal cancer progression at liver metastatic sites.

Colorectal cancer (CRC) liver metastasis, albeit a stage-IV disease, is completely curable by surgical resection in selected patients. In addressing the molecular basics of this phenomenon, differentially expressed genes at primary and liver metastatic sites were screened by RNA sequencing with the use of paraffin-embedded surgical specimens. Chemokine C-C motif ligand 1 (CCL1), a chemotactic factor for a ligand of the chemokine C-C motif receptor 8 (CCR8), was isolated as one of the differentially expressed genes. Histological analysis revealed that the number of CCL1-positive cells, mainly tumour associated macrophages (TAMs) located in the stroma of CRC, decreased significantly at liver metastatic sites, while the expression level of CCR8 on CRC remained unchanged. To explore the biological significance of the CCL1-CCR8 axis in CRC, CCR8-positive CRC cell line Colo320DM was used to assess the effect of the CCL1-CCR8 axis on major signalling pathways, epithelial mesenchymal transition induction and cell motility. Upon stimulation of recombinant CCL1 (rCCL1), phosphorylation of AKT was observed in Colo320DM cells; on the other hand, the corresponding significant increase in MMP-2 levels demonstrated by RT-qPCR was nullified by siRNA (siCCR8). In the scratch test, rCCL1 treatment significantly increased the motility of Colo320DM cells, which was similarly nullified by siCCR8. Thus, the activation of the CCL1-CCR8 axis is a positive regulator of CRC tumour progression. Reduced CCL1 expression of TAMs at liver metastatic sites may partly explain the unique slow tumour progression of CRC, thus providing for a grace period for radical resection of metastatic lesions.

The 5-/12-lipoxygenase-BLT2 cascade induces elevated expression of c-Myc, thus mediating the proliferation and migration of KRAS mutant colorectal cancer cells

ABSTRACTColorectal cancer (CRC) is the most common malignant tumor and one of the leading causes of cancer-related death worldwide. Oncogene KRAS is a commonly mutated in CRC and plays crucial roles in the colorectal tumorigenesis. Emerging evidence suggests that increased expression levels of c-Myc are critical for KRAS-mediated CRC progression. However, it is unclear exactly how c-Myc expression is regulated in CRC. In the present study, we found that blockade of 5/12-lipoxygenase (5/12-LO) or leukotriene B4 receptor (BLT2) markedly reduced c-Myc expression in KRAS-mutant LOVO cells, while the control COLO 320DM cells were not affected. When the 5/12-LO products LTB4 and 12(S)-HETE, ligands of BLT2, were added to LOVO cells, c-Myc expression levels were restored, together suggesting that ‘5/12-LO-BLT2 cascade’ regulates c-Myc expression in KRAS-mutant CRC cells. We also observed that ‘5/12-LO-BLT2’-mediated c-Myc upregulation contributes to cell proliferation by regulating the expression of cyclin D1 in LOVO cells. Moreover, ‘5/12-LO-BLT2-cMyc’ cascade regulates the expression of EMT-related proteins in KRAS-mutant CRC cells, and depletion of both c-Myc and BLT2 using siRNA significantly enhanced the level of E-cadherin and reduced the level of Vimentin in LOVO cells. Taken together, our findings suggest that the BLT2-linked cascade promotes KRAS-mutant CRC cell proliferation and migration through the elevated expression of c-Myc. Thus, our results suggest that BLT2 appears to be an effective therapeutic target for KRAS-mutant CRCs.

Abstract B149: Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity

Abstract Beta-catenin represents a significant challenge as a therapeutic target due to its multifaceted role in tumor development as well as normal cell homeostasis. Dysregulation of the Wnt/beta-catenin signaling pathway is implicated in various cancers, including colorectal, hepatocellular, breast, ovarian, and pancreatic cancers. We developed ST316 as a peptide antagonist of the interaction of beta-catenin with BCL9, a co-activator implicated in oncogenic beta-catenin signaling. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) that is currently enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/beta-catenin signaling pathway. In vitro studies indicate that ST316 selectively attenuates Wnt transcriptional activity in beta-catenin-dependent but not -independent cell lines. Gene expression profiling using RNA sequencing in beta-catenin mutant HCT116 cells and APC mutant Colo320DM cells demonstrates a significant downregulation of Wnt pathway target genes, oncogenic signature genes and pro-tumor immunologic gene sets within 24 hours of ST316 treatment. Consequently, ST316 induced a dose-dependent decrease in viability of beta-catenin-dependent cells while beta-catenin-independent cells were resistant, indicating that the anticancer effects of ST316 are mediated by inhibition of the Wnt/beta-catenin pathway. To evaluate the impact of ST316 in vivo, a 4T1 triple negative breast cancer orthotopic tumor model was employed. Once weekly injection of ST316 (5mg/kg SC) over eight weeks suppressed expression of Wnt target genes CDK4 and cMyc and resulted in a significant 84.3% inhibition of tumor growth compared to the control group. In addition to direct anti-tumor activity, ST316 triggers a pro-inflammatory immune microenvironment. ST316 induces polarization of human macrophages derived from peripheral blood mononuclear cells, resulting in >100-fold shift from immunosuppressive M2 macrophages towards the anti-tumor M1 phenotype. Co-cultures of M2 macrophages with T cells treated with ST316 demonstrate a significant three-fold increase in T-cell activation, as indicated by intracellular IFN-γ staining. Finally, ST316 enhances the anti-tumor activity of anti-PD-1 therapy in a syngeneic orthotopic 4T1 mouse model, where combination ST316 and anti-PD-1 antibody results in enhanced tumor growth inhibition compared to either single agent alone (p<0.01). These findings demonstrate the antitumor and immunostimulatory effects of ST316 and highlight its potential as a therapeutic agent for targeting cancers with aberrantly activated Wnt signaling pathways. Citation Format: Lila Ghamsari, Claudio Scuoppo, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Zachary Mattes, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein, Jim Rotolo. Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B149.

Efficient in vitro oxaliplatin delivery with functionalized single-walled carbon nanotube for enhanced colon cancer treatment

BackgroundSite-specific transport of medicinal products to malignant cells and tissues is an intriguing area since it has an ability to safeguard healthy cells. Selective upregulation of folate receptors on colon cancer cells is usual. Consequently, folate receptors have become one of extensively studied target moieties for targeting the delivery of chemotherapeutics. Hence, the study aimed to anchor folic acid, chitosan and oxaliplatin to the functionalized nanotube (FA-CHI-FSWCNT-OXA) for targeting folate receptors on colon cancer cells. The purification process of single-walled carbon nanotubes (SWCNTs) involved the use of an ultrasonic-assisted acid digestion method. The developed complex was evaluated using FTIR, DSC, SEM, XRD and in vitro dissolution studies. SRB and MTT assays were used to assess in vitro cytotoxicity of oxaliplatin and FA-CHI-FSWCNT-OXA) against HT29 and COLO320DM cell lines. Further, progression of apoptosis in cells was investigated using flow cytometry.ResultsThe FTIR results corroborated drug attachment over carbon nanotube (CNT), whereas the TEM results validated the nanosizing (1–300 nm) of the developed system. Drug entrapment in CNT was found to be 93.43 ± 1.65%, and in vitro drug release was found to be 94.73 ± 0.90% after 24 h. The complex reduced viability of 92.35 ± 0.942% cells than oxaliplatin’s 66.58 ± 0.38% inhibition, revealed by MTT assay. In the SRB assay, the developed system showed 91.22 ± 0.90% inhibition, whereas oxaliplatin showed 76.69 ± 0.52% inhibition against HT29 cells.ConclusionsConclusively, the developed system exhibited better cytotoxicity effects as compared with plain oxaliplatin. Our findings are suggestive of the potential development of CNT-anchored antineoplastic agents for target-specific delivery.

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.

Discovery of diverse sesquiterpenoids from Magnolia grandiflora with cytotoxic activities by inducing cell apoptosis

Phytochemical study of Magnolia grandiflora led to the isolation of 39 sesquiterpenoids, including 15 new compounds (1–15). Compounds 1 and 2 are discovered to be the first 13-norgermacrane type sesquiterpenoids in natural products. Compound 15 is a rare 5,6-seco-guaiane type sesquiterpene and its possible biogenic precursor is presumed to be compound 20. Subsequent structural modification for compound 28 led to 21 derivatives, among which 15 derivatives were new compounds. All compounds were tested for the inhibitory effects on three tumor cell lines, and 17 compounds were active with the IC50 values ranging from 1.91 ± 0.39 μM to 12.29 ± 1.68 μM. The structure–activity relationships implied that an α, β-unsaturated lactone group was an important active group for the cytotoxicity. Two most active compounds (19 and 29) with low toxicity on normal human liver cell line were selected for further mechanism study. Compound 29 could induce apoptosis on Colo320DM cells through influencing the key apoptotic related proteins, such as PARP, Cleaved PARP, cleaved Caspase-3, and pro-Caspase 3. In addition, compound 19 with the best cytotoxic activity on HEL cells also could induce the apoptosis in dose- and time-dependent manners. In summary, our investigation implied that compounds 19 and 29 are two new potential anti-cancer candidates for ongoing study in the future.

Effective combination of biocompatible zinc oxide nanocrystals and high-energy shock waves for the treatment of colorectal cancer

BackgroundColorectal cancer (CRC) is the third most diagnosed tumor worldwide, with a very high mortality rate, second only to lung cancer. Current treatments, such as surgery, chemotherapy or radiotherapy, are not effective enough and show several limitations. Among the emerging strategies, nanomedicine offers very powerful tools in cancer treatment. Recently, the combination of nanoparticle antitumor effect with a triggering external stimulation was formulated to boost up the cytotoxic activity.ResultsIn this work, we show the synergistic effect of oleic acid-capped zinc oxide nanocrystals (ZnO NCs) and mechanical high-energy shock waves (SW) in the treatment for CRC cells, in vitro. We tested two different types of ZnO NCs synthetized in our laboratory, the basal undoped ZnO NCs and the iron-doped ones (Fe:ZnO NCs). The presence of the oleic acid capping and the further amino-propyl functionalization guarantee a high colloidal stability to both NCs, while the iron doping confers to Fe:ZnO NCs interesting magnetic properties useful for imaging applications in a clinical perspective. Thus, the iron-doped ZnO NCs are very attractive as potentially theranostic nanoparticles, allowing both stimuli-responsive therapy and magnetic resonance imaging.Importantly, two colon adenocarcinoma cell lines, the HT-29 and the Dukes’ type C Colo 320DM cells were tested, both showing a good bio-tolerance and internalization rates of NCs. With the aim of eradicating the CRC cells, the possible synergism between the undoped/iron-doped ZnO NCs and an external physical stimulus, i.e., high-energy SW, was then here investigated in vitro. We demonstrated that the combined treatment resulted in an augmentation of the antitumor activity, especially for Colo 320DM cells, when compared to controls. Moreover, a repeated and sequenced SW treatment (three times/day, 3SW) after ZnO NCs exposure resulted in a further increased mortality of CRC cells.ConclusionOur work proposes the combination of the cytotoxic activity of ZnO NCs with the SW external stimulation to obtain a booster of the antitumor activity, which warrants further investigation in vivo on CRC as well as on other tumors.Graphical

Abstract 521: Discovery of novel TNKS inhibitors through structure-based virtual screening

Abstract More than 80% of colorectal cancers (CRCs) have somatic mutations in the adenomatous polyposis coli (APC) gene, which affects abnormal WNT/β-catenin signaling. Since inhibition of tankyrases (TNKS1 and TNKS2) belonging to the poly(ADP-ribose) polymerase family stabilizes AXINs and induces degradation of β-catenin, drug development as a therapeutic target for APC-mutated CRCs is required. To identify selective small molecule inhibitors of TNKS, we performed pharmacophore modeling and structural docking studies targeting nicotinamide, adenosine, and both binding subsites of TNKS. Among the 8 million compounds, 149 of the nicotinamide site binders, 23 of the adenosine site binders, and 15 of the dual-site binders of TNKS were selected through computer-based virtual screening. Using in vitro TNKS1 and TNKS2 enzyme assays, we identified that TI2-N17 and TI3-D11 significantly inhibited TNKS1 and TNKS2 enzyme activities at low nanomolar levels. TI2-N17 and TI3-D11 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM, APC-mutated CRC cells. Both compounds exhibited anticancer effects on COLO320DM and SW403 cells. In addition, the combination treatment of 5-Fluorouracil (5-FU), an anticancer agent for CRC patients, and these compounds synergistically inhibited the proliferation of CRC cells compared to single compound treatment. Our findings demonstrated that TI2-N17 and TI3-D11 are therapeutic candidates targeting TNKS for APC-mutated CRCs treatment and suggested that the combined treatment with the compounds and 5-FU chemotherapy may be an effective therapeutic strategy for APC-mutated CRCs. Based on these compounds, further study will develop potent and selective TNKS inhibitors by designing and synthesizing novel derivative compounds. Citation Format: Hwani Ryu, Hye-Ran Seo, Hyo Jeong Kim, Jiyeon Ahn. Discovery of novel TNKS inhibitors through structure-based virtual screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 521.

Proteomic profiling reveals an association between ALDH and oxidative phosphorylation and DNA damage repair pathways in human colon adenocarcinoma stem cells

Several members of the aldehyde dehydrogenase (ALDH) family, especially ALDH1 isoenzymes, have been identified as biomarkers of cancer stem cells (CSCs), a small subpopulation of oncogenic cells with self-renewal and multipotency capability. Consistent with this contention, cell populations with high ALDH enzymatic activity exhibit greater carcinogenic potential. It has been reported that ALDH1, especially ALDH1A1, serves as a valuable biomarker for colon CSCs. However, the functional roles of ALDHs in CSCs and solid tumors of the colon tissue is not fully understood. The aim of the present study was to identify molecular signature associated with high ALDH activity in human colorectal adenocarcinoma (COLO320DM) cells by proteomics profiling. Aldefluor™ assay was performed to sort COLO320DM cells exhibiting high (ALDHhigh) and low (ALDHlow) ALDH activity. Label-free quantitative proteomics analyses were conducted on these two cell populations. Proteomics profiling revealed a total of 229 differentially expressed proteins (DEPs) in ALDHhigh relative to ALDHlow cells, of which 182 were down-regulated and 47 were up-regulated. In agreement with previous studies, ALDH1A1 appeared to be the principal ALDH isozyme contributing to the Aldefluor™ assay activity in COLO320DM cells. Ingenuity pathway analysis of the proteomic datasets indicated that DEPs were associated with mitochondrial dysfunction, sirtuin signaling, oxidative phosphorylation and nucleotide excision repair. Our proteomics study predicts that high ALDH1A1 activity may be involved in these cellular pathways to promote a metabolic switch and cellular survival of CSCs.

Evaluation of antitumor effects of VEGFR-2 inhibitor F16in a colorectal xenograft model.

Colorectal cancer(CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5μM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 valueof 9.52 ± 1.49µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showeda reduction in CD31 levels also in F16 treated groups. These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.

Development of gold, silver, and iron nanoparticles using isolated berberine: its characterization and antimicrobial and cytotoxic activities against COLO320DM and Hela cells

Development of gold, silver, and iron nanoparticles using isolated berberine: its characterization and antimicrobial and cytotoxic activities against COLO320DM and Hela cells

Wnt Signaling and Cadherin Expressions in Different Staging of Colorectal Cancer as Biomarkers for Metastasis: Study of SW480, COLO 320DM, and HCT116 Cellines

BACKGROUND: Early metastases is still unresolved problem in cancer management, eventually in colorectal cancer (CRC). In addition, many markers are useful just only in the late stage of CRC. AIM: This study evaluates the differences in the expression intensity of nuclear β-catenin, cytoplasmic β-catenin, E-cadherin, and N-cadherin between CRC SW480 cell line as control group and COLO320DM and HCT116 cell lines as case groups. MATERIALS AND METHODS: This study applied experimental research design with the different test methods. Culture growing and subcultures manufacturing for the CRC cell line models were done initially and followed by the immunofluorescence method by administering antibodies on β-catenin, E-cadherin, and N-cadherin, and continued with staining process using fluorescein-5-isothiocyanate and 4’, 6-diamidino-2-phenylindole. Observations were done using an immunofluorescence microscope. Calculation of area density in each cell to perceive the expressions of cytoplasmic and nuclear β-catenin, E-cadherin, and N-cadherin was conducted using ImageJ software, resulted in mean fluorescence intensity. RESULTS: There are significant differences in the expressions of cytoplasmic β-catenin, nuclear β-catenin, E-cadherin, and N-cadherin among SW480, COLO320DM, and HCT116 cell lines (p < 0.05). Despite no significant differences in cytoplasmic and nuclear β-catenin expressions between SW480 and HCT116 cell lines, and in E-cadherin and N-cadherin expressions between COLO320DM and HCT116 cell lines (p > 0.05). SW480 cell line has a higher expression of nuclear β-catenin than the cytoplasm (p < 0.05). CONCLUSION: This study reveals differences in the expression of nucleic and cytoplasmic β-catenin, E-cadherin, and N-cadherin in three stages of CRC (Duke B, C, and D) refer to different activation invasion, migration, and metastatic processes. Furthermore, the high expression of nuclear β-catenin and N-cadherin in the early stage of CRC indicate there is a metastatic process in that stage, so nuclear β-catenin and cadherin can be considered as potential biomarkers in the early stage of this cancer.

Discovery of a Novel Triazolopyridine Derivative as a Tankyrase Inhibitor.

More than 80% of colorectal cancer patients have adenomatous polyposis coli (APC) mutations, which induce abnormal WNT/β-catenin activation. Tankyrase (TNKS) mediates the release of active β-catenin, which occurs regardless of the ligand that translocates into the nucleus by AXIN degradation via the ubiquitin-proteasome pathway. Therefore, TNKS inhibition has emerged as an attractive strategy for cancer therapy. In this study, we identified pyridine derivatives by evaluating in vitro TNKS enzyme activity and investigated N-([1,2,4]triazolo[4,3-a]pyridin-3-yl)-1-(2-cyanophenyl)piperidine-4-carboxamide (TI-12403) as a novel TNKS inhibitor. TI-12403 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM and DLD-1 cells. The antitumor activities of TI-12403 were confirmed by the viability of the colorectal cancer cells and its lack of visible toxicity in DLD-1 xenograft mouse model. In addition, combined 5-FU and TI-12403 treatment synergistically inhibited proliferation to a greater extent than that in a single drug treatment. Our observations suggest that TI-12403, a novel selective TNKS1 inhibitor, may be a suitable compound for anticancer drug development.

Abstract 964: β-catenin antagonist peptide attenuates Wnt-dependent oncogenic activity

Abstract β-catenin is an oncogenic transcription factor that has an essential role in cell development and biology. Increased β-catenin expression or activity has been implicated in many cancers, including breast and colorectal, and is associated with poor prognosis. Despite extensive ongoing research, β-catenin is conventionally considered ‘undruggable', and there are no current drugs available to target Wnt/β-catenin signaling. B-cell CLL/lymphoma 9 protein (BCL9) is a positive regulator of β-catenin transcriptional activity that is overexpressed in tumors. Disruption of β-catenin interaction with BCL9 has been shown genetically to suppress oncogenic Wnt/β-catenin transcription and represents a powerful approach to inhibit this previously undruggable target. Here, we describe the anti-tumor activity of a β-catenin antagonist peptide (BCAP), designed to disrupt the association of β-catenin with co-activator BCL9. Target engagement of BCAP with recombinant β-catenin was demonstrated in biophysical studies by circular dichroism spectroscopy, and inhibition of the β-catenin/BCL9 interaction was demonstrated in transiently transfected A549 cells by NanoBRET assay. Administration of BCAP to HEK293 cells stably transfected with TOPFlash reporter plasmid resulted in dose-dependent inhibition with an EC50 of 1.7 µM. Further, BCAP attenuated oncogenic gene transactivation in colorectal cancer cells (HCT116 and COLO320DM) and HRPOS breast cancer cells (MCF7), significantly decreasing the expression of Axin2 and CDK4 (p<0.05), while not impacting Axin2 or CDK4 gene expression in Wnt-independent RKO and NCI-H460 cells. Administration of BCAP to 4T1-luc triple-negative breast cancer, HCT116 and COLO320DM cells resulted in dose-dependent in vitro cytotoxicity 48 hours post exposure, with EC50 values of 1.4, 4.4 and 1.2 µM, respectively. In in vivo orthotopic 4T1-luc tumor models, administration of BCAP via subcutaneous injection resulted in dose-dependent inhibition of tumor growth and significantly reduced tumor volume compared to control tumors (p<0.05). These data demonstrate the significant in vitro and in vivo anti-cancer activity of BCAP and emphasize it's potential as a potent therapeutic for Wnt/β-catenin-dependent cancers. Citation Format: Jim A. Rotolo, Erin Gallagher, Lila Ghamsari, Siok Leong, Ricardo Ramirez, Mark Koester, Gene Merutka, Barry J. Kappel. β-catenin antagonist peptide attenuates Wnt-dependent oncogenic activity [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 964.

In vitro Assessment of Cytotoxic Activity of Bioactive Peptides from Momordica dioica and Solanum trilobatum against Human Colon Cancer Cells

Plant peptides have gained attention in the medicinal field due to their high anti-microbial and anti-cancer properties. Various plant sources are being used to extract proteins and peptides to be used as a cure for a variety of diseases. The latest studies show that plant peptides are effective in the treatment of cancer due to their ability to preferentially bind to the receptors or membranes of cancer cells leading to tumour growth inhibition, cytotoxicity, decreased proliferation, and apoptosis. The peptides get internalized into the cells causing cancer cell agglutination and aggregation. In addition to acting as therapeutic agents, plant peptides are also used for the targeting of drugs specific to cancer cells. In this study, bioactive peptides were isolated from the seeds of Momordica dioica and leaves of Solanum trilobatum. They were screened and identified using HPLC and MALDI-TOF techniques. In this study, apoptosis was analyzed by the Hoechst 33342 staining method which detected the presence of condensed pycnotic nuclei in apoptotic COLO320DM and COLO205 colon cancer cellsat the maximum concentrations of 150 and 175 µg/mL of plant peptides.Further DCFH-DA staining indicated the intracellular ROS production in the treated COLO320DM and COLO205 colon cancer cells. Thus, the isolated bioactive plant peptides can be formulated towards the development of effective anticancer drugs for the treatment of colon cancer in humans.

In vitro targeting and selective killing of mcf-7 and colo320dm cells by 5-fluorouracil anchored to carboxylated SWCNTs and MWCNTs

The intention of the present work was to synthesize the f-MWCNT and f-SWCNT terminated with proper functional group, loading of 5-Flurouracil and to perform cytotoxic activity. Functionalization of MWCNTs and SWCNTs was achieved through the acid treatment (H2SO4 + HNO3). 5-flurouracil was loaded into the prepared functionalized CNTs, thereafter; in vitro drug loading capacity and % drug release were calculated. Also the prepared f-CNTs, 5-flurouracil loaded CNTs were distinguished by using SEM, TGA, DSC, X-ray diffraction, Raman and FTIR spectroscopy. MCF-7 and COLO320DM cells were treated with selected concentrations of 5-FU loaded f-MWCNTs and f-SWCNTs to estimate the cytotoxic activity. It was observed that 5-FU loaded f-SWCNTs showed good activity against selected cell lines than others. Moreover, apoptosis percentage was reported to be 84.46 ± 4.3515 and 92.78 ± 2.6549 for 5-FU loaded f-SWCNTs against MCF-7 and COLO320DM cells respectively. It is evident from the results that the prepared drug loaded CNTs have comparable antitumor activity in cancer cell lines.

Neratinib reduces CytochromP450 3A enzymes in mouse intestine

Neratinib (NERLYNX®) is a pan-HER tyrosine kinase inhibitor newly approved by FDA in 2017 and post to European market in 2018 to treat HER2-positive breast cancer, but the approval of neratinib has been controversial. Previous retrospective analysis showed that neratinib did not harm patients' quality of life, while another cost-benefit analysis indicated that neratinib's severe side effect comprises its cost-effectiveness. The phase III trial of neratinib showed that 96% of the patients taking neratinib experienced diarrhea. However, very few mechanistic studies have done to explore neratinib-induced gastrointestinal (GI) toxicity. Hereby, we performed toxicity studies in mice to characterize the potential mechanism underlying this adverse effect. C57BL/6 J (female, 5-weeks old) mice were randomly separated into three groups A, B, C. Group A received vehicle; group B was orally dosed with 100mg/kg neratinib once daily for 18 days. Group C was dosed with 100mg/kg neratinib for 12 days and switched to vehicle later for 6 days. Intestine and liver were collected for further analysis. Caco2, HT29 and Colo320DM cells were treated with neratinib in vitro. Our results showed that 12 days treatment of neratinib caused persistent histological damage in mouse GI tract, including blunted villi in small intestine and inflammatory infiltrate in colon. The gene expression of Cyp3a11, the major enzyme metabolizing neratinib in mice was significantly reduced in small intestine. The gene expression of proinflammatory cytokines, TNF-α, IL-6 and IL-1β, increased throughout the GI tract. Such damages were not recovered after 6 days without neratinib treatment. In addition, in vitro data showed that neratinib was potent in killing human intestine derived cell lines but did not reduce CYP3A4 expression in those cells. Based on such findings, we concluded that neratinib triggered inflammatory response in gut. And the release of cytokines downregulated intestinal CYP3A. With lower level of CYP3A, there is likely neratinib accumulation. It eventually leads to high risk of diarrhea. Our findings give new insight into the mechanism of neratinib-induced GI toxicity.

Green synthesis of silver, iron and gold nanoparticles of lycopene extracted from tomato: their characterization and cytotoxicity against COLO320DM, HT29 and Hella cell

Our study aimed at development of Silver, Iron and Gold nanoparticles of Lycopene isolated from tomato by using green synthesis technique and to evaluate its anticancer potential against colorectal and cervical cancer. Lycopene was extracted by benzene extraction method and the silver, iron and gold nanoparticles were developed by green synthesis method. 1% aqueous extract of isolated Lycopene was mixed with 1% solutions of AgNO3, FeCl3 and HAuCl4 solutions and incubated at ambient temperature for 3–4 h separately and observed for the color change which is an indicative of formation of the nanoparticles. The prepared nanoparticles were characterized by FTIR, SEM, XRD analysis and evaluated for their antimicrobial potential. The cytotoxicity studies were carried out by in vitro assay like MTT, SRB and Tryphan blue method against Colo 320 DM, HT 29, and Hella. SEM showed nanosized particles of 50–100 nm range, whereas no antimicrobial activity was exhibited by the prepared nanoparticles. In MTT assay the LyAgNP showed maximum 41.41 ± 0.4124% inhibition against COLO320DM, whereas LyGNP exhibited 41.47 ± 0.4469% inhibition against HT 29 and LyAgNP showed 40.9 ± 0.6908% inhibition against Hella cells. In SRB assay LyAgNP showed maximum 82.68 ± 1.1798% inhibition against COLO320DM, whereas LyGNP exhibited maximum 91.21 ± 0.2372% inhibition against HT29 and 87.98 ± 0.5878% inhibition against Hella cells. In tryphan blue assay against COLO320DM, HT29 and Hella cells, the maximum inhibition exhibited by the prepared nanoparticles were observed as LyGNP 83.45 ± 0.4694%, LyAgNP 88.05 ± 0.1870% and LyAgNP65.47 ± 0.4766%. We conclude that the developed nanoparticles of Lycopene exhibited potential anticancer activity against Colorectal and cervical cancer cell as compared with pure Lycopene.